Multifunctional targeting micelle nanocarriers with both imaging and therapeutic potential for bladder cancer.

BackgroundWe previously developed a bladder cancer-specific ligand (PLZ4) that can specifically bind to both human and dog bladder cancer cells in vitro and in vivo. We have also developed a micelle nanocarrier drug-delivery system. Here, we assessed whether the targeting micelles decorated with PLZ4 on the surface could specifically target dog bladder cancer cells.Materials and methodsMicelle-building monomers (ie, telodendrimers) were synthesized through conjugation of polyethylene glycol with a cholic acid cluster at one end and PLZ4 at the other, which then self-assembled in an aqueous solution to form micelles. Dog bladder cancer cell lines were used for in vitro and in vivo drug delivery studies.ResultsCompared to nontargeting micelles, targeting PLZ4 micelles (23.2 ± 8.1 nm in diameter) loaded with the imaging agent DiD and the chemotherapeutic drug paclitaxel or daunorubicin were more efficient in targeted drug delivery and more effective in cell killing in vitro. PLZ4 facilitated the uptake of micelles together with the cargo load into the target cells. We also developed an orthotopic invasive dog bladder cancer xenograft model in mice. In vivo studies with this model showed the targeting micelles were more efficient in targeted drug delivery than the free dye (14.3×; P < 0.01) and nontargeting micelles (1.5×; P < 0.05).ConclusionTargeting micelles decorated with PLZ4 can selectively target dog bladder cancer cells and potentially be developed as imaging and therapeutic agents in a clinical setting. Preclinical studies of targeting micelles can be performed in dogs with spontaneous bladder cancer before proceeding with studies using human patients

Targeting Tumor-Associated Exosomes with Integrin-Binding Peptides

All cells expel a variety of nanosized extracellular vesicles (EVs), including exosomes, with composition reflecting the cells' biological state. Cancer pathology is dramatically mediated by EV trafficking via key proteins, lipids, metabolites, and microRNAs. Recent proteomics evidence suggests that tumor-associated exosomes exhibit distinct expression of certain membrane proteins, rendering those proteins as attractive targets for diagnostic or therapeutic application, yet it is not currently feasible to distinguish circulating EVs in complex biofluids according to their tissue of origin or state of disease. Here, peptide binding to tumor-associated EVs via overexpressed membrane protein is demonstrated. It is found that SKOV-3 ovarian tumor cells and their released EVs express alpha(3)beta(1) integrin, which can be targeted by the in-house cyclic nonapeptide, LXY30. After measuring bulk SKOV-3 EV association with LXY30 by flow cytometry, Raman spectral analysis of laser-trapped single exosomes with LXY30-dialkyne conjugate enables the differentiation of cancer-associated exosomes from noncancer exosomes. Furthermore, the foundation for a highly specific detection platform for tumor-EVs in solution with biosensor surface-immobilized LXY30 is introduced. LXY30 not only exhibits high specificity and affinity to alpha(3)beta(1) integrin-expressing EVs, but also reduces EV uptake into SKOV-3 parent cells, demonstrating the possibility for therapeutic application.Peer reviewe

Label-free biomolecular sensing using single color near-null reflective interferometry and Brewster Angle Straddle Interferometry

Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2008.Reflective Interferometry uses reflectivity changes at an interface functionalized with molecular probes to detect label-free biomolecular binding. In this thesis, we report Single Color Near-Null Reflective Interferometry (sc-NNRI) that attaches target molecules to the surface and alters the effective thickness of an anti-reflective coating formed by thermal oxidation of a silicon wafer to remove destructive interference of the reflected waves. The thermal oxide thickness is adjusted for precise interference using layer-by-layer electrostatic self-assembly of polyelectrolytes to which the molecular probes can be bound covalently. Reflectivity increases of over a factor of 100 are observed for binding of 2.5 nm of streptavidin to biotinylated polyelectrolytes, considerably more sensitive than surface plasmon resonance detection. Theoretical modeling is in agreement with the experimentally observed reflectivity increases and suggests the sensitivity is at present limited by the roughness of the oxide. We report another novel reflective interferometric method, Brewster Angle Straddle Interferometry (BASI), to detect chemical binding at an interface. The interference layer consists of the thin native oxide on silicon, and we utilize nearly opposite phase shifts of light at the oxide/water and oxide/silicon interfaces to achieve near-complete destructive interference. We measure selective binding of thrombin in solution to DNA aptamers covalently bound to the oxide. The technique can be used to detect and quantitate surface binding of less than 1 Å of material, sensitivity similar to that of surface plasmon resonance imaging and sc-NNRI. Results are in quantitative agreement with what is predicted theoretically. The method is very convenient to implement since it utilizes unmodified silicon wafers as substrates and is extremely insensitive to both probe light bandwidth and collimation. We used under water BASI to detect biomolecular bindings at a water/substrate interface. We measure selective binding of a 47-mer fragment of Has-ra mRNA in solution to six different designed 2’-O-methyl RNA probes covalently immobilized on the silicon oxide surface. The technique can be used to quantitate surface binding since the value of the reflectivity minimum is parabolic with the layer (oxide plus adsorbents) thickness. We were able to see increases in reflectivity of 33%, which is due to binding of 7.5 angstroms of 47-mer Has-ra mRNA with specific probe M 3291 on the surface. The limitation of detection (LOD) is estimated to be ~1.0 angstrom based on signal to noise ratio ~3

Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.

