Search

There is an identified need for point-of-care diagnostic systems for detecting and counting specific rare types of circulating cells in blood. By adequately labeling such cells with immunomagnetic beads and quantum dots, they can be efficiently collected magnetically for quantification using fluorescence methods. Automation of this process requires adequate mixing of the labeling materials with blood samples. A static mixing device can be employed to improve cell labeling efficiency and eliminate error-prone laboratory operations. Computational fluid dynamics (CFD) were utilized to simulate the flow of a labeling-materials/blood mixture through a 20-stage in-line static mixer of the interfacial-surface-generator type. Optimal fluid mixing conditions were identified and tested in a magnetic bead/tumor cell model, and it was found that labeled cells could be produced at 1.0 mL/min flow rate and fed directly into an in-line magnetic trap. The trap design consists of a dual flow channel with three bends and a permanent magnet positioned at the outer curve of each bend. The capture of labeled cells in the device was simulated using CFD, finite-element analysis and magnetophoretic mobility distributions of labeled cells. Testing with cultured CRL14777 human melanoma cells labeled with anti-CD146 1.5 μm diameter beads indicated that 90 ± 10% are captured at the first stage, and these cells can be captured when present in whole blood. Both in-line devices were demonstrated to function separately and together as predicted.

The first introductory book on the subject, this book will provide a complete grounding to this pioneering field for students and professionals across biomedical engineering, biology and medicine. It features a comprehensive overview of original work in this revolutionary field. Topics discussed include drug delivery, cell-material interaction and gene therapy, accompanied by real-world examples and over 100 illustrations. The book teaches readers how to design and test their own nanomedical systems for real-world applications in biomedical engineering, medicine and pharmacy. Presenting a thorough discussion of the science and engineering of nanomedicine, it discusses vital environmental, social and ethical impacts of this revolutionary technology. Including over 200 thought-provoking study questions, allowing the reader to self-assess their understanding, this book is a rich source of information that will be of interest and importance in nanomedicine.

Recent breakthroughs in nanoparticle research have led to improved drug delivery and have overcome problems associated with normal drug delivery methods. Optimizing the design of nanoparticles in terms of controlled size, shape, and surface chemistry of nanoparticles can maximize the therapeutic efficacy. To maximize therapeutic effects, advanced formulation and fabrication methods have been developed. Biomedical applications of nanoparticles produced using the new fabrication techniques, including drug delivery and molecular imaging, have been widely explored. This review highlights the simple and versatile manufacturing techniques that can be used in the development of new types of nanoparticles that have strictly controlled physiochemical properties and their multifaceted advantages in drug delivery and molecular imaging.

small sampling volumes, minimal hydrodynamic focusing avoiding need for most sheath fluid for higher sample throughput, weight/power requirements using super-bright LEDs light sources, “top-hat” focusing optics minimizing cell-positioning requirements, small silicon photomultiplier sensors, with tightly-integrated electronics, all battery-powered and light-weight for portability. Smartphone technology includes CPU, touch-screen, digital speech recognition interface, telecommunications via phone and/or internet, GPS chip to mark the location of the patient using Motorola’s Moto Z3 Play, modular/open-architecture smartphone. The device is software-configurable from a Linux workstation running Android Studio IDE with Java and Python programming. Portable microfluidic-cytometry devices for measurements in the field requires a serious overall systems-level design to face the many engineering tradeoffs encountered for true portability, including: (1) sampling systems of small sample volumes with minimal need for sheath hydrodynamic focusing both to avoid the need for large amounts of sheath fluid and to enable higher volumes of actual sample, (2) weight/power requirements that dictate use of super-bright LEDs as light sources, integrated “top hat” focusing optics to minimize the need for cell positioning and very small silicon photomultiplier sensors, with tightly integrated electronics (3) powered by small batteries or regenerative power sources such as solar, and (4) light-weight and robust enough for portability in potentially extreme environments. Initial prototyping used Raspberry Pi credit-card sized microcomputers, but longer-term development is being geared toward smartphone technologies. Smartphone technology can provide a powerful CPU, touch-screen and digital speech recognition interface, telecommunications via phone and internet, with a GPS chip to mark the location of the patient in the field. We are now using Motorola’s new Moto Z3 Play, a dual 12-megapixel/5-megapixel rear camera with 8-megapixel front camera, modular and open-architecture smartphone which allows building custom applications using hot-swappable accessories via Moto Development Kit modules, including externally-mountable Li-battery packs to power linked cytometry optics and electronics modules and microSD card storage with 512GB addressable memory linkable to other special purpose modules via USB-C connections. The device is software-configurable from a Linux workstation running Android Studio IDE with Java and Python programming.

Design of multifunctional nanoparticles for multimodal in-vivo imaging and advanced targeting to diseased single cells for massive parallel processing nanomedicine approaches requires careful overall design, including particle shape, and a multilayered approach to match the multi-step targeting required. In addition to thermodynamically driven self-assembly, complex patterns can be produced by micro-palette and 3D printing approaches. Initial core materials can include nanomaterials that simultaneously serve as x-ray contrast agents for CAT scan imaging as well as T1 or T2 contrast agents for MRI imaging. Use of superparamagnetic iron oxide allows for convenient magnetic manipulation during manufacturing re-positioning inside the body as well as single-cell hyperthermia therapies. To permit real-time fluorescence-image-guided surgery, fluorescence molecules can also be included. Advanced cell targeting can be achieved by attaching antibodies, peptides, aptamers, or other targeting molecules to the nanoparticle in a multilayered approach mimicking the multi-step targeting required. Addition of “stealth” molecules (e.g. PEG or chitosan) to the outer surfaces of the nanoparticles can permit greatly enhanced circulation times. Nanoscale imaging of these manufactured, multifunctional nanoparticles can be achieved either directly through superresolution microscopy or indirectly through single nanoparticle zeta-sizing or x-ray correlation microscopy. Since these multifunctional nanoparticles are best analyzed by technologies permitting analysis in aqueous environments, superresolution microscopy is, in most cases, the preferred method. This review paper will discuss the importance of specific design criteria as well as advantages and disadvantages of each approach. The overall design required a system engineering approach to the problem.

