Mapping fluorescence resonance energy transfer parameters of a bifunctional agent using time-domain fluorescence diffuse optical tomography

We present a method to map fluorescence resonance energy transfer (FRET) parameters of a bifunctional photodynamic therapy agent, (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a)-cyanine dye (HPPH-CD) conjugate, which consists of a photosensitizer (HPPH) and a fluorescent agent CD. We utilized time-domain fluorescence diffuse optical tomography, the normalized Born ratio model in the Fourier-domain, and an iterative algorithm to map depth-resolved spatial heterogeneities of FRET parameters. Our results exhibited depth-resolved changes of fluorophore's lifetime and the distance maps due to FRET between HPPH and CD. Our model suggests a potential approach of using FRET parameters to monitor efficacies of multifunctional photodynamic therapy agents in deep tissue.     

Chlorophyll-a analogues conjugated with aminobenzyl-DTPA as potential bifunctional agents for magnetic resonance imaging and photodynamic therapy

A clinically relevant photosensitizer, 3-devinyl-3-(1-hexyloxyethyl)pyropheophorbide-a (HPPH, a chlorophyll-a derivative), was conjugated with Gd(III)-aminobenzyl-diethylenetriaminepentaacetic acid (DTPA), an experimental magnetic resonance (MR) imaging agent. In vivo reflectance spectroscopy confirmed tumor uptake of HPPH-aminobenzyl-Gd(III)-DTPA conjugate was higher than free HPPH administered intraveneously (iv) to C3H mice with subcutaneously (sc) implanted radiation-induced fibrosarcoma (RIF) tumor cells. In other experiments, Sprague-Dawley (SD) rats with sc implanted Ward Colon Carcinoma cells yielded markedly increased MR signal intensities from tumor regions-of-interest (ROIs) 24 h post-iv injection of HPPH-aminobenzyl-Gd(III)-DTPA conjugate as compared to unconjugated HPPH. In both in vitro (RIF tumor cells) and in vivo (mice bearing RIF tumors and rats bearing Ward Colon tumors) the conjugate produced significant increases in tumor conspicuity at 1.5 T and retained therapeutic efficacy following PDT. Also synthesized were a series of novel bifunctional agents containing two Gd(III) atoms per HPPH molecule that remained tumor-avid and PDT-active and yielded improved MR tumor conspicuity compared to their corresponding mono-Gd(III) analogues. Administered iv at a MR imaging dose of 10 micromol/kg, these conjugates produced severe skin phototoxicity. However, by replacing the hexyl group of the pyropheophorbide-a with a tri(ethylene glycol) monomethyl ether (PEG-methyl ether), these conjugates produced remarkable MR tumor enhancement at 8 h post-iv injection, significant tumoricidal activity (80% of mice were tumor-free on day 90), and reduced skin phototoxicity compared to their corresponding hexyl ether analogues. The poor water-solubility characteristic of these conjugates was resolved by incorporation into a liposomal formulation. This paper presents the synthesis of tumor-avid contrast enhancing agents for MR imaging and thus represents an important milestone toward improving cancer diagnosis and tumor characterization. More importantly, this paper describes a new family of bifunctional agents that combine two modalities into a single cost-effective "see and treat" approach, namely, a single agent that can be used for contrast agent-enhanced MR imaging followed by targeted photodynamic therapy.     

Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands

We report here the in vivo diagnostic use of a peptide-dye conjugate consisting of a cyanine dye and the somatostatin analog octreotate as a contrast agent for optical tumor imaging. When used in whole-body in vivo imaging of mouse xenografts, indotricarbocyanine-octreotate accumulated in tumor tissue. Tumor fluorescence rapidly increased and was more than threefold higher than that of normal tissue from 3 to 24 h after application. The targeting conjugate was also specifically internalized by primary human neuroendocrine tumor cells. This imaging approach, combining the specificity of ligand/receptor interaction with near-infrared fluorescence detection, may be applied in various other fields of cancer diagnosis.     

