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Metal-organic frameworks (MOFs) are a class of porous, crystalline materials that have been systematically developed for a broad range of applications. Incorporation of two or more metals into a single crystalline phase to generate heterometallic MOFs has been shown to lead to synergistic effects, in which the whole is oftentimes greater than the sum of its parts. Because geometric proximity is typically required for metals to function cooperatively, deciphering and controlling metal distributions in heterometallic MOFs is crucial to establish structure-function relationships. However, determination of short- and long-range metal distributions is nontrivial and requires the use of specialized characterization techniques. Advancements in the characterization of metal distributions and interactions at these length scales is key to rapid advancement and rational design of functional heterometallic MOFs. This perspective summarizes the state-of-the-art in the characterization of heterometallic MOFs, with a focus on techniques that allow metal distributions to be better understood. Using complementary analyses, in conjunction with computational methods, is critical as this field moves toward increasingly complex, multifunctional systems.

Characterizing and identifying cells in multicellular in vitro models remain a substantial challenge. Here, we utilize hyperspectral confocal Raman microscopy and principal component analysis coupled with linear discriminant analysis to form a label-free, noninvasive approach for classifying bone cells and osteosarcoma cells. Through the development of a library of hyperspectral Raman images of the K7M2-wt osteosarcoma cell lines, 7F2 osteoblast cell lines, RAW 264.7 macrophage cell line, and osteoclasts induced from RAW 264.7 macrophages, we built a linear discriminant model capable of correctly identifying each of these cell types. The model was cross-validated using a k-fold cross validation scheme. The results show a minimum of 72% accuracy in predicting cell type. We also utilize the model to reconstruct the spectra of K7M2 and 7F2 to determine whether osteosarcoma cancer cells and normal osteoblasts have any prominent differences that can be captured by Raman. We find that the main differences between these two cell types are the prominence of the β-sheet protein secondary structure in K7M2 versus the α-helix protein secondary structure in 7F2. Additionally, differences in the CH 2 deformation Raman feature highlight that the membrane lipid structure is different between these cells, which may affect the overall signaling and functional contrasts. Overall, we show that hyperspectral confocal Raman microscopy can serve as an effective tool for label-free, nondestructive cellular classification and that the spectral reconstructions can be used to gain deeper insight into the differences that drive different functional outcomes of different cells., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Co-published by Nanjing University and American Chemical Society.)

Herein, we report the synthesis and characterization of a new class of hybrid Wells-Dawson polyoxometalate (POM) containing a diphosphoryl group (P 2 O 6 X) of the general formula [P 2 W 17 O 57 (P 2 O 6 X)] 6- (X=O, NH, or CR 1 R 2 ). Modifying the bridging unit X was found to impact the redox potentials of the POM. The ease with which a range of α-functionalized diphosphonic acids (X=CR 1 R 2 ) can be prepared provides possibilities to access diverse functionalized hybrid POMs. Compared to existing phosphonate hybrid Wells-Dawson POMs, diphosphoryl-substituted POMs offer a wider tunable redox window and enhanced hydrolytic stability. This study provides a basis for the rational design and synthesis of next-generation hybrid Wells-Dawson POMs., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Emerging and re-emerging viral pathogens present a unique challenge for anti-viral therapeutic development. Anti-viral approaches with high flexibility and rapid production times are essential for combating these high-pandemic risk viruses. CRISPR-Cas technologies have been extensively repurposed to treat a variety of diseases, with recent work expanding into potential applications against viral infections. However, delivery still presents a major challenge for these technologies. Lipid-coated mesoporous silica nanoparticles (LCMSNs) offer an attractive delivery vehicle for a variety of cargos due to their high biocompatibility, tractable synthesis, and amenability to chemical functionalization. Here, we report the use of LCMSNs to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) that target the Niemann-Pick disease type C1 gene, an essential host factor required for entry of the high-pandemic risk pathogen Ebola virus, demonstrating an efficient reduction in viral infection. We further highlight successful in vivo delivery of the RNP-LCMSN platform to the mouse liver via systemic administration., (© 2023. The Author(s).)

Lifetime-encoded materials are particularly attractive as optical tags, however examples are rare and hindered in practical application by complex interrogation methods. Here, we demonstrate a design strategy towards multiplexed, lifetime-encoded tags via engineering intermetallic energy transfer in a family of heterometallic rare-earth metal-organic frameworks (MOFs). The MOFs are derived from a combination of a high-energy donor (Eu), a low-energy acceptor (Yb) and an optically inactive ion (Gd) with the 1,2,4,5 tetrakis(4-carboxyphenyl) benzene (TCPB) organic linker. Precise manipulation of the luminescence decay dynamics over a wide microsecond regime is achieved via control over metal distribution in these systems. Demonstration of this platform's relevance as a tag is attained via a dynamic double encoding method that uses the braille alphabet, and by incorporation into photocurable inks patterned on glass and interrogated via digital high-speed imaging. This study reveals true orthogonality in encoding using independently variable lifetime and composition, and highlights the utility of this design strategy, combining facile synthesis and interrogation with complex optical properties., (© 2023. The Author(s).)

