Nikorn Pothayee

Targeting Brucella melitensis with polymeric nanoparticles containing streptomycin and doxycycline

Treatment and eradication of intracellular pathogens such as Brucella is difficult because infections are localized within phagocytic cells and most antibiotics, although highly active in vitro, do not actively pass through cellular membranes. Thus, an optimum strategy to treat these infections should address targeting of active drugs to the intracellular compartment where the bacteria replicate, and should prolong the release of the antibiotics so that the number of doses and associated toxicity can be reduced. We incorporated streptomycin and doxycycline into macromolecular nanoplexes with anionic homo- and block copolymers via cooperative electrostatic interactions among the cationic drugs and anionic polymers. The approach enabled simultaneous binding of both antibiotics into the nanoplexes, and their use resulted in an improvement in performance as compared with the free drugs. Administration of two doses of the nanoplexes significantly reduced the Brucella melitensis load in the spleens and livers of infected BALB/c mice. The nanoplexes were more effective than free drugs in the spleens (0.72-log and 0.51-log reductions, respectively) and in the livers (0.79-log and 0.42-log reductions, respectively) of the infected mice. Further research regarding the design of optimum nanoplex structures will be directed towards alterations in both the core and the shell properties to investigate the effects of the rates and pathways of entry into immune cells where the brucellae replicate.

Synthesis and colloidal properties of polyether-magnetite complexes in water and phosphate-buffered saline.

Biocompatible magnetic nanoparticles show great promise for many biotechnological applications. This paper addresses the synthesis and characterization of magnetite nanoparticles coated with poly(ethylene oxide) (PEO) homopolymers and amphiphilic poly(propylene oxide-b-ethylene oxide) (PPO-b-PEO) copolymers that were anchored through ammonium ions. Predictions and experimental measurements of the colloidal properties of these nanoparticles in water and phosphate-buffered saline (PBS) as functions of the polymer block lengths and polymer loading are reported. The complexes were found to exist as primary particles at high polymer compositions, and most formed small clusters with equilibrium sizes as the polymer loading was reduced. Through implementation of a polymer brush model, the size distributions from dynamic light scattering (DLS) were compared to those from the model. For complexes that did not cluster, the experimental sizes matched the model well. For complexes that clustered, equilibrium diameters were predicted accurately through an empirical fit derived from DLS data and the half-life for doublet formation calculated using the modified Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Deviation from this empirical fit provided insight into possible additional interparticle hydrophobic interactions for select complexes for which the DLVO theory could not account. While the polymers remained bound to the nanoparticles in water, most of them desorbed slowly in PBS. Desorption was slowed significantly at high polymer chain densities and with hydrophobic PPO anchor blocks. By tailoring the PPO block length and the number of polymer chains on the surface, flocculation of the magnetite complexes in PBS was avoided. This allows for in vitro experiments where appreciable flocculation or sedimentation will not take place within the specified time scale requirements of an experiment.

Magnetic Nanoclusters with Hydrophilic Spacing for Dual Drug Delivery and Sensitive Magnetic Resonance Imaging

Magnetic Block Ionomer Clusters (MBIClusters) with hydrophilic ionic cores and nonionic coronas have been prepared that have ultrahigh transverse NMR relaxivities together with capacities for incorporating high concentrations of polar antibiotic payloads. Magnetite-polymer nanoparticles were assembled by adsorbing the polyacrylate block of an aminofunctional poly(ethylene oxide-b-acrylate) (H2N-PEO-b-PAA) copolymer onto magnetite nanoparticles. The PEO blocks extended into aqueous media to keep the nanoparticles dispersed. Amines at the tips of the H2N-PEO corona were then linked through reaction with a PEO diacrylate oligomer to yield MBIClusters where the metal oxide in the precursor nanoparticles were distinctly separated by the hydrophilic polymer. The intensity average spacing between the magnetite nanoparticles within the clusters was estimated to be ~50 nm. These MBIClusters with hydrophilic intra-cluster space had transverse relaxivities (r2s) that increased from 190 to 604 s-1 mM Fe-1 measured at 1.4 T and 37 °C as their average sizes increased. The clusters were loaded with up to ~38 wt% of the multi-cationic drug gentamicin. MRI scans focused on the livers of mice demonstrated that these MBIClusters are sensitive contrast agents.