Synthesis, photophysical, electrochemical, tumor-imaging, and phototherapeutic properties of purpurinimide-N-substituted cyanine dyes joined with variable lengths of linkers

Purpurinimide methyl esters, bearing variable lengths of N-substitutions, were conjugated individually to a cyanine dye with a carboxylic acid functionality. The results obtained from in vitro and in vivo studies showed a significant impact of the linkers joining the phototherapeutic and fluorescence imaging moieties. The photosensitizer-fluorophore conjugate with a PEG linker showed the highest uptake in the liver, whereas the conjugate linked with two carbon units showed excellent tumor-imaging and PDT efficacy at 24 h postinjection. Whole body imaging and biodistribution studies at variable time points portrayed enhanced fluorescent uptake of the conjugates in the tumor compared to that in the skin. Interestingly, the conjugate with the shortest linker and the one joining with two carbon units showed faster clearance from normal organs, e.g., the liver, kidney, spleen, and lung, compared to that in tumors. Both imaging and PDT efficacy of the conjugates were performed in BALB/c mice bearing Colon26 tumors. Compared to the others, the short linker conjugate showed poor tumor fluorescent properties and as a corollary does not exhibit the dual functionality of the photosensitizer-fluorophore conjugate. For this reason, it was not evaluated for in vivo PDT efficacy. However, in Colon26 tumor cells (in vitro), the short linker was highly effective. Among the conjugates with variable linkers, the rate of energy transfer from the purpurinimide moiety to the cyanine moiety increased with deceasing linker length, as examined by femtosecond laser flash photolysis measurements. No electron transfer from the purpurinimide moiety to the singlet excited state of the cyanine moiety or from the singlet excited state of the cyanine moiety to the purpurinimide moiety occurred as indicated by a comparison of transient absorption spectra with spectra of the one-electron oxidized and one-electron reduced species of the conjugate obtained by spectroelectrochemical measurements.     

Synthesis, characterization, and biological properties of cyanine-labeled somatostatin analogues as receptor-targeted fluorescent probes

We present the synthesis and characterization of the somatostatin receptor-specific peptide H(2)N-(D-Phe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, which is labeled with a carboxylated indodicarbo- and an indotricarbocyanine dye at the N-terminal amino group. The preparation was performed by automated solid-phase synthesis, with subsequent attachment of the cyanine dye and cleavage of the entire conjugate from the resin. The compounds display high molar absorbance and fluorescence quantum yields typical for cyanine dyes and are thus suitable receptor-targeted contrast agents for molecular optical imaging. The ability of these agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, in which the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. In contrast, the corresponding linearized derivative of the sequence H(2)N-(D-Phe)-Met-Phe-(D-Trp)-Lys-Thr-Met-Thr-OH produced only minimal cell fluorescence, hence confirming the specificity of the cyclic somatostatin analogue. Intracellular localization could be visualized by near-infrared (NIR) fluorescence microscopy. In conclusion, receptor-specific peptides are promising tools for designing site-directed optical contrast agents for use in molecular optical imaging.     

七甲川花菁染料作为肿瘤靶向和光动力治疗载体的研究进展

七甲川花菁近红外荧光染料(NIRF)可直接被肿瘤细胞特异性吸收,具有肿瘤靶向性。与化疗药物偶联后,该类染料可通过血脑屏障将药物转运至肿瘤部位,不仅可以减少化疗药物使用剂量,降低药物的毒副作用,也可通过近红外荧光成像实现对肿瘤治疗的实时监控。七甲川花菁染料所展示的线粒体毒性和光敏特性,可直接杀死肿瘤细胞,抑制肿瘤新生血管的形成。通过纳米包裹,能够显著增强该类染料的肿瘤靶向能力,实现实时跟踪药物释放情况。七甲川花菁染料特异性识别肿瘤细胞的能力与有机阴离子转运肽的作用密切相关,缺氧和线粒体膜电位也参与了染料吸收的调控。这些发现有利于将近红外荧光染料应用于肿瘤的靶向治疗。     

近红外荧光染料IR-783介导的肿瘤成像

目的评估近红外荧光染料IR-783在犬自发性肿瘤中的特异性成像。方法将IR-783染料(5μmol/kg)腹腔注射荷瘤裸鼠,通过活体成像仪检测IR-783的代谢周期,在此基础上,将IR-783染料(1.5μmol/kg)通过后肢静脉注射入自发肿瘤犬体内,5 d后手术切除肿瘤组织,分别进行荧光成像、组织切片HE染色、冰冻切片荧光观察。结果 IR-783染料注射荷瘤裸鼠后可以在肿瘤部位检测到特异性荧光,连续观察8 d,仍有较强的荧光。IR-783注射自发肿瘤犬5 d后,可在肿瘤组织中检测到特异性荧光。结论近红外荧光染料IR-783能够被肿瘤组织特异性吸收,可用于犬自发性肿瘤的特异性诊断,具有重要的临床应用前景。     

A mitochondria-selective near-infrared-emitting fluorescent dye for cellular imaging studies

This communication details the synthesis, evaluation of photophysical properties, and cellular imaging studies of cyanine chromophore based fluorescent dye 1 as a selective imaging agent for mitochondria.     