We postulate that nanoparticles (NPs) for use in therapeutic applications have largely not realized their clinical potential due to an overall inability to use in vitro results to predict NP performance in vivo . The avian embryo and associated chorioallantoic membrane (CAM) has emerged as an in vivo preclinical model that bridges the gap between in vitro and in vivo , enabling rapid screening of NP behavior under physiologically relevant conditions and providing a rapid, accessible, economical, and more ethical means of qualifying nanoparticles for in vivo use. The CAM is highly vascularized and mimics the diverging/converging vasculature of the liver, spleen, and lungs that serve as nanoparticle traps. Intravital imaging of fluorescently labeled NPs injected into the CAM vasculature enables immediate assessment and quantification of nano-bio interactions at the individual NP scale in any tissue of interest that is perfused with a microvasculature. In this review, we highlight how utilization of the avian embryo and its CAM as a preclinical model can be used to understand NP stability in blood and tissues, extravasation, biocompatibility, and NP distribution over time, thereby serving to identify a subset of NPs with the requisite stability and performance to introduce into rodent models and enabling the development of structure-property relationships and NP optimization without the sacrifice of large populations of mice or other rodents. We then review how the chicken embryo and CAM model systems have been used to accelerate the development of NP delivery and imaging agents by allowing direct visualization of targeted (active) and nontargeted (passive) NP binding, internalization, and cargo delivery to individual cells (of relevance for the treatment of leukemia and metastatic cancer) and cellular ensembles (e.g., cancer xenografts of interest for treatment or imaging of cancer tumors). We conclude by showcasing emerging techniques for the utilization of the CAM in future nano-bio studies.

For workplaces which cannot operate as telework or remotely, there is a critical need for routine occupational SARS-CoV-2 diagnostic testing. Although diagnostic tests including the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (CDC Diagnostic Panel) (EUA200001) were made available early in the pandemic, resource scarcity and high demand for reagents and equipment necessitated priority of symptomatic patients. There is a clearly defined need for flexible testing methodologies and strategies with rapid turnaround of results for (1) symptomatic, (2) asymptomatic with high-risk exposures and (3) asymptomatic populations without preexisting conditions for routine screening to address the needs of an on-site work force. We developed a distinct SARS-CoV-2 diagnostic assay based on the original CDC Diagnostic Panel (EUA200001), yet, with minimum overlap for currently employed reagents to eliminate direct competition for limited resources. As the pandemic progressed with testing loads increasing, we modified the assay to include 5-sample pooling and amplicon target multiplexing. Analytical sensitivity of the pooled and multiplexed assays was rigorously tested with contrived positive samples in realistic patient backgrounds. Assay performance was determined with clinical samples previously assessed with an FDA authorized assay. Throughout the pandemic we successfully tested symptomatic, known contact and travelers within our occupational population with a ~ 24-48-h turnaround time to limit the spread of COVID-19 in the workplace. Our singleplex assay had a detection limit of 31.25 copies per reaction. The three-color multiplexed assay maintained similar sensitivity to the singleplex assay, while tripling the throughput. The pooling assay further increased the throughput to five-fold the singleplex assay, albeit with a subtle loss of sensitivity. We subsequently developed a hybrid 'multiplex-pooled' strategy to testing to address the need for both rapid analysis of samples from personnel at high risk of COVID infection and routine screening. Herein, our SARS-CoV-2 assays specifically address the needs of occupational healthcare for both rapid analysis of personnel at high-risk of infection and routine screening that is essential for controlling COVID-19 disease transmission. In addition to SARS-CoV-2 and COVID-19, this work demonstrates successful flexible assays developments and deployments with implications for emerging highly transmissible diseases and future pandemics., (© 2022. The Author(s).)

A rapid and facile design strategy to create a highly complex optical tag with programmable, multimodal photoluminescent properties is described. This was achieved via intrinsic and DNA-fluorophore hidden signatures. As a first covert feature of the tag, an intricate novel heterometallic near-infrared (NIR)-emitting mesoporous metal-organic framework (MOF) was designed and synthesized. The material is constructed from two chemically distinct, homometallic hexanuclear clusters based on Nd and Yb. Uniquely, the Nd-based cluster is observed here for the first time in a MOF and consists of two staggered Nd μ 3 -oxo trimers. To generate controlled, multimodal, and tailorable emission with difficult to counterfeit features, the NIR-emissive MOF was post-synthetically modified via a fluorescent DNA oligo labeling design strategy. The surface attachment of several distinct fluorophores, including the simultaneous attachment of up to three distinct fluorescently labeled oligos was achieved, with excitation and emission properties across the visible spectrum (480-800 nm). The DNA inclusion as a secondary covert element in the tag was demonstrated via the detection of SYBR Gold dye association. Importantly, the approach implemented here serves as a rapid and tailorable way to encrypt distinct information in a facile and modular fashion and provides an innovative technology in the quest toward complex optical tags.

Optical anticounterfeiting tags utilize the photoluminescent properties of materials to encode unique patterns, enabling identification and validation of important items and assets. These tags must combine optical complexity with ease of production and authentication to both prevent counterfeiting and to remain practical for widespread use. Metal-organic frameworks (MOFs) based on polynuclear, rare earth clusters are ideal materials platforms for this purpose, combining fine control over structure and composition, with tunable, complex energy transfer mechanisms via both linker and metal components. Here we report the design and synthesis of a set of heterometallic MOFs based on combinations of Eu, Nd, and Yb with the tetratopic linker 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene. The energetics of this linker facilitate the intentional concealment of the visible emissions from Eu while retaining the infrared emissions of Nd and Yb, creating an optical tag with multiple covert elements. Unique to the materials system reported herein, we document the occurrence of a previously not observed 11-metal cluster correlated with the presence of Yb in the MOFs, coexisting with a commonly encountered 9-metal cluster. We demonstrate the utility of these materials as intricate optical tags with both rapid and in-depth screening techniques, utilizing orthogonal identifiers across composition, emission spectra, and emission decay dynamics. This work highlights the important effect of linker selection in controlling the resulting photoluminescent properties in MOFs and opens an avenue for the targeted design of highly complex, multifunctional optical tags.

