A cell-penetrating protein designed for bimodal fluorescence and magnetic resonance imaging

Multimodal imaging is a highly desirable biomedical application since it can provide complementary information from each imaging modality. We propose a protein engineering-based strategy for the construction of a bimodal probe for fluorescence and magnetic resonance imaging. A recombinant protein was generated by the fusion of a supercharged green fluorescence protein (GFP³⁶⁺) with a lanthanide-binding tag (dLBT) that can stably bind two Gd³⁺ ions. The GFP³⁶⁺–dLBT fusion protein showed strong fluorescence and exhibited efficient contrast enhancement in magnetic resonance imaging. This protein probe improves the MR relaxation more efficiently than Gd-DTPA (gadopentetate dimeglumine). The superior cell-penetrating activity of GFP³⁶⁺ allows the efficient cellular uptake of this fusion protein and it can thus be used as a cellular imaging probe. Dual imaging was conducted in vitro and in mice. This result indicates that the fusion of different functional domains is a feasible approach for making multi-modal imaging agents.     

Supramolecularly engineered phospholipids constructed by nucleobase molecular recognition: upgraded generation of phospholipids for drug delivery

Despite of great advances of phospholipids and liposomes in clinical therapy, very limited success has been achieved in the preparation of smart phospholipids and controlled-release liposomes for in vivo drug delivery and clinical trials. Here we report a supramolecular approach to synthesize novel supramolecularly engineered phospholipids based on complementary hydrogen bonding of nucleosides, which greatly reduces the need of tedious chemical synthesis, including reducing the strict requirements for multistep chemical reactions, and the purification of the intermediates and the amount of waste generated relative more traditional approaches. These upgraded phospholipids self-assemble into liposome-like bilayer structures in aqueous solution, exhibiting fast stimuli-responsive ability due to the hydrogen bonding connection. In vitro and in vivo evaluations show the resulted supramolecular liposomes from nucleoside phospholipids could effectively transport drug into tumor tissue, rapidly enter tumor cells, and controllably release their payload in response to an intracellular acidic environment, thus resulting in a much higher antitumor activity than conventional liposomes. The present supramolecularly engineered phospholipids represent an important evolution in comparison to conventional covalent-bonded phospholipid systems.     

Encapsulation of Pt(iv) prodrugs within a Pt(ii) cage for drug delivery

This report presents a novel strategy that facilitates delivery of multiple, specific payloads of Pt(iv) prodrugs using a well-defined supramolecular system. This delivery system comprises a hexanuclear Pt(ii) cage that can host four Pt(iv) prodrug guest molecules. Relying on host–guest interactions between adamantyl units tethered to the Pt(iv) molecules and the cage, four prodrugs could be encapsulated within one cage. This host–guest complex, exhibiting a diameter of about 3 nm, has been characterized by detailed NMR spectroscopic measurements. Owing to the high positive charge, this nanostructure exhibits high cellular uptake. Upon entering cells and reacting with biological reductants such as ascorbic acid, the host–guest complex releases cisplatin, which leads to cell cycle arrest and apoptosis. The fully assembled complex displays cytotoxicity comparable to that of cisplatin against a panel of human cancer cell lines, whereas the cage or the Pt(iv) guest alone exhibit lower cytotoxicity. These findings indicate the potential of utilising well-defined supramolecular constructs for the delivery of prodrug molecules.     

Sugar silanes: versatile reagents for stereocontrolled glycosylation via intramolecular aglycone delivery

A new method for the intramolecular glycosylation of alcohols is described. Utilizing carbohydrate-derived silanes, the catalytic dehydrogenative silylation of alcohols is followed by intramolecular glycosylation. Appropriate combinations of silane position and protecting groups allow highly selective access to β-manno, α-gluco, or β-gluco stereochemical relationships as well as 2-azido-2-deoxy-β-gluco- and 2-deoxy-β-glucosides. Intramolecular aglycone delivery from the C-2 or C-6 position provides 1,2-cis or 1,2-trans glycosides, respectively. Multifunctional acceptor substrates such as hydroxyketones and diols are tolerated and are glycosylated in a site-selective manner.     

