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Norman A Lapin,1 Leoncio A Vergara,1,2 Yuri Mackeyev,3,4 Jared M Newton,1,5 Sean A Dilliard,6 Lon J Wilson,3,4 Steven A Curley,1 Rita E Serda1,7 1Michael E DeBakey Department of Surgery, Baylor College of Medicine, 2Institute of Biosciences & Technology, Texas A&M University, 3Department of Chemistry, Rice University, 4The Smalley-Curl Institute for Nanoscale Science and Technology, Rice University, 5Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, 6Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, 7Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM, USA Abstract: [60]Fullerene is a highly versatile nanoparticle (NP) platform for drug delivery to sites of pathology owing to its small size and both ease and versatility of chemical functionalization, facilitating multisite drug conjugation, drug targeting, and modulation of its physicochemical properties. The prominent and well-characterized role of the enhanced permeation and retention (EPR) effect in facilitating NP delivery to tumors motivated us to explore vascular transport kinetics of a water-soluble [60]fullerene derivatives using intravital microscopy in an immune competent murine model of breast adenocarcinoma. Herein, we present a novel local and global image analysis of vascular transport kinetics at the level of individual tumor blood vessels on the micron scale and across whole images, respectively. Similar to larger nanomaterials, [60]fullerenes displayed rapid extravasation from tumor vasculature, distinct from that in normal microvasculature. Temporal heterogeneity in fullerene delivery to tumors was observed, demonstrating the issue of nonuniform delivery beyond spatial dimensions. Trends in local region analysis of fullerene biokinetics by fluorescence quantification were in agreement with global image analysis. Further analysis of intratumoral vascular clearance rates suggested a possible enhanced penetration and retention effect of the fullerene compared to a 70 kDa vascular tracer. Overall, this study demonstrates the feasibility of tracking and quantifying the delivery kinetics and intratumoral biodistribution of fullerene-based drug delivery platforms, consistent with the EPR effect on short timescales and passive transport to tumors. Keywords: fullerene, intravital microscopy, breast cancer, enhanced permeation and retention, drug delivery

Rita Elena Serda Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, TX, USA Abstract: Elevated understanding and respect for the relevance of the immune system in cancer development and therapy has led to increased development of immunotherapeutic regimens that target existing cancer cells and provide long-term immune surveillance and protection from cancer recurrence. This review discusses using particles as immune adjuvants to create vaccines and to augment the anticancer effects of conventional chemotherapeutics. Several particle prototypes are presented, including liposomes, polymer nanoparticles, and porous silicon microparticles, the latter existing as either single- or multiparticle platforms. The benefits of using particles include immune-cell targeting, codelivery of antigens and immunomodulatory agents, and sustained release of the therapeutic payload. Nanotherapeutic-based activation of the immune system is dependent on both intrinsic particle characteristics and on the immunomodulatory cargo, which may include danger signals known as pathogen-associated molecular patterns and cytokines for effector-cell activation. Keywords: adjuvant, particle, immunotherapy, dendritic cell, cancer, vaccine

Guillermo U Ruiz-Esparza,1,3 Jose H Flores-Arredondo,2 Victor Segura-Ibarra,3 Guillermo Torre-Amione,2,3 Mauro Ferrari,1 Elvin Blanco,1,* Rita E Serda1,3,* 1Methodist Hospital Research Institute, 2Methodist DeBakey Heart and Vascular Center, Methodist Hospital, Houston, TX, USA; 3Escuela de Medicina y Ciencias de la Salud, Instituto Tecnológico y de Estudios Superiores de Monterrey, Cátedra de Cardiología y Medicina Vascular, Monterrey, Mexico *Senior authors Abstract: Heart disease remains the major cause of death in males and females, emphasizing the need for novel strategies to improve patient treatment and survival. A therapeutic approach, still in its infancy, is the development of site-specific drug-delivery systems. Nanoparticle-based delivery systems, such as liposomes, have evolved into robust platforms for site-specific delivery of therapeutics. In this review, the clinical impact of cardiovascular disease and the pathophysiology of different subsets of the disease are described. Potential pathological targets for therapy are introduced, and promising advances in nanotherapeutic cardiovascular applications involving liposomal platforms are presented. Keywords: liposomes, cardiovascular disease, therapeutics, nanoparticles, nanomedicine

Intraperitoneal (IP) administration of immunogenic mesoporous silica nanoparticles (iMSN) in a mouse model of metastatic ovarian cancer promotes the development of tumor-specific CD8 + T cells and protective immunity. IP delivery of iMSN functionalized with the Toll-like receptor (TLR) agonists polyethyleneimine (PEI), CpG oligonucleotide, and monophosphoryl lipid A (MPLA) stimulated rapid uptake by all peritoneal myeloid subsets. Myeloid cells quickly transported iMSN to milky spots and fat-associated lymphoid clusters (FALCs) present in tumor-burdened adipose tissues, leading to a reduction in suppressive T cells and an increase in activated memory T cells. Two doses of iMSN cleared or reduced ovarian and colorectal cancer and protected against future tumor engraftment. In contrast, subcutaneous (SC) and intravenous (IV) delivery of iMSN were without therapeutic effect in mice with peritoneal metastases, supporting the need for activation of regional immune cells. Remarkably, intraperitoneal delivery of iMSN cleared subcutaneously implanted ovarian cancer, supporting homing of antigen specific T cells to extraperitoneal tumor sites., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Tunneling nanotubes (TNTs) are open-ended, membrane-encased extensions that connect neighboring cells. They have diameters up to 1 μm but are able to expand to convey large cargos. Lengths vary depending on the distance of the cells but have been reported to be capable of extending beyond 300 μm. They have actin cytoskeletons that are essential for their formation, and may or may not have microtubule networks. It is thought that thin TNTs lack microtubules, while thicker TNTs have microtubular highways that use motor proteins to convey materials, including proteins, mitochondria, and nanoparticles between cells. Specifically, the presence of dynein and myosin support trafficking of cargo in both directions. The purpose of these connections is to enable cells to work as a unit or to extend cell life by diluting cytotoxic agents or acquiring biological material needed to survive., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