Near-infrared pH-activatable fluorescent probes for imaging primary and metastatic breast tumors

Highly tumor selective near-infrared (NIR) pH-activatable probe was developed by conjugating pH-sensitive cyanine dye to a cyclic arginine-glycine-aspartic acid (cRGD) peptide targeting α(v)β(3) integrin (ABIR), a protein that is highly overexpressed in endothelial cells during tumor angiogenesis. The NIR pH-sensitive dye used to construct the probe exhibits high spectral sensitivity with pH changes. It has negligible fluorescence above pH 6 but becomes highly fluorescent below pH 5, with a pK(a) of 4.7. This probe is ideal for imaging acidic cell organelles such as tumor lysosomes or late endosomes. Cell microscopy data demonstrate that binding of the cRGD probe to ABIR facilitated the endocytosis-mediated lysosomal accumulation and subsequent fluorescence enhancement of the NIR pH-activatable dye in tumor cells (MDA-MB-435 and 4T1/luc). A similar fluorescence enhancement mechanism was observed in vivo, where the tumors were evident within 4 h post injection. Moreover, lung metastases were also visualized in an orthotopic tumor mouse model using this probe, which was further confirmed by histologic analysis. These results demonstrate the potential of using the new integrin-targeted pH-sensitive probe for the detection of primary and metastatic cancer.     

Lipopolysaccharide neutralizing Peptide-porphyrin conjugates for effective photoinactivation and intracellular imaging of gram-negative bacteria strains

A simple and specific strategy based on the bioconjugation of a photosensitizer protophophyrin IX (PpIX) with a lipopolysaccharide (LPS) binding antimicrobial peptide YI13WF (YVLWKRKRKFCFI-Amide) has been developed for the effective fluorescent imaging and photodynamic inactivation of Gram-negative bacterial strains. The intracellular fluorescent imaging and photodynamic antimicrobial chemotherapy (PACT) studies supported our hypothesis that the PpIX-YI13WF conjugates could serve as efficient probes to image the bacterial strains and meanwhile indicated the potent activities against Gram-negative bacterial pathogens especially for those with antibiotics resistance when exposed to the white light irradiation. Compared to the monomeric PpIX-YI13WF conjugate, the dimeric conjugate indicated the stronger fluorescent imaging signals and higher photoinactivation toward the Gram-negative bacterial pathogens throughout the whole concentration range. In addition, the photodynamic bacterial inactivation also demonstrated more potent activity than the minimum inhibitory concentration (MIC) values of dimeric PpIX-YI13WF conjugate itself observed for E. coli DH5a (～4 times), S. enterica (～8 times), and other Gram-negative strains including antibiotic-resistant E. coli BL21 (～8 times) and K. pneumoniae (～16 times). Moreover, both fluorescent imaging and photoinactivation measurements also demonstrated that the dimeric PpIX-YI13WF conjugate could selectively recognize bacterial strains over mammalian cells and generate less photo damage to mammalian cells. We believed that the enhanced fluorescence and bacterial inactivation were probably attributed to the higher binding affinity between dimeric photosensitizer peptide conjugate and LPS components on the surface of bacterial strains, which were the results of efficient multivalent interactions.     

Targeted photodynamic therapy agent with a built-in apoptosis sensor for in vivo near-infrared imaging of tumor apoptosis triggered by its photosensitization in situ

Imaging apoptotic cells or tissues after cancer therapy in situ would be a very useful tool for assessing proper treatment conditions and therapeutic outcome. By combining therapeutic and imaging functions, we have designed a multifunctional, membrane-permeable, and cancer-specific agent that triggers and images apoptosis in targeted cells. We chose photodynamic therapy (PDT) as an appropriate cancer treatment modality and caspase 3 as an apoptosis-specific imaging target. This targeted photodynamic therapy agent with a built-in apoptosis sensor (TaBIAS) induces photodamage only to target cells and simultaneously identifies those that are apoptotic by its near-infrared fluorescence. It contains a fluorescent photosensitizer used as an anticancer drug and a cancer-associated folate receptor homing molecule connected to a caspase 3 cleavable peptide linker that has a fluorescence quencher on the opposing site. We demonstrated that PDT-triggered cleavage of the peptide linker by caspase 3, one of the key executioner caspases, results in a detectable increase in fluorescence in folate receptor-overexpressing cancer cells and tumors. The presence of apoptosis was confirmed in vitro by flow cytometry and ex vivo by Apoptag assay, supporting the ability of TaBIAS to specifically induce and image apoptosis in situ.     

