Dual-Function Antibody Conjugate-Enabled Photoimmunotherapy Complements Fluorescence and Photoacoustic Imaging of Head and Neck Cancer Spheroids.

Several molecular-targeted imaging and therapeutic agents are in clinical trials for image-guided surgery and photoimmunotherapy (PIT) for head and neck cancers. In this context, we have previously reported the development, characterization, and specificity of a dual-function antibody conjugate (DFAC) for multimodal imaging and photoimmunotherapy (PIT) of EGFR-overexpressing cancer cells. The DFAC reported previously and used in the present study comprises an EGFR-targeted antibody, cetuximab, conjugated to benzoporphyrin derivative (BPD) for fluorescence imaging and PIT and a Si-centered naphthalocyanine dye for photoacoustic imaging. We report here the evaluation and performance of DFAC in detecting microscopic cancer spheroids by fluorescence and photoacoustic imaging along with their treatment by PIT. We demonstrate that while fluorescence imaging can detect spheroids with volumes greater than 0.049 mm3, photoacoustic imaging-based detection was possible even for the smallest spheroids (0.01 mm3) developed in the study. When subjected to PIT, the spheroids showed a dose-dependent response, with smaller spheroids (0.01 and 0.018 mm3) showing a complete response with no recurrence when treated with 100 J/cm2. Together our results demonstrate the complementary imaging and treatment capacity of DFAC. This potentially enables fluorescence imaging to assess the presence of tumor on a macroscopic scale, followed by photoacoustic imaging for delineating tumor margins guiding surgical resection and elimination of any residual microscopic disease by PIT, in a single intraoperative setting.

Superhydrophobic Tipped Antimicrobial Photodynamic Therapy Device for the In Vivo Treatment of Periodontitis Using a Wistar Rat Model.

Limited options exist for treatment of periodontitis; scaling and root planing (SRP) are not sufficient to eradicate P. gingivalis and the resulting inflammatory disease. Chlorhexidine (CHX), used as an adjuvant to SRP, may reduce bacterial loads but leads to pain and staining, while evidence for its efficacy is lacking. Antibiotics are effective but can lead to drug-resistance. The rising concern of antibiotic resistance limits the future use of this treatment approach. This study evaluates the efficacy of a novel superhydrophobic (SH) antimicrobial photodynamic therapy (aPDT) device as an adjuvant to SRP for the treatment of periodontitis induced in a Wistar rat in vivo model relative to CHX. The SH-aPDT device comprises an SH silicone rubber strip coated with verteporfin photosensitizer (PS), sterilized, and secured onto a tapered plastic optical fiber tip connected to a red diode laser. The superhydrophobic polydimethylsiloxane (PDMS) strips were fabricated by using a novel soluble template method that creates a medical-grade elastomer with hierarchical surface roughness without the use of nanoparticles. Superhydrophobicity minimizes direct contact of the PS-coated surface with bacterial biofilms. Upon insertion of the device tip into the pocket and energizing the laser, the device generates singlet oxygen that effectively targets and eliminates bacteria within the periodontal pocket. SH-aPDT treatment using 125 J/cm2 of red light on three consecutive days reduced P. gingivalis significantly more than SRP-CHX controls (p < 0.05). Clinical parameters significantly improved (p < 0.05), and histology and stereometry results demonstrated SH-aPDT to be the most effective treatment for improving healing and reducing inflammation, with an increase in fibroblast cells and extracellular matrix and a reduction in vascularization, inflammatory cells, and COX-2 expression. The SH-aPDT approach resulted in complete disease clearance assessed 30 days after treatment initiation with significant reduction of the periodontal pocket and re-formation of the junctional epithelium at the enamel-cementum junction. PS isolation on a SH strip minimizes the potential for bacteria to develop resistance, where the treatment may be aided by the oxygen supply retained within the SH surface.

Fluorescence-guided photoimmunotherapy using targeted nanotechnology and ML7710 to manage peritoneal carcinomatosis.