Mesoporous silica and chitosan based pH-sensitive smart nanoparticles for tumor targeted drug delivery

This study investigated inclusion formation and the physicochemical properties of naringin/cyclodextrin through a combined computational and experimental approach. Molecular dynamics simulations were applied to investigate the thermodynamics and geometry of naringin/cyclodextrin cavity docking. The complexes were investigated by UV, FT-IR, DSC, XRD, SEM, 2D-NOSEY and ¹H-NMR analyses. Clearly visible protons belonging to naringin and chemical shift displacements of the H3 and H5 protons in cyclodextrin were anticipated in the formation of an inclusion complex. Naringin solubility increased linearly with increasing cyclodextrin concentration (displaying an A_L profile). The simulations indicated that the phenyl group of naringin was located deep within the cyclodextrin cavity, while the glycoside group of naringin was on the plane of the wider rim of cyclodextrin. The simulation and molecular modeling results indicate that (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) provided the more stable inclusion complex. This result was also in good concordance with the stability constants that had been determined by the phase solubility method. The consistency of the computational and experimental results indicates their reliability.     

Multifunctional nanoparticles possessing magnetic, long-lived fluorescence and bio-affinity properties for time-resolved fluorescence cell imaging

Multifunctional nanoparticles possessing magnetic, long-lived fluorescence and bio-affinity properties have been prepared by copolymerization of a conjugate of (3-aminopropyl)triethoxysilane bound to a fluorescent Eu³⁺ complex, 4,4′-bis(1″,1″,1″-trifluoro- 2″,4″-butanedion-4″-yl)chlorosulfo-o-terphenyl-Eu³⁺ (APS-BTBCT-Eu³⁺), free (3-aminopropyl)triethoxysilane (APS) and tetraethyl orthosilicate (TEOS) in the presence of poly(vinylpyrrolidone) (PVP) stabilized magnetic Fe₃O₄ nanoparticles (10 nm) with aqueous ammonia in ethanol. The nanoparticles were characterized by transmission electron microscopy (TEM), spectrofluorometry and vibrating sample magnetometry methods. The direct-introduced amino groups on the nanoparticle's surface by using free APS in nanoparticle preparation facilitated the surface modification and bioconjugation of the nanoparticles. The nanoparticle-labeled transferrin was prepared and used for staining the cultured Hela cells. A time-resolved fluorescence imaging technique that can fully eliminate the fast-decaying background noises was developed and used for the fluorescence imaging detection of the cells. A distinct image with the high ratio of signal to noise (S/N) was obtained.     

Multifunctional nanoprobe for cancer cell targeting and simultaneous fluorescence/magnetic resonance imaging

Multifunctional nanoprobes with distinctive magnetic and fluorescent properties are highly useful in accurate and early cancer diagnosis. In this study, nanoparticles of Fe₃O₄ core with fluorescent SiO₂ shell (MFS) are synthesized by a facile improved Stöber method. These nanoparticles owning a significant core-shell structure exhibit good dispersion, stable fluorescence, low cytotoxicity and excellent biocompatibility. TLS11a aptamer (Apt1), a specific membrane protein for human liver cancer cells which could be internalized into cells, is conjugated to the MFS nanoparticles through the formation of amide bond working as a target-specific moiety. The attached TLS11a aptamers on nanoparticles are very stable and can't be hydrolyzed by DNA hydrolytic enzyme in vivo. Both fluorescence and magnetic resonance imaging show significant uptake of aptamer conjugated nanoprobe by HepG2 cells compared to 4T1, SGC-7901 and MCF-7 cells. In addition, with the increasing concentration of the nanoprobe, T₂-weighted MRI images of the as-treated HepG2 cells are significantly negatively enhanced, indicating that a high magnetic field gradient is generated by MFS-Apt1 which has been specifically captured by HepG2 cells. The relaxivity of nanoprobe is calculated to be 11.5 mg⁻¹s⁻¹. The MR imaging of tumor-bearing nude mouse is also confirmed. The proposed multifunctional nanoprobe with the size of sub-100 nm has the potential to provide real-time imaging in early liver cancer cell diagnosis.     

Responsive fluorinated lanthanide probes for 19F magnetic resonance spectroscopy

The introduction of CF(3) reporter groups close to the paramagnetic centre in macrocyclic lanthanide(iii) complexes allows faster acquisition of (19)F magnetic resonance data, and amplifies chemical shift non-equivalence, as exemplified by the definition of ratiometric chemical shift probes for pH and, in principle, enzyme activity.     

