RESUMO
Because of the recent increasing demand for the synthetic biomimetic nanoparticles as in vivo carriers of drugs and imaging probes, it is very important to develop reliable, stable, and orthogonal methods for surface functionalization of the particles. To address these issues, in this study, a recently reported chemoselective amide-forming ligation reaction [potassium acyltrifluoroborate (KAT) ligation] was employed for the first time, as a mean to provide the surface functionalization of particles for creating covalent attachments of bioactive molecules. A KAT derivative of oleic acid (OA-KAT, 1) was added to a mixture of three lipid components (triolein, phosphatidyl choline, and cholesteryl oleate), which have been commonly used as substrates for lipid nanoparticles. After sonication and extrusion in a buffer, successfully obtained lipid nanoparticles containing OA-KAT (NP-KAT) resulted to be well-dispersed with mean diameters of about 40-70 nm by dynamic light scattering. After preliminary confirmation of the fast and efficient KAT ligation in a solution phase using the identical reaction substrates, the "on-surface (on-particle)" KAT ligation on the NP-KAT was tested with an N-hydroxylamine derivative of fluorescein 2. The ligation was carried out in a phosphate buffer (10 mM, pH 5.2) at room temperature with reactant concentration ranges of 250 µM. Reaction efficiency was evaluated based on the amount of boron (determined by inductively coupled plasma mass spectrometry) and fluorescein (determined by fluorescence emission) in the particles before and after the reaction. As a result, the reaction proceeded in a significantly efficient way with ca. 40-50% conversion of the OA-KAT incorporated in the particles. Taken together with the fact that KAT ligation does not require any additional coupling reagents, these results indicated that the "on-surface" chemical functionalization of nanoparticles by KAT ligation is a useful method and represents a powerful and potentially versatile tool for the production of nanoparticles with a variety of covalently functionalized biomolecules and probes.
RESUMO
A polymeric FRET probe for the detection of MMP2 was prepared using a new N-hydroxylamine derivative of lysine (1), which was successfully incorporated into the natural peptide sequence by solid phase peptide synthesis (SPPS). Following the attachment of a PEG group to the N-terminus, a peptide was cleaved from the resin. The fully-deprotected peptide-PEG conjugate was subsequently subjected to the α-ketoacid-hydroxylamine (KAHA) ligation and Michael addition of FRET donor (MCA) and acceptor (DNP) moieties, respectively. The successfully synthesized polymeric FRET probes with an MMP2-reactive peptide and a negative control peptide with a random sequence were subjected to an in vitro test with MMP2. This methodology under mild conjugation conditions of KAHA ligation can be applicable for the preparation of NIR probes with sensitive fluorophore moieties.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Hidroxilamina/química , Metaloproteinase 2 da Matriz/análise , Peptídeos/química , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Peptídeos/síntese química , Técnicas de Síntese em Fase SólidaRESUMO
To allow visualization of macrophage-rich and miniature-sized atheromas by magnetic resonance (MR) imaging, we have converted low-density lipoprotein (LDL) into MR-active nanoparticles via the intercalation of a 1,4,7,10-tetraazacyclodecane-1,4,7-triacetic acid (DO3A) derivative and the subsequent coordination reaction with Gd(3+). After careful removal of nonchelated Gd(3+), an MR-active LDL (Gd(3+)-LDL) with a remarkably high payload of Gd(3+) (in excess of 200 Gd(3+) atoms per particle) and a high relaxivity (r(1) = 20.1 s(-1) mM(-1) per Gd(3+) or 4040 s(-1) mM(-1) per LDL) was obtained. Dynamic light-scattering photon correlation spectroscopy (DLS) and cryo transmission electron microscope (cryoTEM) images showed that Gd(3+)-LDL particles did not aggregate and remained of a similar size (25-30 nm) to native LDL. Intravenous injection of Gd(3+)-LDL into an atherosclerotic mouse model (ApoE(-/-)) resulted in an extremely high enhancement of the atheroma-bearing aortic walls at 48 h after injection. Free Gd(3+) dissociation from Gd(3+)-LDL was not detected over the imaging time window (96 h). Because autologous LDL can be isolated, modified, and returned to the same patient, our results suggest that MR-active LDL can potentially be used as a noninfectious and nonimmunogenic imaging probe for the enhancement of atheroplaques presumably via the uptake into macrophages inside the plaque.