As cancer progresses, tumor cells adapt to evade immune cells. To counter this, cancer cells can be silicified ex vivo, creating surface masks that can be decorated with microbial-associated molecules that are readily recognized by antigen-presenting cells (APCs). The transformation process renders the tumor cells nonviable and preserves the integrity of the cell and associated tumor antigens. The resulting personalized cancer vaccine, when returned to the patient, engages molecules on the surface of APC, activating signaling pathways that lead to immune cell activation, vaccine internalization, processing of tumor antigens, and major histocompatibility complex peptide presentation to T cells. The cancer-specific T cells then circulate throughout the body, killing tumor cells. This chapter presents detailed methods for the cryogenic precipitation of silica on cellular structures (cryo-silicification), creating vaccines that are potent immune activators. Further, silicified cells can be dehydrated for shelf storage, eliminating the need for costly cryogenic storage., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Immunogenic lipid-coated mesoporous silica nanoparticles (ILM) present pathogen-associated molecular patterns (PAMPs) on the nanoparticle surface to engage pathogen-associated receptors on immune cells. The mesoporous core is capable of loading additional immunogens, antigens or drugs. In this study, the impact of lipid composition, surface potential and intercalation of lipophilic monophosphoryl lipid A (MPL-A) in the lipid coat on nanoparticle properties and cellular interactions is presented. Loading and retention of the model antigen ovalbumin into the mesoporous silica core were found to be similar for all nanoparticle formulations, with presentation of ova peptide (SIINFEKL) by major histocompatibility complex (MHC) evaluated to facilitate the selection of an anionic nanoparticle composition. ILM were able to induce lysosomal tubulation and streaming of lysosomes towards the cell surface in dendritic cells, leading to an enhanced surface presentation of MHC. Myeloid cells robustly internalized all ILM formulations; however, non-myeloid cells selectively internalized cationic ILM in vitro in the presence of 20% serum. Interestingly, ILM administration to the peritoneal cavity of mice with disseminated ovarian cancer resulted in selective accumulation of ILM in tumor-associated tissues (>80%), regardless of nanoparticle surface charge or the presence of MPL-A. Immunofluorescence analysis of the omental tumor showed that ILMs, regardless of surface charge, were localized within clusters of CD11b + myeloid cells 24 h post administration. Selective uptake of ILMs by myeloid cells in vivo indicates that these cells outcompete other cell populations in the ovarian tumor microenvironment, making them a strong target for therapeutic interventions.

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).)

Current imaging approaches used to monitor tumor progression can lack the ability to distinguish true progression from pseudoprogression. Simultaneous metabolic 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) positron emission tomography (PET) and magnetic resonance imaging (MRI) offers new opportunities to overcome this challenge by refining tumor identification and monitoring therapeutic responses to cancer immunotherapy. In the current work, spatial and quantitative analysis of tumor burden were performed using simultaneous [ 18 F]FDG-PET/MRI to monitor therapeutic responses to a novel silicified cancer cell immunotherapy in a mouse model of disseminated serous epithelial ovarian cancer. Tumor progression was validated by bioluminescence imaging of luciferase expressing tumor cells, flow cytometric analysis of immune cells in the tumor microenvironment, and histopathology. While PET demonstrated the presence of metabolically active cancer cells through [ 18 F]FDG uptake, MRI confirmed cancer-related accumulation of ascites and tissue anatomy. This approach provides complementary information on disease status without a confounding signal from treatment-induced inflammation. This work provides a possible roadmap to facilitate accurate monitoring of therapeutic responses to cancer immunotherapies.

The production of personalized cancer vaccines made from autologous tumour cells could benefit from mechanisms that enhance immunogenicity. Here we show that cancer vaccines can be made via the cryogenic silicification of tumour cells, which preserves tumour antigens within nanoscopic layers of silica, followed by the decoration of the silicified surface with pathogen-associated molecular patterns. These pathogen-mimicking cells activate dendritic cells and enhance the internalization, processing and presentation of tumour antigens to T cells. In syngeneic mice with high-grade ovarian cancer, a cell-line-based silicified cancer vaccine supported the polarization of CD4 + T cells towards the T-helper-1 phenotype in the tumour microenvironment, and induced tumour-antigen-specific T-cell immunity, resulting in complete tumour eradication and in long-term animal survival. In the setting of established disease and a suppressive tumour microenvironment, the vaccine synergized with cisplatin. Silicified and surface-modified cells from tumour samples are amenable to dehydration and room-temperature storage without loss of efficacy and may be conducive to making individualized cancer vaccines across tumour types., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

This study examines intra- and intercellular trafficking of mesoporous silica nanoparticles along microtubular highways, with an emphasis on intercellular bridges connecting interphase and telophase cells. The study of nanoparticle trafficking within and between cells during all phases of the cell cycle is relevant to payload destination and dilution, and impacts delivery of therapeutic or diagnostic agents. Super-resolution stochastic optical reconstruction and sub-airy unit image acquisition, the latter combined with Huygens deconvolution microscopy, enable single nanoparticle and microtubule resolution. Combined structural and functional data provide enhanced details on biological processes, with an example of mitotic inheritance during cancer cell trivision.

Macrophages line the walls of microvasculature, extending processes into the blood flow to capture foreign invaders, including nano-scale materials. Using mesoporous silica nanoparticles (MSNs) as a model nano-scale system, we show the interplay between macrophages and MSNs from initial uptake to intercellular trafficking to neighboring cells along microtubules. The nature of cytoplasmic bridges between cells and their role in the cell-to-cell transfer of nano-scale materials is examined, as is the ability of macrophages to function as carriers of nanomaterials to cancer cells. Both direct administration of nanoparticles and adoptive transfer of nanoparticle-loaded splenocytes in mice resulted in abundant localization of nanomaterials within macrophages 24 h post-injection, predominately in the liver. While heterotypic, trans-species nanomaterial transfer from murine macrophages to human HeLa cervical cancer cells or A549 lung cancer cells was robust, transfer to syngeneic 4T1 breast cancer cells was not detected in vitro or in vivo. Cellular connections and nanomaterial transfer in vivo were rich among immune cells, facilitating coordinated immune responses.

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.)

