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Thirteen red-shifted pentamethine dimethyl and diethylamino tetrahydroxanthylium derivatives have been successfully synthesized

Highly stable symmetric and asymmetric squaraine fluorophores have been synthesized featuring an internal salt bridge between a quaternary ammonium cation and the central oxycyclobutenolate ring of the chromophore. Some of our newly synthesized symmetric and asymmetric compounds display increased molar absorptivity, quantum yield in serum, and thermal/photochemical stability over previously reported squaraine-based dyes. Consequently, both classes show great promise in resurfacing the normal environment-labile squaraine dyes as novel imaging agents and scaffolds for fluorescence sensing. Furthermore, incorporating a covalent attachment point away from the conjugated system allows for biological tagging applications without disturbing the optimum optical characteristics of the newly designed fluorophore.

Some heptamethine cyanine dyes accumulate in solid tumors in vivo and persist there for several days. The reasons why they accumulate and persist in tumors were incompletely defined, but explanations based on uptake into cancer cells via organic anion transporting polypeptides (OATPs) have been widely discussed. All cyanine-based “tumor-seeking dyes” have a chloride centrally placed on the heptamethine bridge (a “meso-chloride”). We were intrigued and perplexed by the correlation between this particular functional group and tumor uptake, so the following study was designed. It features four dyes (1-Cl, 1-Ph, 5-Cl, and 5-Ph) with complementary properties. Dye 1-Cl is otherwise known as MHI-148, and 1-Ph is a close analog wherein the meso-chloride has been replaced by a phenyl group. Data presented here shows that both 1-Cl and 1-Ph form noncovalent adducts with albumin, but only 1-Cl can form a covalent one. Both dyes 5-Cl and 5-Ph have a methylene (CH(2)) unit replaced by a dimethylammonium functionality (N(+)Me(2)). Data presented here shows that both these dyes 5 do not form tight noncovalent adducts with albumin, and only 5-Cl can form a covalent one (though much more slowly than 1-Cl). In tissue culture experiments, uptake of dyes 1 is more impacted by the albumin in the media than by the pan-OATP uptake inhibitor (BSP) that has been used to connect uptake of tumor-seeking dyes in vivo with the OATPs. Uptake of 1-Cl in media containing fluorescein-labeled albumin gave a high degree of colocalization of intracellular fluorescence. No evidence was found for the involvement of OATPs in uptake of the dyes into cells in media containing albumin. In an in vivo tumor model, only the two dyes that can form albumin adducts (1-Cl and 5-Cl) gave intratumor fluorescence that persisted long enough to be clearly discerned over the background (~4 h); this fluorescence was still observed at 48 h. Tumors could be imaged with a higher contrast if 5-Cl is used instead of 1-Cl, because 5-Cl is cleared more rapidly from healthy tissues. Overall, the evidence is consistent with in vitro and in vivo results and indicates that the two dyes in the test series that accumulate in tumors and persist there (1-Cl and 5-Cl, true tumor-seeking dyes) do so as covalent albumin adducts trapped in tumor tissue via uptake by some cancer cells and via the enhanced permeability and retention (EPR) effect.