Design of portable microfluidic-cytometry devices for measurements in the field (e.g. initial medical diagnostics and recommended actions for first responders and search and rescue teams) requires careful design in terms of power requirements and weight to allow for true portability. True portability with high-throughput microfluidic systems also requires sampling systems with minimal need for sheath hydrodynamic focusing both to avoid the need for large amounts of sheath fluid and to enable higher volumes of actual sample, rather than conventional sheath/sample combinations. Weight/power requirements dictate use of super-bright LEDs as light sources and very small silicon photomultiplier sensors, with tightly integrated electronics that can both be powered by small batteries or regenerative power sources such as solar. GPS-based positioning, and telecommunications (including possible satellite–based, if cellphone towers are not nearby) to export data to other medical facilities. Microfluidic-cytometry also requires judicious use of small sample volumes and appropriate statistical sampling to permit real-time (typically in less than 10-15 minutes) initial medical decisions, not just raw data, for first responders in the field who may need results which include on-board expert medical systems. The portable system should be robust for extreme environments and should be modular and flexible to allow for multiple applications and for plug-in repairs if subsystems should become damaged. For example, one or two drops of blood obtained by pin-prick should be able to provide statistically meaningful results for use in making real-time medical decisions without the need for blood fractionation, which is not realistic in the field.

Design of multifunctional nanoparticles for multimodal in-vivo imaging and advanced targeting to diseased single cells for massive parallel processing nanomedicine approaches requires careful overall design and a multilayered approach. Initial core materials can include non-toxic metals which not only serve as an x-ray contrast agent for CAT scan imaging, but can contain T1 or T2 contrast agents for MRI imaging. One choice is superparamagnetic iron oxide NPs which also allow for convenient magnetic manipulation during manufacturing but also for re-positioning inside the body and for single cell hyperthermia therapies. To permit real-time fluorescence-guided surgery, fluorescence molecules can be included. Advanced targeting can be achieved by attaching antibodies, peptides, aptamers, or other targeting molecules to the nanoparticle in a multilayered approach producing “programmable nanoparticles” whereby the “programming” means controlling a sequence of multi-step targeting methods. Addition of membrane permeating peptides can facilitate uptake by the cell. Addition of “stealth” molecules (e.g. PEG or chitosan) to the outer surfaces of the nanoparticles can permit greatly enhanced circulation times in-vivo which in turn lead to lower amounts of drug exposure to the patient which can reduce undesirable side effects. Nanoparticles with incomplete layers can be removed by affinity purification methods to minimize mistargeting events in-vivo. Nanoscale imaging of these manufactured, multifunctional nanoparticles can be achieved either directly through superresolution microscopy or indirectly through single nanoparticle zeta-sizing or x-ray correlation microscopy. Since these multifunctional nanoparticles are best analyzed by technologies permitting analysis in aqueous environments, superresolution microscopy is, in most cases, the preferred method.

Design of portable microfluidic flow/image cytometry devices for measurements in the field (e.g. initial medical diagnostics) requires careful design in terms of power requirements and weight to allow for realistic portability. True portability with high-throughput microfluidic systems also requires sampling systems without the need for sheath hydrodynamic focusing both to avoid the need for sheath fluid and to enable higher volumes of actual sample, rather than sheath/sample combinations. Weight/power requirements dictate use of super-bright LEDs with top-hat excitation beam architectures and very small silicon photodiodes or nanophotonic sensors that can both be powered by small batteries. Signal-to-noise characteristics can be greatly improved by appropriately pulsing the LED excitation sources and sampling and subtracting noise in between excitation pulses. Microfluidic cytometry also requires judicious use of small sample volumes and appropriate statistical sampling by microfluidic cytometry or imaging for adequate statistical significance to permit real-time (typically in less than 15 minutes) initial medical decisions for patients in the field. This is not something conventional cytometry traditionally worries about, but is very important for development of small, portable microfluidic devices with small-volume throughputs. It also provides a more reasonable alternative to conventional tubes of blood when sampling geriatric and newborn patients for whom a conventional peripheral blood draw can be problematical. Instead one or two drops of blood obtained by pin-prick should be able to provide statistically meaningful results for use in making real-time medical decisions without the need for blood fractionation, which is not realistic in the doctor’s office or field.

Design of inexpensive and portable hand-held microfluidic flow/image cytometry devices for initial medical diagnostics at the point of initial patient contact by emergency medical personnel in the field requires careful design in terms of power/weight requirements to allow for realistic portability as a hand-held, point-of-care medical diagnostics device. True portability also requires small micro-pumps for high-throughput capability. Weight/power requirements dictate use of super-bright LEDs and very small silicon photodiodes or nanophotonic sensors that can be powered by batteries. Signal-to-noise characteristics can be greatly improved by appropriately pulsing the LED excitation sources and sampling and subtracting noise in between excitation pulses. The requirements for basic computing, imaging, GPS and basic telecommunications can be simultaneously met by use of smartphone technologies, which become part of the overall device. Software for a user-interface system, limited real-time computing, real-time imaging, and offline data analysis can be accomplished through multi-platform software development systems that are well-suited to a variety of currently available cellphone technologies which already contain all of these capabilities. Microfluidic cytometry requires judicious use of small sample volumes and appropriate statistical sampling by microfluidic cytometry or imaging for adequate statistical significance to permit real-time (typically < 15 minutes) medical decisions for patients at the physician’s office or real-time decision making in the field. One or two drops of blood obtained by pin-prick should be able to provide statistically meaningful results for use in making real-time medical decisions without the need for blood fractionation, which is not realistic in the field.