Targeted drug delivery remains at the forefront of biomedical research but remains a challenge to date. Herein, the first superassembly of nanosized metal-organic polyhedra (MOP) and their biomimetic coatings of lipid bilayers are described to synergistically combine the advantages of micelles and supramolecular coordination cages for targeted drug delivery. The superassembly technique affords unique hydrophobic features that endow individual MOP to act as nanobuilding blocks and enable their superassembly into larger and well-defined nanocarriers with homogeneous sizes over a broad range of diameters. Various cargos are controllably loaded into the MOP with high payloads, and the nanocages are then superassembled to form multidrug delivery systems. Additionally, functional nanoparticles are introduced into the superassemblies via a one-pot process for versatile bioapplications. The MOP superassemblies are surface-engineered with epidermal growth factor receptors and can be targeted to cancer cells. In vivo studies indicated the assemblies to have a substantial circulation half-life of 5.6 h and to undergo renal clearance-characteristics needed for nanomedicines., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Beyond heterogeneous cancer cells, the tumor microenvironment includes stromal and immune cells, blood vessels, extracellular matrix and biologically active molecules. Abnormal signaling, uncontrolled proliferation and high interstitial pressure all contribute to a chaotic, non-hierarchical vascular organization. Using an immune competent 4T1 breast adenocarcinoma murine model, this study fully characterizes the architecture and immunocyte milieu of the tumor microenvironment. Heterogeneous vessel distribution, chaotic connectivity, limited perfusion, cancer cell density, immune phenotype, and biological responses to immune therapy are presented. Cancer cell density mirrored the distribution of large, perfusable vessels, both predominately in the tumor periphery. Intratumoral administration of the proinflammatory cytokine IL-12 led to an increase in CD45 + leukocytes, with a specific increase in CD4 + and CD8 + T cells, and a decrease in the percentage of Gr-l lo myeloid-derived suppressor cells. Concomitantly, serum G-CSF, IL-10 and VEGF decreased, while CXCR9 and interferon gamma increased. The distribution pattern of infiltrating monocytes/macrophages, visualized using a fluorescent perfluorocarbon emulsion, indicated that macrophages predominately localize in the vicinity of large blood vessels. Electron microscopy supports the presence of dense tumor cell masses throughout the tumor, with the largest vessels present in the surrounding mammary fat pad. Overall, large vessels in the 4T1 tumor periphery support high, localized vascular perfusion and myeloid accumulation. The pro-inflammatory cytokine IL-12 stimulated a transition towards T helper 1 cytokines in serum, supporting suppression of tumor growth and angiostatic conditions.

Venezuelan equine encephalitis virus (VEEV) poses a major public health risk due to its amenability for use as a bioterrorism agent and its severe health consequences in humans. ML336 is a recently developed chemical inhibitor of VEEV, shown to effectively reduce VEEV infection in vitro and in vivo. However, its limited solubility and stability could hinder its clinical translation. To overcome these limitations, lipid-coated mesoporous silica nanoparticles (LC-MSNs) were employed. The large surface area of the MSN core promotes hydrophobic drug loading while the liposome coating retains the drug and enables enhanced circulation time and biocompatibility, providing an ideal ML336 delivery platform. LC-MSNs loaded 20 ± 3.4 μg ML336/mg LC-MSN and released 6.6 ± 1.3 μg/mg ML336 over 24 hours. ML336-loaded LC-MSNs significantly inhibited VEEV in vitro in a dose-dependent manner as compared to unloaded LC-MSNs controls. Moreover, cell-based studies suggested that additional release of ML336 occurs after endocytosis. In vivo safety studies were conducted in mice, and LC-MSNs were not toxic when dosed at 0.11 g LC-MSNs/kg/day for four days. ML336-loaded LC-MSNs showed significant reduction of brain viral titer in VEEV infected mice compared to PBS controls. Overall, these results highlight the utility of LC-MSNs as drug delivery vehicles to treat VEEV.

Background: Applied radiofrequency (RF) energy induces hyperthermia in tissues, facilitating vascular perfusion This study explores the impact of RF radiation on the integrity of the luminal endothelium, and then predominately explores the impact of altering the conductivity of biologically-relevant solutions on RF-induced heating rates and cell death. The ability of cells to survive high sucrose (i.e. hyperosmotic conditions) to achieve lower conductivity as a mechanism for directing hyperthermia is evaluated., Methods: RF radiation was generated using a capacitively-coupled radiofrequency system operating at 13.56 MHz. Temperatures were recorded using a FLIR SC 6000 infrared camera., Results: RF radiation reduced cell-to-cell connections among endothelial cells and altered cell morphology towards a more rounded appearance at temperatures reported to cause in vivo vessel deformation. Isotonic solutions containing high sucrose and low levels of NaCl displayed low conductivity and faster heating rates compared to high salt solutions. Heating rates were positively correlated with cell death. Addition of sucrose to serum similarly reduced conductivity and increased heating rates in a dose-dependent manner. Cellular proliferation was normal for cells grown in media supplemented with 125 mM sucrose for 24 hours or for cells grown in 750 mM sucrose for 10 minutes followed by a 24 h recovery period., Conclusions: Sucrose is known to form weak hydrogen bonds in fluids as opposed to ions, freeing water molecules to rotate in an oscillating field of electromagnetic radiation and contributing to heat induction. The ability of cells to survive temporal exposures to hyperosmotic (i.e. elevated sucrose) conditions creates an opportunity to use sucrose or other saccharides to selectively elevate heating in specific tissues upon exposure to a radiofrequency field., Competing Interests: Declaration Statement: The authors report no declarations of interest.

