Development of light and pH-dual responsive self-quenching theranostic SPION to make EGFR overexpressing micro tumors glow and destroy.

Drug resistant and undetectable tumors easily escape treatment leading metastases and/or recurrence of the lethal disease. Therefore, it is vital to diagnose and destroy micro tumors using simple yet novel approaches. Here, we present fluorescence-based detection and light-based destruction of cancer cells that are known to be resistant to standard therapies. We developed a superparamagnetic iron oxide nanoparticle (SPION)-based theranostic agent that is composed of self-quenching light activated photosensitizer (BPD) and EGFR targeting ligand (Anti-EGFR ScFv or GE11 peptide). Photosensitizer (BPD) was immobilized to PEG-PEI modified SPION with acid-labile linker. Prior to stimulation of the theranostic system by light its accumulation within cancer cells is vital since BPD phototoxicity and fluorescence is activated by lysosomal proteolysis. As BPD is cleaved, the system switches from off to on position which triggers imaging and therapy. Targeting, therapeutic and diagnostic features of the theranostic system were evaluated in high and moderate level EGFR expressing pancreatic cancer cell lines. Our results indicate that the system distinguishes high and moderate EGFR expression levels and yields up to 4.3-fold increase in intracellular fluorescence intensity. Amplification of fluorescence signal was as low as 1.3-fold in the moderate or no EGFR expressing cell lines. Anti-EGFR ScFv targeted SPION caused nearly 2-fold higher cell death via apoptosis in high EGFR expressing Panc-1 cell line. The developed system, possessing advanced targeting, enhanced imaging and effective therapeutic features, is a promising candidate for multi-mode detection and destruction of residual drug-resistant cancer cells.

Stimuli-responsive theranostic system: A promising approach for augmented multimodal imaging and efficient drug release.

Destruction of drug resistant and invisible micro-tumors requires innovative screening and treatment modalities. Theranostic nanosystems offering multimodal imaging and therapy are attractive platforms with potential to make micro-tumors visible to clinicians. Gold nanoparticles (AuNPs) are intrinsic theranostic agents and act as fluorescence quenchers. They can be easily transformed to multimodal imaging and combination therapy agents by combining them with various adjuvant therapies such as photodynamic therapy. In this study, we developed a highly specific, hybrid theranostic agent that is only activated when it meets with its stimuli at the site of interest. Surface-coated AuNPs were modified with Cathepsin B cleavable peptide (stimuli responsive linker) and Verteporfin (photosensitizer and fluorescence imaging agent). Unless the theranostic system meets with the internal stimuli in tumor cells, fluorescence is quenched due to AuNP-Verteporfin and Verteporfin-Verteporfin interactions. Following cellular internalization of the theranostic agent, fluorescence is gained by Cathepsin B cleavage and phototoxicity is initiated by light. The system was efficiently internalized by SKOV-3 cells and demonstrated high specificity towards its stimuli. In comparison to Verteporfin, ∼14-fold fluorescence increase, 81% fluorescence recovery and comparable toxicity were achieved. The system is a promising candidate for multimodal imaging and dual treatment to destroy the micro-tumors.

A Rational design of multi-functional nanoplatform: Fluorescent-based "off-on" theranostic gold nanoparticles modified with D-α-Tocopherol succinate.

