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Low-cost Custom Fabrication and Mode-locked Operation of an All-normal-dispersion Femtosecond Fiber Laser for Multiphoton Microscopy

A protocol is presented to build a custom low-cost yet high-performance femtosecond (fs) fiber laser. This all-normal-dispersion (ANDi) ytterbium-doped ...

Northeastern University

Nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour-selective drug release. However, few are effective because cancer cells develop ways to resist and evade treatment. Here, we introduce a photoactivable multi-inhibitor nanoliposome (PMIL) that imparts light-induced cytotoxicity in synchrony with a photoinitiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways. The PMIL consists of a nanoliposome doped with a photoactivable chromophore (benzoporphyrin derivative, BPD) in the lipid bilayer, and a nanoparticle containing cabozantinib (XL184)-a multikinase inhibitor-encapsulated inside. Near-infrared tumour irradiation, following intravenous PMIL administration, triggers photodynamic damage of tumour cells and microvessels, and simultaneously initiates release of XL184 inside the tumour. A single PMIL treatment achieves prolonged tumour reduction in two mouse models and suppresses metastatic escape in an orthotopic pancreatic tumour model. The PMIL offers new prospects for cancer therapy by enabling spatiotemporal control of drug release while reducing systemic drug exposure and associated toxicities.

A photoactivable multi-inhibitor nanoliposome for tumour control and simultaneous inhibition of treatment escape pathways.

The emergence of fluorescently labeled therapeutic antibodies has given rise to molecular probes for image-guided surgery. However, the extraneous interstitial presence of an unbound and nonspecifically accumulated probe gives rise to false-positive detection of tumor tissue and margins. Thus, the concept of tumor-cell activation of smart probes provides a potentially superior mechanism of delineating tumor margins as well as small tumor deposits. The combination of molecular targeting with intracellular activation circumvents the presence of extracellular, nonspecific signals of targeted probe accumulation. Here, we present a demonstration of the clinical antibodies cetuximab (cet, anti-EGFR mAb) and trastuzumab (trast, anti-HER-2 mAb) conjugated to Alexa Fluor molecules and IRDye QC-1 quencher optimized at the ratio of 1∶2∶6 to provide the greatest degree of proteolytic fluorescence activation, synonymous with intracellular lysosomal degradation. The cet-AF-Q-C1 conjugate (1∶2∶6) provides up to 9.8-fold proteolytic fluorescence activation. By preparing a spectrally distinct, irrelevant sham IgG-AF-QC-1 conjugate, a dual-activatable probe approach is shown to enhance the specificity of imaging within an orthotopic AsPC-1 pancreatic cancer xenograft model. The dual-activatable approach warrants expedited clinical translation to improve the specificity of image-guided surgery by spectrally decomposing specific from nonspecific probe accumulation, binding, and internalization.

Activatable clinical fluorophore-quencher antibody pairs as dual molecular probes for the enhanced specificity of image-guided surgery.

In this issue, Cramer et al. introduce 3D culture models of metastatic mesothelioma to investigate basic cancer biology and new combination therapies for combating this complex and lethal disease. The results suggest that erlotinib-enhanced photodynamic therapy could further improve the efficacy of intraoperative light-activation to mop up residual tumor deposits in the clinic following surgical removal of macroscopic mesothelioma metastases.

3D Culture Models of Malignant Mesothelioma Reveal a Powerful Interplay Between Photodynamic Therapy and Kinase Suppression Offering Hope to Reduce Tumor Recurrence.