The use of non-invasive radiofrequency (RF) energy to induce mild thermal and non-thermal effects in cancer tissue is under study as an adjuvant to chemo, radio or immuno therapy. This study examines cell specific sensitivities to RF exposure and the potential of nanoparticles to elevate heating rates or enhance biological effects. Increases in the heating rate of water in an RF field operating at 13.56MHz (0.004-0.028°C/s) were positively correlated with concentration of hybrid nanoparticles (1-10mg/ml) consisting of water soluble malonodiserinolamide [60]fullerene (C 60 -ser) conjugated to the surface of mesoporous silica nanoparticles (SiO 2 -C 60 ). The heating rate of highly conductive cell culture media (0.024°C/s) was similar to that of the highest concentration of nanoparticles in water, with no significant increase due to addition of nanoparticles at relevant doses (<100μg/ml). With respect to cell viability, anionic (SiO 2 and SiO 2 -C 60 ) or neutral (C 60 ) nanoparticles did not influence RF-induced cell death, however, cationic nanoparticles (4-100μg/ml) caused dose-dependent increases in RF-induced cell death (24-42% compared to RF only). The effect of cell type, size and immortalization on sensitivity of cells to RF fields was examined in endothelial (HUVEC and HMVEC), fibroblast (primary dermal and L939) and cancer cells (HeLa and 4T1). While the state of cellular immortalization itself did not consistently influence the rate of RF-induced cell death compared to normal cell counter parts, cell size (ranging from 7 to 30μm) negatively correlated with cell sensitivity to RF (21-97% cell death following 6min irradiation). In summary, while nanoparticles do not alter the heating rate of biologically-relevant solutions, they can increase RF-induced cell death based on intrinsic cytotoxicity; and cells with smaller radii, and thereby greater surface membrane, are more susceptible to cell damage in an RF field than larger cells., Statement of Significance: The ability of nanoparticles to either direct heating or increase susceptibility of cancer cells to radiofrequency (RF) energy remains controversial, as is the impact of cell attributes on susceptibility of cells to RF-induced cell death. This manuscript examines the impact of nanoparticle charge, size, and cellular localization on RF-induced cell death and the influence of nanoparticles on the heating rates of water and biologically-relevant media. Susceptibility of immortalized or primary cells to RF energy and the impact of cell size are also examined. The ability to selectively modulate RF heating rates in specific biological locations or in specific cell populations would enhance the therapeutic potential of RF therapy., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Aims: Ongoing inflammation and endothelial dysfunction occurs within the local microenvironment of heart failure, creating an appropriate scenario for successful use and delivery of nanovectors. This study sought to investigate whether cardiovascular cells associate, internalize, and traffic a nanoplatform called mesoporous silicon vector (MSV), and determine its intravenous accumulation in cardiac tissue in a murine model of heart failure., Methods and Results: In vitro cellular uptake and intracellular trafficking of MSVs was examined by scanning electron microscopy, confocal microscopy, time-lapse microscopy, and flow cytometry in cardiac myocytes, fibroblasts, smooth muscle cells, and endothelial cells. The MSVs were internalized within the first hours, and trafficked to perinuclear regions in all the cell lines. Cytotoxicity was investigated by annexin V and cell cycle assays. No significant evidence of toxicity was found. In vivo intravenous cardiac accumulation of MSVs was examined by high content fluorescence and confocal microscopy, with results showing increased accumulation of particles in failing hearts compared with normal hearts. Similar to observations in vitro, MSVs were able to associate, internalize, and traffic to the perinuclear region of cardiomyocytes in vivo., Conclusions: Results show that MSVs associate, internalize, and traffic in cardiovascular cells without any significant toxicity. Furthermore, MSVs accumulate in failing myocardium after intravenous administration, reaching intracellular regions of the cardiomyocytes. These findings represent a novel avenue to develop nanotechnology-based therapeutics and diagnostics in heart failure., Competing Interests: Conflicts of Interest: None declared., (© 2016 The Authors European Journal of Heart Failure © 2016 European Society of Cardiology.)

Functionalization of nanoparticles with cationic moieties, such as polyethyleneimine (PEI), enhances binding to the cell membrane; however, it also disrupts the integrity of the cell's plasma and vesicular membranes, leading to cell death. Primary fibroblasts were found to display high surface affinity for cationic iron oxide nanoparticles and greater sensitivity than their immortalized counterparts. Treatment of cells with cationic nanoparticles in the presence of incremental increases in serum led to a corresponding linear decrease in cell death. The surface potential of the nanoparticles also decreased linearly as serum increased and this was strongly and inversely correlated with cell death. While low doses of nanoparticles were rendered non-toxic in 25% serum, large doses overcame the toxic threshold. Serum did not reduce nanoparticle association with primary fibroblasts, indicating that the decrease in nanoparticle cytotoxicity was based on serum masking of the PEI surface, rather than decreased exposure. Primary endothelial cells were likewise more sensitive to the cytotoxic effects of cationic nanoparticles than their immortalized counterparts, and this held true for cellular responses to cationic microparticles despite the much lower toxicity of microparticles compared to nanoparticles.

Herein, we present a novel imaging platform to study the biological effects of non-invasive radiofrequency (RF) electric field cancer hyperthermia. This system allows for real-time in vivo intravital microscopy (IVM) imaging of radiofrequency-induced biological alterations such as changes in vessel structure and drug perfusion. Our results indicate that the IVM system is able to handle exposure to high-power electric-fields without inducing significant hardware damage or imaging artifacts. Furthermore, short durations of low-power (< 200 W) radiofrequency exposure increased transport and perfusion of fluorescent tracers into the tumors at temperatures below 41°C. Vessel deformations and blood coagulation were seen for tumor temperatures around 44°C. These results highlight the use of our integrated IVM-RF imaging platform as a powerful new tool to visualize the dynamics and interplay between radiofrequency energy and biological tissues, organs, and tumors.

Acute lung injury (ALI) and its most severe manifestation, acute respiratory distress syndrome (ARDS), is a clinical syndrome defined by acute hypoxemic respiratory failure and bilateral pulmonary infiltrates consistent with edema. In-hospital mortality is 38.5% for AL, and 41.1% for ARDS. Activation of alveolar macrophages in the donor lung causes the release of pro-inflammatory chemokines and cytokines, such as TNF-α. To determine the relevance of TNF-α in disrupting bronchial endothelial cell function, we stimulated human THP-1 macrophages with lipopolysaccharide (LPS) and used the resulting cytokine-supplemented media to disrupt normal endothelial cell functions. Endothelial tube formation was disrupted in the presence of LPS-activated THP- 1 conditioned media, with reversal of the effect occurring in the presence of 0.1µg/ml Enbrel, indicating that TNF-α was the major serum component inhibiting endothelial tube formation. To facilitate lung conditioning, we tested liposomal and porous silicon (pSi) delivery systems for their ability to selectively silence TNFR1 using siRNA technology. Of the three types of liposomes tested, only cationic liposomes had substantial endothelial uptake, with human cells taking up 10-fold more liposomes than their pig counterparts; however, non-specific cellular activation prohibited their use as immunosuppressive agents. On the other hand, pSi microparticles enabled the accumulation of large amounts of siRNA in endothelial cells compared to standard transfection with Lipofectamine(®) LTX, in the absence of non-specific activation of endothelia. Silencing of TNFR1 decreased TNF-α mediated inhibition of endothelial tube formation, as well as TNF-α-induced upregulation of ICAM-1, VCAM, and E-selection in human lung microvascular endothelial cells.