Neurological injury, such as spinal cord injury, has a secondary injury associated with it. The secondary injury results from the biological cascade after the primary injury and affects previous uninjured, healthy tissue. Therefore, the mitigation of such a cascade would benefit patients suffering a primary injury and allow the body to recover more quickly. Unfortunately, the delivery of effective therapeutics is quite limited. Due to the inefficient delivery of therapeutic drugs, nanoparticles have become a major field of exploration for medical applications. Based on their material properties, they can help treat disease by delivering drugs to specific tissues, enhancing detection methods, or a mixture of both. Incorporating nanomedicine into the treatment of neuronal injury and disease would likely push nanomedicine into a new light. This review highlights the various pathological issues involved in secondary spinal cord injury, current treatment options, and the improvements that could be made using a nanomedical approach.

The concept of personalized medicine has recently emerged as a promising way to address unmet medical needs. Due to the limitations of standard diagnostic and therapeutic strategies, the disease treatment is moving towards tailored treatment for individual patients, considering the inter-individual variability in therapeutic response. Theranostics, which involves the combination of therapy and diagnostic imaging into a single system, may fulfill the promise of personalized medicine. By integrating molecular imaging functionalities into therapy, theranostic approach could be advantageous in therapy selection, treatment planning, objective response monitoring and follow-up therapy planning based on the specific molecular characteristics of a disease. Although the field of therapy and imaging of its response have been independently developed thus far, developing imaging strategies can be fully exploited to revolutionize the theranostic systems in combination with the therapy modality. In this review, we describe the recent advances in molecular imaging technologies that have been specifically developed to evaluate the therapeutic efficacy for theranostic purposes.

Iron oxide nanoparticles (IOs) are intrinsically theranostic agents that could be used for magnetic resonance imaging (MRI) and local hyperthermia or tissue thermal ablation. Yet, effective hyperthermia and high MR contrast have not been demonstrated within the same nanoparticle configuration. Here, magnetic nanoconstructs are obtained by confining multiple, ∼ 20 nm nanocubes (NCs) within a deoxy-chitosan core. The resulting nanoconstructs-magnetic nanoflakes (MNFs)-exhibit a hydrodynamic diameter of 156 ± 3.6 nm, with a polydispersity index of ∼0.2, and are stable in PBS up to 7 days. Upon exposure to an alternating magnetic field of 512 kHz and 10 kA m-1, MNFs provide a specific absorption rate (SAR) of ∼75 W gFe-1, which is 4-15 times larger than that measured for conventional IOs. Moreover, the same nanoconstructs provide a remarkably high transverse relaxivity of ∼500 (mM s)-1, at 1.41T. MNFs represent a first step toward the realization of nanoconstructs with superior relaxometric and ablation properties for more effective theranostics. © 2014 American Chemical Society.

This study examines student perceived gains from an undergraduate research experience (URE) program, using data from pre-, mid-, and postparticipation surveys. Results suggest that students experienced different gains at developmentally different stages of their UREs and reported gains in fewer areas at the end of the Summer segment than at end of the yearlong experience., The current study examines the trajectories of student perceived gains as a result of time spent in an undergraduate research experience (URE). Data for the study come from a survey administered at three points over a 1-yr period: before participation in the program, at the end of a Summer segment of research, and at the end of the year. Repeated-measures analysis of variance was used to examine the effect of time on perceived gains in student research skills, research confidence, and understanding of research processes. The results suggest that the students experienced different gains/benefits at developmentally different stages of their UREs. Participants reported gains in fewer areas at the end of the Summer segment compared with the end of the yearlong experience, thus supporting the notion that longer UREs offer students more benefit.

Imaging techniques including computed tomography, magnetic resonance imaging, and positron emission tomography (PET) offer many potential benefits to diagnosis and treatment of cancers. Each method has its own strong and weak points. Therefore, multimodal imaging techniques have been highlighted as an alternative method for overcoming the limitations of each respective imaging method. In this study, we fabricated PET/optical activatable imaging probe based on glycol chitosan nanoparticles (CNPs) for multimodal imaging. To prepare the dual PET/optical probes based on CNPs, both (64)Cu radiolabeled DOTA complex and activatable matrix metalloproteinase (MMP)-sensitive peptide were chemically conjugated onto azide-functionalized CNPs via bio-orthogonal click chemistry, which was a reaction between azide group and dibenzyl cyclooctyne. The PET/optical activatable imaging probes were visualized by PET and optical imaging system. Biodistribution of probes and activity of MMP were successfully measured in tumor-bearing mice.

Preventive actions for chronic diseases hold the promise of improving lives and reducing healthcare costs. For several diseases, including breast cancer, multiple risk and protective factors have been identified by epidemiologists. The impact of most of these factors has yet to be fully understood at the organism, tissue, cellular and molecular levels. Importantly, combinations of external and internal risk and protective factors involve cooperativity thus, synergizing or antagonizing disease onset. Models are needed to mechanistically decipher cancer risks under defined cellular and microenvironmental conditions. Here, we briefly review breast cancer risk models based on 3D cell culture and propose to improve risk modeling with lab-on-a-chip approaches. We suggest epithelial tissue polarity, DNA repair and epigenetic profiles as endpoints in risk assessment models and discuss the development of 'risks-on-chips' integrating biosensors of these endpoints and of general tissue homeostasis. Risks-on-chips will help identify biomarkers of risk, serve as screening platforms for cancer preventive agents, and provide a better understanding of risk mechanisms, hence resulting in novel developments in disease prevention.

