Samir V. Jenkins

Synergistic Photothermal and Antibiotic Killing of Biofilm-Associated Staphylococcus aureus Using Targeted Antibiotic-Loaded Gold Nanoconstructs

Resistance to conventional antibiotics is a growing public health concern that is quickly outpacing the development of new antibiotics. This has led the Infectious Diseases Society of America (IDSA) to designate Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species as “ESKAPE pathogens” on the basis of the rapidly decreasing availability of useful antibiotics. This emphasizes the urgent need for alternative therapeutic strategies to combat infections caused by these and other bacterial pathogens. In this study, we used Staphylococcus aureus (S. aureus) as a proof-of-principle ESKAPE pathogen to demonstrate that an appropriate antibiotic (daptomycin) can be incorporated into polydopamine-coated gold nanocages (AuNC@PDA) and that daptomycin-loaded AuNC@PDA can be conjugated to antibodies targeting a species-specific surface protein (staphylococcal protein A; Spa) as a means of achieving selective delivery of the nanoconstructs directly to the bacterial cell surface. Targeting specificity was confirmed by demonstrating a lack of binding to mammalian cells, reduced photothermal and antibiotic killing of the Spa-negative species Staphylococcus epidermidis, and reduced killing of S. aureus in the presence of unconjugated anti-Spa antibodies. We demonstrate that laser irradiation at levels within the current safety standard for use in humans can be used to achieve both a lethal photothermal effect and controlled release of the antibiotic, thus resulting in a degree of therapeutic synergy capable of eradicating viable S. aureus cells. The system was validated using planktonic bacterial cultures of both methicillin-sensitive and methicillin-resistant S. aureus strains and subsequently shown to be effective in the context of an established biofilm, thus indicating that this approach could be used to facilitate the effective treatment of intrinsically resistant biofilm infections.

Rapid determination of plasmonic nanoparticle agglomeration status in blood

Plasmonic nanomaterials as drug delivery or bio-imaging agents are typically introduced to biological systems through intravenous administration. However, the potential for agglomeration of nanoparticles in biological systems could dramatically affect their pharmacokinetic profile and toxic potential. Development of rapid screening methods to evaluate agglomeration is urgently needed to monitor the physical nature of nanoparticles as they are introduced into blood. Here, we establish novel methods using darkfield microscopy with hyperspectral detection (hsDFM), single particle inductively-coupled plasma mass spectrometry (spICP-MS), and confocal Raman microscopy (cRM) to discriminate gold nanoparticles (AuNPs) and their agglomerates in blood. Rich information about nanoparticle agglomeration in situ is provided by hsDFM monitoring of the plasmon resonance of primary nanoparticles and their agglomerates in whole blood; cRM is an effective complement to hsDFM to detect AuNP agglomerates in minimally manipulated samples. The AuNPs and the particle agglomerates were further distinguished in blood for the first time by quantification of particle mass using spICP-MS with excellent sensitivity and specificity. Furthermore, the agglomeration status of synthesized and commercial NPs incubated in blood was successfully assessed using the developed methods. Together, these complementary methods enable rapid determination of the agglomeration status of plasmonic nanomaterials in biological systems, specifically blood.

The unusual visible photothermal response of free standing multilayered films based on plasmonic bimetallic nanocages

An optical phenomena based on the incident light absorption and transduction to the detectable thermal signal by plasmonic bimetallic Ag and Au nanocages (Ag@AuNCs) has been researched on free standing layer-by-layer (LbL) films of poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVPON), (PVA/PVPON_Ag@AuNCs). Unlike well-studied monometallic Au nanocages (NCs), which possess a photothermal response in the near-infrared region, the bimetallic nanocages show a pronounced photothermal response in the visible range (532 nm) and near-infrared range (780 nm) due to the presence of two characteristic peaks at both wavelength ranges. The photothermal response in the visible range (532 nm) is distinguishable. Specifically increasing the laser power to 100 mW led to visual burning of the free standing film (temperature increased greater than >150 °C). The photothermal response by (PVA/PVPON_Ag@AuNCs)n films increases in proportion to the number (n) of bilayers (bl). It also increases as the molar concentration of the Ag@AuNCs introduced to the PVPON layer is increased. Therefore the molar concentration of the plasmonic Ag@AuNCs in (PVA/PVPON_Ag@AuNCs)n films is a primary factor that affects the photothermal dynamic response along with Ag@AuNCs distribution. This is supposed to result from the Ag@AuNCs assembled in a layer that leads to electromagnetic field enhancement. The unusual observation in multilayered (PVA/PVPON_Ag@AuNCs)n films is that the UV-visible spectra (extinction efficiency) and photothermal response (Tmax) do not rely on the content of the adjacent layer of PVA and show a comparable (by value in magnitude) photothermal response at a different PVA composition of 2 mg ml−1 and 20 mg ml−1 at the same Ag@AuNCs concentration in the PVPON layer.