Fluorescence-guided intervention can bolster standard therapies by detecting and treating microscopic tumors before lethal recurrence. Tremendous progress in photoimmunotherapy and nanotechnology has been made to treat metastasis. However, many are lost in translation due to heterogeneous treatment effects. Here, we integrate three technological advances in targeted photo-activable multi-agent liposome (TPMAL), fluorescence-guided intervention, and laser endoscopy (ML7710) to improve photoimmunotherapy. TPMAL consists of a nanoliposome chemotherapy labeled with fluorophores for tracking and photosensitizer immunoconjugates for photoimmunotherapy. ML7710 is connected to Modulight Cloud to capture and analyze multispectral emission from TPMAL for fluorescence-guided drug delivery (FGDD) and fluorescence-guided light dosimetry (FGLD) in peritoneal carcinomatosis mouse models. FGDD revealed that TPMAL enhances drug delivery to metastases by 14-fold. ML7710 captured interpatient variability in TPMAL uptake and prompted FGLD in >50% of animals. By combining TPMAL, ML7710, and fluorescence-guided intervention, variation in treatment response was substantially reduced and tumor control improved without side effects.

Significant improvement of facial actinic keratoses after blue light photodynamic therapy with oral vitamin D pretreatment: An interventional cohort-controlled trial.

BACKGROUND: In mouse models of skin cancer, high-dose oral vitamin D3 (VD3; cholecalciferol) combined with photodynamic therapy (PDT) can improve the clearance of squamous precancers (actinic keratoses [AKs]). OBJECTIVE: To determine whether oral VD3 can improve the clinical efficacy of a painless PDT regimen in humans with AK. METHODS: The baseline lesion counts and serum 25-hydroxyvitamin D3 levels were determined. In group 1, 29 patients underwent gentle debridement and 15-minute aminolevulinic acid preincubation with blue light (30 minutes; 20 J/cm2). In group 2, 29 patients took oral VD3 (10,000 IU daily for 5 or 14 days) prior to debridement and PDT. Lesion clearance was assessed at 3 to 6 months. RESULTS: In group 1, the mean clearance rates of facial AK were lower in patients with VD3 deficiency (25-hydroxyvitamin D3 level < 31 ng/dL; clearance rate, 40.9% ± 42%) than in patients with normal 25-hydroxyvitamin D3 levels (62.6% ± 14.2%). High-dose VD3 supplementation (group 2) significantly improved the overall AK lesion response (72.5% ± 13.6%) compared with that in group 1 (54.4% ± 22.8%). No differences in side effects were noted. LIMITATIONS: Nonrandomized trial design (interventional cohort matched to registry-based controls). CONCLUSIONS: Oral VD3 pretreatment significantly improves AK clinical responses to PDT. The regimen appears promising and well tolerated.

Dual Function Antibody Conjugates for Multimodal Imaging and Photoimmunotherapy of Cancer Cells.

Precision imaging, utilizing molecular targeted agents, is an important tool in cancer diagnostics and guiding therapies. While there are limitations associated with single mode imaging probes, multimodal molecular imaging probes enabling target visualization through complementary imaging technologies provides an attractive alternative. However, there are several challenges associated with designing molecular probes carrying contrast agents for complementary multimodal imaging. Here, we propose a dual function antibody conjugate (DFAC) comprising an FDA approved photosensitizer Benzoporphyrin derivative (BPD) and a naphthalocyanine-based photoacoustic dye (SiNc(OH)) for multimodal infrared (IR) imaging. While fluorescence imaging, through BPD, provides sensitivity, complementing it with photoacoustic imaging, through SiNc(OH), provides a depth-resolved spatial resolution much beyond the optical diffusion limits of fluorescence measurements. Through a series of in vitro experiments, we demonstrate the development and utilization of DFACs for multimodal imaging and photodynamic treatment of squamous cell carcinoma (A431) cell line. The proposed DFACs have potential use in precision imaging applications such as guiding tumor resection surgeries and photodynamic treatment of residual microscopic disease thereby minimizing local recurrence. The data demonstrated in this study merits further investigation for its preclinical and clinical translation.

Vitamin D Receptor Activation and Photodynamic Priming Enables Durable Low-dose Chemotherapy.

Patients with cancer often confront the decision of whether to continue high-dose chemotherapy at the expense of cumulative toxicities. Reducing the dose of chemotherapy regimens while preserving efficacy is sorely needed to preserve the performance status of these vulnerable patients, yet has not been prioritized. Here, we introduce a dual pronged approach to modulate the microenvironment of desmoplastic pancreatic tumors and enable significant dose deescalation of the FDA-approved chemotherapeutic nanoliposomal irinotecan (nal-IRI) without compromising tumor control. We demonstrate that light-based photodynamic priming (PDP) coupled with vitamin D3 receptor (VDR) activation within fibroblasts increases intratumoral nal-IRI accumulation and suppresses protumorigenic CXCL12/CXCR7 crosstalk. Combined photodynamic and biochemical modulation of the tumor microenvironment enables a 75% dose reduction of nal-IRI while maintaining treatment efficacy, resulting in improved tolerability. Modifying the disease landscape to increase the susceptibility of cancer, via preferentially modulating fibroblasts, represents a promising and relatively underexplored strategy to enable dose deescalation. The approach presented here, using a combination of three clinically available therapies with nonoverlapping toxicities, can be rapidly translated with minimal modification to treatment workflow, and challenges the notion that significant improvements in chemotherapy efficacy can only be achieved at the expense of increased toxicity.