The earliest impact of nanomedicine in ophthalmology is likely to involve the areas of biopharmaceuticals, implantable materials (e.g. tissue regeneration scaffolds, bioresorbable materials), implantable devices (e.g. glaucoma drainage valves), and diagnostic tools (e.g. intraocular pressure (IOP) monitors). Nanotechnology will bring about the development of regenerative medicine (i.e. replacement and improvement of cells, tissues, and organs) and artificial vision. In this chapter, we review ophthalmic applications of nanotechnology in the following areas: drug and trophic factor therapy for glaucoma, retinal degenerative, and retinal vascular disease; gene therapy for retinal degenerative disease; regenerative medicine, including optogenetics and optic nerve regeneration; and diagnostics (minimally invasive IOP monitoring). Nanotechnology will play an important role in both early-stage and late-stage intervention in the management of blinding diseases.

Since current cancer therapies do not work effectively for all patients, there is a pressing need for alternate, affordable, yet effective treatments for inoperable and chemo-resistant tumors. Presented in this paper are our investigations on the application of short duration (milli and micro seconds) electrical voltage pulses for the treatment of breast carcinoma. For this purpose, electroporation, a pulse power technology, was evaluated for its effect upon the viability and proliferation of human breast cancer cells using a number of voltages and pulse durations. The results indicate the promising potential of electrical pulses to effectively and economically enhance drug transport across the normally impermeable plasma cell membranes and to reduce proliferation.

Jaehong Key,1,2 Deepika Dhawan,3 Christy L Cooper,3,4 Deborah W Knapp,3 Kwangmeyung Kim,5 Ick Chan Kwon,5 Kuiwon Choi,5 Kinam Park,1,6 Paolo Decuzzi,7–9 James F Leary1,3,41Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA; 2Department of Biomedical Engineering, Yonsei University, Wonju, Republic of Korea; 3School of Veterinary Medicine-Department of Basic Medical Sciences, Purdue University, West Lafayette, 4Birck Nanotechnology Center at Discovery Park, Purdue University, West Lafayette, IN, USA; 5Biomedical Research Center, Korea Institute of Science and Technology, Sungbook-Gu, Seoul, Republic of Korea; 6Department of Pharmaceutics, Purdue University, West Lafayette, IN, 7Department of Translational Imaging, 8Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX USA; 9Laboratory of Nanotechnology for Precision Medicine, Fondazione Istituto Italiano di Tecnologia (IIT), Genova, Italy Abstract: While current imaging modalities, such as magnetic resonance imaging (MRI), computed tomography, and positron emission tomography, play an important role in detecting tumors in the body, no single-modality imaging possesses all the functions needed for a complete diagnostic imaging, such as spatial resolution, signal sensitivity, and tissue penetration depth. For this reason, multimodal imaging strategies have become promising tools for advanced biomedical research and cancer diagnostics and therapeutics. In designing multimodal nanoparticles, the physicochemical properties of the nanoparticles should be engineered so that they successfully accumulate at the tumor site and minimize nonspecific uptake by other organs. Finely altering the nano-scale properties can dramatically change the biodistribution and tumor accumulation of nanoparticles in the body. In this study, we engineered multimodal nanoparticles for both MRI, by using ferrimagnetic nanocubes (NCs), and near infrared fluorescence imaging, by using cyanine 5.5 fluorescence molecules. We changed the physicochemical properties of glycol chitosan nanoparticles by conjugating bladder cancer-targeting peptides and loading many ferrimagnetic iron oxide NCs per glycol chitosan nanoparticle to improve MRI contrast. The 22 nm ferrimagnetic NCs were stabilized in physiological conditions by encapsulating them within modified chitosan nanoparticles. The multimodal nanoparticles were compared with in vivo MRI and near infrared fluorescent systems. We demonstrated significant and important changes in the biodistribution and tumor accumulation of nanoparticles with different physicochemical properties. Finally, we demonstrated that multimodal nanoparticles specifically visualize small tumors and show minimal accumulation in other organs. This work reveals the importance of finely modulating physicochemical properties in designing multimodal nanoparticles for bladder cancer imaging.Keywords: MRI, NIRF, multimodal imaging, chitosan, iron oxide, bladder cancer

Flow and image cytometry are mature single-cell analysis technologies that have been developed over the past 50 years. Fluorescence has been the primary mode of generating single-cell signals. There are now hundreds of different color fluorescent probes linked to thousands of different molecules including antibodies, ligands, and other molecules. Increasingly sophisticated commercial flow cytometry instruments measure multiple colors (e.g., 17 or more colors per cell) of fluorescence per single-cell. Often these fluorescent labels are being used to identify combinations of biomarkers in many human diseases. Recently, microfluidic systems have shown the advantage of either reexamining the same cells at different time points or capturing and treating those cells and measuring their response. These microfluidic structures can combine flow and imaging technologies in the same structure. Other technologies such as PCR can be added to these multifaceted microfluidic structures. The microfluidic devices can be built to provide low-cost, point-of-care medicine. Making multiple correlated measurements per cell is extremely powerful leading to very large amounts of data of high dimensionality. Recently, bioinformatics techniques are being employed to study “big data” from large ensembles of single-cell data. This is leading to questions whether any two single-cells are really ever the same if you are able to measure enough parameters at sufficient accuracy? When are two cells similar enough to have similar biology, functions and predictable outcomes when challenged with drugs and/or environmental factors? Single-cell analyses provide us with the tools to answer these intriguing questions.