It is crucial to develop nanocarrier systems to detect and treat drug-resistant micro tumors to prevent recurrence and/or metastasis of cancer. Due to their exceptional features such as biocompatibility, easy surface modification, serving as imaging and therapeutic agent, gold nanoparticles (AuNPs) draw attention as theranostic agents. It is beneficial to combine AuNPs with a second imaging and/or treatment modality such as photodynamic therapy (PDT). PDT is a non-mutagenic treatment approach in which photosensitizer is activated with light, generating reactive oxygen species and/or free radicals to destroy tumor cells. With the aim of developing "off-on" theranostic system, citrate stabilized spherical 13 nm AuNPs were densely coated with polyethylene glycol (PEG). To advance the theranostic feature of PEGylated AuNPs, they were further functionalized with FDA-Approved photosensitizer, Verteporfin (BPD-MA). Due to static quenching between BPD-MA and AuNPs as well as in between nearby BPD-MA molecules, the fluorescence of the ground state complex is quenched and the system is in "off" state. When BPD-MA molecules are cleaved from the AuNPs surface and diffuse away, fluorescence is recovered. Consequently, the system switches to the "on" state. Among the various mole ratios of BPD-MA carrying conjugates prepared, the most promising candidate was selected based on stability, quenching factor, and fluorescence recovery rate. The conjugate was further decorated with D-α-Tocopherol succinate (VitES) to increase the therapeutic efficacy of the theranostic agent via enhancing cellular uptake. Our results showed that it was possible to achieve as high as 80 times fluorescence quenching when the system was "off". As the system switched from "off" to "on" state, 51% of the fluorescence was recovered. When BPD-MA was immobilized on the PEGylated AuNPs, the phototoxic effect of BPD-MA increased twice against the MCF-7 cell line. Moreover, the developed system showed four times more phototoxicity than BPD-MA alone after it was decorated with VitES. Since the developed system is capable of dual imaging (computed tomography and fluorescence) and dual treatment (PDT and hyperthermia), it potentially offers superior imaging and therapy options for various types of in vitro/in vivo applications.

Design of Dense Brush Conformation Bearing Gold Nanoparticles as Theranostic Agent for Cancer.

Dense brush conformation-bearing theranostic agents are emerging as drug delivery systems due to their higher ability to escape from reticuloendothelial system uptake which prolongs their in vivo circulation time. With the aim of developing dual therapy agent, 13-nm gold nanoparticles' (AuNPs) surfaces were coated with different amounts of polyethylene glycol (PEG) (SH-PEG-NH2) to obtain dense brush conformation-bearing theranostic agents. Among the 14 different theranostic agent candidates prepared, the one hosting 1819 PEG per particle was selected as the most promising theranostic agent candidate based on structural conformation, stability, size, zeta potential, hemocompatibility, cell inhibition, and cell death pathway towards MCF-7 cell line. To test drug delivery efficiency of the developed PEGylated AuNP and to improve efficacy of the treatment, apoptotic peptide (AP) was covalently conjugated to NH2 terminus of the PEG in various ratios to yield AuNP-AP conjugate. Among the prepared conjugates, the one having 1 nmol of peptide per milliliter of AuNP yielded the most promising results based on the same criteria as employed for PEGylated AuNPs. Besides, incorporation of AP to AuNP returned in superior efficacy of AP since it was possible to achieve 50% cell death with 1000 times less amount of AP alone.

Verteporfin mediated sequence dependent combination therapy against ovarian cancer cell line.

Ovarian Cancer is one of the deadliest gynecological cancer showing high resistance to chemotherapy. Non-overlapping and synergistic combination therapies are the best option to overcome this multi-pathological silent disease. Cationic peptides (CPs) with high targeting feature and ability to pass through cell membrane induce apoptosis via disruption of cancer cell membrane. Photodynamic Therapy (PDT) is a noninvasive clinically approved treatment modality combining light activated photosensitizer, light and oxygen. In this study we present, combination therapy composed of 9-mer +4 charge bearing CP and Benzoporphyrin derivative monoacid, (BPD-MA, Verteporfin) mediated PDT. In order to evaluate the effect of sequence on the outcome of the therapy, CP and BPD-MA mediated PDT was applied in two different sequence: 'CP first' 'BPD-MA first'. Treatment efficacy of combination therapy in SKOV-3 ovarian cancer cell line has been evaluated based on cell inhibition, cell death pathway, Combination index (CI), and Dose Reduction Index (DRI) values. When SKOV-3 ovarian cancer cell line treated with BPD-MA mediated PDT (5 J/cm2) and CP individually, IC30 values for each drug were determined as 1.1 µM and 240 µM respectively and apoptosis was the major death cell pathway for both of the drugs. In the case of combination therapy, SKOV-3 cell line treated with drugs in constant ratio yet on different sequence. Drugs were used in constant ratio so that one of them would not de-emphasize the effect of other in any concentration point. Our theoretical and experimental results were in agreement and showed that the treatment outcome significantly depends on the order of the treatment. For instance, while BPD-MA mediated PDT was applied prior to CP, cell inhibition at IC30 value of BPD-MA was roughly 28% with CI =3.3 suggesting antagonistic interaction between each therapy. When the sequence of treatment was changed to CP first, cell inhibition at IC30 concentration of CP was determined as 98% with CI = 0.3 creating substantial synergism between the drugs. Moreover, synergistic interactions were observed at all concentration points at CP first scenario. DRI value for CP first treatment option was much higher compared to BPD-MA first treatment making the former treatment sequence more attractive option for clinically relevant combination therapies. Based on our results, we strongly believe that 9-mer CP and BPD-MA-PDT based combination therapy, offering synergistic therapeutic outcome, may increase chances of treatment of ovarian cancer in comparison to 9-mer CP and/or BPD-MA alone case.

