Nanoparticle-mediated targeting of autoantigen peptide to cross-presenting liver sinusoidal endothelial cells protects from CD8 T-cell-driven autoimmune cholangitis.

Autoimmune diseases are caused by adaptive immune responses to self-antigens. The development of antigen-specific therapies that suppress disease-related, but not unrelated immune responses in general, is an important goal of biomedical research. We have previously shown that delivery of myelin peptides to liver sinusoidal endothelial cells (LSECs) using LSEC-targeting nanoparticles provides effective protection from CD4 T-cell-driven autoimmune encephalomyelitis. Here, we investigated whether this methodology might also serve antigen-specific treatment of a CD8 T-cell-driven autoimmune disease. As a model for CD8 T-cell-mediated autoimmunity, we used OT-1 T-cell-driven cholangitis in K14-OVAp mice expressing the cognate MHC I-restricted SIINFEKL peptide in cholangiocytes. To study whether peptide delivery to LSECs could modulate cholangitis, SIINFEKL peptide-conjugated nanoparticles were administered intravenously one day before transfer of OT-1 T cells; five days after cell transfer, liver pathology and hepatic infiltrates were analysed. SIINFEKL peptide-conjugated nanoparticles were rapidly taken up by LSECs in vivo, which effectively cross-presented the delivered peptide on MHC I molecules. Intriguingly, K14-OVAp mice receiving SIINFEKL-loaded nanoparticles manifested significantly reduced liver damage compared with vehicle-treated K14-OVAp mice. Mechanistically, treatment with LSEC-targeting SIINFEKL-loaded nanoparticles significantly reduced the number of liver-infiltrating OT-1 T cells, which up-regulated expression of the co-inhibitory receptor PD-1 and down-regulated cytotoxic effector function and inflammatory cytokine production. These findings show that tolerogenic LSECs can effectively internalize circulating nanoparticles and cross-present nanoparticle-bound peptides on MHC I molecules. Therefore, nanoparticle-mediated autoantigen peptide delivery to LSECs might serve the antigen-specific treatment of CD8 T-cell-driven autoimmune disease.

Indocyanine green molecular angiography of choroidal neovascularization.

Retinal diseases such as proliferative diabetic retinopathy and neovascular AMD are characterized by the formation of new blood vessels. Current imaging techniques such as fluorescein and ICG angiography help to identify areas of vascular leakage but are limited in their applicability due to their nonspecific nature. However, as new treatment paradigms emerge in an effort to have patient specific treatments, the development of new imaging techniques that are capable of identifying patient specific biomarkers will become crucial for the success of these approaches. In this study, we create and characterize an endoglin (CD105) targeted imaging probe that can be used for indocyanine green (ICG) molecular angiography. This anti-endoglin-ICG bioconjugate has a self-quenching "off-on" capacity to enable high contrast imaging of proliferative blood vessels at a molecular level in vivo. Using the laser CNV mouse model we demonstrate an approximate 3-fold increase in lesion visualization compared to non-targeting controls.

Theranostic Liposome-Nanoparticle Hybrids for Drug Delivery and Bioimaging.

Advanced theranostic nanomedicine is a multifunctional approach which combines the diagnosis and effective therapy of diseased tissues. Here, we investigated the preparation, characterization and in vitro evaluation of theranostic liposomes. As is known, liposome-quantum dot (L-QD) hybrid vesicles are promising nanoconstructs for cell imaging and liposomal-topotecan (L-TPT) enhances the efficiency of TPT by providing protection against systemic clearance and allowing extended time for it to accumulate in tumors. In the present study, hydrophobic CdSe/ZnS QD and TPT were located in the bilayer membrane and inner core of liposomes, respectively. Dynamic light scattering (DLS), zeta potential (ζ) measurements and fluorescence/absorption spectroscopy were performed to determine the vesicle size, charge and spectroscopic properties of the liposomes. Moreover, drug release was studied under neutral and acidic pH conditions. Fluorescence microscopy and flow cytometry analysis were used to examine the cellular uptake and intracellular distribution of the TPT-loaded L-QD formulation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to investigate the in vitro cytotoxicity of the formulations on HeLa cells. According to the results, the TPT-loaded L-QD hybrid has adequate physicochemical properties and is a promising multifunctional delivery vehicle which is capable of a simultaneous co-delivery of therapeutic and diagnostic agents.

Nanostructured Amphiphilic Star-Hyperbranched Block Copolymers for Drug Delivery.