One difficulty of diagnosing and treating cancer is that it is very challenging to detect cancers in the early stages before metastasis occurs. A variety of imaging modalities needs to be used from non-invasive, moderate resolution modalities, such as magnetic resonance imaging (MRI) to very high-resolution (e.g. fluorescence) imaging that can help guide surgeons during a surgical operation. While MRI can have relatively high resolution and deep penetration to visualize soft tissues, low sensitivity of MRI frequently requires tumor imaging agents to enhance the MRI contrast at the tumor site. At the other end of the resolution spectrum, near infrared fluorescence (NIRF) imaging has very high sensitivity but frequently cannot be utilized for initial human in vivo imaging due to its very limited penetration depth. To combine the advantages of each imaging modality we have constructed MRI and NIRF dual-modality nanoparticles using glycol chitosan, Cy5.5, and superparamagnetic iron oxide nanoparticles (SPIOs). We have demonstrated these advantages for dual-modality, in vivo tumor imaging in mice. Our studies suggest the potential use of NIRF and MR dual modality imaging for human cancer diagnosis. (c) 2012 Elsevier B.V. All rights reserved.

Tumor-targeted imaging and therapy have been the challenging issue in the clinical field. Herein, we report tumor-targeting hyaluronic acid nanoparticles (HANPs) as the carrier of the hydrophobic photosensitizer, chlorin e6 (Ce6) for simultaneous photodynamic imaging and therapy. First, self-assembled HANPs were synthesized by chemical conjugation of aminated 5β-cholanic acid, polyethylene glycol (PEG), and black hole quencher3 (BHQ3) to the HA polymers. Second, Ce6 was readily loaded into the HANPs by a simple dialysis method resulting in Ce6-loaded hyaluronic acid nanoparticles (Ce6-HANPs), wherein in the loading efficiency of Ce6 was higher than 80%. The resulting Ce6-HANPs showed stable nano-structure in aqueous condition and rapid uptake into tumor cells. In particular Ce6-HANPs were rapidly degraded by hyaluronidases abundant in cytosol of tumor cells, which may enable intracellular release of Ce6 at the tumor tissue. After an intravenous injection into the tumor-bearing mice, Ce6-HANPs could efficiently reach the tumor tissue via the passive targeting mechanism and specifically enter tumor cells through the receptor-mediated endocytosis based on the interactions between HA of nanoparticles and CD44, the HA receptor on the surface of tumor cells. Upon laser irradiation, Ce6 which was released from the nanoparticles could generate fluorescence and singlet oxygen inside tumor cells, resulting in effective suppression of tumor growth. Overall, it was demonstrated that Ce6-HANPs could be successfully applied to in vivo photodynamic tumor imaging and therapy simultaneously.

Regenerative medicine deals with the repair or the replacement of tissues and organs using advanced materials and methodologies. Regenerative nanomedicine uses nanoparticles containing gene transcription factors and other modulating molecules that allow reprogramming of cells in vivo as well as nanomaterials to induce selective differentiation of neural progenitor cells and to create neural-mechanical interfaces. In this article, we consider some applications of nanotechnology that may be useful for the treatment of degenerative retinal diseases, for example, use of nanoparticles for drug and gene therapy, use of nanomaterials for neural interfaces and extracellular matrix construction for cell-based therapy and neural prosthetics, and the use of bionanotechnology to re-engineer proteins and cell behavior for regenerative medicine.

ErbB4 is a member of the ErbB family of receptor tyrosine kinases. This family includes ErbB2 (HER2/Neu), a validated therapeutic target in breast cancer. Several studies indicate that ErbB4 functions as a tumor suppressor in breast cancer, whereas others indicate that ErbB4 functions as an oncogene. Here the authors explore the context in which ErbB4 functions as an oncogene. Silencing expression of either ErbB2 or ErbB4 in breast tumor cell lines results in reduced stimulation of anchorage independence and cell motility by the ErbB4 agonist neuregulin 2β. ErbB2 tyrosine kinase activity, but not ErbB4 tyrosine kinase activity, is required for neuregulin 2β to stimulate cell proliferation. Moreover, sites of ErbB4 tyrosine phosphorylation, but not sites of ErbB2 tyrosine phosphorylation, are required for neuregulin 2β to couple to cell proliferation. These data suggest that targeting ErbB2 expression or tyrosine kinase activity may be effective in treating ErbB4-dependent breast tumors, even those tumors that lack ErbB2 overexpression.

Cancer prevention is a broad field that crosses many disciplines; therefore, educational efforts to enhance cancer prevention research focused on interdisciplinary approaches to the field are greatly needed. In order to hasten progress in cancer prevention research, the Cancer Prevention Internship Program (CPIP) at Purdue University was designed to develop and test an interdisciplinary curriculum for undergraduate and graduate students. The hypothesis was that course curriculum specific to introducing interdisciplinary concepts in cancer prevention would increase student interest in and ability to pursue advanced educational opportunities (e.g., graduate school, medical school). Preliminary results from the evaluation of the first year which included ten undergraduate and five graduate students suggested that participation in CPIP is a positive professional development experience, leading to a significant increase in understanding of interdisciplinary research in cancer prevention. In its first year, the CPIP project has created a successful model for interdisciplinary education in cancer prevention research.

