ALA/Ag2S/MnO2 Hybrid Nanoparticles for Near-Infrared Image-Guided Long-Wavelength Phototherapy of Breast Cancer.

The combination of photothermal therapy (PTT) and photodynamic therapy (PDT) based on temperature increase and the formation of reactive oxygen species (ROS), respectively, is an exciting avenue to provide local and improved therapy of tumors with minimal off-site toxicity. 5-Aminolevulinic acid (ALA) is one of the most popular PDT pro-drugs, and its efficiency improves significantly when delivered to tumors with nanoparticles (NPs). But the tumor site's hypoxic environment is a handicap for the oxygen-consuming PDT process. In this work, highly stable, small, theranostic NPs composed of Ag2S quantum dots and MnO2, electrostatically loaded with ALA, were developed for enhanced PDT/PTT combination of tumors. MnO2 catalyzes endogenous H2O2 to O2 conversion and glutathione depletion, enhancing ROS generation and ALA-PDT efficiency. Ag2S quantum dots (AS QDs) conjugated with bovine serum albumin (BSA) support MnO2 formation and stabilization around Ag2S. AS-BSA-MnO2 provided a strong intracellular near-infrared (NIR) signal and increased the solution temperature by 15 °C upon laser irradiation at 808 nm (215 mW, 10 mg/mL), proving the hybrid NP as an optically trackable, long-wavelength PTT agent. In the in vitro studies, no significant cytotoxicity was observed in the absence of laser irradiation in healthy (C2C12) or breast cancer cell lines (SKBR3 and MDA-MB-231). The most effective phototoxicity was observed when AS-BSA-MnO2-ALA-treated cells were co-irradiated for 5 min with 640 nm (300 mW) and 808 nm (700 mW) due to enhanced ALA-PDT combined with PTT. The viability of cancer cells decreased to approximately 5-10% at 50 µg/mL [Ag], corresponding to 1.6 mM [ALA], whereas at the same concentration, individual PTT and PDT treatments decreased the viability to 55-35%, respectively. The late apoptotic death of the treated cells was mostly correlated with high ROS levels and lactate dehydrogenase. Overall, these hybrid NPs overcome tumor hypoxia, deliver ALA to tumor cells, and provide both NIR tracking and enhanced PDT + PTT combination therapy upon short, low-dose co-irradiation at long wavelengths. These agents that may be utilized for treating other cancer types are also highly suitable for in vivo investigations.

Image-Guided Enhanced PDT/PTT Combination Therapy Using Brominated Hemicyanine-Loaded Folate Receptor-Targeting Ag2S Quantum Dots.

Tumor-targeting nanoparticles and phototherapies are the two major trends in tumor-specific, local cancer therapy with minimal side effects. Organic photosensitizers (PSs) usually offer effective photodynamic therapy (PDT) but require enhanced solubility and tumor-targeting, which may be provided by a nanoparticle. Near-infrared (NIR)-emitting Ag2S quantum dots may act as a delivery vehicle for the PS, NIR tracking agent, and as a phototherapy (PTT) agent. A combination of the two provides luminescent dual-phototherapy agents with tumor-specificity and image-guided and enhanced cytotoxicity as a result of synergistic PDT and PTT. In this study, brominated hemicyanine (Hemi-Br), a photosensitizer, was loaded onto folic acid (FA)-tagged, glutathione (GSH)-coated Ag2S quantum dots (AS-GSH QDs) to provide enhanced phototoxicity via a photodynamic and mild photothermal effect in folate receptor(+) cancer cell lines at clinically relevant 640 nm irradiation. Final particles (AS-GSH-FA/Hemi-Br) had a hydrodynamic size of 75.5 nm, dual emission at both 705 and 910 nm, and a 93% light-to-heat conversion efficiency under 640 nm laser irradiation. In vitro cytotoxicity studies were conducted with folate receptor (FR)-positive HeLa and -negative A549 cell lines to differentiate receptor-mediated uptake. Enhanced phototoxicity on HeLa cells was observed with AS-GSH-FA/Hemi-Br compared to free Hemi-Br and AS-GSH-FA QDs due to increased uptake of the photosensitizer via active targeting and combination therapy, which is especially visible at the safe dose of single agents. Upon irradiation with a 640 nm (300 mW, 0.78 W/cm2) laser for 5 min, the viability of the HeLa cells decreased from 64% to 42 and 25% when treated with free Hemi-Br, AS-GSH-FA, and AS-GSH-FA/Hemi-Br, respectively. Overall, AS-GSH-FA/Hemi-Br provides image-guided enhanced PDT/PTT, which may be adopted for different FR(+) tumors.

