Intravital microscopy for evaluating tumor perfusion of nanoparticles exposed to non-invasive radiofrequency electric fields

Poor biodistribution and accumulation of chemotherapeutics in tumors due to limitations on diffusive transport and high intra-tumoral pressures (Jain RK, Nat Med. 7(9):987–989, 2001) have prompted the investigation of adjunctive therapies to improve treatment outcomes. Hyperthermia has been widely applied in attempts to meet this need, but it is limited in its ability to reach tumors in deeply located body regions. High-intensity radiofrequency (RF) electric fields have the potential to overcome such barriers enhancing delivery and extravasation of chemotherapeutics. However, due to factors, including tumor heterogeneity and lack of kinetic information, there is insufficient understanding of time-resolved interaction between RF fields and tumor vasculature, drug molecules and nanoparticle (NP) vectors. Intravital microscopy (IVM) provides time-resolved high-definition images of specific tumor microenvironments, overcoming heterogeneity issues, and can be integrated with a portable RF device to enable detailed observation over time of the effects of the RF field on kinetics and biodistribution at the microvascular level. Herein, we provide a protocol describing the safe integration of IVM with a high-powered non-invasive RF field applied to 4T1 orthotopic breast tumors in live mice. Results show increased perfusion of NPs in microvasculature upon RF hyperthermia treatment and increased perfusion, release and spreading of injected reagents preferentially in irregular vessels during RF exposure

Chemical and biomolecular functionalization of silicon surfaces for biosensing applications

The reliable functioning of biosensors and bioassays is dependent on the robust attachment of active biomolecules to device substrates, such that the structural integrity, organization and appropriate orientation of these molecules at the surface is maintained. To a significant extent, the underlying surface chemistry and molecular organization that these biomolecules come in contact with and attach to affect the properties of the functional overlayer that they compose. In this work, primarily through the use of infrared spectroscopy, we characterize two main types of biosensor platforms including biotin-streptavidin linkage and surface attachment and covalent attachment of protein to sensor surfaces via amines and sulfhydryls. We further observe the effects of several variations in processing conditions on these platforms including initial atmospheric humidity, use of anhydrous versus aqueous solvents in molecular adsorption and the effect of primary molecular layer stability on the organization and characteristics of subsequently adsorbed biolayers. With infrared spectroscopy, not only do we identify the formation and breaking of chemical bonds in each of the attachment steps, we also monitor changes in the moieties of each layer with changing environmental conditions. We find that changes in ureido moiety vibrational modes of the biotinylated surface occur near 1250 and 1700 cm-1 dependent on the stability of an underlying layer of aminopropyltriethoxysiloxane, on the type of solvent used in biotinylation itself, and on subsequent protein adsorption to and/or rinsing of the biotinylated surface. In covalent attachment studies, we use small molecules in lieu of protein to characterize amine and sulfhydryl chemical bonding to maleimide-terminated surfaces and using infrared polarization techniques, we find that molecular orientation may be restricted upon covalent attachment. Ellipsometry is used in conjuction with infrared absorption area measurements to determine the relative composition of silane and maleimide prior to attachment of protein. Additionally, fluorescence measurements of labeled protein are used to quantify protein desorption by surface acoustic wave streaming (SAWS). These measurements are correlated with power dose of SAWS operation. In all, these surface characterization methods are found to successfully monitor chemical and biomolecular layer formation and change under a variety of conditions.Ph.D.Includes bibliographical referencesby Norman A. Lapi

Physical Approaches to Prevent and Treat Bacterial Biofilm

Prosthetic joint infection (PJI) presents several clinical challenges. This is in large part due to the formation of biofilm which can make infection eradication exceedingly difficult. Following an extensive literature search, this review surveys a variety of non-pharmacological methods of preventing and/or treating biofilm within the body and how they could be utilized in the treatment of PJI. Special attention has been paid to physical strategies such as heat, light, sound, and electromagnetic energy, and their uses in biofilm treatment. Though these methods are still under study, they offer a potential means to reduce the morbidity and financial burden related to multiple stage revisions and prolonged systemic antibiotic courses that make up the current gold standard in PJI treatment. Given that these options are still in the early stages of development and offer their own strengths and weaknesses, this review offers an assessment of each method, the progress made on each, and allows for comparison of methods with discussion of future challenges to their implementation in a clinical setting

Attachment Of Streptavidin-Biotin On 3-Aminopropyltriethoxysilane (APTES) Modified Porous Silicon Surfaces

Nanostructured porous silicon (PS) has a large surface area that can be well controlled and modified to have a high specificity for biomolecules. These properties make it a very promising biomaterial, in particular for sensing devices that need to be linked to the biological system and completely compatible with standard integrated circuit processes. We report the formation of nanostructured PS on boron doped, p-type silicon (100) wafers by electrochemical anodization, using aqueous hydrofluoric acid and isopropyl alcohol solutions and a constant current density 50 mA/cm2. The pore diameter can be tuned by varying the etching conditions. The interaction of streptavidin with biotin was studied on 3-aminopropyltriethoxysilane (APTES) functionalized PS surfaces using infrared absorption spectroscopy to characterize the surface at each step, including subsequent reaction steps. These studies show that the streptavidin-biotin interaction on modified PS surfaces depends on the details of the APTES adsorption process

Biotransport kinetics and intratumoral biodistribution of malonodiserinolamide-derivatized [60]fullerene in a murine model of breast adenocarcinoma

[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

Intermittent alternating magnetic fields diminish metal-associated biofilm in vivo

Abstract Prosthetic joint infection (PJI) is a complication of arthroplasty that results in significant morbidity. The presence of biofilm makes treatment difficult, and removal of the prosthesis is frequently required. We have developed a non-invasive approach for biofilm eradication from metal implants using intermittent alternating magnetic fields (iAMF) to generate targeted heating at the implant surface. The goal of this study was to determine whether iAMF demonstrated efficacy in an in vivo implant biofilm infection model. iAMF combined with antibiotics led to enhanced reduction of biofilm on metallic implants in vivo compared to antibiotics or untreated control. iAMF-antibiotic combinations resulted in a > 1 − log further reduction in biofilm burden compared to antibiotics or iAMF alone. This combination effect was seen in both S. aureus and P. aeruginosa and seen with multiple antibiotics used to treat infections with these pathogens. In addition, efficacy was temperature dependent with increasing temperatures resulting in a greater reduction of biofilm. Tissue damage was limited (< 1 mm from implant-tissue interface). This non-invasive approach to eradicating biofilm could serve as a new paradigm in treating PJI