Near-infrared (NIR) fluorescence light has been widely utilized in clinical imaging by providing surgeons highly specific images of target tissue. The "NIR window" from 650 to 900 nm is especially useful due to several special features such as minimal autofluorescence and absorption of biomolecules in tissue, as well as low light scattering. Compared with visible wavelengths, NIR fluorescence light is invisible, thus allowing highly sensitivity real-time image guidance in human surgery without changing the surgical field. The benefit of using NIR fluorescence light as a clinical imaging technology can be attributed to its molecular fluorescence as an exogenous contrast agent. Indeed, whole body preoperative imaging of single-photon emission computed tomography (SPECT) and positron emission tomography (PET) remains important in diagnostic utility, but they lack the efficacy of innocuous and targeted NIR fluorophores to simultaneously facilitate the real-time delineation of diseased tissue while preserving vital tissues. Admittedly, NIR imaging technology has been slow to enter clinical use mostly due to the late-coming development of truly breakthrough contrast agents for use with current imaging systems. Therefore, clearly defining the physical margins of tumorous tissue remains of paramount importance in bioimaging and targeted therapy. An equally noteworthy yet less researched goal is the ability to outline healthy vital tissues that should be carefully navigated without transection during the intraoperative surgery. Both of these paths require optimizing a gauntlet of design considerations to obtain not only an effective imaging agent in the NIR window but also high molecular brightness, water solubility, biocompatibility, and tissue-specific targetability. The imaging community recognizes three strategic approaches which include (1) passive targeting via the EPR effect, (2) active targeting using the innate overall biodistribution of known molecules, and (3) activatable targeting through an internal stimulus, which turns on fluorescence from an off state. Recent advances in nanomedicine and bioimaging offer much needed promise toward fulfilling these stringent requirements as we develop a successful catalog of targeted contrast agents for illuminating both tumors and vital tissues in the same surgical space by employing spectrally distinct fluorophores in real time. These tissue-specific contrast agents can be versatile arsenals to physicians for real-time intraoperative navigation as well as image-guided targeted therapy. There is a versatile library of tissue-specific fluorophores available in the literature, with many discussed herein, which offers clinicians an array of possibilities that will undoubtedly improve intraoperative success and long-term postoperation prognosis.

Despite all the advances in multimodal imaging, it remains a significant challenge to acquire both magnetic resonance and nuclear imaging in a single dose because of the enormous difference in sensitivity. Indeed, nuclear imaging is almost 10(6)-fold more sensitive than magnetic resonance imaging (MRI); thus, repeated injections are generally required to obtain sufficient MR signals after nuclear imaging. Here, we show that strategically engineered magnetoferritin nanoprobes can image tumors with high sensitivity and specificity using SPECT and MRI in living mice after a single intravenous injection. The magnetoferritin nanoprobes composed of (125)I radionuclide-conjugated human H-ferritin iron nanocages ((125)I-M-HFn) internalize robustly into cancer cells via a novel tumor-specific HFn-TfR1 pathway. In particular, the endocytic recycling characteristic of TfR1 transporters solves the nuclear signal blocking issue caused by the high dose nanoprobes injected for MRI, thus enabling simultaneous functional and morphological tumor imaging without reliance on multi-injections.

We conducted real-time bioimaging of the hyaluronate–interferon α (HA–IFNα) conjugate using a biologically inert zwitterionic fluorophore of ZW800-1 for the treatment of hepatitis C virus (HCV) infection. ZW800-1 was labeled on the IFNα molecule of the HA–IFNα conjugate to investigate its biodistribution and clearance without altering its physicochemical and targeting characteristics. Confocal microscopy clearly visualized the effective in vitro cellular uptake of the HA–IFNα conjugate to HepG2 cells. After verifying the biological activity in Daudi cells, we conducted the pharmacokinetic analysis of the HA–IFNα conjugate, which confirmed its target-specific delivery to the liver with a prolonged residence time longer than that of PEGylated IFNα. In vivo and ex vivo bioimaging of the ZW800-1-labeled HA–IFNα conjugate directly showed real-time biodistribution and clearance of the conjugate that are consistent with the biological behaviors analyzed by an enzyme-linked immunosorbent assay. Furthermore, the elevated level of OAS1 mRNA in the liver confirmed in vivo antiviral activity of HA–IFNα conjugates. With the data taken together, we could confirm the feasibility of ZW800-1 as a biologically inert fluorophore and target-specific HA–IFNα conjugate for the treatment of HCV infection.