A new optical and nuclear dual-labeled imaging agent targeting interleukin 11 receptor alpha-chain

Optical imaging has great potential for studying molecular recognitions both in vivo and in vitro, yet nuclear imaging is the most effective clinical molecular imaging modality. The combination of optical and nuclear imaging modalities may provide complementary information for improving diagnosis and management of diseases. In this study we developed an optical and nuclear dual-labeled imaging agent, 111In-DTPA-Bz-NH-SA-K(IR-783-S-Ph-CO)-c(CGRRAGGSC)NH2, called DLIA-IL11Ralpha. 111In-DTPA-Bz-NH-SA is the radiotracer moiety; a near-infrared dye IR-783-S-Ph-COOH serves as the fluorescent emitter; and the cyclic peptide c(CGRRAGGSC), which is known to target interleukin 11 receptor alpha-chain (IL-11Ralpha), delivers the desired imaging agent to its target. Experiments revealed that the cyclic peptide c(CGRRAGGSC) continued to possess the targeting capability to IL-11Ralpha after the conjugation of the optical and nuclear tracers. Furthermore, the presence of the metal isotope chelator did not cause quenching of fluorescence emission. The cross validation and direct comparison of optical and nuclear imaging of a tumor was achieved using a single injection, and the preliminary results show the conjugate has tumor targeting capabilities in vivo.     

Pyropheophorbide 2-deoxyglucosamide: a new photosensitizer targeting glucose transporters

To prepare near-infrared fluorescence imaging and photodynamic therapy agents targeted at glucose transporters, pyropheophorbide 2-deoxyglucosamide (Pyro-2DG) was synthesized and evaluated in a 9L glioma rat model. Fluorescence imaging studies demonstrate that Pyro-2DG is selectively accumulated in the tumor. Upon its photoactivation, we demonstrate that this agent efficiently causes selective mitochondrial damage to the region of a tumor that was photoirradiated after administration of this agent, but does not affect tissues photoirradiated in the absence of the agent or tissues treated with the agent that are not photoirradiated. Preliminary confocal microscopy studies suggest that Pyro-2DG is delivered and trapped in tumor cells via the GLUT/hexokinase pathway and therefore is useful both as a tumor-targeted NIR fluorescence imaging probe and as a PDT agent for the destruction of cancer.     

Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer

ABSTRACT: BACKGROUND: The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. METHODS: The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA) in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR). Tumor-targeting ligands such as peanut agglutinin (PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. RESULTS AND DISCUSSION: Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. CONCLUSIONS: These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA nanoparticles, in order to use them for both detection as well as therapy of colon cancer and others.     

Conjugation of a water-soluble gadolinium endohedral fulleride with an antibody as a magnetic resonance imaging contrast agent

Water-soluble gadofullerides exhibited high efficiency as magnetic resonance imaging (MRI) contrast agents. In this paper, we report the conjugation of the newly synthesized gadofulleride, Gd@C82O6(OH) 16(-)(NHCH2CH2COOH)8, with the antibody of green fluorescence protein (anti-GFP), as a model for "tumor targeted" imaging agents based on endohedral metallofullerenes. In this model system, the activity of the anti-GFP conjugate can be conveniently detected by green fluorescence protein (GFP), leading to in vitro experiments more direct and facile than those of tumor antibodies. Objective-type total internal reflection fluorescence microscopy revealed that each gadofulleride aggregate conjugated on average five anti-GFPs, and the activity of anti-GFPs was preserved after conjugation. In addition, the gadofulleride/antibody conjugate exhibited higher water proton relaxivity (12.0 mM (-1) s (-1)) than the parent gadofulleride aggregate (8.1 mM (-1) s (-1)) in phosphate buffered saline at 0.35 T, as also confirmed by T1-weighted images of phantoms. These observations clearly indicate that the synthesized gadofulleride/antibody conjugate not only has targeting potential, but also exhibits higher efficiency as an MRI contrast agent.     

Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag

Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAP(f) is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAP(f). This property makes SNAP(f) a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAP(f)-Beta-2 adrenergic receptor (SNAP(f)-ADRβ2) fusion protein were created. The ADRβ2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAP(f)-ADRβ2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAP(f)-ADRβ2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAP(f)-ADRβ2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAP(f)-ADRβ2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.     

Comparative study of the optical and heat generation properties of IR820 and indocyanine green

AbstractNear-infrared (NIR) fluorophores are the focus of extensive research for combined molecular imaging and hyperthermia. In this study, we showed that the cyanine dye IR820 has optical and thermal generation properties similar to those of indocyanine green (ICG) but with improved in vitro and in vivo stability. The fluorescent emission of IR820 has a lower quantum yield than ICG but less dependence of the emission peak location on concentration. IR820 demonstrated degradation half-times approximately double those of ICG under all temperature and light conditions in aqueous solution. In hyperthermia applications, IR820 generated lower peak temperatures than ICG (4-9%) after 3-minute laser exposure. However, there was no significant difference in hyperthermia cytotoxicity, with both dyes causing significant cell growth inhibition at concentrations ≥ 5 μM. Fluorescent images of cells with 10 μM IR820 were similar to ICG images. In rats, IR820 resulted in a significantly more intense fluorescence signal and significantly higher organ dye content than for ICG 24 hours after intravenous dye administration (p < .05). Our study shows that IR820 is a feasible agent in experimental models of imaging and hyperthermia and could be an alternative to ICG when greater stability, longer image collection times, or more predictable peak locations are desirable.     

Quantitative imaging of lymph function

Functional lymphatic imaging was demonstrated in the abdomen and anterior hindlimb of anesthetized, intact Yorkshire swine by using near-infrared (NIR) fluorescence imaging following intradermal administration of 100-200 microl of 32 microM indocyanine green (ICG) and 64 microM hyaluronan NIR imaging conjugate to target the lymph vascular endothelial receptor (LYVE)-1 on the lymph endothelium. NIR fluorescence imaging employed illumination of 780 nm excitation light ( approximately 2 mW/cm(2)) and collection of 830 nm fluorescence generated from the imaging agents. Our results show the ability to image the immediate trafficking of ICG from the plexus, through the vessels and lymphangions, and to the superficial mammary, subiliac, and middle iliac lymph nodes, which were located as deep as 3 cm beneath the tissue surface. "Packets" of ICG-transited lymph vessels of 2-16 cm length propelled at frequencies of 0.5-3.3 pulses/min and velocities of 0.23-0.75 cm/s. Lymph propulsion was independent of respiration rate. In the case of the hyaluronan imaging agent, lymph propulsion was absent as the dye progressed immediately through the plexus and stained the lymph vessels and nodes. Lymph imaging required 5.0 and 11.9 microg of ICG and hyaluronan conjugate, respectively. Our results suggest that microgram quantities of NIR optical imaging agents and their conjugates have a potential to image lymph function in patients suffering from lymph-related disorders.     

Targeted cowpea chlorotic mottle virus-based nanoparticles with tumor-homing peptide F3 for photothermal therapy

Our aim was to devise targeted drug delivery systems using genetically modified cowpea chlorotic mottle virus (CCMV) capsids by fusion expression with tumorhoming peptide F3 for efficient delivery of therapeutic substances into tumor cells. The RNA-binding domain at the N terminus (amino acid residues 1–25) of CCMV capsid protein (CP) was selectively deleted, and F3 was inserted for the expression in Pichia pastoris. After chromatographic purification, F3-CCMV capsids were obtained via selfassembly of the F3-CP fusion protein and then analyzed by transmission electron microscopy and dynamic light scattering analysis, which revealed spherical nanoparticles (NPs) ca. 18 nm in diameter with regular monodispersity. Near-infrared fluorescent dye IR780 iodide, which has been applied for cancer imaging, photodynamic therapy, and photothermal therapy, was encapsulated in F3-CCMV NPs. The resultant F3-CCMV-IR780 NPs showed excellent molecular targeting to nucleolin receptor overexpressed on the surface of MCF-7 tumor cells. Furthermore, the in vitro cellular uptake and cell viability assay proved a photothermal effect by a single dose of near-infrared laser irradiation. The present system may offer a programmable nanoscaffoldbased drug delivery system vehicle for fabrication of promising therapeutic substances for cancer therapy.