Mesenchymal stromal cells (MSCs) have broad-ranging therapeutic properties, including the ability to inhibit bacterial growth and resolve infection. However, the genetic mechanisms regulating these antibacterial properties in MSCs are largely unknown. Here, we utilized a systems-based approach to compare MSCs from different genetic backgrounds that displayed differences in antibacterial activity. Although both MSCs satisfied traditional MSC-defining criteria, comparative transcriptomics and quantitative membrane proteomics revealed two unique molecular profiles. The antibacterial MSCs responded rapidly to bacterial lipopolysaccharide (LPS) and had elevated levels of the LPS co-receptor CD14. CRISPR-mediated overexpression of endogenous CD14 in MSCs resulted in faster LPS response and enhanced antibacterial activity. Single-cell RNA sequencing of CD14-upregulated MSCs revealed a shift in transcriptional ground state and a more uniform LPS-induced response. Our results highlight the impact of genetic background on MSC phenotypic diversity and demonstrate that overexpression of CD14 can prime these cells to be more responsive to bacterial challenge., Competing Interests: The authors do not have any competing interests to report., (© 2022 The Authors.)

Covering: 2016 to 2021With genetic information available for hundreds of thousands of organisms in publicly accessible databases, scientists have an unprecedented opportunity to meticulously survey the diversity and inner workings of life. The natural product research community has harnessed this breadth of sequence information to mine microbes, plants, and animals for biosynthetic enzymes capable of producing bioactive compounds. Several orthogonal genome mining strategies have been developed in recent years to target specific chemical features or biological properties of bioactive molecules using biosynthetic, resistance, or transporter proteins. These "biosynthetic hooks" allow researchers to query for biosynthetic gene clusters with a high probability of encoding previously undiscovered, bioactive compounds. This review highlights recent case studies that feature orthogonal approaches that exploit genomic information to specifically discover bioactive natural products and their gene clusters.

Owing to their facile synthesis, tailorable porosity, diverse compositions, and low toxicity, zeolitic imidazolate framework (ZIF) nanoparticles (NPs) have emerged as attractive platforms for a variety of biologically relevant applications. To date, a small subset of ZIFs representing only two topologies and very few linker chemistries have been studied in this realm. We seek to expand the bio-design space for ZIF NPs through the targeted synthesis of a hierarchically complex ZIF based on two types of cages, ZIF-20, with lta topology. This study demonstrates the rapid synthesis and size tunability of ZIF-20 particles across the micro and nanoregimes via microwave heating and the use of a modulating agent. To evaluate the utility of ZIF particles for biological applications, we examine their stability in biologically relevant media and demonstrate biocompatibility with A549 human epithelial cells. Further, the ability to encapsulate and release methylene blue, a therapeutic and bioimaging agent, is validated. Importantly, ZIF-20 NPs display a unique behavior relative to previously studied ZIFs based on their specific structural and chemical features. This finding highlights the need to expand the design space across the broader ZIFs family, to exploit a wider range of relevant properties for biological applications and beyond.

Optical tags provide a way to quickly and unambiguously identify valuable assets. Current tag fluorophore options lack the tunability to allow combined methods of encoding in a single material. Herein we report a design strategy to encode multilayer complexity in a family of heterometallic rare-earth metal-organic frameworks based on highly connected nonanuclear clusters. To impart both intricacy and security, a synergistic approach was implemented resulting in both overt (visible) and covert (near-infrared, NIR) properties, with concomitant multi-emissive spectra and tunable luminescence lifetimes. Tag authentication is validated with a variety of orthogonal detection methodologies. Importantly, the effect induced by subtle compositional changes on intermetallic energy transfer, and thus on the resulting photophysical properties, is demonstrated. This strategy can be widely implemented to create a large library of highly complex, difficult-to-counterfeit optical tags., (© 2020 Wiley-VCH GmbH.)

Competing Interests: The authors declare no competing interest.

CRISPR gene editing technology is strategically foreseen to control diseases by correcting underlying aberrant genetic sequences. In order to overcome drawbacks associated with viral vectors, the establishment of an effective non-viral CRISPR delivery vehicle has become an important goal for nanomaterial scientists. Herein, we introduce a monosized lipid-coated mesoporous silica nanoparticle (LC-MSN) delivery vehicle that enables both loading of CRISPR components [145 µg ribonucleoprotein (RNP) or 40 µg plasmid/mg nanoparticles] and efficient release within cancer cells (70%). The RNP-loaded LC-MSN exhibited 10% gene editing in both in vitro reporter cancer cell lines and in an in vivo Ai9-tdTomato reporter mouse model. The structural and chemical versatility of the mesoporous silica core and lipid coating along with framework dissolution-assisted cargo delivery open new prospects towards safe CRISPR component delivery and enhanced gene editing. STATEMENT OF SIGNIFICANCE: After the discovery of CRISPR gene-correcting technology in bacteria. The translation of this technology to mammalian cells may change the face of cancer therapy within the next years. This was first made possible through the use of viral vectors; however, such systems limit the safe translation of CRISPR into clinics because its difficult preparation and immunogenicity. Therefore, biocompatible non-viral nanoparticulate systems are required to successfully deliver CRISPR into cancer cells. The present study presents the use of biomimetic lipid-coated mesoporous silica nanoparticles showing successful delivery of CRISPR ribonucleoprotein and plasmid into HeLa cervical and A549 lung cancer cells as well as successful gene editing in mice brain., Competing Interests: Declaration of Competing Interest No conflict of interest to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The design and synthesis of artificial materials that mimic the structures, mechanical properties, and ultimately functionalities of biological cells remains a current holy grail of materials science. Here, based on a silica cell bioreplication approach, we report the design and construction of synthetic rebuilt red blood cells (RRBCs) that fully mimic the broad properties of native RBCs: size, biconcave shape, deformability, oxygen-carrying capacity, and long circulation time. Four successive nanoscale processing steps (RBC bioreplication, layer-by-layer polymer deposition, and precision silica etching, followed by RBC ghost membrane vesicle fusion) are employed for RRBC construction. A panel of physicochemical analyses including zeta-potential measurement, fluorescence microscopy, and antibody-mediated agglutination assay proved the recapitulation of RBC shape, size, and membrane structure. Flow-based deformation studies carried out in a microfluidic blood capillary model confirmed the ability of RRBCs to deform and pass through small slits and reconstitute themselves in a manner comparable to native RBCs. Circulation studies of RRBCs conducted ex ovo in a chick embryo and in vivo in a mouse model demonstrated the requirement of both deformability and native cell membrane surface to achieve long-term circulation. To confer additional non-native functionalities to RRBCs, we developed modular procedures with which to load functional cargos such as hemoglobin, drugs, magnetic nanoparticles, and ATP biosensors within the RRBC interior to enable various functions, including oxygen delivery, therapeutic drug delivery, magnetic manipulation, and toxin biosensing and detection. Taken together, RRBCs represent a class of long-circulating RBC-inspired artificial hybrid materials with a broad range of potential applications.