There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticle-nanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Dendritic cells (DC) process and present antigens to T lymphocytes, inducing potent immune responses when encountered in association with activating signals, such as pathogen-associated molecular patterns. Using the 4T1 murine model of breast cancer, cationic liposomes containing monophosphoryl lipid A (MPL) and interleukin (IL)-12 were administered by intratumoral injection. Combination multivalent presentation of the Toll-like receptor-4 ligand MPL and cytotoxic 1,2-dioleoyl-3-trmethylammonium-propane lipids induced cell death, decreased cellular proliferation, and increased serum levels of IL-1β and tumor necrosis factor (TNF)-α. The addition of recombinant IL-12 further suppressed tumor growth and increased expression of IL-1β, TNF-α, and interferon-γ. IL-12 also increased the percentage of cytolytic T cells, DC, and F4/80(+) macrophages in the tumor. While single agent therapy elevated levels of nitric oxide synthase 3-fold above basal levels in the tumor, combination therapy with MPL cationic liposomes and IL-12 stimulated a 7-fold increase, supporting the observed cell cycle arrest (loss of Ki-67 expression) and apoptosis (TUNEL positive). In mice bearing dual tumors, the growth of distal, untreated tumors mirrored that of liposome-treated tumors, supporting the presence of a systemic immune response.

Understanding the effect of variability in the interaction of individual cells with nanoparticles on the overall response of the cell population to a nanoagent is a fundamental challenge in bionanotechnology. Here, we show that the technique of time-resolved, high-throughput microscopy can be used in this endeavor. Mass measurement with single-cell resolution provides statistically robust assessments of cell heterogeneity, while the addition of a temporal element allows assessment of separate processes leading to deconvolution of the effects of particle supply and biological response. We provide a specific demonstration of the approach, in vitro, through time-resolved measurement of fibroblast cell (HFF-1) death caused by exposure to cationic nanoparticles. The results show that heterogeneity in cell area is the major source of variability with area-dependent nanoparticle capture rates determining the time of cell death and hence the form of the exposure–response characteristic. Moreover, due to the particulate nature of the nanoparticle suspension, there is a reduction in the particle concentration over the course of the experiment, eventually causing saturation in the level of measured biological outcome. A generalized mathematical description of the system is proposed, based on a simple model of particle depletion from a finite supply reservoir. This captures the essential aspects of the nanoparticle–cell interaction dynamics and accurately predicts the population exposure–response curves from individual cell heterogeneity distributions.

Porous silicon (pSi) microparticles, in diverse sizes and shapes, can be functionalized to present pathogen-associated molecular patterns that activate dendritic cells. Intraperitoneal injection of MPL-adsorbed pSi microparticles, in contrast to free MPL, resulted in the induction of local inflammation, reflected in the recruitment of neutrophils, eosinophils and proinflammatory monocytes, and the depletion of resident macrophages and mast cells at the injection site. Injection of microparticle-bound MPL resulted in enhanced secretion of the T helper 1 associated cytokines IFN-γ and TNF-α by peritoneal exudate and lymph node cells in response to secondary stimuli while decreasing the anti-inflammatory cytokine IL-10. MPL-pSi microparticles independently exhibited anti-tumor effects and enhanced tumor suppression by low dose doxorubicin nanoliposomes. Intravascular injection of the MPL-bound microparticles increased serum IL-1β levels, which was blocked by the IL-1 receptor antagonist Anakinra. The microparticles also potentiated tumor infiltration by dendritic cells, cytotoxic T lymphocytes, and F4/80+ macrophages, however, a specific reduction was observed in CD204+ macrophages.

Aims: Endothelial cells are dynamic cells tasked with selective transport of cargo from blood vessels to tissues. Here we demonstrate the potential for nanoparticle transport across endothelial cells in membrane-bound vesicles., Materials & Methods: Cell-free endothelial-derived biovesicles were characterized for cellular and nanoparticle content by electron microscopy. Confocal microscopy was used to evaluate biovesicles for organelle-specific proteins, and to monitor biovesicle engulfment by naive cells., Results: Nanoparticle-laden biovesicles containing low-density polyethyleneimine nanoparticles appear to be predominately of endosomal origin, combining features of multivesicular bodies, lysosomes and autophagosomes. Conversely, high-density polyethyleneimine nanoparticles stimulate the formation of biovesicles associated with cellular apoptotic breakdown. Secreted LAMP-1-positive biovesicles are internalized by recipient cells, either of the same origin or of novel phenotype., Conclusion: Cellular biovesicles, rich in cellular signals, present an important mode of cell-to-cell communication either locally or through broadcasting of biological messages.

Modulation of the cytokine milieu is one approach for vaccine development. However, therapy with pro-inflammatory cytokines, such as IL-12, is limited in practice due to adverse systemic effects. Spatially-restricted gene expression circumvents this problem by enabling localized amplification. Intracellular co-delivery of gold nanorods (AuNR) and a heat shock protein 70 (HSP70) promoter-driven expression vector enables gene expression in response to near infrared (NIR) light. AuNRs absorb the light, convert it into heat and thereby stimulate photothermal expression of the cytokine. As proof-of-concept, human HeLa and murine B16 cancer cells were transfected with a HSP70-Enhanced Green Fluorescent Protein (EGFP) plasmid and polyethylenimine (PEI)-conjugated AuNRs. Exposure to either 42 °C heat-shock or NIR light induced significant expression of the reporter gene. In vivo NIR driven expression of the reporter gene was confirmed at 6 and 24 h in mice bearing B16 melanoma tumors using in vivo imaging and flow-cytometric analysis. Overall, we demonstrate a novel opportunity for site-directed, heat-inducible expression of a gene based upon the NIR-absorbing properties of AuNRs and a HSP70 promoter-driven expression vector.

Tracking vaccine components from the site of injection to their destination in lymphatic tissue, and simultaneously monitoring immune effects, sheds light on the influence of vaccine components on particle and immune cell trafficking and therapeutic efficacy. In this study, we create a hybrid particle vaccine platform comprised of porous silicon (pSi) and superparamagnetic iron oxide nanoparticles (SPIONs). The impact of nanoparticle size and mode of presentation on magnetic resonance contrast enhancement are examined. SPION-enhanced relaxivity increased as the core diameter of the nanoparticle increased, while encapsulation of SPIONs within a pSi matrix had only minor effects on T2 and no significant effect on T2* relaxation. Following intravenous injection of single and hybrid particles, there was an increase in negative contrast in the spleen, with changes in contrast being slightly greater for free compared to silicon encapsulated SPIONs. Incubation of bone marrow-derived dendritic cells (BMDC) with pSi microparticles loaded with SPIONs, SIINFEKL peptide, and lipopolysaccharide stimulated immune cell interactions and interferon gamma production in OT-1 TCR transgenic CD8 + T cells. Overall, the hybrid particle platform enabled presentation of a complex payload that was traceable, stimulated functional T cell and BMDC interactions, and resolved in cellular activation of T cells in response to a specific antigen.