The protein family known as G-protein coupled receptors (GPCRs) comprises an important class of membrane-associated proteins, which remains a difficult family of proteins to characterize because their function requires a native-like lipid membrane environment. This paper focuses on applying a single step method leading to the formation of nanolipoprotein particles (NLPs) capable of solubilizing functional GPCRs for biophysical characterization. NLPs were used to demonstrate increased solubility for multiple GPCRs such as the Neurokinin 1 Receptor (NK1R), the Adrenergic Receptor â2 (ADRB2) and the Dopamine Receptor D1 (DRD1). All three GPCRs showed affinity for their specific ligands using a simple dot blot assay. The NK1R was characterized in greater detail to demonstrate correct folding of the ligand pocket with nanomolar specificity. Electron paramagnetic resonance (EPR) spectroscopy validated the correct folding of the NK1R binding pocket for Substance P (SP). Fluorescence correlation spectroscopy (FCS) was used to identify SP-bound NK1R-containing NLPs and measure their dissociation rate in an aqueous environment. The dissociation constant was found to be 83 nM and was consistent with dot blot assays. This study represents a unique combinational approach involving the single step de novo production of a functional GPCR combined with biophysical techniques to demonstrate receptor association with the NLPs and binding affinity to specific ligands. Such a combined approach provides a novel path forward to screen and characterize GPCRs for drug discovery as well as structural studies outside of the complex cellular environment

Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs

Gao T, Petrlova J, He W, et al. Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs. PLoS ONE. 2012;7(9): e44911

Single-Bead Quantification of Peptide Loading Distribution for One-Bead One-Compound Library Synthesis Using Confocal Raman Spectroscopy

We report an analytical method to determine peptide loading of “one-bead one-compound” (OBOC) combinatorial peptide libraries at single-bead level. The quantification is based on a linear relationship between the amount of N-terminal amino groups on individual peptide beads and the intensity of Raman signal obtained from a specifically designed reporter labeled on amino groups. Confocal Raman spectroscopy was employed to characterize peptide loading of beads with defined peptide sequences and from OBOC combinatorial peptide libraries. Although amine loading of blank TentaGel beads was found to be uniform, peptide loading among beads of OBOC peptide libraries varied substantially, particularly for those libraries with long sequences. Construction of OBOC libraries can be monitored with this novel analytical technique so that synthetic conditions can be optimized for the preparation of high-quality OBOC peptide libraries. As the variability of peptide loading of individual library beads can significantly influence the screening results, quantitative information obtained by this method will allow us to gain insight into the complexity and challenge of OBOC library synthesis and screening

Saturation binding assay of FAM-SP on filter paper after interacting with NK1R-NLPs.

<p>The fluorescence intensity was averaged through 3 replicates with the error bar showing the standard deviation. Each data point represented intensity of different amounts of FAM-SP interacting with NK1R-NLPs subtracted by non-specific adsorption of the same amounts of FAM-SP to the paper. The solid curve represents specific binding. The dashed curve represents non-specific binding (NK1R-NLPs saturated by excessive amount of non-labeled SP). The binding curve was fit to an “OneSiteBind” model Y  =  B_max × X/(K_d + X), where Y represents fluorescence intensity caused by binding and X represents concentration of FAM-SP in the solution after reaction. The fitting results gave (3.5±0.3) ×10⁶ (fluorescence intensity) for B_max and 34±7.8 nM for K_d (dissociation constant).</p

Diffusion curves of lipid vesicles, NK1R and NK1R-NLP complexes.

<p>The lipids and NK1R were labeled by Texas Red and GFP respectively. The cross correlation of Texas Red and GFP represents the interaction between lipids and NK1R, indicating the formation of NK1R-associated NLPs. The diffusion times of lipid vesicles, NK1R and NK1R- NLP complexes are 4.46, 0.17, and 0.51 ms respectively.</p

Multifunctional targeting micelle nanocarriers with both imaging and therapeutic potential for bladder cancer.

BackgroundWe previously developed a bladder cancer-specific ligand (PLZ4) that can specifically bind to both human and dog bladder cancer cells in vitro and in vivo. We have also developed a micelle nanocarrier drug-delivery system. Here, we assessed whether the targeting micelles decorated with PLZ4 on the surface could specifically target dog bladder cancer cells.Materials and methodsMicelle-building monomers (ie, telodendrimers) were synthesized through conjugation of polyethylene glycol with a cholic acid cluster at one end and PLZ4 at the other, which then self-assembled in an aqueous solution to form micelles. Dog bladder cancer cell lines were used for in vitro and in vivo drug delivery studies.ResultsCompared to nontargeting micelles, targeting PLZ4 micelles (23.2 ± 8.1 nm in diameter) loaded with the imaging agent DiD and the chemotherapeutic drug paclitaxel or daunorubicin were more efficient in targeted drug delivery and more effective in cell killing in vitro. PLZ4 facilitated the uptake of micelles together with the cargo load into the target cells. We also developed an orthotopic invasive dog bladder cancer xenograft model in mice. In vivo studies with this model showed the targeting micelles were more efficient in targeted drug delivery than the free dye (14.3×; P &lt; 0.01) and nontargeting micelles (1.5×; P &lt; 0.05).ConclusionTargeting micelles decorated with PLZ4 can selectively target dog bladder cancer cells and potentially be developed as imaging and therapeutic agents in a clinical setting. Preclinical studies of targeting micelles can be performed in dogs with spontaneous bladder cancer before proceeding with studies using human patients