Tetraphenylporphyrin‐based dual‐functional medical agent for magnetic resonance and fluorescence imaging

A bimodal magnetic resonance imaging contrast agent, TPP‐M‐Gd, was developed by modifying tetraphenylporphyrin (TPP) with a small dendritic molecule as a ligand (M) to chelate gadolinium (Gd) ions. The ligand featured four carboxylate groups, which contributed to good water solubility and a strong combination with metal ions. The longitudinal relaxivity (R₁) of the resulting agent was calculated to be 12.45 mM^?1 s^?1, which is much higher than that of DTPA‐Gd (4.49 mM^?1 s^?1). The magnetic resonance imaging experiments showed that the newly synthesized contrast agent could enhance T₁‐weighted magnetic resonance imaging quality both in vitro and in vivo. In addition, TPP‐M‐Gd exhibited good fluorescent property as shown in cell imaging experiments. The cytotoxicity of TPP‐M‐Gd was even better than that of clinically approved DTPA‐Gd, which makes it a promising dual‐functional medical imaging agent to provide more detailed information about biological and disease‐related events.     

Photosensitizer-incorporated G-quadruplex DNA-functionalized magnetofluorescent nanoparticles for targeted magnetic resonance/fluorescence multimodal imaging and subsequent photodynamic therapy of cancer

A smart heteronanostructure has been constructed for targeted photodynamic therapy and magnetic fluorescent imaging of cancer cells using photosensitizer-incorporated G-quadruplex DNA functionalized magnetic nanoparticles.     

Synthesis of oxygen-deficient luminescent mesoporous silica nanoparticles for synchronous drug delivery and imaging

Oxygen-deficient luminescent mesoporous silica nanoparticles with uniform morphology/size and integrated mesoporosity-luminescent property in a single nanoparticle are successfully synthesized by a bottom-up self-assembly route followed by a post-calcination process, and can be used to facilely load/deliver drugs into cells and luminescently image cells.     

Biofunctional magnetic nanoparticles as contrast agents for magnetic resonance imaging of pancreas cancer

We report on the fabrication and characterization of biofunctional magnetic nanoparticles as contrast agents for magnetic resonance imaging. The anti-cancer antigen 19-9 monoclonal antibody (a cancer-targeting antibody) was conjugated onto the magnetic contrast agents in an effort to detect pancreatic tumor. The structure, size, morphology and magnetic property of the biofunctional magnetic nanoparticles are characterized systematically by means of transmission electron microscopy and X-ray diffractometry. Furthermore, the interaction between the nanoparticles and pancreas cancers cells are investigated by atomic force microscope and transmission electron microscopy. Magnetic resonance imaging demonstrates that the conjugated nanoparticles can effectively target cancer cells both in vitro and in vivo, suggesting that they potentially can be used as contrast agents for magnetic resonance imaging of pancreas cancer.     

Design of chemical shift-switching 19F magnetic resonance imaging probe for specific detection of human monoamine oxidase A

Monoamine oxidase (MAO) A is a flavoenzyme that catalyzes the oxidation of biologically important monoamines and is thought to be associated with psychiatric disorders. Here, we report a strategy for rationally designing a (19)F magnetic resonance imaging probe for the specific detection of human MAO-A (hMAO-A) activity. Our designed (19)F probe was oxidized expeditiously by hMAO-A to produce 2-fluoro-4-nitrophenol via a spontaneous β-elimination mechanism. Concomitant with the structural change of the probe to the product, the (19)F chemical shift changed by 4.2 ppm, which was enough to visualize the probe and enzymatic product separately. Importantly, our probe achieved excellent discrimination of hMAO-A from its isoform hMAO-B.     

Expanding discriminative dimensions for analysis and imaging

Eliminating the contribution of interfering compounds is a key step in chemical analysis. In complex media, one possible approach is to perform a preliminary separation. However purification is often demanding, long, and costly; it may also considerably alter the properties of interacting components of the mixture (e.g. in a living cell). Hence there is a strong interest for developing separation-free non-invasive analytical protocols. Using photoswitchable probes as labelling and titration contrast agents, we demonstrate that the association of a modulated monochromatic light excitation with a kinetic filtering of the overall observable is much more attractive than constant excitation to read-out the contribution from a target probe under adverse conditions. An extensive theoretical framework enabled us to optimize the out-of-phase concentration first-order response of a photoswitchable probe to modulated illumination by appropriately matching the average light intensity and the radial frequency of the light modulation to the probe dynamics. Thus, we can selectively and quantitatively extract from an overall signal the contribution from a target photoswitchable probe within a mixture of species, photoswitchable or not. This simple titration strategy is more specifically developed in the context of fluorescence imaging, which offers promising perspectives.