Biomedical applications of porous silicon include drug delivery, imaging, diagnostics and immunotherapy. This review summarizes new silicon particle fabrication techniques, dynamics of cellular transport, advances in the multistage vector approach to drug delivery, and the use of porous silicon as immune adjuvants. Recent findings support superior therapeutic efficacy of the multistage vector approach over single particle drug delivery systems in mouse models of ovarian and breast cancer. With respect to vaccine development, multivalent presentation of pathogen-associated molecular patterns on the particle surface creates powerful platforms for immunotherapy, with the porous matrix able to carry both antigens and immune modulators., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

New insights into the intra- and intercellular trafficking of drug delivery particles challenges the dogma of particles as static intracellular depots for sustained drug release. Recent discoveries in the cell-to-cell transfer of cellular constituents, including proteins, organelles, and microparticles sheds light on new ways to propagate signals and therapeutics. While beneficial for the dispersion of therapeutics at sites of pathologies, propagation of biological entities advancing disease states is less desirable. Mechanisms are presented for the transfer of porous silicon microparticles between cells. Direct cell-to-cell transfer of microparticles by means of membrane adhesion or using membrane extensions known as tunneling nanotubes is presented. Cellular relays, or shuttle cells, are also shown to mediate the transfer of microparticles between cells. These microparticle-transfer events appear to be stimulated by environmental cues, introducing a new paradigm of environmentally triggered propagation of cellular signals and rapid dispersion of particle-delivered therapeutics. The opportunity to use microparticles to study cellular transfer events and biological triggers that induce these events may aid in the discovery of therapeutics that limit the spread of disease., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Porous silicon microparticles presenting pathogen-associated molecular patterns mimic pathogens, enhancing internalization of the microparticles and activation of antigen presenting dendritic cells. We demonstrate abundant uptake of microparticles bound by the TLR-4 ligands LPS and MPL by murine bone marrow-derived dendritic cells (BMDC). Labeled microparticles induce concentration-dependent production of IL-1β, with inhibition by the caspase inhibitor Z-VAD-FMK supporting activation of the NLRP3-dependent inflammasome. Inoculation of BALB/c mice with ligand-bound microparticles induces a significant increase in circulating levels of IL-1β, TNF-α, and IL-6. Stimulation of BMDC with ligand-bound microparticles increases surface expression of costimulatory and MHC molecules, and enhances migration of BMDC to the draining lymph node. LPS-microparticles stimulate in vivo C57BL/6 BMDC and OT-1 transgenic T cell interactions in the presence of OVA SIINFEKL peptide in lymph nodes, with intact nodes imaged using two-photon microscopy. Formation of in vivo and in vitro immunological synapses between BMDC, loaded with OVA peptide and LPS-microparticles, and OT-1 T cells are presented, as well as elevated intracellular interferon gamma levels in CD8(+) T cells stimulated by BMDC carrying peptide-loaded microparticles. In short, ligand-bound microparticles enhance (1) phagocytosis of microparticles; (2) BMDC inflammasome activation and upregulation of costimulatory and MHC molecules; (3) cellular migration of BMDC to lymphatic tissue; and (4) cellular interactions leading to T cell activation in the presence of antigen.

Purpose: To evaluate the effects of near-infrared (NIR) laser irradiation of microspheres (MS) containing hollow gold nanospheres (HAuNS) and paclitaxel (PTX) administered intraarterially in an animal model., Materials and Methods: For the ex vivo experiments, VX2 tumor-bearing rabbits underwent administration of MS-HAuNS or MS via the hepatic artery (HA). The animals were killed, the liver tumors were subjected to NIR irradiation, and temperature changes were estimated with magnetic resonance (MR) imaging. For the in vivo study, VX2 tumor-bearing rabbits were randomly assigned to three groups: MS-HAuNS-PTX-plus-NIR, MS-HAuNS-PTX, and saline-plus-NIR. Laser irradiation was delivered at 1 hour and at 3 days after administration of saline or MS-HAuNS-PTX via the HA. Animals were euthanized, and tumors were analyzed for necrosis and apoptosis. Plasma samples were collected from the MS-HAuNS-PTX-plus-NIR animals for PTX analysis., Results: Ex vivo experiments showed intratumoral heating in animals that received MS-HAuNS but no temperature change in animals that received MS. Animals treated with MS-HAuNS-PTX-plus-NIR showed a transient increase in plasma PTX levels after each NIR irradiation and significantly greater tumor necrosis than animals that received MS-HAuNS-PTX or saline-plus-NIR (44.9% vs 13.8% or 23.7%; P < .0001). The mean apoptotic index in the MS-HAuNS-PTX-plus-NIR group (5.01 ± 1.66) was significantly higher than the mean apoptotic index in the MS-HAuNS-PTX (2.99 ± 0.97) or saline-plus-NIR (1.96 ± 0.40) groups (P = .0013)., Conclusions: NIR laser irradiation after MS-HAuNS-PTX administration results in intratumoral heating and increases the efficacy of treatment. Further studies are required to evaluate the optimal laser settings to maximize therapeutic efficacy., (Copyright © 2012 SIR. Published by Elsevier Inc. All rights reserved.)