We report on the availability and chemical utility of primary amines within metal-organic frameworks (MOFs) for cell targeting. Primary amine groups represent one of the most versatile chemical moieties for conjugation to biologically relevant molecules, including antibodies and enzymes. Specifically, we used two different chemical conjugations schemes, utilizing the amino functionality on the organic linker: first, carbodiimide chemistry was used to link the primary amine to available carboxyl groups on the protein neutravidin; second, sulfhydryl cross-linking chemistry was used via Traut's reagent scheme. Importantly, this is the first report that documents this methodology implemented with MOF systems. Finally, the ability of the EpCAM antibody targeted MOFs to bind to a human epithelial cell line (A549), a common target for imaging studies, was confirmed with confocal microscopy.

Blood flowing through microvascular bifurcations has been an active research topic for many decades, while the partitioning pattern of nanoscale solutes in the blood remains relatively unexplored. Here we demonstrate a multiscale computational framework for direct numerical simulation of the nanoparticle (NP) partitioning through physiologically relevant vascular bifurcations in the presence of red blood cells (RBCs). The computational framework is established by embedding a particulate suspension inflow-outflow boundary condition into a multiscale blood flow solver. The computational framework is verified by recovering a tubular blood flow without a bifurcation and validated against the experimental measurement of an intravital bifurcation flow. The classic Zweifach-Fung (ZF) effect is shown to be well captured by the method. Moreover, we observe that NPs exhibit a ZF-like heterogeneous partition in response to the heterogeneous partition of the RBC phase. The NP partitioning prioritizes the high-flow-rate daughter branch except for extreme (large or small) suspension flow partition ratios under which the complete phase separation tends to occur. By analyzing the flow field and the particle trajectories, we show that the ZF-like heterogeneity in the NP partition can be explained by the RBC-entrainment effect caused by the deviation of the flow separatrix preceded by the tank treading of RBCs near the bifurcation junction. The recovery of homogeneity in the NP partition under extreme flow partition ratios is due to the plasma skimming of NPs in the cell-free layer. These findings, based on the multiscale computational framework, provide biophysical insights to the heterogeneous distribution of NPs in microvascular beds that are observed pathophysiologically.

Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application., (© 2020 The Author(s).)

Long-term antimicrobial therapies are necessary to treat infections caused by virulent intracellular pathogens, including biothreat agents. Current treatment plans include injectable therapeutics given multiple times daily over a period for up to 8 weeks. Here, we present a metal-organic framework (MOF), zeolitic imidazolate framework-8 (ZIF-8), as a robust platform to support the sustained release of ceftazidime, an important antimicrobial agent for many critical bacterial infections. Detailed material characterization confirms the successful encapsulation of ceftazidime within the ZIF-8 matrix, indicating sustained drug release for up to a week. The antibacterial properties of ceftazidime@ZIF-8 particles were confirmed against Escherichia coli, chosen here as a representative of Gram-negative bacteria infection model in a proof-of-concept study. Further, we showed that this material system is compatible with macrophage and lung epithelial cell lines, relevant targets for antibacterial therapy for pulmonary and intracellular infections. A promising methodology to enhance the treatment of intracellular infections is to deliver the antibiotic cargo intracellularly. Importantly, this is the first study to unequivocally demonstrate direct MOF particle internalization using confocal microscopy via 3D reconstructions of z-stacks, taking advantage of the intrinsic emission properties of ZIF-8. This is an important development as it circumvents the need to use any staining dyes and addresses current methodology limitations concerning false impression of cargo uptake in the event of the carrier particle breakdown within biological media.

The progress of nanoparticle (NP)-based drug delivery has been hindered by an inability to establish structure-activity relationships in vivo. Here, using stable, monosized, radiolabeled, mesoporous silica nanoparticles (MSNs), we apply an integrated SPECT/CT imaging and mathematical modeling approach to understand the combined effects of MSN size, surface chemistry and routes of administration on biodistribution and clearance kinetics in healthy rats. We show that increased particle size from ~32- to ~142-nm results in a monotonic decrease in systemic bioavailability, irrespective of route of administration, with corresponding accumulation in liver and spleen. Cationic MSNs with surface exposed amines (PEI) have reduced circulation, compared to MSNs of identical size and charge but with shielded amines (QA), due to rapid sequestration into liver and spleen. However, QA show greater total excretion than PEI and their size-matched neutral counterparts (TMS). Overall, we provide important predictive functional correlations to support the rational design of nanomedicines.

Herein, we describe a novel multifunctional metal-organic framework (MOF) materials platform that displays both porosity and tunable emission properties as a function of the metal identity (Eu, Nd, and tuned compositions of Nd/Yb). Their emission collectively spans the deep red to near-infrared (NIR) spectral region (∼614-1350 nm), which is highly relevant for in vivo bioimaging. These new materials meet important prerequisites as relevant to biological processes: they are minimally toxic to living cells and retain structural integrity in water and phosphate-buffered saline. To assess their viability as optical bioimaging agents, we successfully synthesized the nanoscale Eu analog as a proof-of-concept system in this series. In vitro studies show that it is cell-permeable in individual RAW 264.7 mouse macrophage and HeLa human cervical cancer tissue culture cells. The efficient discrimination between the Eu emission and cell autofluorescence was achieved with hyperspectral confocal fluorescence microscopy, used here for the first time to characterize MOF materials. Importantly, this is the first report that documents the long-term conservation of the intrinsic emission in live cells of a fluorophore-based MOF to date (up to 48 h). This finding, in conjunction with the materials' very low toxicity, validates the biocompatibility in these systems and qualifies them as promising for use in long-term tracking and biodistribution studies.

Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentially overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. The modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Many nanocarrier cancer therapeutics currently under development, as well as those used in the clinical setting, rely upon the enhanced permeability and retention (EPR) effect to passively accumulate in the tumor microenvironment and kill cancer cells. In leukemia, where leukemogenic stem cells and their progeny circulate within the peripheral blood or bone marrow, the EPR effect may not be operative. Thus, for leukemia therapeutics, it is essential to target and bind individual circulating cells. Here, we investigate mesoporous silica nanoparticle (MSN)-supported lipid bilayers (protocells), an emerging class of nanocarriers, and establish the synthesis conditions and lipid bilayer composition needed to achieve highly monodisperse protocells that remain stable in complex media as assessed in vitro by dynamic light scattering and cryo-electron microscopy and ex ovo by direct imaging within a chick chorioallantoic membrane (CAM) model. We show that for vesicle fusion conditions where the lipid surface area exceeds the external surface area of the MSN and the ionic strength exceeds 20 mM, we form monosized protocells (polydispersity index <0.1) on MSN cores with varying size, shape, and pore size, whose conformal zwitterionic supported lipid bilayer confers excellent stability as judged by circulation in the CAM and minimal opsonization in vivo in a mouse model. Having established protocell formulations that are stable colloids, we further modified them with anti-EGFR antibodies as targeting agents and reverified their monodispersity and stability. Then, using intravital imaging in the CAM, we directly observed in real time the progression of selective targeting of individual leukemia cells (using the established REH leukemia cell line transduced with EGFR) and delivery of a model cargo. Overall, we have established the effectiveness of the protocell platform for individual cell targeting and delivery needed for leukemia and other disseminated disease.

Mesoporous silica nanoparticle-supported lipid bilayers, termed 'protocells,' represent a potentially transformative class of therapeutic and theranostic delivery vehicle. The field of targeted drug delivery poses considerable challenges that cannot be addressed with a single 'magic bullet'. Consequently, the protocell has been designed as a modular platform composed of interchangeable biocompatible components. The mesoporous silica core has variable size and shape to direct biodistribution and a controlled pore size and surface chemistry to accommodate diverse cargo. The encapsulating supported lipid bilayer can be modified with targeting and trafficking ligands as well as polyethylene glycol (PEG) to effect selective binding, endosomal escape of cargo, drug efflux prevention, and potent therapeutic delivery, while maintaining in vivo colloidal stability. This review describes the individual components of the platform, including the mesoporous silica nanoparticle core and supported lipid bilayer, their assembly (by multiple techniques) into a protocell, and the combined, often synergistic, performance of the protocell based on in vitro and in vivo studies, including the assessment of biocompatibility and toxicity. In closing, the many emerging variations of the protocell theme and the future directions for protocell research are commented on., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Next-Generation Sequencing is a rapidly advancing technology that has research and clinical applications. For many cancers, it is important to know the precise mutation(s) present, as specific mutations could indicate or contra-indicate certain treatments as well as be indicative of prognosis. Using the Ion Torrent Personal Genome Machine and the AmpliSeq Cancer Hotspot panel v2, we sequenced two pancreatic cancer cell lines, BxPC-3 and HPAF-II, alone or in mixtures, to determine the error rate, sensitivity, and reproducibility of this system. The system resulted in coverage averaging 2000× across the various amplicons and was able to reliably and reproducibly identify mutations present at a rate of 5%. Identification of mutations present at a lower rate was possible by altering the parameters by which calls were made, but with an increase in erroneous, low-level calls. The panel was able to identify known mutations in these cell lines that are present in the COSMIC database. In addition, other, novel mutations were also identified that may prove clinically useful. The system was assessed for systematic errors such as homopolymer effects, end of amplicon effects and patterns in NO CALL sequence. Overall, the system is adequate at identifying the known, targeted mutations in the panel., (Published by Elsevier Inc.)

The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage. AgNPs of all sizes tested (10, 20, 50 and 100nm), along with silver nitrate (AgNO3), were negative for mutagenicity in bacteria. No AgNPs could be identified within the bacteria cells using transmission electron microscopy (TEM), indicating these bacteria lack the ability to actively uptake AgNPs 10nm or larger. Clastogenicity (flow cytometry-based micronucleus assay) and intermediate DNA damage (DNA strand breaks as measured in the Comet assay) were assessed in two mammalian white blood cell lines: Jurkat Clone E6-1 and THP-1. It was observed that micronucleus and Comet assay end points were inversely correlated with AgNP size, with smaller NPs inducing a more genotoxic response. TEM results indicated that AgNPs were confined within intracellular vesicles of mammalian cells and did not penetrate the nucleus. The genotoxicity test results and the effect of AgNO3 controls suggest that silver ions may be the primary, and perhaps only, cause of genotoxicity. Furthermore, since AgNO3 was not mutagenic in the gram-negative bacterial Ames strains tested, the lack of bacterial uptake of the AgNPs may not be the major reason for the lack of genotoxicity observed., (Published by Oxford University Press on behalf of the UK Environmental Mutagen Society 2015.)