Intravascular fibrin molecular imaging improves the detection of unhealed stents assessed by optical coherence tomography in vivo.

AIMS: Fibrin deposition and absent endothelium characterize unhealed stents that are at heightened risk of stent thrombosis. Optical coherence tomography (OCT) is increasingly used for assessing stent tissue coverage as a measure of healed stents, but cannot precisely identify whether overlying tissue represents physiological neointima. Here we assessed and compared fibrin deposition and persistence on bare metal stent (BMS) and drug-eluting stent (DES) using near-infrared fluorescence (NIRF) molecular imaging in vivo, in combination with simultaneous OCT stent coverage. METHODS AND RESULTS: Rabbits underwent implantation of one BMS and one DES without overlap in the infrarenal aorta (N = 20 3.5 × 12 mm). At Days 7 and/or 28, intravascular NIRF-OCT was performed following the injection of fibrin-targeted NIRF molecular imaging agent FTP11-CyAm7. Intravascular NIRF-OCT enabled high-resolution imaging of fibrin overlying stent struts in vivo, as validated by histopathology. Compared with BMS, DES showed greater fibrin deposition and fibrin persistence at Days 7 and 28 (P < 0.01 vs. BMS). Notably, for edge stent struts identified as covered by OCT on Day 7, 92.8 ± 9.5% of DES and 55.8 ± 23.6% of BMS struts were NIRF fibrin positive (P < 0.001). At Day 28, 18.6 ± 10.6% (DES) and 5.1 ± 8.7% (BMS) of OCT-covered struts remained fibrin positive (P < 0.001). CONCLUSION: Intravascular NIRF fibrin molecular imaging improves the detection of unhealed stents, using clinically translatable technology that complements OCT. A sizeable percentage of struts deemed covered by OCT are actually covered by fibrin, particularly in DES, and therefore such stents might remain prothrombotic. These findings have implications for the specificity of standalone clinical OCT assessments of stent healing.

Blood Accessibility to Fibrin in Venous Thrombosis is Thrombus Age-Dependent and Predicts Fibrinolytic Efficacy: An In Vivo Fibrin Molecular Imaging Study.

Fibrinolytic therapy of venous thromboembolism (VTE) is increasingly utilized, yet limited knowledge is available regarding in vivo mechanisms that govern fibrinolytic efficacy. In particular, it is unknown how age-dependent thrombus organization limits direct blood contact with fibrin, the target of blood-based fibrinolytic agents. Utilizing high-resolution in vivo optical molecular imaging with FTP11, a near-infrared fluorescence (NIRF) fibrin-specific reporter, here we investigated the in vivo interrelationships of blood accessibility to fibrin, thrombus age, thrombus neoendothelialization, and fibrinolysis in murine venous thrombosis (VT). In both stasis VT and non-stasis VT, NIRF microscopy showed that FTP11 fibrin binding was thrombus age-dependent. FTP11 localized to the luminal surface of early-stage VT, but only minimally to subacute VT (p<0.001). Transmission electron microscopy of early stage VT revealed direct blood cell contact with luminal fibrin-rich surfaces. In contrast, subacute VT exhibited an encasing CD31+ neoendothelial layer that limited blood cell contact with thrombus fibrin in both VT models. Next we developed a theranostic strategy to predict fibrinolytic efficacy based on the in vivo fibrin accessibility to blood NIRF signal. Mice with variably aged VT underwent FTP11 injection and intravital microscopy (IVM), followed by tissue plasminogen activator infusion to induce VT fibrinolysis. Fibrin molecular IVM revealed that early stage VT, but not subacute VT, bound FTP11 (p<0.05), and experienced higher rates of fibrinolysis and total fibrinolysis (p<0.05 vs. subacute VT). Before fibrinolysis, the baseline FTP11 NIRF signal predicted the net fibrinolysis at 60 minutes (p<0.001). Taken together, these data provide novel insights into the temporal evolution of VT and its susceptibility to therapeutic fibrinolysis. Fibrin molecular imaging may provide a theranostic strategy to identify venous thrombi amenable to fibrinolytic therapies.