Nanotechnology involves the creation and use of materials and devices at the size scale of intracellular structures and molecules, and involves systems and constructs in the order of

Matrix metalloproteinases (MMPs) have been shown to be abundant in pathological conditions such as cancer, osteoarthritis (OA), and rheumatoid arthritis (RA). The extent of MMPs detected in biological samples provides important clinical information for diagnosis, prognosis, and therapeutic monitoring of various diseases relating with MMPs. Herein, we developed a new high-throughput MMP diagnostic kit (MMP-D-KIT) based on a 96-well plate by immobilizing MMP-13 specific fluorogenic peptide probes (MMP peptide probe), which is a pair consisting of a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3), onto the biocompatible glycol chitosan (GC) polymer anchored 96-well plate. When MMP enzymes were simply added and incubated in a MMP-D-KIT, the fluorescence of each well was recovered and the fluorescence intensity showed distinct difference within minutes through NIR fluorescence imaging system. The fluorescence was recovered not only by MMP-13 activity, but also by other MMPs activity. Furthermore, recovery of NIR fluorescent signals in MMP-D-KIT was proportional to concentrations of immobilized MMP peptide probe-GC conjugates and, importantly, MMP concentration. The MMP-D-KIT is most specific for target MMP, compared with other enzymes including caspase-3 and 20s proteasome. Additionally, the MMP-D-KIT was used to detect MMP activity in biological samples such as synovial fluid from 12 OA patients (grades 1-4 based on the Kellgren-Lawrence grading scale). It was found that the fluorescence intensity measured using MMP-D-KIT decidedly correlates with the progression of OA. The MMP-D-KIT could be applicable in detecting MMP activities in various biological samples and evaluating the effects of MMP inhibitors in a rapid and easy fashion.

A polymeric thermosensor composed of the thermo-responsive block copolymer Pluronic F127 (PF127) and the near-infrared (NIR) dye Cy5.5 can simply monitor, image, and analyze temperature changes. The thermoprobe exhibited linear NIR fluorescent emission changes (see figure) over a broad temperature range (0–80 °C).

Oxidative stress plays a major role in many disease pathologies, notably in the central nervous system (CNS). For instance, after initial spinal cord injury, the injury site tends to increase during a secondary chemical injury process based on oxidative stress from necrotic cells and the inflammatory response. Prevention of this secondary chemical injury would represent a major advance in the treatment of people with spinal cord injuries. Few therapeutics are useful in combating such stress in the CNS due to side effects, low efficacy, or half-life. Mesoporous silica nanoparticles show promise for delivering therapeutics based on the formation of a porous network during synthesis. Ideally, they increase the circulation time of loaded therapeutics to increase the half-life while reducing overall concentrations to avoid side effects. The current study explored the use of silica nanopar ticles for therapeutic delivery of anti-oxidants, in particular, the neutralization of acrolein which can lead to extensive tissue damage due to its ability to generate more and more copies of itself when it interacts with normal tissue. Both an FDA-approved therapeu tic, hydralazine, and natural product, epigallocatechin gallate, were explored as anti-oxidants for acrolein with nanoparticles for increased efficacy and stability in ne uronal cell cultures. Not only were the nanoparticles explored in neuronal cells, but also in a co-cultured in vitro model with microglial cells to study potential immune responses to near-infrared (NIRF)-labeled nanoparticles and uptake. Studies included nanoparticle toxicity, uptake, and therapeutic response using fluorescence-based techniques with both dormant and activated immune microglia co-cultured with neuronal cells. Keywords: Silica nanoparticles, NIRF, nanom edicine, nanotoxicity, ROS, inflammation, spinal cord injury

Aneurysms are pockets of blood that collect outside blood vessel walls forming dilatations and leaving arterial walls very prone to rupture. There is little information concerning the causes of intracranial aneurysm formation, growth, and rupture. Current treatments include: (1) clipping, and (2) coil embolization, including stent-assisted coiling. Further, the evolution of any aneurysm is assumed to be caused by the remodeling of the affected blood vessel’s material constituents (tunica intima, tunica media, or tunica adventitia). Velocity, pressure, and wall shear stresses aid in the disease development of aneurysmal growth, while the shear force mechanisms effecting wound closure are elusive. To study aneurysm pathogenesis, a lab-on-a-chip device is the key to discovering the underlying mechanisms of these lesions. A two-dimensional microfluidic model, the Aneurysm-on-a-Chip™ (AOC), was the logical answer to study particle flow within an aneurysm “sac”. The AOC apparatus can track particles/cells when it is coupled to particle image velocimetry software (PIV) package. The AOC fluid flow was visualized using standard microscopy techniques with commercial microparticles and human aortic smooth muscle cells (HASMC). Images were taken during fluid flow experiments and PIV was utilized to monitor the flow of particles within the “sac” region, as well as particles entering and exiting the device. Quiver plots were generated from fluid flow experiments using standard 7 μm latex particles and fixed HASMC in PBS. PIV analysis shows that the particles flowed nicely from input to output. Wall shear stress provided evidence that there was some back flow at the edges of the “sac” – an indicator of aneurysm development in human patients.

Thioaptamers offer advantages over normal phosphate ester backbone aptamers due to their enhanced affinity, specificity, and higher stability, largely due to the properties of the sulfur backbone modifications. Over the past several years, in vitro thioaptamer selection and bead-based thioaptamer selection techniques have been developed in our laboratory. Furthermore, several thioaptamers targeting specific proteins such as transcription factor NF-kappaB and AP-1 proteins have been identified. Selected thioaptamers have been shown diagnostic promise in proteome screens. Moreover, some promising thioaptamers have been shown in preliminary animal therapeutic dosing to increase survival in animal models of infection with West Nile virus.