Logic-embedded vectors (LEVs) have been introduced as a means to overcome sequential, biological barriers that prevent particle-based drug delivery systems from reaching their targets. In this chapter, we address the challenge of fabricating and optimizing LEVs to reach non-endosomal targets. We describe the general preparation, characterization, and cellular association of porous silicon-based LEVs. A specific example of LEV fabrication from start to finish, along with optimization and troubleshooting information, is presented to serve as a template for future designs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Over the last few decades a great variety of nanotechnology based platforms have been synthesized and fabricated to improve the delivery of active compounds to a disease site. Nanoparticles currently used in the clinic, and the majority of nanotherapeutics/nanodiagnostics under investigation, accommodate single- or multiple- functionalities on the same entity. Because many heterogeneous biological barriers can prevent therapeutic and imaging agents from reaching their intended targets in sufficient concentrations, there is an emerging requirement to develop a multimodular nanoassembly, in which different components with individual specific functions act in a synergistic manner. The multistage nanovectors (MSVs) were introduced in 2008 as the first system of this type. It comprises several nanocomponents or "stages", each of which is designed to negotiate one or more biological barriers. Stage 1 mesoporous silicon particles (S1MPs) were rationally designed and fabricated in a nonspherical geometry to enable superior blood margination and to increase cell surface adhesion. The main task of S1MPs is to efficiently transport nanoparticles that are loaded into their porous structure and to protect them during transport from the administration site to the disease lesion. Semiconductor fabrication techniques including photolithography and electrochemical etching allow for the exquisite control and precise reproducibility of S1MP physical characteristics such as geometry and porosity. Furthermore, S1MPs can be chemically modified with negatively/positively charged groups, PEG and other polymers, fluorescent probes, contrast agents, and biologically active targeting moieties including antibodies, peptides, aptamers, and phage. The payload nanoparticles, termed stage 2 nanoparticles (S2NPs), can be any currently available nanoparticles such as liposomes, micelles, inorganic/metallic nanoparticles, dendrimers, and carbon structures, within the approximate size range of 5-100 nm in diameter. Depending upon the physicochemical features of the S1MP (geometry, porosity, and surface modifications), a variety of S2NPs or nanoparticle "cocktails" can be loaded and efficiently delivered to the disease site. As demonstrated in the studies reviewed here, once the S2NPs are loaded into the S1MPs, a variety of novel properties emerge, which enable the design of new and improved imaging contrast agents and therapeutics. For example, the loading of the MRI Gd-based contrast agents onto hemispherical and discoidal S1MPs significantly increased the longitudal relaxivity (r1) to values of up to 50 times larger than those of clinically available gadolinium-based agents (~4 mM(-1) s(-1)/Gd(3+) ion). Furthermore, administration of a single dose of MSVs loaded with neutral nanoliposomes containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein enabled sustained EphA2 gene silencing for at least 21 days. As a result, the tumor burden was reduced in an orthotopic mouse model of ovarian cancer. We envision that the versatility of the MSV platform and its emerging properties will enable the creation of personalized solutions with broad clinical implications within and beyond the realm of cancer theranostics.

Background: The daunting task for drug molecules to reach pathological lesions has fueled rapid advances in Nanomedicine. The progressive evolution of nanovectors has led to the development of multi-stage delivery systems aimed at overcoming the numerous obstacles encountered by nanovectors on their journey to the target site., Scope of Review: This review summarizes major findings with respect to silicon-based drug delivery vectors for cancer therapeutics and imaging. Based on rational design, well-established silicon technologies have been adapted for the fabrication of nanovectors with specific shapes, sizes, and porosities. These vectors are part of a multi-stage delivery system that contains multiple nano-components, each designed to achieve a specific task with the common goal of site-directed delivery of therapeutics., Major Conclusions: Quasi-hemispherical and discoidal silicon microparticles are superior to spherical particles with respect to margination in the blood, with particles of different shapes and sizes having unique distributions in vivo. Cellular adhesion and internalization of silicon microparticles is influenced by microparticle shape and surface charge, with the latter dictating binding of serum opsonins. Based on in vitro cell studies, the internalization of porous silicon microparticles by endothelial cells and macrophages is compatible with cellular morphology, intracellular trafficking, mitosis, cell cycle progression, cytokine release, and cell viability. In vivo studies support superior therapeutic efficacy of liposomal encapsulated siRNA when delivered in multi-stage systems compared to free nanoparticles. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine., (2010 Elsevier B.V. All rights reserved.)

Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.

A new generation of nanocarriers, logic-embedded vectors (LEVs), is endowed with the ability to localize components at multiple intracellular sites, thus creating an opportunity for synergistic control of redundant or dual-hit pathways. LEV encoding elements include size, shape, charge, and surface chemistry. In this study, LEVs consist of porous silicon nanocarriers, programmed for cellular uptake and trafficking along the endosomal pathway, and surface-tailored iron oxide nanoparticles, programmed for endosomal sorting and partitioning of particles into unique cellular locations. In the presence of persistent endosomal localization of silicon nanocarriers, amine-functionalized nanoparticles are sorted into multiple vesicular bodies that form novel membrane-bound compartments compatible with cellular secretion, while chitosan-coated nanoparticles escape from endosomes and enter the cytosol. Encapsulation within the porous silicon matrix protects these nanoparticle surface-tailored properties, and enhances endosomal escape of chitosan-coated nanoparticles. Thus, LEVs provide a mechanism for shielded transport of nanoparticles to the lesion, cellular manipulation at multiple levels, and a means for targeting both within and between cells., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Magnetic resonance imaging contrast agents are currently designed by modifying their structural and physiochemical properties to improve relaxivity and to enhance image contrast. Here, we show a general method for increasing relaxivity by confining contrast agents inside the nanoporous structure of silicon particles. Magnevist, gadofullerenes and gadonanotubes were loaded inside the pores of quasi-hemispherical and discoidal particles. For all combinations of nanoconstructs, a boost in longitudinal proton relaxivity r(1) was observed: Magnevist, r(1) ≈ 14 mM(-1) s(-1)/Gd(3+) ion (∼ 8.15 × 10(+7) mM(-1) s(-1)/construct); gadofullerenes, r(1) ≈ 200 mM(-1) s(-1)/Gd(3+) ion (∼ 7 × 10(+9) mM(-1) s(-1)/construct); gadonanotubes, r(1) ≈ 150 mM(-1) s(-1)/Gd(3+) ion (∼ 2 × 10(+9) mM(-1) s(-1)/construct). These relaxivity values are about 4 to 50 times larger than those of clinically available gadolinium-based agents (∼ 4 mM(-1) s(-1)/Gd(3+) ion). The enhancement in contrast is attributed to the geometrical confinement of the agents, which influences the paramagnetic behaviour of the Gd(3+) ions. Thus, nanoscale confinement offers a new and general strategy for enhancing the contrast of gadolinium-based contrast agents.

Injectable and implantable porosified silicon (pSi) carriers and devices for prolonged and controlled delivery of biotherapeutics offer great promise for treatment of various chronic ailments and acute conditions. Polyethylene glycols (PEGs) are important surface modifiers currently used in clinic mostly to avoid uptake of particulates by reticulo-endothelial system (RES). In this work we show for the first time that covalent attachment of PEGs to the pSi surface can be used as a means to tune degradation kinetics of silicon structures. Seven PEGs with varying molecular weights (245, 333, 509, 686, 1214, 3400, and 5000 Da) were employed and the degradation of PEGylated pSi hemispherical microparticles in simulated physiological conditions was monitored by means of ICP-AES, SEM, and fluorimetry. Biocompatibility of the systems with human macrophages in vitro was also evaluated. The results clearly indicate that controlled PEGylation of silicon microparticles can offer a sensitive tool to finely tune their degradation kinetics and that the systems do not induce release of proinflammatory cytokines IL-6 and IL-8 in THP1 human macrophages., ((c) 2010 Wiley Periodicals, Inc.)