Assuntos
Aterosclerose/diagnóstico , Lipoproteínas LDL , Imageamento por Ressonância Magnética , Nanopartículas , Compostos Organometálicos , Compostos Radiofarmacêuticos , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Modelos Animais de Doenças , Gadolínio/química , Gadolínio/farmacocinética , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/farmacocinética , Humanos , Lipoproteínas LDL/química , Lipoproteínas LDL/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Nanopartículas/química , Compostos Organometálicos/química , Compostos Organometálicos/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
Membrane receptors for steroid hormones are currently a subject of considerable debate. One approach to selectively target these putative receptors has been to couple ligands to substances that restrict cell permeability. Using this approach, an analogue of the estrogen receptor ligand 4-hydroxytamoxifen was attached to fluorescent dyes with differing degrees of predicted cell permeability. The conjugates bound to estrogen receptor in vitro, but all three conjugates, including one predicted to be cell-impermeable, inhibited estradiol-induced transcriptional activation. Fluorescence microscopy revealed cytoplasmic localization for all three conjugates. We further characterized a 4-hydroxytamoxifen analogue conjugated to a BODIPY fluorophore in breast cancer cell lines. Those experiments suggested a similar, but not identical, mode of action to 4-hydroxytamoxifen, as the fluorescent conjugate was equally effective at inhibiting proliferation of both tamoxifen-sensitive and tamoxifen-resistant breast cancer cell lines. While these findings point to significant complicating factors in designing steroid hormone mimics targeted to the plasma membrane, the results also reveal a possible new direction for designing estrogen receptor modulators.
Assuntos
Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/farmacologia , Corantes Fluorescentes/química , Receptores de Estrogênio/metabolismo , Tamoxifeno/análogos & derivados , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Antagonistas de Estrogênios/síntese química , Antagonistas de Estrogênios/farmacocinética , Corantes Fluorescentes/síntese química , Humanos , Tamoxifeno/síntese química , Tamoxifeno/química , Tamoxifeno/farmacocinética , Tamoxifeno/farmacologiaRESUMO
Low-density lipoprotein (LDL)-mimetic lipid nanoparticles (LNPs), decorated with MRI contrast agents and fluorescent dyes, were prepared by the covalent attachment of apolipoprotein-mimetic peptide (P), Gd(iii)-chelate (Gd), and sulforhodamine B (R) moieties on the LNP surface. The functionalized LNPs were prepared using the amide-forming potassium acyltrifluoroborate (KAT) ligation reaction. The KAT groups on the surface of LNPs were allowed to react with the corresponding hydroxylamine (HA) derivatives of P and Gd to provide bi-functionalized LNPs (PGd-LNP). The reaction proceeded with excellent yields, as observed by ICP-MS (for B and Gd amounts) and MALDI-TOF-MS data, and did not alter the morphology of the LNPs (mean diameter: ca. 50 nm), as shown by DLS and cryoTEM analyses. With the help of the efficient KAT ligation, a high payload of Gd(iii)-chelate on the PGd-LNP surface (ca. 2800 Gd atoms per LNP) was successfully achieved and provided a high r 1 relaxivity (r 1 = 22.0 s-1 mM-1 at 1.4 T/60 MHz and 25 °C; r 1 = 8.2 s-1 mM-1 at 9.4 T/400 MHz and 37 °C). This bi-functionalized PGd-LNP was administered to three atherosclerotic apoE -/- mice to reveal the clear enhancement of atherosclerotic plaques in the brachiocephalic artery (BA) by MRI, in good agreement with the high accumulation of Gd in the aortic arch as shown by ICP-MS. The parallel in vivo MRI and ex vivo studies of whole mouse cryo-imaging were performed using triply functionalized LNPs with P, Gd, and R (PGdR-LNP). The clear presence of atherosclerotic plaques in BA was observed by ex vivo bright field cryo-imaging, and they were also observed by high emission fluorescent imaging. These directly corresponded to the enhanced tissue in the in vivo MRI of the identical mouse.
RESUMO
Highly water-soluble fullerene polymers were successfully prepared by a simple direct free-radical copolymerization of N-vinylpyrrolidone and intact C60 or C70 as a radical-capping agent. Using AIBN as a radical initiator, the polymers (C60- or C70-PVP) with significantly high molecular weight (~30 kDa) and with efficient (1)O2 generation were obtained.