As new magnetic nanoparticle-based technologies are developed and new target cells are identified, there is a critical need to understand the features important for magnetic isolation of specific cells in fluids, an increasingly important tool in disease research and diagnosis. To investigate magnetic cell collection, cell-sized spherical microparticles, coated with superparamagnetic nanoparticles, were suspended in (1) glycerine-water solutions, chosen to approximate the range of viscosities of bone marrow, and (2) water in which 3, 5, 10 and 100% of the total suspended microspheres are coated with magnetic nanoparticles, to model collection of rare magnetic nanoparticle-coated cells from a mixture of cells in a fluid. The magnetic microspheres were collected on a magnetic needle, and we demonstrate that the collection efficiency versus time can be modeled using a simple, heuristically-derived function, with three physically-significant parameters. The function enables experimentally-obtained collection efficiencies to be scaled to extract the effective drag of the suspending medium. The results of this analysis demonstrate that the effective drag scales linearly with fluid viscosity, as expected. Surprisingly, increasing the number of non-magnetic microspheres in the suspending fluid results increases the collection of magnetic microspheres, corresponding to a decrease in the effective drag of the medium.

Due to their unique properties, the use of nanoparticles (NPs) is expanding; these same properties may affect their potential risk to humans. However, standard methods for genotoxicity assessment may not be adequate for NPs; altered tests reported here have been developed to address perceived inadequacies. The bacterial reverse mutation assay is an essential part of the battery of tests to determine genotoxicity. The utility of this test for assessing NPs is currently questioned, due to negative results seemingly caused by failure of particle uptake. To probe uptake issues, we examined the physical state in different media, dose and time dependent association, uptake and mutagenicity of titanium dioxide (TiO2) NPs in Salmonella typhimurium and Escherichia coli. The NPs suspended in water were characterized using dynamic light scattering, NP tracking analysis and transmission electron microscopy. NP association with bacteria was assessed by flow cytometry. Association was found to be time and dose dependent, with maximal association by 60 min. Therefore mutagenicity was assessed after a 60 min pre-incubation in a miniaturized assay demonstrating enhanced sensitivity. To assess potential indirect effects on bacterial mutagenicity, the effect of TiO2 NPs on the action of standard mutagens or on DNA repair capability was also investigated. TiO2 NPs did not affect mutant yields in standard strains of S. typhimurium or E. coli, including those detecting oxidative damage, using the modified methods. Nor did TiO2 NPs affect the action of standard mutagens or DNA excision repair capability. Despite particle association with the bacteria, subsequent analysis using electron microscopy and energy dispersive x-ray spectroscopy indicated that the NPs were not internalized. This work demonstrates that additional studies, including flow cytometry, are valuable tools for understanding the action of NPs in biological systems., (Published by Elsevier B.V.)

Organ transplantation is a life-saving procedure and the preferred method of treatment for a growing number of disease states. The advent of new immunosuppressants and improved care has led to great advances in both patient and graft survival. However, acute T-cell-mediated graft rejection occurs in a significant quantity of recipients and remains a life-threatening condition. Acute rejection is associated with decrease in long-term graft survival, demonstrating a need to carefully monitor transplant patients. Current diagnostic criteria for transplant rejection rely on invasive tissue biopsies or relatively nonspecific clinical features. A noninvasive way is needed to detect, localize, and monitor transplant rejection. Capitalizing on advances in targeted contrast agents and magnetic-based detection technology, we developed anti-CD3 antibody-tagged nanoparticles. T cells were found to bind preferentially to antibody-tagged nanoparticles, as identified through light microscopy, transmission electron microscopy, and confocal microscopy. Using mouse skin graft models, we were also able to demonstrate in vivo vascular delivery of T-cell targeted nanoparticles. We conclude that targeting lymphocytes with magnetic nanoparticles is conducive to developing a novel, noninvasive strategy for identifying transplant rejection.

Telomere dysregulation occurs in both the in situ and invasive stages of many carcinomas, including breast. Knockout experiments have identified several telomere-associated proteins required for proper telomere function and maintenance, including telomere repeat-binding factor 1 and 2 (TRF1 and TRF2), protection of telomeres (POT1), and TRF1-interacting nuclear factor 2 (TIN2). Using telomere content assays and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we examined the relationship between telomere length and the mRNA levels of telomere-associated proteins in breast tumors. The levels of TRF2, TRF1, TIN2, and POT1 mRNA, but not telomerase reverse transcriptase (TERT) RNA, are inversely correlated with telomere content in breast tumors. Significant associations were identified between the mRNA levels of TRF1, TIN2, and POT1; however, there were no significant associations with the mRNA levels of TRF2 or TERT. These associations suggest that a complex transcriptional program coordinately regulates the expression of these mRNAs. We examined the promoter regions of the telomere-associated proteins to identify transcription factors consistent with the observed patterns of presumed coordinate expression. We demonstrated in human breast cancer cell lines that expressions of TRF1, TIN2, and POT1 are upregulated by dexamethasone, suggesting activation of the glucocorticoid receptor, whereas TERT, TRF2, TRF1, TIN2, and POT1 are upregulated by tumor necrosis factor-α (TNF-α), suggesting activation of the nuclear factor kappa B transcription factor. These findings link telomere content in breast tumors to the coordinate expression of several telomere-associated proteins previously shown to be negative regulators of telomere length in cell lines. The results further suggest a possible link between the expressions of the telomere-associated proteins and mediators of stress and inflammation.Telomere content assays and quantitative RT-PCR demonstrate that the levels of TRF2, TRF1, TIN2, and POT1 mRNA, but not telomerase reverse transcriptase (TERT) RNA, are inversely correlated with telomere content in breast tumors. Within human breast cancer cell lines, expressions of TRF1, TIN2, and POT1 are upregulated by dexamethasone, suggesting activation of the glucocorticoid receptor, whereas TERT, TRF2, TRF1, TIN2, and POT1 are upregulated by TNF-α, suggesting activation of the NFκB transcription factor. These findings link telomere content in breast tumors to the expression of several telomere-associated proteins previously shown to be negative regulators of telomere length in cell lines and suggest a link between the expressions of the telomere-associated proteins and mediators of stress and inflammation.