Development of a cysteine responsive chlorinated hemicyanine for image-guided dual phototherapy.

A cysteine (Cys) activatable chlorinated hemicyanine (Cl-Cys) was introduced as a tumour selective image-guided dual phototherapy agent. Cl-Cys exhibited a significant turn on response in its near-IR emission signal and activated its singlet oxygen generation as well as photothermal conversion potentials upon reacting with Cys. The laser irradiation of Cl-Cys induced significant cell death in cancer cells with high Cys level, while it stayed deactivated and non-emissive in a healthy cell line. A profound synergistic PDT/PTT effect was observed at high doses. Remarkably, Cl-Cys marks the first ever example of Cys-responsive small organic-based therapeutic agent and holds a great promise to develop new activity-based photosensitizers for dual phototherapy action.

Determination of the Wavelength-Dependent Photothermal Conversion Efficiency of Photosensitizers for Photothermal Therapy: Application to Ag2S-Glutathione Quantum Dots.

Nanoparticles have become popular photosensitizers for photothermal therapy (PTT), as they can be targeted to specific cancer tissues and deliver a chemotherapeutic drug, providing a multimodal therapeutic approach. Photothermal conversion efficiency of nanoparticles is critical in the assessment of their therapeutic use in PTT. We describe an accurate calorimetric method for the determination of the photothermal conversion efficiency of nanoparticles in solution. A tightly focused laser beam was used to irradiate a cuvette containing a solution of silver sulfide-glutathione quantum dots (Ag2S-GSH QDs), and the maximum steady-state temperature rise was measured with an infrared camera. The data were analyzed using two different photothermal conversion efficiencies, the intrinsic and external conversion efficiencies, to relate the induced heating power of the nanoparticles to the absorbed and incident optical powers, respectively. Measurements with a tunable Ti3+:sapphire laser showed that the intrinsic photothermal conversion efficiency of Ag2S-GSH QDs exceeded 91% over the 720-810 nm wavelength range. The method was also used to analyze poly(acrylic acid)-coated superparamagnetic iron oxide nanoparticles (PAA/SPIONs), and the intrinsic photothermal conversion efficiency was determined to be 83.4% at 810 nm. This approach is useful for the evaluation of various potential nanoparticles for photothermal therapy applications.

Cetuximab-Ag2S quantum dots for fluorescence imaging and highly effective combination of ALA-based photodynamic/chemo-therapy of colorectal cancer cells.

Colorectal cancer (CRC) has a poor prognosis and urgently needs better therapeutic approaches. 5-Aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX) based photodynamic therapy (PDT) is already used in the clinic for several cancers but not yet well investigated for CRC. Currently, systemic administration of ALA offers a limited degree of tumour selectivity, except for intracranial tumours, limiting its wider use in the clinic. The combination of effective ALA-PDT and chemotherapy may provide a promising alternative approach for CRC treatment. Herein, theranostic Ag2S quantum dots (AS-2MPA) optically trackable in near-infrared (NIR), conjugated with endothelial growth factor receptor (EGFR) targeting Cetuximab (Cet) and loaded with ALA for PDT monotherapy or ALA/5-fluorouracil (5FU) for the combination therapy are proposed for enhanced treatment of EGFR(+) CRC. AS-2MPA-Cet exhibited excellent targeting of the high EGFR expressing cells and showed a strong intracellular signal for NIR optical detection in a comparative study performed on SW480, HCT116, and HT29 cells, which exhibit high, medium and low EGFR expression, respectively. Targeting provided enhanced uptake of the ALA loaded nanoparticles by strong EGFR expressing cells and formation of higher levels of PpIX. Cells also differ in their efficiency to convert ALA to PpIX, and SW480 was the best, followed by HT29, while HCT116 was determined as unsuitable for ALA-PDT. The therapeutic efficacy was evaluated in 2D cell cultures and 3D spheroids of SW480 and HT29 cells using AS-2MPA with either electrostatically loaded, hydrazone or amide linked ALA to achieve different levels of pH or enzyme sensitive release. Most effective phototoxicity was observed in SW480 cells using AS-2MPA-ALA-electrostatic-Cet due to enhanced uptake of the particles, fast ALA release and effective ALA-to-PpIX conversion. Targeted delivery reduced the effective ALA concentration significantly which was further reduced with codelivery of 5FU. Delivery of ALA via covalent linkages was also effective for PDT, but required a longer incubation time for the release of ALA in therapeutic doses. Phototoxicity was correlated with high levels of reactive oxygen species (ROS) and apoptotic/necrotic cell death. Hence, both AS-2MPA-ALA-Cet based PDT and AS-2MPA-ALA-Cet-5FU based chemo/PDT combination therapy coupled with strong NIR tracking of the nanoparticles demonstrate an exceptional therapeutic effect on CRC cells and excellent potential for synergistic multistage tumour targeting therapy.