Imaging Granzyme B Activity Assesses Immune-Mediated Myocarditis.

RATIONALE: The development of molecular imaging approaches that assess specific immunopathologic mechanisms can advance the study of myocarditis. OBJECTIVE: This study validates a novel molecular imaging tool that enables the in vivo visualization of granzyme B activity, a major effector of cytotoxic CD8+ T lymphocytes. METHODS AND RESULTS: We synthesized and optimized a fluorogenic substrate capable of reporting on granzyme B activity and examined its specificity ex vivo in mice hearts with experimental cytotoxic CD8+ T lymphocyte-mediated myocarditis using fluorescence reflectance imaging, validated by histological examination. In vivo experiments localized granzyme B activity in hearts with acute myocarditis monitored by fluorescent molecular tomography in conjunction with coregistered computed tomography imaging. A model anti-inflammatory intervention (dexamethasone administration) in vivo reduced granzyme B activity (vehicle versus dexamethasone: 504±263 versus 194±77 fluorescence intensities in hearts; P=0.002). CONCLUSIONS: Molecular imaging of granzyme B activity can visualize T cell-mediated myocardial injury and monitor the response to an anti-inflammatory intervention.

Rapid, low-cost fluorescent assay of ß-lactamase-derived antibiotic resistance and related antibiotic susceptibility.

Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of ß -lactamase resistance (the most common form of AR) that uses a modified ß -lactam structure decorated with two fluorophores quenched due to their close proximity. When the ß -lactam core is cleaved by ß -lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the ß -lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of ß -lactamase resistance, both for epidemiological purposes as well as individualized patient care.

Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates.

Drug-resistant micrometastases that escape standard therapies often go undetected until the emergence of lethal recurrent disease. Here, we show that it is possible to treat microscopic tumors selectively using an activatable immunoconjugate. The immunoconjugate is composed of self-quenching, near-infrared chromophores loaded onto a cancer cell-targeting antibody. Chromophore phototoxicity and fluorescence are activated by lysosomal proteolysis, and light, after cancer cell internalization, enabling tumor-confined photocytotoxicity and resolution of individual micrometastases. This unique approach not only introduces a therapeutic strategy to help destroy residual drug-resistant cells but also provides a sensitive imaging method to monitor micrometastatic disease in common sites of recurrence. Using fluorescence microendoscopy to monitor immunoconjugate activation and micrometastatic disease, we demonstrate these concepts of "tumor-targeted, activatable photoimmunotherapy" in a mouse model of peritoneal carcinomatosis. By introducing targeted activation to enhance tumor selectively in complex anatomical sites, this study offers prospects for catching early recurrent micrometastases and for treating occult disease.

Photodynamic therapy: one step ahead with self-assembled nanoparticles.