Triple-negative breast cancer targeting and killing by EpCAM-directed, plasmonically active nanodrug systems

An ongoing need for new cancer therapeutics exists, especially ones that specifically home and target triple-negative breast cancer. Because triple-negative breast cancer express low or are devoid of estrogen, progesterone, or Her2/Neu receptors, another target must be used for advanced drug delivery strategies. Here, we engineered a nanodrug delivery system consisting of silver-coated gold nanorods (AuNR/Ag) targeting epithelial cell adhesion/activating molecule (EpCAM) and loaded with doxorubicin. This nanodrug system, AuNR/Ag/Dox-EpCAM, was found to specifically target EpCAM-expressing tumors compared to low EpCAM-expressing tumors. Namely, the nanodrug had an effective dose (ED50) of 3 μM in inhibiting 4T1 cell viability and an ED50 of 110 μM for MDA-MD-231 cells. Flow cytometry data indicated that 4T1 cells, on average, express two orders of magnitude more EpCAM than MDA-MD-231 cells, which correlates with our ED50 findings. Moreover, due to the silver coating, the AuNR/Ag can be detected simultaneously by surface-enhanced Raman spectroscopy and photoacoustic microscopy. Analysis by these imaging detection techniques as well as by inductively coupled plasma mass spectrometry showed that the targeted nanodrug system was taken up by EpCAM-expressing cells and tumors at significantly higher rates than untargeted nanoparticles (p < 0.05). Thus, this approach establishes a plasmonically active nanodrug theranostic for triple-negative breast cancer and, potentially, a delivery platform with improved multimodal imaging capability for other clinically relevant chemotherapeutics with dose-limiting toxicities, such as platinum-based or taxane-based therapies.Targeted drug delivery for triple-negative breast cancerSilver-coated gold nanorods deliver drugs to a difficult-to-treat breast cancer by targeting an over-expressed antigen on its surface. Ruud Dings and colleagues at the University of Arkansas in the USA loaded the chemotherapeutic drug doxorubicin onto silver-coated gold nanorods that were conjugated with an antibody that specifically targets an over-expressed antigen on many types of ‘triple-negative breast cancers’ (TNBCs). Unlike other breast cancers, TNBCs lack certain receptors, making them difficult to target with cancer therapies. The team found that one of the two TNBC cell lines studied over-expressed the epithelial antigen EpCAM 100 times more than the other. Their drug-loaded silver-coated gold nanorods specifically targeted the EpCAM over-expressing cells over the low-expressing ones. The nanorods’ coatings also allowed them to be easily detected by two different imaging techniques: surfaced-enhanced Raman spectroscopy and photoacoustic microscopy.

Optical imaging of radiation-induced metabolic changes in radiation-sensitive and resistant cancer cells

Abstract. Radiation resistance remains a significant problem for cancer patients, especially due to the time required to definitively determine treatment outcome. For fractionated radiation therapy, nearly 7 to 8 weeks can elapse before a tumor is deemed to be radiation-resistant. We used the optical redox ratio of FAD/(FAD+NADH) to identify early metabolic changes in radiation-resistant lung cancer cells. These radiation-resistant human A549 lung cancer cells were developed by exposing the parental A549 cells to repeated doses of radiation (2 Gy). Although there were no significant differences in the optical redox ratio between the parental and resistant cell lines prior to radiation, there was a significant decrease in the optical redox ratio of the radiation-resistant cells 24 h after a single radiation exposure (p=0.01). This change in the redox ratio was indicative of increased catabolism of glucose in the resistant cells after radiation and was associated with significantly greater protein content of hypoxia-inducible factor 1 (HIF-1α), a key promoter of glycolytic metabolism. Our results demonstrate that the optical redox ratio could provide a rapid method of determining radiation resistance status based on early metabolic changes in cancer cells.

Thermoresponsive nanoparticle agglomeration/aggregation in salt solutions: Dependence on graft density

Gold nanoparticles with a graft density of 0.09, 0.30 and 0.40chains/nm2 of poly(N-isopropylacrylamide) were reproducibly synthesized by varying the ratio of disulfide terminated poly(N-isopropylacrylamide) to gold nanoparticle. The polymer coated nanoparticles were stable at room temperature in 50mM NaCl, yet agglomerated at 37°C. Previous studies have observed conflicting results as to the reversibility of this agglomeration. Particle agglomeration with three different graft densities was studied in 50mM NaCl by measurements of their localized surface plasmon resonance and hydrodynamic diameter, and imaging with electron microscopy. Agglomerates with a polymer graft density of 0.30 and 0.40chains/nm2 could be dispersed with sonication, while particles with a graft density of 0.09chains/nm2 irreversibly aggregated. The graft density dependence on whether agglomeration or aggregation occurred is due to changes in collapsed polymer steric effects. Localized surface plasmon resonance measurements of agglomerates were discordant with hydrodynamic diameter measurements in determining agglomeration reversibility, which shed light on reasons previous reports yielded different interpretations on the reversibility of this agglomeration. This work demonstrates how polymer graft density affects thermoresponsive nanoparticle stability in salt solutions and the need for use of complementary techniques when determining agglomeration.