Changing the Enzyme Reaction Rate in Magnetic Nanosuspensions by a Non‐Heating Magnetic Field

Weak magnetic fields (< 1 T) are known to change the paths and kinetics of some radical chemical reactions at room temperature through spin conversion of short-lived radical pairs. [1] Magnetic fields were also used to tune enzyme activity (oxidation of glucose). Specifically, exposure of magnetic nanoparticles functionalized with enzymes to magnetic field allowed the movement of particles in proximity to electrode, thus activating the enzyme. [2] Moreover, radio-frequency (RF) alternating current (AC) magnetic fields can induce the heating of single-domain magnetic nanoparticles (MNPs), and this can be used in magnetic hyperthermia to kill tumors. [3] Herein we describe a distinct effect of non-heating superlow-frequency magnetic fields as well as more conventional RF magnetic fields on the kinetics of chemical reactions catalyzed by the enzymes a-chymotrypsin (ChT) or bgalactosidase (b-GaL) immobilized on nanoscale MNP aggregates. Magnetite MNPs (7 to 12 nm diameter) were synthesized by reducing tris(acetylacetonato)iron(III), then they were coated with anionic poly(ethylene glycol)-bpolyacrylate (PEG-PAA), or poly(ethylene glycol)-b-polymethacrylate (PEG-PMA) copolymers. The polymers were bound to the magnetite surfaces by the carboxylate groups. [4] According to thermogravimetric analysis, the content of Fe3O4 in both coated MNP materials was about 35 wt %, and this was in excellent agreement with that measured by inductively coupled plasma-mass spectrometry. Upon dispersion in water (pH 6.5), they formed nanoscale, negatively charged aggregates (hydrodynamic intensity average diameters and zeta potentials of 194 nm, 70 mV and 39 nm, 49 mV for PEG-PAA/MNP and PEG-PMA/MNP, respectively, as determined by dynamic light scattering). Based on TEM, they contained clusters of 7 to 20 MNP grains. Enzymes were conjugated to these aggregates using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide and N-hydroxysulfosuccinimide to couple amines on the protein to carboxylic acids on the copolymer (Scheme 1).

Antibacterial efficacy of core-shell nanostructures encapsulating gentamicin against an in vivo intracellular Salmonella model

Pluronic based core-shell nanostructures encapsulating gentamicin were designed in this study. Block copolymers of (PAA−+Na-b-(PEO-b-PPO-b-PEO)-b-PAA−+Na) were blended with PAA− Na+ and complexed with the polycationic antibiotic gentamicin to form nanostructures. Synthesized nanostructures had a hydrodynamic diameter of 210 nm, zeta potentials of −0.7 (±0.2), and incorporated ∼20% by weight of gentamicin. Nanostructures upon co-incubation with J774A.1 macrophage cells showed no adverse toxicity in vitro. Nanostructures administered in vivo either at multiple dosage of 5 μg g−1 or single dosage of 15 μg g−1 in AJ-646 mice infected with Salmonella resulted in significant reduction of viable bacteria in the liver and spleen. Histopathological evaluation for concentration-dependent toxicity at a dosage of 15 μg g−1 revealed mineralized deposits in 50% kidney tissues of free gentamicin-treated mice which in contrast was absent in nanostructure-treated mice. Thus, encapsulation of gentamicin in nanostructures may reduce toxicity and improve in vivo bacterial clearance.

Wireless Amplified Nuclear MR Detector (WAND) for High-Spatial-Resolution MR Imaging of Internal Organs: Preclinical Demonstration in a Rodent Model