Cadmium-Free and Efficient Type-II InP/ZnO/ZnS Quantum Dots and Their Application for LEDs.

It is a generally accepted perspective that type-II nanocrystal quantum dots (QDs) have low quantum yield due to the separation of the electron and hole wavefunctions. Recently, high quantum yield levels were reported for cadmium-based type-II QDs. Hence, the quest for finding non-toxic and efficient type-II QDs is continuing. Herein, we demonstrate environmentally benign type-II InP/ZnO/ZnS core/shell/shell QDs that reach a high quantum yield of ∼91%. For this, ZnO layer was grown on core InP QDs by thermal decomposition, which was followed by a ZnS layer via successive ionic layer adsorption. The small-angle X-ray scattering shows that spherical InP core and InP/ZnO core/shell QDs turn into elliptical particles with the growth of the ZnS shell. To conserve the quantum efficiency of QDs in device architectures, InP/ZnO/ZnS QDs were integrated in the liquid state on blue light-emitting diodes (LEDs) as down-converters that led to an external quantum efficiency of 9.4% and a power conversion efficiency of 6.8%, respectively, which is the most efficient QD-LED using type-II QDs. This study pointed out that cadmium-free type-II QDs can reach high efficiency levels, which can stimulate novel forms of devices and nanomaterials for bioimaging, display, and lighting.

Dual laser activatable brominated hemicyanine as a highly efficient and photostable multimodal phototherapy agent.

Dual phototherapy agents have attracted great interest in recent years as they offer enhanced cytotoxicity on cancer cells due to the synergistic effect of photodynamic and photothermal therapies (PDT/PTT). In this study, we demonstrate a brominated hemicyanine (HC-1), which is previously shown as mitochondria targeting PDT agent, can also serve as an effective photosensitizer for PTT for the first time under a single (640 nm or 808 nm) and dual laser (640 nm + 808 nm) irradiation. Generation of reactive oxygen species and photothermal conversion as a function of irradiation wavelength and power were studied. Both single wavelength irradiations caused significant phototoxicity in colon and cervical cancer cells after 5 min of irradiation. However, co-irradiation provided near-complete elimination of cancer cells due to synergistic action. This work introduces an easily accessible small molecule-based synergistic phototherapy agent, which holds a great promise towards the realization of local, rapid and highly efficient treatment modalities against cancer.

Multimodal image-guided folic acid targeted Ag-based quantum dots for the combination of selective methotrexate delivery and photothermal therapy.

Multifunctional quantum dots (QDs) with photothermal therapy (PTT) potential loaded with an anticancer drug and labelled with a targeting agent can be highly effective nano-agents for tumour specific, image-guided PTT/chemo combination therapy of cancer. Ag-chalcogenides are promising QDs with good biocompatibility. Ag2S QDs are popular theranostic agents for imaging in near-infrared with PTT potential. However, theranostic applications of AgInS2 QDs emitting in the visible region and its PTT potential need to be explored. Here, we first present a simple synthesis of small, glutathione (GSH) coated AgInS2 QDs with peak emission at 634 nm, 21% quantum yield, and excellent long-term stability without an inorganic shell. Ag2S-GSH QDs emitting in the near-infrared region (peak emission = 822 nm) were also produced. Both QDs were tagged with folic acid (FA) and conjugated with methotrexate (MTX). About 3-fold higher internalization of FA-tagged QDs by folate-receptor (FR) overexpressing HeLa cells than HT29 and A549 cells was observed. Delivery of MTX by QD-FA-MTX reduced the IC50 of the drug from 10 µg/mL to 2.5-5 µg/mL. MTX release was triggered at acidic pH, which was further enhanced with local temperature increase created by laser irradiation. Irradiation of AgInS2-GSH QDs at 640 nm (300 mW) for 10 min, caused about 10 °C temperature increase but did not cause any thermal ablation of cells. On the other hand, Ag2S-GSH-FA based PTT effectively and selectively killed HeLa cells with 10 min 808 nm laser irradiation via mostly necrosis with an IC50 of 5 µg Ag/mL. Under the same conditions, IC50 of MTX was reduced to 0.21 µg/mL if Ag2S-GSH-FA.