Complex, multilayered nanoparticles hold great promise for more sophisticated drug/gene delivery systems to single cells. Outermost layers can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest. Molecular biosensors can be used to confirm the presence of expected molecules (for example, reactive oxygen species (ROS) as a surrogate molecule for signs of infection, or for activation in radiation damage, etc.) prior to delivery of counter-measure molecules such as drugs or gene therapy. They can also be used as a feedback control mechanism to control the proper amount of drug/gene delivery for each cell. Importantly, the full nanoparticle system can be used to prevent any cells from encountering the drug unless that cell is specifically targeted. Thus, if a cell is initially non-specifically targeted, a secondary check for other molecular targets which must also be present inside the target cell of interest can be used to catch initial targeting mistakes and prevent subsequent delivery of treatment molecules to the wrong cells. The precise intracellular location of nanoparticles within specific regions of a cell can be confirmed by 3D multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.

Background Molecular cell systems research (cytomics) aims at the understanding of the molecular architecture and functionality of cell systems (cytomes) by single-cell analysis in combination with exhaustive bioinformatic knowledge extraction. In this way, loss of information as a consequence of molecular averaging by cell or tissue homogenisation is avoided. Progress The cytomics concept has been significantly advanced by a multitude of current developments. Amongst them are confocal and laser scanning microscopy, multiphoton fluorescence excitation, spectral imaging, fluorescence resonance energy transfer (FRET), fast imaging in flow, optical stretching in flow, and miniaturised flow and image cytometry within laboratories on a chip or laser microdissection, as well as the use of bead arrays. In addition, biomolecular analysis techniques like tyramide signal amplification, single-cell polymerase chain reaction (PCR), and the labelling of biomolecules by quantum dots, magnetic nanobeads, or aptamers open new horizons of sensitivity and molecular specificity at the single-cell level. Data sieving or data mining of the vast amounts of collected multiparameter data for exhaustive multilevel bioinformatic knowledge extraction avoids the inadvertent loss of information from unknown molecular relations being inaccessible to an a priori hypothesis. Challenge It seems important to address the challenge of a human cytome project using hypothesis-driven molecular information collection from disease associated cell systems, supplemented by systematic and exhaustive knowledge extraction. This will allow the description of the molecular setup of normal and abnormal cell systems within a relational knowledge system, permitting the standardised discrimination of abnormal cell states in disease. As one of the consequences, individualised predictions of further disease course in patients (predictive medicine by cytomics) by characteristic discriminatory data patterns will permit individualised therapies, identification of new pharmaceutical targets, and establishment of a standardised framework of relevant molecular alterations in disease. This special issue of Cytometry, on new technologies in cytomics, focuses on prominent examples of this presently fast-moving scientific field, and represents one of the preconditions for the formulation of a human cytome project. © 2004 Wiley-Liss, Inc.

Oligonucleotide-functionalized nanoparticles (NPs) are promising agents for nanomedicine, but the potential in vitro nanotoxicity that may arise from such conjugates has yet to be evaluated in a dose response manner. Since nanomedicine functions on the single-cell level, measurements of nanotoxicity should also be performed as such. In vitro single-cell nanotoxicity assays based on scanning image cytometry are used to study a specific type of oligo-functionalized NP, "nanobarcoded" superparamagnetic iron oxide NPs (NB-SPIONs). The selected panel of single-cell assays measures well-known modes of nanotoxicity--apoptosis, necrosis, generation of reactive oxygen species (ROS), and cell number. Using these assays, the cytotoxicity of two sizes of NB-SPIONs (10 nm and 30 nm core size) was compared to the parent NP, carboxylated SPIONs (COOH-SPIONs). The results suggest that the conjugated NB confers a biocompatible coating that protects against cytotoxicity at very high SPION doses, but both NB- and COOH-SPIONs of either size generally have low in vitro cytotoxicity at physiologically relevant doses.

Acrolein, a very reactive aldehyde, is a culprit in the biochemical cascade after primary, mechanical spinal cord injury (SCI), which leads to the destruction of tissue initially unharmed, referred to as “secondary injury”. Additionally, in models of multiple sclerosis (MS) and some clinical research, acrolein levels are significantly increased. Due to its ability to make more copies of itself in the presence of tissue via lipid peroxidation, researchers believe that acrolein plays a role in the increased destruction of the central nervous system in both SCI and MS. Hydralazine, an FDAapproved hypotensive drug, has been shown to scavenge acrolein, but its side effects and short half life at the appropriate dose for acrolein scavenging must be improved for beneficial clinical translation. Therefore, a nanomedical approach has been designed using silica nanoparticles as a porous delivery vehicle hydralazine. The silica particles are formed in a one-step method that incorporates poly(ethylene) glycol (PEG), a stealth molecule, directly onto the nanoparticles. As an additional avenue for study, a natural product in green tea, epigallocatechin gallate (EGCG), has been explored for its ability to react with acrolein, disabling its reactive capabilities. Upon demonstration of attenuating acrolein, EGCG's delivery may also be improved using the nanomedical approach. The current work exposes the potential of using silica nanoparticles as a delivery vehicle and EGCG's antioxidant capabilities in B35 neuroblastoma cells exposed to acrolein. We also measure nanotoxicity to individual rat neurons using high-throughput image scanning cytometry.