PURPOSE To assess the feasibility of imaging deep-lying internal organs at high spatial resolution by imaging kidney glomeruli in a rodent model with use of a newly developed, wireless amplified nuclear magnetic resonance (MR) detector. MATERIALS AND METHODS This study was approved by the Animal Care and Use Committee at the National Institutes of Health/National Institute of Neurologic Disorder and Stroke. As a preclinical demonstration of this new detection technology, five different millimeter-scale wireless amplified nuclear MR detectors configured as double frequency resonators were chronically implanted on the medial surface of the kidney in five Sprague-Dawley rats for MR imaging at 11.7 T. Among these rats, two were administered gadopentetate dimeglumine to visualize renal tubules on T1-weighted gradient-refocused echo (GRE) images, two were administered cationized ferritin to visualize glomeruli on T2*-weighted GRE images, and the remaining rat was administered both gadopentetate dimeglumine and cationized ferritin to visualize the interleaved pattern of renal tubules and glomeruli. The image intensity in each pixel was compared with the local tissue signal intensity average to identify regions of hyper- or hypointensity. RESULTS T1-weighted images with 70-μm in-plane resolution and 200-μm section thickness were obtained within 3.2 minutes to image renal tubules, and T2*-weighted images of the same resolution were obtained within 5.8 minutes to image the glomeruli. Hyperintensity from gadopentetate dimeglumine enabled visualization of renal tubules, and hypointensity from cationic ferritin enabled visualization of the glomeruli. CONCLUSION High-spatial-resolution images have been obtained to observe kidney microstructures in vivo with a wireless amplified nuclear MR detector.

Nanomedicine for intracellular therapy

Intracellular pathogens like Salmonella evade host phagocytic killing by various mechanisms. Classical antimicrobial therapy requires multiple dosages and frequent administration of drugs for a long duration. Intracellular delivery of antimicrobials using nanoparticle may effectively devise therapies for bacterial infections. This review will address the mechanisms used by Salmonella to avoid host pathogenic killing, reasons for therapeutic failure and advances in nanoparticle drug delivery technology for efficient intracellular bacterial clearance.

In Vitro Trafficking and Efficacy of Core-Shell Nanostructures for Treating Intracellular Salmonella Infections

ABSTRACT Nanostructures encapsulating gentamicin and having either amphiphilic (N1) or hydrophilic (N2) surfaces were designed. Flow cytometry and confocal microscopy studies demonstrated a higher rate of uptake for amphiphilic surfaces. A majority of N1 were localized in the cytoplasm, whereas N2 colocalized with the endosomes/lysosomes. Colocalization was not observed between nanostructures and intracellular Salmonella bacteria. However, significant in vitro reductions in bacterial counts (0.44 log10) were observed after incubation with N1, suggesting that the surface property of the nanostructure influences intracellular bacterial clearance.

Live nephron imaging by MRI

The local sensitivity of MRI can be improved with small MR detectors placed close to regions of interest. However, to maintain such sensitivity advantage, local detectors normally need to communicate with the external amplifier through cable connections, which prevent the use of local detectors as implantable devices. Recently, an integrated wireless amplifier was developed that can efficiently amplify and broadcast locally detected signals, so that the local sensitivity was enhanced without the need for cable connections. This integrated detector enabled the live imaging of individual glomeruli using negative contrast introduced by cationized ferritin, and the live imaging of renal tubules using positive contrast introduced by gadopentetate dimeglumine. Here, we utilized the high blood flow to image individual glomeruli as hyperintense regions without any contrast agent. These hyperintense regions were identified for pixels with signal intensities higher than the local average. Addition of Mn(2+) allowed the simultaneous detection of both glomeruli and renal tubules: Mn(2+) was primarily reabsorbed by renal tubules, which would be distinguished from glomeruli due to higher enhancement in T1-weighted MRI. Dynamic studies of Mn(2+) absorption confirmed the differential absorption affinity of glomeruli and renal tubules, potentially enabling the in vivo observation of nephron function.

Remote Actuation of Magnetic Nanoparticles For Cancer Cell Selective Treatment Through Cytoskeletal Disruption

Motion of micron and sub-micron size magnetic particles in alternating magnetic fields can activate mechanosensitive cellular functions or physically destruct cancer cells. However, such effects are usually observed with relatively large magnetic particles (>250 nm) that would be difficult if at all possible to deliver to remote sites in the body to treat disease. Here we show a completely new mechanism of selective toxicity of superparamagnetic nanoparticles (SMNP) of 7 to 8 nm in diameter to cancer cells. These particles are coated by block copolymers, which facilitates their entry into the cells and clustering in the lysosomes, where they are then magneto-mechanically actuated by remotely applied alternating current (AC) magnetic fields of very low frequency (50 Hz). Such fields and treatments are safe for surrounding tissues but produce cytoskeletal disruption and subsequent death of cancer cells while leaving healthy cells intact.