Photodynamic therapy (PDT) is a promising treatment modality for cancer with possible advantages over current treatment alternatives. It involves combination of light and a photosensitizer (PS), which is activated by absorption of specific wavelength light and creates local tissue damage through generation of reactive oxygen species (ROS) that induce a cascade of cellular and molecular events. However, as of today, PDT is still in need of improvement and nanotechnology may play a role. PDT frequently employs PS with molecular structures that are highly hydrophobic, water insoluble and prone to aggregation. Aggregation of PS leads to reduced ROS generation and thus lowers the PDT activity. Some PS such as 5-aminolevulinic acid (ALA) cannot penetrate through the stratum corneum of the skin and systemic administration is not an option due to frequently encountered side effects. Therefore PS are often encapsulated or conjugated in/on nano-drug delivery vehicles to allow them to be better taken up by cells and to more selectively deliver them to tumors or other target tissues. Several nano-drug delivery vehicles including liposomes, fullerosomes and nanocells have been tested and reviewed. Here we cover non-liposomal self-assembled nanoparticles consisting of polymeric micelles including block co-polymers, polymeric micelles, dendrimers and porphysomes.

Detection and treatment of intravascular thrombi with magnetofluorescent nanoparticles.

Thrombosis, the formation of a clot within a blood vessel, underlies a number of life-threatening cardiovascular disorders such as heart attack, ischemic stroke, pulmonary embolism, and deep vein thrombosis. These conditions affect the lives of millions of people worldwide and result in significant morbidity and mortality. It is thus crucial to develop novel methodologies to enhance the detection and treatment of these disorders. Thrombolysis, or the dissolution of blood clots, relies upon the administration of exogenous plasminogen activators (PAs) that lyse fibrin. Yet, there are several drawbacks to the use of current PAs, including significant risks of uncontrolled bleeding and suboptimal efficacy and pharmacokinetics. Nanomaterials are well positioned to address these priority issues in thrombolysis, via the alteration of PA pharmacokinetics and biodistribution. Additionally, due to the multifunctional nature of nanoparticles, these thrombolytics may be targeted to the site of occlusion, effectively concentrating the drug where it is most needed. Herein, we describe the methodology associated with the synthesis of a novel thrombus-targeted fibrinolytic nanoagent. At each step of the synthesis, we analyze the nanomaterials, including their physical properties and their ability to bind to thrombosis targets of interest. The effect of the conjugation of tPA to the nanoparticle surface on the amidolytic and fibrinolytic activities of nanoagents is also investigated. Lastly, the in vivo binding of the targeted thrombolytic to intravascular thrombi is examined.

Multifunctional nanoagent for thrombus-targeted fibrinolytic therapy.

BACKGROUND: Current thrombolytic therapies utilize exogenous plasminogen activators (PAs) to effectively lyse clots, restoring blood flow, and preventing tissue and organ death. These PAs may also impair normal hemostasis, leading to life-threatening bleeding, including intracerebral hemorrhage. AIMS: This study aims to develop new thrombus-targeted fibrinolytic agents that harness the multifunctional theranostic capabilities of nanomaterials, potentially allowing for the generation of efficacious thrombolytics while minimizing deleterious side effects. MATERIALS & METHODS: A thrombus-targeted nano-fibrinolytic agent was synthesized using a magnetofluorescent crosslinked dextran-coated iron oxide nanoparticle platform that was conjugated to recombinant tissue PA (tPA). Thrombus-targeting was achieved by derivatizing the nanoparticle with an activated factor XIII (FXIIIa)-sensitive peptide. Human plasma clot binding ability of the targeted and control agents was assessed by fluorescence reflectance imaging. Next, the in vitro enzymatic activity of the agents was assessed by S2288-based amidolytic activity, and an ELISA D-dimer assay for fibrinolysis. In vivo targeting of the nanoagent was next examined by intravital fluorescence microscopy of murine arterial and venous thrombosis. The fibrinolytic activity of the targeted nanoagent compared to free tPA was then evaluated in vivo in murine pulmonary embolism. RESULTS: In vitro, the targeted thrombolytic nanoagent demonstrated superior binding to fresh-frozen plasma clots compared to control nanoagents (analysis of variance, p < 0.05). When normalized by S2288-based amidolytic activity, targeted, control and free tPA samples demonstrated equivalent in vitro fibrinolytic activity against human plasma clots, as determined by ELISA D-dimer assays. The FXIIIa targeted fibrinolytic nanoagent efficiently bound the margin of intravascular thrombi as detected by intravital fluorescence microscopy. In in vivo fibrinolysis studies the FXIIIa-targeted agent lysed pulmonary emboli with similar efficacy as free tPA (p > 0.05). CONCLUSION: The applicability of a FXIIIa-targeted thrombolytic nanoagent in the treatment of thromboembolism was demonstrated in vitro and in vivo. Future studies are planned to investigate the safety profile and overall efficacy of this class of nanoagents.