Leukemic cancer stem cells are both stem-like and leukemic-like. This complicates their detection as rare circulating tumor cells in peripheral blood of leukemia patients. The leukemic stem cells are also highly resistant to standard chemotherapeutic regimens so new therapeutic strategies need to be designed to kill the leukemic stem cells without killing normal stem cells. In these initial studies we have designed an antibody-targeted and fluorescent (Cy5.5) nanoparticle for targeting these leukemic stem cells and then introducing new strategies for killing them. Multicolor flow cytometric analyses were performed on a BD FACS Aria III. Human leukemic stem cell-like cell line RS4;11 (with putative immunophenotype CD123+/CD24+/CD38-/CD10-/Flt-3-) was used as a model human leukemic stem cell systems and were spiked into normal human peripheral blood cells containing normal blood stem-progenitor cells (immunophenotype CD123-/CD34+/CD38-) and Cy5.5-labeled nanoparticles with targeting molecule anti-CD123 antibody. An irrelevant antibody (CD71) which should not bind to any live leukemic stem cell or normal stem cell (binds erythrocytes) was used as a way of distinguishing between true-positive live and false-positive damaged/dead cells, the latter occurring at much higher frequencies than the very rare (e.g. 0.001 to 0.0001 percent frequency true leukemic stem cells). These studies are designed to measure the targeting sensitivity and specificity of the fluorescent nanoparticles to the putative rare leukemic stem cells with the eventual design to use the nanoparticles to direct killing therapeutic doses to the leukemic stem cells but not to the normal stem-progenitor cells.

The death of a woman every 71 seconds in the world and every 12 minutes in the US, from breast cancer, indicate that the current standard of cure does not work. In addition, these therapies are expensive and have many side effects. This calls for safe, effective, alternate, affordable and physical therapies. Electrochemotherapy (ECT) has promising potential for treating inoperable, chemo-resistant and radio-resistant tumors that do not respond to the current standard of cure. Clinical trials show the successful applications of ECT for chest wall breast carcinomas. In this research, the efficacy of FDA approved, commonly prescribed and administered breast cancer drugs, including doxorubicin, paclitaxol, tamoxifen and bleomycin are studied using electroporation. In addition, the efficacy of curcumin is also studied using high and low voltage electroporation. MCF-7 breast cancer cells were used for this study at various pulse parameter conditions, including both low and high voltages. The positive results indicate that ECT is a good vehicle to efficiently upload these drugs intratumorally.

RNA interference (RNAi) is a natural phenomenon existing in plants, insects, and many mammals involved in gene silencing or the inhibition of gene expression. Small interfering RNAs (siRNAs) could be used as potential tools to implement this process that could lead to treatments in diseases such as cancer. However, the delivery of siRNA to eukaryotic cells is a challenge due to its lower cellular uptake and easy degradation by enzymes in blood, tissues, and cells. Electroporation-based siRNA delivery is an upcoming therapeutic tool to deliver various siRNAs, both in vitro and in vivo . Discussed in this chapter is the review of various successful deliveries of siRNA using electroporation.

Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens. G0/1 doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G2 1 M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns. G0/1 doublets were easily discernible from G2 1 M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G0/1 doublet estimates were observed in breast tumor specimens (n 5 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G0/1 doublets and G2 1 M singlet populations in biologically heterogeneous specimens. To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory comparison difficult. Cytometry (Comm. Clin. Cytometry) 46:296 ‐306, 2001. Published 2001 Wiley-Liss, Inc. †

Background: As flow cytometric data becomes more complex, it becomes increasingly difficult to classify cells using conventional flow cytometry data techniques based on visual classification of the data by user-drawn regions. This paper shows some simple applications of multivariate statistical classification to classify flow cytometric data. Methods: Discriminant Function Analysis (DFA) and Logistic Regression (LR) analysis techniques were evaluated with respect to their potential utility in the problem of detecting human breast cancer cells within normal bone marrow cells. Data sets having defined properties were employed to evaluate the potential utility of these statistical classification techniques whose performance was measured by ROC analysis. Results: Two extreme but reasonable situations are presented: (1) data where the separation of cells was obvious by visual inspection and (2) data where major overlaps in the values of the individual FCM parameters made intuitive classification improbable. Both DFA and LR analysis were able to classify the cells of each type with acceptable accuracy and yield. Conclusions: The excellent empirical performance of both DFA and LR techniques, suggests that they offer promising approaches for classifying multiparameter FCM data using objective rules that may represent an improvement over commonly employed ad hoc approaches. Cytometry 36:60‐70, 1999. r 1999 Wiley-Liss, Inc.

This paper describes new approaches to calculating the number of cells that need to be processed using flow cytometry (FCM) techniques and the subsequent time required in order to isolate a specific number of cells having selected characteristics. The methods proposed use probabilistic assumptions about the contents of the sample to be sorted, logarithmic/exponential transformations to avert the computer ‘‘underflow’’ and ‘‘overflow’’ limitations of brute force calculations for the parameters of the binomial distribution imposed by existing computer hardware, and an established mathematical procedure for calculating error bounds for the normal approximation to the binomial distribution. Estimates are derived for the total number of cells in the FCM sample volume that must be available for processing and, for given FCM cell sorting decision speeds, the total elapsed times necessary to conduct particular experiments. The proposed approach obviates the need to resort to calculation expediencies such as the theoretically limited Poisson approximation for what can be considered a Bernoulli process mathematically characterized by the binomial distribution. Tables and graphs illustrate the projected times required to complete FCM experiments as a function of ‘‘effective’’ cell sorting decision speeds. Results from this paper also demonstrate that, as the ‘‘effective’’ cell sorting decision speed increases, there may not be a corresponding linear decrease in the time required to sort a given number of cells with selected statistical properties. The focus of this paper is on the use of innovative mathematical techniques for the design of experiments involving rare cell sorting.However,thesesamecomputationalapproaches may also prove useful for the high-speed enrichment sorting of non-rare cell subpopulations. Cytometry 27:233‐238,1997. r1997 Wiley-Liss, Inc.