Plasmonic Nanostructures for Biomedical and Sensing Applications

Noble metal nanostructures are appealing for therapeutic, diagnostics, and sensing applications because of their unique optical properties and biocompatibility. The collective oscillation of electrons in a metal nanostructure resonates with particular wavelengths of light, generating the localized surface plasmon resonance (LSPR). The LSPR results in absorption and scattering of incoming photons. Absorption leads to photothermal generation of heat, photoluminescence, and quenching of fluorophores in close proximity. Scattering results in reflected photons and its amplification of the local electromagnetic field can enhance fluorescence, phosphorescence, and Raman scattering. These optical properties make plasmonic nanostructures ideal candidates for theranostic applications including light-induced thermal therapy, drug/gene delivery, and biomedical imaging. The use of plasmonic nanostructures for ex vivo detection of chemicals and biomolecules are also discussed in this chapter.

A Radiosensitizing Inhibitor of HIF-1 alters the Optical Redox State of Human Lung Cancer Cells In Vitro

Treatment failure caused by a radiation-resistant cell phenotype remains an impediment to the success of radiation therapy in cancer. We recently showed that a radiation-resistant isogenic line of human A549 lung cancer cells had significantly elevated expression of hypoxia-inducible factor (HIF-1α), and increased glucose catabolism compared with the parental, radiation-sensitive cell line. The objective of this study was to investigate the longitudinal metabolic changes in radiation-resistant and sensitive A549 lung cancer cells after treatment with a combination of radiation therapy and YC-1, a potent HIF-1 inhibitor. Using label-free two-photon excited fluorescence microscopy, we determined changes in the optical redox ratio of FAD/(NADH and FAD) over a period of 24 hours following treatment with YC-1, radiation, and both radiation and YC-1. To complement the optical redox ratio, we also evaluated changes in mitochondrial organization, glucose uptake, reactive oxygen species (ROS), and reduced glutathione. We observed significant differences in the optical redox ratio of radiation-resistant and sensitive A549 cells in response to radiation or YC-1 treatment alone; however, combined treatment eliminated these differences. Our results demonstrate that the optical redox ratio can elucidate radiosensitization of previously radiation-resistant A549 cancer cells, and provide a method for evaluating treatment response in patient-derived tumor biopsies.

Hypoxia-derived exosomes induce putative altered pathways in biosynthesis and ion regulatory channels in glioblastoma cells

Hypoxia, a hallmark characteristic of glioblastoma (GBM) induces changes in the transcriptome and the proteome of tumor cells. We discovered that hypoxic stress produces significant qualitative and quantitative changes in the protein content of secreted exosomes from GBM cells. Among the proteins found to be selectively elevated in hypoxic exosomes were protein-lysine 6-oxidase (LOX), thrombospondin-1 (TSP1), vascular derived endothelial factor (VEGF) and a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), well studied contributors to tumor progression, metastasis and angiogenesis. Our findings demonstrate that hypoxic exosomes induce differential gene expression in recipient glioma cells. Glioma cells stimulated with hypoxic exosomes showed a marked upregulation of small nucleolar RNA, C/D box 116–21 (SNORD116-21) transcript among others while significantly downregulated the potassium voltage-gated channel subfamily J member 3 (KCNJ3) message. This differential expression of certain genes is governed by the protein cargo being transferred via exosomes. Additionally, compared to normoxic exosomes, hypoxic exosomes increased various angiogenic related parameters vis-à-vis, overall tube length, branching intervals and length of isolated branches studied in tube formation assay with endothelial progenitor cells (EPCs). Thus, the intercellular communication facilitated via exosomes secreted from hypoxic GBM cells induce marked changes in the expression of genes in neighboring normoxic tumor cells and possibly in surrounding stromal cells, many of which are involved in cancer progression and treatment resistance mechanisms.

Biodistribution and toxicity assessment of photoactivatable antibody-conjugated, antibiotic loaded gold nanocages for the treatment of bacterial infections (Conference Presentation)

We previously explored the use of antibody-conjugated, antibiotic-loaded gold nanocages for the treatment of bacterial infections. Using Staphylococcus aureus as a proof-of-principle pathogen, we confirmed that nanocages coated with polydopamine and loaded with daptomycin could be effectively targeted to bacterial cells using an antibody targeting S. aureus surface-associated protein A. We also confirmed that laser irradiation could then be used to achieve a lethal photothermal effect and localized release of the antibiotic, the synergistic effect of which was capable of eradicating viable bacteria even from a therapeutically recalcitrant biofilm. To assess the possibility that this comes at the cost of adverse side effects, we used multispectral optoacoustic tomography (MSOT) to track the biodistribution of our nanocages following intravenous administered and determined whether their administration was associated with toxic side effects. The results of our MSOT analysis confirmed that our nanocages accumulate primarily in the liver, spleen and kidney irrespective of infection status. However, in an infected animal, they also confirmed that nanocages ultimately do reach the site of infection. MSOT results were consistent with studies involving the direct analysis of these tissues, which confirmed the correlation between MSOT signals and the presence of gold nanocages. More importantly, they also demonstrated that the presence of nanocages was not associated with appreciable histopathology in the spleen, liver, kidney, lung or heart. This suggests that our use of antibody-conjugated, antibiotic-loaded gold nanocages for the treatment of infection offers significant promise that would not be compromised by systemic toxicity.