One-bead-one-compound (OBOC) solid-phase combinatorial chemistry has been used extensively in drug discovery. However, a major bottleneck has been the sorting of individual beads, while still swollen in organic solvent, into individual wells of a microwell plate. To solve this problem, we have constructed an automated bead sorting system with integrated quality control that is capable of sorting and placing large numbers of beads in bulk to single wells of a 384-well plate, all in an organic solvent. The bead sorter employs a unique, reciprocating fluidic design capable of depositing 1 bead every 1.5 s, with an average accuracy of 97%. We quantified the performance of this instrument by sorting over 8500 beads, followed by cleaving the conjugated compound and confirming the chemical identity of each by liquid chromatography/mass spectrometry (LC/MS). This instrument should enable more efficient screening of combinatorial small molecule libraries without the need to dry beads or otherwise change the chemical environment.

Functional polymeric films with antireflective and hydrophobic properties have been widely used for electronic device displays. However, the design of such functional films with an antimicrobial characteristic has been a challenge. We designed a nanostructured surface using a rigorous coupled-wave analysis to obtain a period of 300 nm and an aspect ratio of 3.0 on a flat poly(methyl methacrylate) film. The fabricated nanostructure was hydrophobic and exhibited the desired optical characteristics with a reflectance of less than 0.5% over the visible wavelength range. Furthermore, the nanoimprinted polymer film exhibited antimicrobial characteristics and low adhesion when compared with the corresponding flat surface. The results suggest that the nanostructured surface designed in this study is multifunctional and should be suitable for the production of protective optical and hygienic polymer films for the displays of portable electronic devices.

The emergence of photoluminescent carbon-based nanomaterials has shown exciting potential in the development of benign nanoprobes. However, the in vivo kinetic behaviors of these particles that are necessary for clinical translation are poorly understood to date. In this study, fluorescent carbon dots (C-dots) were synthesized and the effect of three injection routes on their fate in vivo was explored by using both near-infrared fluorescence (NIRF) and positron emission tomography (PET) imaging techniques. We found that C-dots are efficiently and rapidly excreted from the body after all three injection routes. The clearance rate of C-dots is ranked as: intravenous > intramuscular > subcutaneous. The particles had relatively low retention in the reticuloendothelial system (RES) and showed high tumor-to-background contrast. Furthermore, different injection routes also resulted in different blood clearance patterns and tumor uptakes of C-dots. These results satisfy the need for clinical translation and should promote efforts to further investigate the possibility of using carbon-based nanoprobes in a clinical setting. More broadly, we provide a testing blueprint for in vivo behavior of nanoplatforms under various injection routes, an important step forward towards safety and efficacy analysis of nanoparticles.

Encoderless combinatorial chemistry requires high-throughput product identification without the use of chemical or other tags. We developed a novel nano-layered substrate plate and combined it with a microarraying robot, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, and custom software to produce a high-throughput small molecule identification system. To optimize system performance, we spotted 5 different chemical entities, spanning a m/z range of 195 to 1338, in 20,304 spots for a total of 101,520 molecules. The initial spot identification rate was 99.85% (20,273 spots), and after a proofreading algorithm was added, 100% of 20,304 spots and 101,520 molecules were identified. An internal recalibration algorithm also significantly improved mass accuracy to as low as 45 ppm. Using this optimized system, 47 different chemical entities, spanning a m/z range of 138 to 1,592, were spotted over 5,076 spots and could be identified with 100% accuracy. Our study lays the foundation for improved encoderless combinatorial chemistry.