Near-IR single fluorophore quenching system based on phthalocyanine (Pc) aggregation and its application for monitoring inhibitor/activator action on a therapeutic target: L1-EN.

Controlled H-aggregation of single Pc-labeled oligonucleotides is utilized as a fluorescence quenching system to discern changes in enzyme activity for the discovery of inhibitors for Long Interspersed Element 1 endonuclease (L1-EN), which is involved in genome instability and implicated in many different diseases.

Mono-amine functionalized phthalocyanines: microwave-assisted solid-phase synthesis and bioconjugation strategies.

Phthalocyanines (Pcs) are excellent candidates for use as fluors for near-infrared (near-IR) fluorescent tagging of biomolecules for a wide variety of bioanalytical applications. Monofunctionalized Pcs, having two different types of peripheral substitutents, one for covalent conjugation of the Pc to biomolecules and others to improve the solubility of the macrocycle, are ideally suited for the desired applications. To date, difficulties faced during the purification of monofunctionalized Pcs limited their usage in various types of applications. Herein are reported a new synthetic method for rapid synthesis of the target Pcs and bioconjugation techniques for labeling of the oligonucleotides with the near-IR fluors. A novel synthetic route was developed utilizing a hydrophilic, poly(ethylene glycol) (PEG)-based support with an acid-labile Rink Amide linker. The Pcs were functionalized with an amine group for covalent conjugation purposes and were decorated with short PEG chains, serving as solubilizing groups. Microwave-assisted solid-phase synthetic method was successfully applied to obtain pure asymmetrically substituted monoamine functionalized Pcs in a short period of time. Three different bioconjugation techniques, reductive amination, amidation, and Huisgen cycloaddition, were employed for covalent conjugation of Pcs to oligonucleotides. The described microwave-assisted bioconjugation methods give an opportunity to synthesize and isolate the Pc-oligonucleotide conjugate in a few hours.

Phthalocyanine dimerization-based molecular beacons using near-IR fluorescence.

Herein we demonstrate the use of a novel dimerization-based molecular beacon (MB) probe consisting of two metallo-phthalocyanine (Pc) fluorophores that use near-IR fluorescence, appropriate for highly specific and sensitive in vivo and/or in vitro DNA/RNA detection. Pc's possess a propensity to form nonfluorescent H-dimers that is utilized as the molecular "off" switch in the closed MB conformation. The "on" switch, which is generated when the solution target binds to the loop of the MB forming the open form, also provides two fluorophores for transduction resulting in a doubling of the extinction coefficient and improving the resulting fluorescence yield compared to a classical single-fluorophore/quencher MB system. In addition, the Pc-based MBs possess high thermal, photo, and chemical stabilities that are essential for many highly sensitive applications, such as molecular imaging. The dimer-based MBs were obtained using a simple single-step synthesis procedure and demonstrated excellent quenching efficiencies (98%) as well as a high signal-to-background ratio (approximately 60) exceeding the performance characteristics of many conventionally available MB probes.

Solid-phase synthesis of asymmetrically substituted "AB3-type" phthalocyanines.

Synthesis of phthalocyanines with asymmetrical substitution on the periphery is often difficult due the problems in purification of the phthalocyanine mixtures obtained. Using a poly(ethylene glycol) (PEG)-based support with a Wang-type linker, we have developed the synthesis of monohydroxylated, oligoethylene glycol substituted phthalocyanines utilizing an amidine-base-promoted phthalonitrile tetramerization reaction. The use of a hydrophilic support allows symmetrical phthalocyanine product formed in solution to be readily and completely removed by washing while leaving the "AB3" product on the support. Acid cleavage with 10% trifluoroacetic acid provides the pure unsymmetrically substituted Pc. This method was applied to several metallo Pcs. Additionally, methods to avoid premature reactions on-resin that give A2B2 products are provided.