Both magnetic relaxometry and magnetic resonance imaging (MRI) can be used to detect and locate targeted magnetic nanoparticles, noninvasively and without ionizing radiation. Magnetic relaxometry offers advantages in terms of its specificity (only nanoparticles are detected) and the linear dependence of the relaxometry signal on the number of nanoparticles present. In this study, detection of single-core iron oxide nanoparticles by superconducting quantum interference device (SQUID)-detected magnetic relaxometry and standard 4.7 T MRI are compared. The nanoparticles were conjugated to a Her2 monoclonal antibody and targeted to Her2-expressing MCF7/Her2-18 (breast cancer cells); binding of the nanoparticles to the cells was assessed by magnetic relaxometry and iron assay. The same nanoparticle-labeled cells, serially diluted, were used to assess the detection limits and MR relaxivities. The detection limit of magnetic relaxometry was 125 000 nanoparticle-labeled cells at 3 cm from the SQUID sensors. T(2)-weighted MRI yielded a detection limit of 15 600 cells in a 150 µl volume, with r(1) = 1.1 mm(-1) s(-1) and r(2) = 166 mm(-1) s(-1). Her2-targeted nanoparticles were directly injected into xenograft MCF7/Her2-18 tumors in nude mice, and magnetic relaxometry imaging and 4.7 T MRI were performed, enabling direct comparison of the two techniques. Co-registration of relaxometry images and MRI of mice resulted in good agreement. A method for obtaining accurate quantification of microgram quantities of iron in the tumors and liver by relaxometry was also demonstrated. These results demonstrate the potential of SQUID-detected magnetic relaxometry imaging for the specific detection of breast cancer and the monitoring of magnetic nanoparticle-based therapies., (Copyright © 2012 John Wiley & Sons, Ltd.)

Introduction: Breast cancer detection using mammography has improved clinical outcomes for many women, because mammography can detect very small (5 mm) tumors early in the course of the disease. However, mammography fails to detect 10 - 25% of tumors, and the results do not distinguish benign and malignant tumors. Reducing the false positive rate, even by a modest 10%, while improving the sensitivity, will lead to improved screening, and is a desirable and attainable goal. The emerging application of magnetic relaxometry, in particular using superconducting quantum interference device (SQUID) sensors, is fast and potentially more specific than mammography because it is designed to detect tumor-targeted iron oxide magnetic nanoparticles. Furthermore, magnetic relaxometry is theoretically more specific than MRI detection, because only target-bound nanoparticles are detected. Our group is developing antibody-conjugated magnetic nanoparticles targeted to breast cancer cells that can be detected using magnetic relaxometry., Methods: To accomplish this, we identified a series of breast cancer cell lines expressing varying levels of the plasma membrane-expressed human epidermal growth factor-like receptor 2 (Her2) by flow cytometry. Anti-Her2 antibody was then conjugated to superparamagnetic iron oxide nanoparticles using the carbodiimide method. Labeled nanoparticles were incubated with breast cancer cell lines and visualized by confocal microscopy, Prussian blue histochemistry, and magnetic relaxometry., Results: We demonstrated a time- and antigen concentration-dependent increase in the number of antibody-conjugated nanoparticles bound to cells. Next, anti Her2-conjugated nanoparticles injected into highly Her2-expressing tumor xenograft explants yielded a significantly higher SQUID relaxometry signal relative to unconjugated nanoparticles. Finally, labeled cells introduced into breast phantoms were measured by magnetic relaxometry, and as few as 1 million labeled cells were detected at a distance of 4.5 cm using our early prototype system., Conclusions: These results suggest that the antibody-conjugated magnetic nanoparticles are promising reagents to apply to in vivo breast tumor cell detection, and that SQUID-detected magnetic relaxometry is a viable, rapid, and highly sensitive method for in vitro nanoparticle development and eventual in vivo tumor detection.

Previous studies have shown that a field of genetically altered but histologically normal tissue extends 1 cm or more from the margins of human breast tumors. The extent, composition and biological significance of this field are only partially understood, but the molecular alterations in affected cells could provide mechanisms for limitless replicative capacity, genomic instability and a microenvironment that supports tumor initiation and progression. We demonstrate by microarray, qRT-PCR and immunohistochemistry a signature of differential gene expression that discriminates between patient-matched, tumor-adjacent histologically normal breast tissues located 1 cm and 5 cm from the margins of breast adenocarcinomas (TAHN-1 and TAHN-5, respectively). The signature includes genes involved in extracellular matrix remodeling, wound healing, fibrosis and epithelial to mesenchymal transition (EMT). Myofibroblasts, which are mediators of wound healing and fibrosis, and intra-lobular fibroblasts expressing MMP2, SPARC, TGF-β3, which are inducers of EMT, were both prevalent in TAHN-1 tissues, sparse in TAHN-5 tissues, and absent in normal tissues from reduction mammoplasty. Accordingly, EMT markers S100A4 and vimentin were elevated in both luminal and myoepithelial cells, and EMT markers α-smooth muscle actin and SNAIL were elevated in luminal epithelial cells of TAHN-1 tissues. These results identify cellular processes that are differentially activated between TAHN-1 and TAHN-5 breast tissues, implicate myofibroblasts as likely mediators of these processes, provide evidence that EMT is occurring in histologically normal tissues within the affected field and identify candidate biomarkers to investigate whether or how field cancerization contributes to the development of primary or recurrent breast tumors., (Copyright © 2011 UICC.)