Individualized medicine is the healthcare strategy that rebukes the idiomatic dogma of 'losing sight of the forest for the trees'. We are entering a new era of healthcare where it is no longer acceptable to develop and market a drug that is effective for only 80% of the patient population. The emergence of "-omic" technologies (e.g. genomics, transcriptomics, proteomics, metabolomics) and advances in systems biology are magnifying the deficiencies of standardized therapy, which often provide little treatment latitude for accommodating patient physiologic idiosyncrasies. A personalized approach to medicine is not a novel concept. Ever since the scientific community began unraveling the mysteries of the genome, the promise of discarding generic treatment regimens in favor of patient-specific therapies became more feasible and realistic. One of the major scientific impediments of this movement towards personalized medicine has been the need for technological enablement. Nanotechnology is projected to play a critical role in patient-specific therapy; however, this transition will depend heavily upon the evolutionary development of a systems biology approach to clinical medicine based upon "-omic" technology analysis and integration. This manuscript provides a forward looking assessment of the promise of nanomedicine as it pertains to individualized medicine and establishes a technology "snapshot" of the current state of nano-based products over a vast array of clinical indications and range of patient specificity. Other issues such as market driven hurdles and regulatory compliance reform are anticipated to "self-correct" in accordance to scientific advancement and healthcare demand. These peripheral, non-scientific concerns are not addressed at length in this manuscript; however they do exist, and their impact to the paradigm shifting healthcare transformation towards individualized medicine will be critical for its success., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon particles to be optimized for specific applications such as vascular targeting and avoidance of biological barriers commonly found between the site of drug injection and the final destination. In this study, the intracellular trafficking of unloaded carrier silicon particles and carrier particles loaded with secondary iron oxide nanoparticles was investigated. Following cellular uptake, membrane-encapsulated silicon particles migrated to the perinuclear region of the cell by a microtubule-driven mechanism. Surface charge, shape (spherical and hemispherical) and size (1.6 and 3.2 microm) of the particle did not alter the rate of migration. Maturation of the phagosome was associated with an increase in acidity and acquisition of markers of late endosomes and lysosomes. Cellular uptake of iron oxide nanoparticle-loaded silicon particles resulted in sorting of the particles and trafficking to unique destinations. The silicon carriers remained localized in phagosomes, while the second stage iron oxide nanoparticles were sorted into multi-vesicular bodies that dissociated from the phagosome into novel membrane-bound compartments. Release of iron from the cells may represent exocytosis of iron oxide nanoparticle-loaded vesicles. These results reinforce the concept of multi-functional nanocarriers, in which different particles are able to perform specific tasks, in order to deliver single- or multi-component payloads to specific sub-cellular compartments.

The realization that blood-borne delivery systems must overcome a multiplicity of biological barriers has led to the fabrication of a multistage delivery system (MDS) designed to temporally release successive stages of particles or agents to conquer sequential barriers, with the goal of enhancing delivery of therapeutic and diagnostic agents to the target site. In its simplest form, the MDS comprises stage-one porous silicon microparticles that function as carriers of second-stage nanoparticles. Cellular uptake of nontargeted discoidal silicon microparticles by macrophages is confirmed by electron and atomic force microscopy (AFM). Using superparamagnetic iron oxide nanoparticles (SPIONs) as a model of secondary nanoparticles, successful loading of the porous matrix of silicon microparticles is achieved, and retention of the nanoparticles is enhanced by aminosilylation of the loaded microparticles with 3-aminopropyltriethoxysilane. The impact of silane concentration and reaction time on the nature of the silane polymer on porous silicon is investigated by AFM and X-ray photoelectron microscopy. Tissue samples from mice intravenously administered the MDS support co-localization of silicon microparticles and SPIONs across various tissues with enhanced SPION release in spleen, compared to liver and lungs, and enhanced retention of SPIONs following silane capping of the MDS. Phantom models of the SPION-loaded MDS display negative contrast in magnetic resonance images. In addition to forming a cap over the silicon pores, the silane polymer provides free amines for antibody conjugation to the microparticles, with both VEGFR-2- and PECAM-specific antibodies leading to enhanced endothelial association. This study demonstrates the assembly and cellular association of a multiparticle delivery system that is biomolecularly targeted and has potential for applications in biological imaging.

Nanomedicine is an emerging field that utilizes nanotechnology concepts for advanced therapy and diagnostics. This convergent discipline merges research areas such as chemistry, biology, physics, mathematics and engineering. It therefore bridges the gap between molecular and cellular interactions, and has the potential to revolutionize medicine. This review presents recent developments in nanomedicine research poised to have an important impact on the treatment of cardiovascular disease. This will occur through improvement of the diagnosis and therapy of cardiovascular disorders as atherosclerosis, restenosis and myocardial infarction. Specifically, we discuss the use of nanoparticles for molecular imaging and advanced therapeutics, specially designed drug eluting stents and in vivo/ex vivo early detection techniques., (Published by Elsevier Ltd.)

Multistage carriers were recently introduced by our laboratory, with the concurrent objectives of co-localized delivery of multiple therapeutic agents, the "theranostic" integration of bioactive moieties with imaging contrast, and the selective, potentially personalized bypassing of the multiplicity of biological barriers that adversely impact biodistribution of vascularly injected particulates. Mesoporous ("nanoporous") silicon microparticles were selected as primary carriers in multi-stage devices, with targets including vascular endothelia at pathological lesions. The objective of this study was to evaluate biocompatibility of mesoporous silicon microparticles with endothelial cells using in vitro assays with an emphasis on microparticle compatibility with mitotic events. We observed that vascular endothelial cells, following internalization of silicon microparticles, maintain cellular integrity, as demonstrated by cellular morphology, viability and intact mitotic trafficking of vesicles bearing silicon microparticles. The presence of gold or iron oxide nanoparticles within the porous matrix did not alter the cellular uptake of particles or the viability of endothelial cells subsequent to engulfment of microparticles. Endothelial cells maintained basal levels of IL-6 and IL-8 release in the presence of silicon microparticles. This is the first study that demonstrates polarized, ordered partitioning of endosomes based on tracking microparticles. The finding that mitotic sorting of endosomes is unencumbered by the presence of nanoporous silicon microparticles advocates the use of silicon microparticles for biomedical applications.