Marek Romanowski

Optical coherence tomography with plasmon resonant nanorods of gold

We explored plasmon resonant nanorods of gold as a contrast agent for optical coherence tomography (OCT). Nanorod suspensions were generated through wet chemical synthesis and characterized with spectrophotometry, transmission electron microscopy, and OCT. Polyacrylamide-based phantoms were generated with appropriate scattering and anisotropy coefficients (30 cm(-1) and 0.89, respectively) to image distribution of the contrast agent in an environment similar to that of tissue. The observed signal was dependent on whether the plasmon resonance peak overlapped the source bandwidth of the OCT, confirming the resonant character of enhancement. Gold nanorods with plasmon resonance wavelengths overlapping the OCT source yielded a signal-to-background ratio of 4.5 dB, relative to the tissue phantom. Strategies for OCT imaging with nanorods are discussed.

Nanoparticle drug delivery system for intravenous delivery of topoisomerase inhibitors.

Camptothecin-based drugs, because of their poor solubility and labile lactone ring, pose challenges for drug delivery. The purpose of this research was to develop a nanoparticle delivery system for camptotheca alkaloids. After initial investigations SN-38 was selected as the candidate camptotheca alkaloid for further development. Nanoparticles comprising SN-38, phospholipids and polyethylene glycol were developed and studied in vitro and in vivo. The SN-38 formulations were stable in human serum albumin and high lactone concentrations were observed even after 3 h. In vivo studies in nude mice showed prolonged half-life of the active (lactone form) drug in whole blood and increased efficacy compared to Camptosar in a mouse xenograft tumor model.

Light-Activated Content Release from Liposomes

Successful integration of diagnostic and therapeutic actions at the level of individual cells requires new materials that combine biological compatibility with functional versatility. This review focuses on the development of liposome-based functional materials, where payload release is activated by light. Methods of sensitizing liposomes to light have progressed from the use of organic molecular moieties to the use of metallic plasmon resonant structures. This development has facilitated application of near infrared light for activation, which is preferred for its deep penetration and low phototoxicity in biological tissues. Presented mechanisms of light-activated liposomal content release enable precise in vitro manipulation of minute amounts of reagents, but their use in clinical diagnostic and therapeutic applications will require demonstration of safety and efficacy.

Gold nanorods targeted to delta opioid receptor: plasmon-resonant contrast and photothermal agents.

Molecularly targeted gold nanorods were investigated for applications in both diagnostic imaging and disease treatment with cellular resolution. The nanorods were tested in two genetically engineered cell lines derived from the human colon carcinoma HCT-116, a model for studying ligand-receptor interactions. One of these lines was modified to express delta opioid receptor (δOR) and green fluorescent protein, whereas the other was receptor free and expressed a red fluorescent protein, to serve as the control. Deltorphin, a high-affinity ligand for δOR, was stably attached to the gold nanorods through a thiol-terminated linker. In a mixed population of cells, we demonstrated selective imaging and destruction of receptor-expressing cells while sparing those cells that did not express the receptor. The molecularly targeted nanorods can be used as an in vitro ligand-binding and cytotoxic treatment assay platform and could potentially be applied in vivo for diagnostic and therapeutic purposes with endoscopic technology.

NIR-Activated Content Release from Plasmon Resonant Liposomes for Probing Single-Cell Responses

Technological limitations have prevented the interrogation and manipulation of cellular activity in response to bioactive molecules within model and living systems that is required for the development of diagnostic and treatment modalities for diseases, such as cancer. In this work, we demonstrate that gold-coated liposomes are capable of encapsulation and on-demand release of signaling molecules with a spatial and temporal resolution leading to activation of individual cells. As a model system, we used cells modified to overexpress a certain G-protein coupled receptor, the CCK2 receptor, and achieved its activation in a single cell via the localized release of its agonist. This content release was triggered by illumination of the liposomes at wavelengths corresponding to the plasmon resonance of the gold coating. The use of plasmon resonant liposomes may enable on-demand release of a broad range of molecules using biologically safe near-infrared light and without molecule chemical modification. In combination with the spectral tunability of plasmon resonant coating, this technology may allow for multiplexed interrogation of complex and diverse signaling pathways in model or living tissues with unprecedented spatial and temporal control.