Optimizing the sensitivity of SQUID (superconducting quantum interference device) relaxometry for detecting cell-targeted magnetic nanoparticles for in vivo diagnostics requires nanoparticles with a narrow particle size distribution to ensure that the Néel relaxation times fall within the measurement timescale (50 ms-2 s, in this work). To determine the optimum particle size, single-core magnetite nanoparticles (with nominal average diameters 20, 25, 30 and 35 nm) were characterized by SQUID relaxometry, transmission electron microscopy, SQUID susceptometry, dynamic light scattering and zeta potential analysis. The SQUID relaxometry signal (detected magnetic moment/kg) from both the 25 nm and 30 nm particles was an improvement over previously studied multi-core particles. However, the detected moments were an order of magnitude lower than predicted based on a simple model that takes into account the measured size distributions (but neglects dipolar interactions and polydispersity of the anisotropy energy density), indicating that improved control of several different nanoparticle properties (size, shape and coating thickness) will be required to achieve the highest detection sensitivity. Antibody conjugation and cell incubation experiments show that single-core particles enable a higher detected moment per cell, but also demonstrate the need for improved surface treatments to mitigate aggregation and improve specificity.

Acute leukemia is a hematopoietic malignancy for which the accurate measurement of minimal residual disease is critical to determining prognosis and treatment. Although bone marrow aspiration and light microscopy remain the current standard of care for detecting residual disease, these approaches cannot reliably discriminate less than 5% lymphoblast cells. To improve the detection of leukemia cells in the marrow, we developed a novel apparatus that utilizes antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPION) and directed against the acute leukemia antigen CD34, coupled with a "magnetic needle" biopsy. Leukemia cell lines expressing high or minimal CD34 were incubated with anti-CD34-conjugated SPIONs. Three separate approaches including microscopy, superconducting quantum interference device magnetometry, and in vitro magnetic needle extraction were then used to assess cell sampling. We found that CD34-conjugated nanoparticles preferentially bind high CD34-expressing cell lines. Furthermore, the magnetic needle enabled identification of both cell line and patient leukemia cells diluted into normal blood at concentrations below those normally found in remission marrow samples. Finally, the magnetic needle enhanced the percentage of lymphoblasts detectable by light microscopy by 10-fold in samples of fresh bone marrow aspirate approximating minimal residual disease. These data suggest that bone marrow biopsy using antigen-targeted magnetic nanoparticles and a magnetic needle for the evaluation of minimal residual disease in CD34-positive acute leukemias can significantly enhance sensitivity compared with the current standard of care.

Field cancerization denotes the occurrence of aberrant cells in tumor adjacent histologically normal tissues (TAHN). To characterize field cancerization in prostate cancer, we used RNA from paired patient tumor and TAHN tissues excised at 1 cm from the tumor margin and subjected them to microarray expression analysis comparative to RNA from normal cancer-free prostatic tissues. Eleven novel transcripts were significantly up-regulated in TAHN tissues and also in tumors. Expression of early growth response protein 1, tristetraprolin, testican, and fatty acid synthase, mutually up-regulated at different levels in tumors and TAHN tissues was confirmed by quantitative reverse transcriptase PCR in the experimental and in an independent validation set. This study offers proof of expressional changes in field cancerized prostatic TAHN tissues at defined distances from tumor margins. Markers of field cancerized prostatic tissues could be early diagnostic indicators in biopsies after abnormal prostate-specific antigen and digital rectal examination and independent of cancerous histology and/or early targets for chemo-preventive intervention in pre-malignant disease.

Genomic instability can generate chromosome breakage and fusion randomly throughout the genome, frequently resulting in allelic imbalance, a deviation from the normal 1:1 ratio of maternal and paternal alleles. Allelic imbalance reflects the karyotypic complexity of the cancer genome. Therefore, it is reasonable to speculate that tissues with more sites of allelic imbalance have a greater likelihood of having disruption of any of the numerous critical genes that cause a cancerous phenotype and thus may have diagnostic or prognostic significance. For this reason, it is desirable to develop a robust method to assess the frequency of allelic imbalance in any tissue. To address this need, we designed an economical and high-throughput method, based on the Applied Biosystems AmpFlSTR Identifiler multiplex polymerase chain reaction system, to evaluate allelic imbalance at 16 unlinked, microsatellite loci located throughout the genome. This method provides a quantitative comparison of the extent of allelic imbalance between samples that can be applied to a variety of frozen and archival tissues. The method does not require matched normal tissue, requires little DNA (the equivalent of approximately 150 cells) and uses commercially available reagents, instrumentation, and analysis software. Greater than 99% of tissue specimens with >or=2 unbalanced loci were cancerous.

Young and aged rats were subjected to cerebrovascular insufficiency (CVI) for 3 and 9 weeks. At the end of each time period, local cerebral blood flow (lCBF), spatial memory function, 31P- and 1H-NMR spectroscopy and imaging of the brains were evaluated in vivo. Morphometric counts of CA1 hippocampal neuron damage and staining for glial fibrillary acidic protein (GFAP) were done post-mortem. Results show that after 3 weeks of CVI, cortical and hippocampal lCBF was significantly reduced in young and aged animals respectively. In addition, young and aged rats at 3 weeks following CVI showed spatial memory deficits in the Morris water maze and elevation of 31P-phosphomonoester as measured by non-invasive NMR spectroscopy. At the same time period, in vivo 1H-microimaging (MRI) of brains showed areas of high signal intensity (suggesting local edema) localized asymmetrically to the right hippocampal region in young and aged CVI rats. Morphometry of the hippocampal CA1 sector at post-mortem confirmed the in vivo MRI changes and demonstrated that a significant percentage of the CA1 pyramidal cells were damaged after CVI. Nine weeks after CVI, hippocampal CBF reductions, spatial memory impairment, spectroscopic-microimaging changes and CA1 sector cell damage continued to be observed in the aged animals but were resolved in the young rat brains. In addition, GFAP immunoreaction progressively increased in the hippocampus of aged rats subjected to CVI for 9 weeks. It is concluded that cognitive, metabolic and morphologic damage was significantly more severe and longer lasting in aged than young rat brain after chronic CVI. The deficits observed in this rat model appear to mimic the early pathology reported in Alzheimer's disease and suggest that the present model could provide fundamental clues relative to the etiology and possible management of this dementia.