Indocyanine green loaded APTMS coated SPIONs for dual phototherapy of cancer.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been recently recognized as highly efficient photothermal therapy (PTT) agents. Here, we demonstrate, for the first time to our knowledge, dose and laser intensity dependent PTT potential of small, spherical, 3-aminopropyltrimethoxysilane coated cationic superparamagnetic iron oxide nanoparticles (APTMS@SPIONs) in aqueous solutions upon irradiation at 795 nm. Indocyanine green (ICG) which has been recently used for photodynamic therapy (PDT), was loaded to APTMS@SPIONs to improve the stability of ICG and to achieve an effective mild PTT and PDT (dual therapy) combination for synergistic therapeutic effect on cancer cells via a single laser treatment in the near infrared (NIR). Neither APTMS@SPIONs nor ICG-APTMS@SPIONs showed dark toxicity on MCF7 breast and HT29 colon cancer cell lines. A safe laser procedure was determined as 10 min irradiation at 795 nm with 1.8 W/cm2 of laser intensity, at which APTMS@SPION did not cause a significant cell death. However, free ICG reduced cell viability at and above 10 µg/ml under these conditions along with generation of reactive oxygen species (ROS), more effectively in MCF7. ICG-APTMS@SPION treated cells showed 2-fold increase in ROS generation and near complete cell death at and below 5 µg/ml ICG dose, even in less sensitive HT29 cells after a single laser treatment at NIR, which would be safe for the healthy tissue and provide a longer penetration depth. Besides, both components can be utilized for diagnosis and the overall composition may be used for optical-image guided phototherapy in the NIR region.

Development of a thulium (Tm:YAP) laser system for brain tissue ablation.

In this study, a thulium (Tm:YAP) laser system was developed for brain surgery applications. As the Tm:YAP laser is a continuous-wave laser delivered via silica fibers, it would have great potential for stereotaxic neurosurgery with highest local absorption in the IR region. The laser system developed in this study allowed the user to set the power level, exposure time, and modulation parameters (pulse width and on-off cycles). The Tm:YAP laser beam (200-600 mW, 69-208 W/cm(2)) was delivered from a distance of 2 mm to cortical and subcortical regions of ex-vivo Wistar rat brain tissue samples via a 200-µm-core optical fiber. The system performance, dosimetry study, and ablation characteristics of the Tm:YAP laser were tested at different power levels by maximizing the therapeutic effects and minimizing unwanted thermal side-effects. The coagulation and ablation diameters were measured under microscope. The maximum ablation efficiency (100 × ablation diameter/coagulation diameter) was obtained when the Tm:YAP laser system was operated at 200 mW for 10 s. At this laser dose, the ablation efficiency was found to be 71.4% and 58.7% for cortical and subcortical regions, respectively. The fiber-coupled Tm:YAP laser system in hence proposed for the delivery of photothermal therapies in medical applications.

Fluence of thulium laser system in skin ablation.

Tm:YAP laser system at power levels up to 1.2 W at 1980 nm was established in both continuous-wave and modulated modes of operation. The fluence effect of the laser system for skin ablation was analyzed by histology analysis with Wistar rat skin tissues. Thermally altered length, thermally altered area, ablation area, and ablation depth parameters were measured on histology images of skin samples just after the laser operation and after four-day healing period. Continuous-wave mode of operation provided higher thermal effects on the skin samples. Lower fluence levels were found for efficient ablation effect.

Modulated and continuous-wave operations of low-power thulium (Tm:YAP) laser in tissue welding.

Our aim is to explore the welding capabilities of a thulium (Tm:YAP) laser in modulated and continuous-wave (CW) modes of operation. The Tm:YAP laser system developed for this study includes a Tm:YAP laser resonator, diode laser driver, water chiller, modulation controller unit, and acquisition/control software. Full-thickness incisions on Wistar rat skin were welded by the Tm:YAP laser system at 100 mW and 5 s in both modulated and CW modes of operation (34.66 Wcm(2)). The skin samples were examined during a 21-day healing period by histology and tensile tests. The results were compared with the samples closed by conventional suture technique. For the laser groups, immediate closure at the surface layers of the incisions was observed. Full closures were observed for both modulated and CW modes of operation at day 4. The tensile forces for both modulated and CW modes of operation were found to be significantly higher than the values found by conventional suture technique. The 1980-nm Tm:YAP laser system operating in both modulated and CW modes maximizes the therapeutic effect while minimizing undesired side effects of laser tissue welding. Hence, it is a potentially important alternative tool to the conventional suturing technique.