Control of green and red upconversion in NaYF4:Yb3+,Er3+ nanoparticles by excitation modulation

Control of the two strongest upconversion emission lines in NaYF4:Yb3+, Er3+ nanoparticles is demonstrated by varying the excitation repetition rate. This technique may enable new multiplexed sensing modalities based on multicolor luminescent nanoparticles, currently contemplated for biomedical imaging and diagnostics.

Polarized absorption and emission spectra of stilbazolium merocyanines

Abstract Natural light and polarized absorption and fluorescence spectra of six stilbazolium merocyanines (with different side groups and molecule lengths) embedded in polyvinyl alcohol film were measured. In a stretched film, dye molecules are highly oriented. Different spectra are obtained for the same dye in isotropic and anisotropic polymer films. Conclusions concerning the degree of dye orientation and the distribution of molecules in the direction of film stretching are made on the basis of different combinations of polarized fluorescence components. Dyes occur in the polymer matrix in protonated and free base forms. The protonation of merocyanines molecules influences their degree of orientation in an anisotropic matrix and the aggregation of the dye. Aggregation of protonated and free base forms competes with the interaction of the dye with the matrix. Aggregates are oriented in a different way from monomers. The side groups attached to the dye chain have a strong influence on the orientation of the molecules and their aggregation properties.

Structural domains of phytochrome deduced from homologies in amino acid sequences

A method of semiempirical identification of structural domains is proposed. The procedure is based on the comparison of amino acid sequences in groups of homologous proteins. This approach was tested using 32 known protein sequences from different cytochromeb5, cytochromec, lysozyme, hemoglobin, and myoglobin proteins. The method presented was able to identify all structural domains of these reference proteins. A consensus secondary structure provided information on structural content of these domains predicting correctly 21 of 23 (91%) of α-helices. We applied this method to six homologous phytochrome sequences fromAvena, Arabadopsis, Cucurbita, Maize, Oryza, andPisum. Some of the identified domains can be assigned to the known tertiary structure categories. For example, an α/β domain is localized in the region known to stabilize the phytochrome chromophore in the red light absorbing form (Pr). One α-helical and one α/β domains are localized in regions important for the chromophore stabilization in the far-red absorbing form (Pfr). From an analysis of noncovalent interaction patterns in another domain it is proposed that a phytochrome dimer contact involves two segments localized between residues 730 and 821 (using numbering of aligned sequences). Also, a possible antiparallel β-sheet structure of this region has been suggested. According to this model, the long axis of the interacting structures is perpendicular to a twofold symmetry axis of the phytochrome dimer.

Scanning two-photon microscopy with upconverting lanthanide nanoparticles via Richardson-Lucy deconvolution

The use of upconverting lanthanide nanoparticles in fast-scanning microscopy is hindered by a long luminescence decay time, which greatly blurs images acquired in a nondescanned mode. We demonstrate herein an image processing method based on Richardson-Lucy deconvolution that mitigates the detrimental effects of their luminescence lifetime. This technique generates images with lateral resolution on par with the systems performance, ∼1.2  μm, while maintaining an axial resolution of 5 μm or better at a scan rate comparable with traditional two-photon microscopy. Remarkably, this can be accomplished with near infrared excitation power densities of 850 W/cm(2), several orders of magnitude below those used in two-photon imaging with molecular fluorophores. By way of illustration, we introduce the use of lipids to coat and functionalize these nanoparticles, rendering them water dispersible and readily conjugated to biologically relevant ligands, in this case epidermal growth factor receptor antibody. This deconvolution technique combined with the functionalized nanoparticles will enable three-dimensional functional tissue imaging at exceptionally low excitation power densities.

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.