Breaking the selectivity-uptake trade-off of photoimmunoconjugates with nanoliposomal irinotecan for synergistic multi-tier cancer targeting.

BACKGROUND: Photoimmunotherapy involves targeted delivery of photosensitizers via an antibody conjugate (i.e., photoimmunoconjugate, PIC) followed by light activation for selective tumor killing. The trade-off between PIC selectivity and PIC uptake is a major drawback limiting the efficacy of photoimmunotherapy. Despite ample evidence showing that photoimmunotherapy is most effective when combined with chemotherapy, the design of nanocarriers to co-deliver PICs and chemotherapy drugs remains an unmet need. To overcome these challenges, we developed a novel photoimmunoconjugate-nanoliposome (PIC-Nal) comprising of three clinically used agents: anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody cetuximab (Cet), benzoporphyrin derivative (BPD) photosensitizer, and irinotecan (IRI) chemotherapy. RESULTS: The BPD photosensitizers were first tethered to Cet at a molar ratio of 6:1 using carbodiimide chemistry to form PICs. Conjugation of PICs onto nanoliposome irinotecan (Nal-IRI) was facilitated by copper-free click chemistry, which resulted in monodispersed PIC-Nal-IRI with an average size of 158.8 ± 15.6 nm. PIC-Nal-IRI is highly selective against EGFR-overexpressing epithelial ovarian cancer cells with 2- to 6-fold less accumulation in low EGFR expressing cells. Successful coupling of PIC onto Nal-IRI enhanced PIC uptake and photoimmunotherapy efficacy by up to 30% in OVCAR-5 cells. Furthermore, PIC-Nal-IRI synergistically reduced cancer viability via a unique three-way mechanism (i.e., EGFR downregulation, mitochondrial depolarization, and DNA damage). CONCLUSION: It is increasingly evident that the most effective therapies for cancer will involve combination treatments that target multiple non-overlapping pathways while minimizing side effects. Nanotechnology combined with photochemistry provides a unique opportunity to simultaneously deliver and activate multiple drugs that target all major regions of a cancer cell-plasma membrane, cytoplasm, and nucleus. PIC-Nal-IRI offers a promising strategy to overcome the selectivity-uptake trade-off, improve photoimmunotherapy efficacy, and enable multi-tier cancer targeting. Controllable drug compartmentalization, easy surface modification, and high clinical relevance collectively make PIC-Nal-IRI extremely valuable and merits further investigations in living animals.

Combination of oral vitamin D3 with photodynamic therapy enhances tumor cell death in a murine model of cutaneous squamous cell carcinoma.

Photodynamic therapy (PDT), in which 5-ALA (a precursor for protoporphyrin IX, PpIX) is administered prior to exposure to light, is a nonscarring treatment for skin cancers. However, for deep tumors, ALA-PDT is not always effective due to inadequate production of PpIX. We previously developed and reported a combination approach in which the active form of vitamin D3 (calcitriol) is given systemically prior to PDT to improve PpIX accumulation and to enhance PDT-induced tumor cell death; calcitriol, however, poses a risk of hypercalcemia. Here, we tested a possible strategy to circumvent the problem of hypercalcemia by substituting natural dietary vitamin D3 (cholecalciferol; D3 ) for calcitriol. Oral D3 supplementation (10 days of a 10-fold elevated D3 diet) enhanced PpIX levels 3- to 4-fold, and PDT-mediated cell death 20-fold, in subcutaneous A431 tumors. PpIX levels and cell viability in normal tissues were not affected. Hydroxylated metabolic forms of D3 were only modestly elevated in serum, indicating minimal hypercalcemic risk. These results show that brief oral administration of cholecalciferol can serve as a safe neoadjuvant to ALA-PDT. We suggest a clinical study, using oral vitamin D3 prior to PDT, should be considered to evaluate this promising new approach to treating human skin cancer.