The synthesis, characterization, and degradation kinetics of three alpha-cyclodextrin (alpha-CD)-poly(ethylene glycol) (PEG) polyrotaxanes with endcaps that were installed using Cu(I)-catalyzed Huisgen cyclization is reported. PEG1500, azidated with azidoacetic acid, was threaded with alpha-CD to form a pseudopolyrotaxane that was then capped in up to 82% yield with three different substituents to provide polyrotaxanes that were either acid-, base-, or fluoride-sensitive. NMR, GPC, XRD, and AFM methods were used to characterize the polyrotaxanes. Dethreading rates upon exposure to mild deprotection conditions were monitored by turbidity analysis. The vinyl ether-endcapped polyrotaxane is stable at pH 7 for 16 h but is solubilized at approximately 0.0211 min(-1) at pH 4. The ester-endcapped polyrotaxane is solubilized at 0.0122 min(-1) at pH 12.1. Our results show that pH-triggerable polyrotaxanes can be readily and efficiently prepared from pseudopolyrotaxanes in high yield by Huisgen cyclization of azido- and alkynyl-modified precursors in the presence of Cu(I).

Supramolecular assembling systems governed by intermolecular cooperative host−guest complexation and ionic interactions were investigated based on β-cyclodextrin-conjugated poly(e-lysine) as a supr...

An ABA triblock copolymer, consisting of linear polyethylenimine (PEI) and poly(ethylene glycol) (PEG) (PEI-b-PEG-b-PEI), was examined for pH-dependent polypseudorotaxane formation with α-cyclodext...

A series of linear polyethylenimines (LPEIs) with various molecular weights were synthesized and used for pH-dependent polypseudorotaxane formation with α- or γ-cyclodextrins (α-, γ-CDs). The polypseudorotaxane formation was significantly dependent on the pH of the aqueous media. The maximum yield of the recovered polypseudorotaxane precipitates was observed at pH 11.0, whereas no complexation was observed in the pH range below 8.0 due to the protonation of secondary amine groups in LPEI backbones. This suggests that the ionization of the secondary amine groups leads to dethreading of CD molecules because PEI chains with cationic nature disfavor the hydrophobic cavities of CDs. The solid-state 13C CP/MAS NMR and X-ray diffraction measurements verified the successful formation of crystalline polypseudorotaxanes through the inclusion complexation. In addition, 1H NMR analysis showed that LPEI formed a 2:1 inclusion complex with α-CD ([EI]:[α-CD] = 2:1) and a 4:1 complex with γ-CD ([EI]:[γ-CD] = 4:1) as repo...

Various types of cyclodextrin-conjugated poly(e-lysine)s (CDPLs) were prepared as polymeric hosts, and the inclusion properties with 6-(p-toluidino)-2-naphthalenesulfonate (TNS) were investigated at a low concentration under various environmental stimuli. Fluorescence studies revealed that the ability of CDPLs for inclusion complexation was much stronger than that of the corresponding CDs due to their cooperative binding. This property can be controlled by the chemical composition of polymeric hosts such as changing the ring size and varying the degree of substitution of CDs. In addition, the inclusion property of the polymeric host can be modulated by environmental stimuli such as temperature, pH, and ionic strength of aqueous media. Such an interesting functionality results from the combination of the cooperative host−guest interactions and the ionic interaction between the negatively charged TNS moieties and the positive amino groups of poly(e-lysine).

A variation of A−B−A-type triblock copolymers consisting of poly(l-lactide) (PLLA) and poly(ethylene glycol) (PEG) was synthesized and examined for complexation with α-cyclodextrins (α-CDs). Although the PLLA block has bulky methyl groups as side chains, stable polypseudorotaxanes of PLLA−PEG−PLLA triblock copolymers as well as PLLA were obtained and confirmed by 1H NMR, solid-state 13C CP/MAS NMR, FT-IR, and X-ray spectroscopies. From the results, it was hypothesized that the guest molecules threaded into the hydrophobic CD cavities, and they form stable pseudorotaxanes in both PEG and PLLA blocks. The α-CDs slide over the flanking bulky PLLA blocks to form an inclusion complex with PEG block; in addition, they form very stick pseudorotaxanes with the end-blocks of PLLA parts. The copolymers confined to the CD channels lost their original crystalline properties but formed a channel-type hydrophobic crystalline structure with CDs due to long chain nature of the copolymers. Such a polymeric inclusion compl...