Corneal tissue welding with infrared laser irradiation after clear corneal incision.

PURPOSE: The aim of this study was to investigate the potential of infrared lasers for corneal welding to seal corneal cuts done in an experimental animal model. MATERIALS AND METHODS: Full-thickness corneal cuts on freshly enucleated bovine eyes were irradiated with infrared (809-nm diode, 980-nm diode, 1070-nm YLF, and 1980-nm Tm:YAP) lasers to get immediate laser welding. An 809-nm laser was used with the topical application of indocyanine green to enhance the photothermal interaction at the weld site. In total, 60 bovine eyes were used in this study; 40 eyes were used in the first part of the study for the determination of optimal welding parameters (15 eyes were excluded because of macroscopic carbonization, opacification, or corneal shrinkage; 2 eyes were used for control), and 20 eyes were used for further investigation of more promising lasers (YLF and Tm:YAP). Laser wavelength, irradiating power, exposure time, and spot size were the dose parameters, and optimal dose for immediate closure with minimal thermal damage was estimated through histological examination of welded samples. RESULTS: In the first part of the study, results showed that none of the applications was satisfactory. Full-thickness success rates were 28% (2 of 7) for 809-nm and for 980-nm diode lasers and 67% (2 of 3) for 1070-nm YLF and (4 of 6) for 1980-nm Tm:YAP lasers. In the second part of the study, YLF and Tm:YAP lasers were investigated with bigger sample size. Results were not conclusive but promising again. Five corneal incisions were full-thickness welded out of 10 corneas with 1070-nm laser, and 4 corneal incisions were partially welded out of 10 corneas with 1980-nm laser in the second part of the study. CONCLUSIONS: Results showed that noteworthy corneal welding could be obtained with 1070-nm YLF laser and 1980-nm Tm:YAP laser wavelengths. Furthermore, in vitro and in vivo studies will shed light on the potential usage of corneal laser welding technique.

Multi-photon microscopy with a low-cost and highly efficient Cr:LiCAF laser.

Multi-photon microscopy (MPM) is a powerful tool for biomedical imaging, enabling molecular contrast and integrated structural and functional imaging on the cellular and subcellular level. However, the cost and complexity of femtosecond laser sources that are required in MPM are significant hurdles to widespread adoption of this important imaging modality. In this work, we describe femtosecond diode pumped Cr:LiCAF laser technology as a low cost alternative to femtosecond Ti:Sapphire lasers for MPM. Using single mode pump diodes which cost only $150 each, a diode pumped Cr:LiCAF laser generates approximately 70-fs duration, 1.8-nJ pulses at approximately 800 nm wavelengths, with a repetition rate of 100 MHz and average output power of 180 mW. Representative examples of MPM imaging in neuroscience, immunology, endocrinology and cancer research using Cr:LiCAF laser technology are presented. These studies demonstrate the potential of this laser source for use in a broad range of MPM applications.

Synthesis and characterization of poly(acrylic acid) stabilized cadmium sulfide quantum dots.

Cadmium sulfide (CdS) nanoparticles (NPs) capped with poly(acrylic acid) (PAA) were prepared in aqueous solutions from Cd(NO3)2 and Na2S. Influence of the COOH/Cd ratio (0.8-12.5), reaction pH (5.5 and 7.5), and PAA molecular weight (2100 and 5100 g/mol) on the particle size, colloidal stability, and photoluminescence were investigated. A Cd/S ratio of <1 causes ineffective passivization of the surface with the carboxylate and therefore results in a red shift of the absorption band and a significant drop in photoluminescence. Therefore, the Cd/S ratio was fixed at 1.1 for all experiments studying the mentioned variables. PAA coating provided excellent colloidal stability at a COOH/Cd ratio above 1. Absorption edges of PAA-coated CdS NPs are in the range of 460-508 nm. The size of the NPs increases slightly with increasing PAA molecular weight and COOH/Cd ratio at pH 7.5. It is demonstrated that there is a critical COOH/Cd ratio (1.5-2) that maximizes the photoluminescence intensity and quantum yield (QY, 17%). Above this critical ratio, which corresponds to smaller crystal sizes (3.7-4.1 nm) for each reaction set, the quantum yield decreases and the crystal size increases. Moreover, CdS NPs prepared at pH 7.5 have significantly higher QY and absorb at lower wavelengths in comparison with those prepared at pH 5.5. Luminescence quenching has not been observed over 8 months.