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.

Development and applications of photo-triggered theranostic agents.

Theranostics, the fusion of therapy and diagnostics for optimizing efficacy and safety of therapeutic regimes, is a growing field that is paving the way towards the goal of personalized medicine for the benefit of patients. The use of light as a remote-activation mechanism for drug delivery has received increased attention due to its advantages in highly specific spatial and temporal control of compound release. Photo-triggered theranostic constructs could facilitate an entirely new category of clinical solutions which permit early recognition of the disease by enhancing contrast in various imaging modalities followed by the tailored guidance of therapy. Finally, such theranostic agents could aid imaging modalities in monitoring response to therapy. This article reviews recent developments in the use of light-triggered theranostic agents for simultaneous imaging and photoactivation of therapeutic agents. Specifically, we discuss recent developments in the use of theranostic agents for photodynamic-, photothermal- or photo-triggered chemotherapy for several diseases.

A mechanism-based combination therapy reduces local tumor growth and metastasis in an orthotopic model of prostate cancer.

Therapy-induced stimulation of angiogenic molecules can promote tumor angiogenesis leading to enhanced tumor growth and cancer metastasis. Several standard and emerging therapies, such as radiation and photodynamic therapy (PDT), can induce angiogenic molecules, thus limiting their effectiveness. PDT is approved for the treatment of several cancers; however, its induction of vascular endothelial growth factor (VEGF) creates conditions favorable to enhanced tumor growth and metastasis, therefore mitigating its cytotoxic and antivascular effects. This is the first report showing that subcurative PDT in an orthotopic model of prostate cancer (LNCaP) increases not only VEGF secretion (2.1-fold) but also the fraction of animals with lymph node metastases. PDT followed by administration of an antiangiogenic agent, TNP-470, abolished this increase and reduced local tumor growth. On the other hand, administration of TNP-470 before PDT was less effective at local tumor control. In addition, animals in all groups, except in the PDT + TNP-470 group, had a weight loss of >3 g at the time of sacrifice; the weight of the animals in the PDT + TNP-470 group did not change. The significant reduction (P < 0.05) in tumor weight and volume observed between the PDT + TNP-470 group and the control group suggests that the combination of PDT and antiangiogenic treatment administered in the appropriate sequence was not only more effective at controlling local tumor growth and metastases but also reduced disease-related toxicities. Such molecular response-based combinations merit further investigations as they enhance both monotherapies and lead to improved treatment outcomes.

Tumor vascular permeabilization by vascular-targeting photosensitization: effects, mechanism, and therapeutic implications.

PURPOSE: Loss of vascular barrier function has been observed shortly following vascular-targeting photodynamic therapy. However, the mechanism involved in this event is still not clear, and the therapeutic implications associated with this pathophysiologic change have not been fully explored. EXPERIMENTAL DESIGN: The effect of vascular-targeting photodynamic therapy on vascular barrier function was examined in both s.c. and orthotopic MatLyLu rat prostate tumor models and endothelial cells in vitro, using photosensitizer verteporfin. Vascular permeability to macromolecules (Evans blue-albumin and high molecular weight dextran) was assessed with dye extraction (ex vivo) and intravital microscopy (in vivo) methods. Intravital microscopy was also used to monitor tumor vascular functional changes after vascular-targeting photodynamic therapy. The effects of photosensitization on monolayer endothelial cell morphology and cytoskeleton structures were studied with immunofluorescence staining. RESULTS: Vascular-targeting photodynamic therapy induced vascular barrier dysfunction in the MatLyLu tumors. Thus, tumor uptake of macromolecules was significantly increased following photodynamic therapy treatments. In addition to vascular permeability increase, blood cell adherence to vessel wall was observed shortly after treatment, further suggesting the loss of endothelial integrity. Blood cell adhesion led to the formation of thrombi that can occlude blood vessels, causing vascular shutdown. However, viable tumor cells were often detected at tumor periphery after vascular-targeting photodynamic therapy. Endothelial cell barrier dysfunction following photodynamic therapy treatment was also observed in vitro by culturing monolayer endothelial cells on Transwell inserts. Immunofluorescence study revealed microtubule depolymerization shortly after photosensitization treatment and stress actin fiber formation thereafter. Consequently, endothelial cells were found to retract, and this endothelial morphologic change led to the formation of intercellular gaps. CONCLUSIONS: Vascular-targeting photodynamic therapy permeabilizes blood vessels through the formation of endothelial intercellular gaps, which are likely induced via endothelial cell microtubule depolymerization following vascular photosensitization. Loss of endothelial barrier function can ultimately lead to tumor vascular shutdown and has significant implications in drug transport and tumor cell metastasis.