Solid-state femtosecond lasers have stimulated the broad adoption of multiphoton microscopy in the modern laboratory. However, these devices remain costly. Fiber lasers offer promise as a means to inexpensively produce ultrashort pulses of light suitable for nonlinear microscopy in compact, robust and portable devices. Although encouraging, the initial methods reported in the biomedical engineering community to construct home-built femtosecond fiber laser systems overlooked fundamental aspects that compromised performance and misrepresented the significant financial and intellectual investments required to build these devices. Here, we present a practical protocol to fabricate an all-normal-dispersion ytterbium (Yb)-doped femtosecond fiber laser oscillator using commercially-available parts (plus standard optical components and extra-cavity accessories) as well as basic fiber splicing and laser pulse characterization equipment. We also provide a synthesis of established protocols in the laser physics community, but often overlooked in other fields, to verify true versus seemingly (partial or noise-like) mode-locked performance. The approaches described here make custom fabrication of femtosecond fiber lasers more accessible to a wide range of investigators and better represent the investments required for the proper laser design, fabrication and operation.

Custom fabrication and mode-locked operation of a femtosecond fiber laser for multiphoton microscopy.

Cancer photomedicine offers unique mechanisms for inducing local tumor damage with the potential to stimulate local and systemic anti-tumor immunity. Optically-active nanomedicine offers these features as well as spatiotemporal control of tumor-focused drug release to realize synergistic combination therapies. Achieving quantitative dosimetry is a major challenge, and dosimetry is fundamental to photomedicine for personalizing and tailoring therapeutic regimens to specific patients and anatomical locations. The challenge of dosimetry is perhaps greater for photomedicine than many standard therapies given the complexity of light delivery and light-tissue interactions as well as the resulting photochemistry responsible for tumor damage and drug-release, in addition to the usual intricacies of therapeutic agent delivery. An emerging multidisciplinary approach in oncology utilizes mathematical and computational models to iteratively and quantitively analyze complex dosimetry, and biological response parameters. These models are parameterized by preclinical and clinical observations and then tested against previously unseen data. Such calibrated and validated models can be deployed to simulate treatment doses, protocols, and combinations that have not yet been experimentally or clinically evaluated and can provide testable optimal treatment outcomes in a practical workflow. Here, we foresee the utility of these computational approaches to guide adaptive therapy, and how mathematical models might be further developed and integrated as a novel methodology to guide precision photomedicine.

Illuminating the Numbers: Integrating Mathematical Models to Optimize Photomedicine Dosimetry and Combination Therapies.

Photodynamic therapy (PDT) is an established therapeutic modality that uses nonionizing near-infrared light to activate photocytotoxicity of endogenous or exogenous photosensitizers (PSs). An ongoing avenue of cancer research involves leveraging PDT to stimulate antitumor immune responses; however, these effects appear to be best elicited in low-dose regimens that do not provide significant tumor reduction using conventional, nonspecific PSs. The loss of immune enhancement at higher PDT doses may arise in part from indiscriminate damage to local immune cell populations, including tumor-infiltrating T cells. We previously introduced "tumor-targeted, activatable photoimmunotherapy" (taPIT) using molecular-targeted and cell-activatable antibody-PS conjugates to realize precision tumor photodamage with microscale fidelity. Here, we investigate the immune cell sparing effect provided by taPIT in a 3D model of the tumor immune microenvironment. We report that high-dose taPIT spares 25% of the local immune cell population, five times more than the conventional PDT regimen, in a 3D coculture model incorporating epithelial ovarian cancer cells and T cells. These findings suggest that the enhanced selectivity of taPIT may be utilized to achieve local tumor reduction with sparing of intratumor effector immune cells that would otherwise be lost if treated with conventional PDT.

Bryan Q. Spring

Translational Biophotonics Cluster,
Department of Physics,
Department of Bioengineering

Northeastern University

Low-cost Custom Fabrication and Mode-locked Operation of an All-normal-dispersion Femtosecond Fiber Laser for Multiphoton Microscopy

Bryan Q. Spring

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Translational Biophotonics Cluster,Department of Physics,Department of Bioengineering

Northeastern University

Low-cost Custom Fabrication and Mode-locked Operation of an All-normal-dispersion Femtosecond Fiber Laser for Multiphoton Microscopy

Translational Biophotonics Cluster,
Department of Physics,
Department of Bioengineering