A robust drug delivery system based on nanosized amphiphilic star-hyperbranched block copolymer, namely, poly(methyl methacrylate-block-poly(hydroxylethyl methacrylate) (PMMA-b-PHEMA) is described. PMMA-b-PHEMA was prepared by sequential visible light induced self-condensing vinyl polymerization (SCVP) and conventional vinyl polymerization. All of the synthesis and characterization details of the conjugates are reported. To accomplish tumor cell targeting property, initially cell-targeting (arginylglycylaspactic acid; RGD) and penetrating peptides (Cys-TAT) were binding to each other via the well-known EDC/NHS chemistry. Then, the resulting peptide was further incorporated to the surface of the amphiphilic hyperbranched copolymer via a coupling reaction between the thiol (-SH) group of the peptide and the hydroxyl group of copolymer by using N-(p-maleinimidophenyl) isocyanate as a heterolinker. The drug release property and targeting effect of the anticancer drug (doxorobucin; DOX) loaded nanostructures to two different cell lines were evaluated in vitro. U87 and MCF-7 were chosen as integrin αvß3 receptor positive and negative cells for the comparison of the targeting efficiency, respectively. The data showed that drug-loaded copolymers exhibited enhanced cell inhibition toward U87 cells in compared to MCF-7 cells because targeting increased the cytotoxicity of drug-loaded copolymers against integrin αvß3 receptor expressing tumor cells.

A novel DAD type and folic acid conjugated fluorescent monomer as a targeting probe for imaging of folate receptor overexpressed cells.

We describe a modification and post-functionalization technique for a donor-acceptor-donor type monomer; 6-(4,7-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-2H-benzo[d][1,2, 3]triazol-2-yl)hexan-1-amine. Folic acid was attached to the fluorescent structure. The conjugation was confirmed via NMR and Fourier transform infrared analyses. Cytotoxicity was investigated and the comparison of association of targeted monomeric structures in tumor cells was monitored via fluorescence microscopy.

Biofunctional quantum dots as fluorescence probe for cell-specific targeting.

We describe here the synthesis, characterization, bioconjugation, and application of water-soluble thioglycolic acid TGA-capped CdTe/CdS quantum dots (TGA-QDs) for targeted cellular imaging. Anti-human epidermal growth factor receptor 2 (HER2) antibodies were conjugated to TGA-QDs to target HER2-overexpressing cancer cells. TGA-QDs and TGA-QDs/anti-HER2 bioconjugates were characterized by fluorescence and UV-Vis spectroscopy, X-ray diffraction (XRD), hydrodynamic sizing, electron microscopy, and gel electrophoresis. TGA-QDs and TGA-QDs/anti-HER2 were incubated with cells to examine cytotoxicity, targeting efficiency, and cellular localization. The cytotoxicity of particles was measured using an MTT assay and the no observable adverse effect concentration (NOAEC), 50% inhibitory concentration (IC50), and total lethal concentration (TLC) were calculated. To evaluate localization and targeting efficiency of TGA-QDs with or without antibodies, fluorescence microscopy and flow cytometry were performed. Our results indicate that antibody-conjugated TGA-QDs are well-suited for targeted cellular imaging studies.

From invisible structures of SWCNTs toward fluorescent and targeting architectures for cell imaging.

Single-walled carbon nanotubes (SWNTs) are unique nanostructures used as cargo systems for variety of diagnostic and therapeutic agents. For taking advantage of these structures in biological processes, they should be visible. Therefore, fluorescence labeling of SWCNTs with various probes is a significant issue. Herein, we demonstrate a simple approach for cell specific imaging and diagnosis by combining SWCNTs with a copolymer poly(para-phenylene) (PPP) containing polystyrene (PSt) and poly(ε-caprolactone) (PCL) side chains (PPP-g-PSt-PCL). In this approach PPP-g-PSt-PCL is noncovalently attached on carboxyl functional SWCNTs. The obtained fluorescent probe is bound to folic acid (FA) for targeted imaging of folate receptor (FR) positive HeLa cells. In vitro studies demonstrate that this conjugate can specifically bind to HeLa cells and indicate great potential for targeting and imaging studies.

Folic acid-modified clay: targeted surface design for cell culture applications.

Here we report the preparation, characterization and application of folic acid modified Montmorillonite clay (FA-Mont) as a cell culture material. Clays exhibit unique properties such as good mechanical and chemical stabilities, high surface area, low toxicity and easy combination with functional organic groups. FA was used as a modifier to facilitate adhesion of folate positive cells on the clay surface. FA-Mont was characterized using FT-IR, XRD, zeta potential measurements as well as thermogravimetric analyses. Finally, the usage potential of FA-Mont as a receptor mediated cell adhesion material was successfully proved by using folate receptor (FR) rich HeLa and FR poor A-549 cells. The cell adhesion studies were monitored and imaged by fluorescence and scanning electron microscopy techniques.