Synergism of epidermal growth factor receptor-targeted immunotherapy with photodynamic treatment of ovarian cancer in vivo.

BACKGROUND: Epithelial ovarian cancer often develops resistance to standard treatments, which is a major reason for the high mortality associated with the disease. We examined the efficacy of a treatment regimen that combines immunotherapy to block the activity of epidermal growth factor receptor (EGFR), overexpression of which is associated with the development of resistant ovarian cancer, and photodynamic therapy (PDT), a mechanistically distinct photochemistry-based modality that is effective against chemo- and radioresistant ovarian tumors. METHODS: We tested a combination regimen consisting of C225, a monoclonal antibody that inhibits the receptor tyrosine kinase activity of EGFR, and benzoporphyrin derivative monoacid A (BPD)-based PDT in a mouse model of human ovarian cancer. Therapeutic efficacy was evaluated in acute treatment response and survival studies that used 9-19 mice per group. Analysis of variance and Wilcoxon statistics were used to analyze the data. All statistical tests were two-sided. RESULTS: Mice treated with PDT + C225 had the lowest mean tumor burden compared with that in the no-treatment control mice (mean percent tumor burden = 9.8%, 95% confidence interval [CI] = 2.3% to 17.3%, P < .001). Mean percent tumor burden for mice treated with C225 only or PDT only was 66.6% (95% CI = 58.7% to 74.4%, P < .001) and 38.2% (95% CI = 29.3% to 47.0%, P < .001), respectively. When compared with PDT only or C225 only, PDT + C225 produced synergistic reductions in mean tumor burden (P < .001, analysis of variance) and improvements in survival (P = .0269, Wilcoxon test). Median survival was approximately threefold greater for mice in the PDT + C225 group than for mice in the no-treatment control group (80 days versus 28 days), and more mice in the PDT + C225 group were alive at 180 days (3/9; 33% [95% CI = 7% to 70%]) than mice in the C225-only (0/12; 0% [95% CI = 0% to 22%]) or PDT-only (1/10; 10% [95% CI = 0.2% to 44%]) groups. CONCLUSION: A mechanistically nonoverlapping combination modality consisting of receptor tyrosine kinase inhibition with C225 and BPD-PDT is well tolerated, effective, and synergistic in mice.

Detection of female lower genital tract dysplasia using orally administered 5-aminolevulinic acid induced protoporphyrin IX: a preliminary study.

OBJECTIVE: Previous studies have suggested that 5-aminolevulinic acid (ALA) may be used topically on the cervix to allow optical detection of cervical dysplasia, based on the fluorescence of protoporphyrin IX (PpIX) synthesized in situ from ALA. However, the uniformity of distribution of topically applied PpIX and the sensitivity and specificity of detection are not optimal. The current study was undertaken to demonstrate the feasibility of administering ALA by mouth (po) with the hypothesis that systemic administration might provide a more reliable diagnostic tool. METHODS: Oral ALA was administered to 14 patients with abnormal Pap smears in a dose- and time-intensity design. Institutional review board approval was obtained. A starting dose of 10 mg/kg of po ALA was administered and colposcopy was performed in 3 patients at 1 h, 3 patients at 2 h, 6 patients at 3 h, and 2 patients at 4 h. The study was written with the intent to increase the dose in 10 mg/kg increments if fluorescence was not detected; however, fluorescence was detected at the first dose level. Liver function tests were checked pre and post ALA and follow-up telephone calls were made regarding possible side effects. Both white and blue light colposcopy examinations were performed by two blinded clinicians and biopsies of all abnormal areas were performed. RESULTS: All patients tolerated po ALA well, with no systemic side effects. At the 10 mg/kg dose there was no reported nausea or photosensitivity. Optimal fluorescence was achieved at the 3-h time point, with quenching noted at the 4-h time point. Excellent absorption was documented with fluorescence of the lip demonstrated with Wood's lamp. In some cases fluorescence correlated with dysplasia on biopsy. CONCLUSION: 5-ALA given via the po route and at the dose and time period studied is well tolerated and affords fluorescence of the cervix. Future study is needed to demonstrate the successful identification of dysplastic lesions, with the ultimate goal of treating dysplasia of the lower genital tract with 5-ALA and light therapy.