Photodynamic drug delivery enhancement in tumours does not depend on leukocyte-endothelial interaction in a human mesothelioma xenograft model†

OBJECTIVES The pre-treatment of tumour neovessels by low-level photodynamic therapy (PDT) improves the distribution of concomitantly administered systemic chemotherapy. The mechanism by which PDT permeabilizes the tumour vessel wall is only partially known. We have recently shown that leukocyte-endothelial cell interaction is essential for photodynamic drug delivery to normal tissue. The present study investigates whether PDT enhances drug delivery in malignant mesothelioma and whether it involves comparable mechanisms of actions. METHODS Human mesothelioma xenografts (H-meso-1) were grown in the dorsal skinfold chambers of 28 nude mice. By intravital microscopy, the rolling and recruitment of leukocytes were assessed in tumour vessels following PDT (Visudyne® 400μg/kg, fluence rate 200mW/cm2and fluence 60J/cm2) using intravital microscopy. Likewise, the distribution of fluorescently labelled macromolecular dextran (FITC-dextran, MW 2000kDa) was determined after PDT. Study groups included no PDT, PDT, PDT plus a functionally blocking anti-pan-selectin antibody cocktail and PDT plus isotype control antibody. RESULTS PDT significantly enhanced the extravascular accumulation of FITC-dextran in mesothelioma xenografts, but not in normal tissue. PDT significantly increased leukocyte-endothelial cell interaction in tumour. While PDT-induced leukocyte recruitment was significantly blunted by the anti-pan-selectin antibodies in the tumour xenograft, this manipulation did not affect the PDT-induced extravasation of FITC-dextran. CONCLUSIONS Low-level PDT pre-treatment selectively enhances the uptake of systemically circulating macromolecular drugs in malignant mesothelioma, but not in normal tissue. Leukocyte-endothelial cell interaction is not required for PDT-induced drug delivery to malignant mesotheliom

Characterization and therapeutic exploitation of the vascular permeability induced by photodynamic therapy

Photodynamic therapy (PDT) can be used to cause vascular collapse and blood flow stasis of the irradiated pathological neovascularisation appearing in several diseases such as age-related macular degeneration (AMD) and cancer. We hypothesized that this PDT-related interruption of vessel integrity may lead to an increased transvascular passage of drugs that could be used as a drug delivery pathway. Thus, preceding the occlusion of the pathological vasculature, PDT could be used for instance as a local drug delivery pathway to administrate an anti-angiogenic drug in the case of AMD or chemotherapy in the case of cancer, potentially improving combination therapies. In the case of chorioneovessels (CNV) due to AMD, the recurrence of the exudative AMD component of the weeks/months after PDT, due to the re-opening and/or re-growth of neovessels might be avoided by adding an anti-angiogenic factor such as anti-VEGF or anti-inflammatory drug before, during or shortly after PDT. In the case of cancer, the starvation of tumour cells induced by the PDT occlusion of blood vessels feeding the tumour might be combined with a chemotherapeutic agent for the direct kill of the cancer cells themselves. It has been reported that following the light application in PDT, a physiological cascade of responses on the one hand leads to vascular occlusion but may also induce a vascular permeability enhancement. The aim of this thesis is to find conditions where this increase in leakage due to PDT can be observed, to characterize it and to take advantage of this phenomenon to develop the basis of a novel combination therapy approach. Hence in this thesis, pre-clinical experiments were performed in the vasculature of the chorioallantoic membrane model (CAM) of the chicken embryo and in the dorsal skinfold optical chamber of nude mice observed by intravital microscopy (IVM). In the CAM, no PDT-induced leakage of a fluorescent dye (FITC-dextran) was observed unless an anti-aggregating factor, such as aspirin was added. In the chicken embryo model, delaying the blood clot appears to be an essential process to allow effective potential drug delivery. In the dorsal skinfold of the nude mouse, the inflammatory response after PDT was observed and quantified. This revealed that PDT induces a time dependent acute inflammatory response as shown by increased number of leukocytes "rolling" along the vessel wall after treatment. This was observed over a 2 hour period following PDT. The quantification of the microvascular leakage showed a continuous FITC-dextran leakage from the vasculature treated by PDT to the interstitial space. This local leakage was clearly increased by the inflammatory status of the tissue (observed by quantifying the rolling leukocytes and by histology). This concept has the potential to improve the drug delivery of anti-angiogenic drugs in the eyes of patients treated for AMD and could also be applied to improve the uptake of cytostatic drugs in tumours

Combination Therapy Using Verteporfin and Ranibizumab; Optimizing the Timing in the CAM Model

Combining photodynamic therapy (PDT) using verteporfin (Visudyne(R)) with ranibizumab (Lucentis(R)) can optimize the overall treatment outcome by providing more efficacy in vessel closure, and thus reduce the need for retreatment in patients with wet age-related macular degeneration. In this preclinical study in the chorioallantoic membrane (CAM) of the chicken embryo, we compare the vascular occlusion effects of verteporfin and ranibizumab as monotherapies with those observed in the combined therapy. In order to optimize the combination therapy, we varied the timing and sequence of the PDT and antivascular endothelial growth factor modalities. We observed that 1 day after PDT, the smaller blood vessels (null set < 70 mu m) of the CAM were selectively occluded, but as early as 2 days after PDT, both significant reperfusion and regrowth of new vessels were observed. Both these phenomena could be significantly delayed by application of ranibizumab. Ranibizumab itself did not induce any vascular occlusion. Under the applied conditions of combination therapy, the occlusion of the targeted blood vessels could be significantly extended to 3 days in this model compared with 1 day in the case of verteporfin monotherapy. Thus, in the present preclinical study, we demonstrate that for the applied conditions, the optimal time to administer ranibizumab is 24 h after PDT

Effect of nanoparticle size on the extravasation and the photothrombic activity of meso(p-tetracarboxyphenyl)porphyrin

Particle size should be optimized to achieve targeted and extended drug delivery to the affected tissues. We describe here the effects of the mean particle size on the pharmacokinetics and photothrombic activity of meso-tetra(carboxyphenyl)porphyrin (TCPP), which is encapsulated into biodegradable nanoparticles based on poly(d,l-lactic acid). Four batches of nanoparticles with different mean sizes ranging from 121 to 343 nm, were prepared using the emulsification-diffusion technique. The extravasations of each TCPP-loaded nanoparticle formulation from blood vessels were measured, as well as the extent of photochemically induced vascular occlusion. These preclinical tests were carried out in the chorioallantoic membrane (CAM) of the chicken's embryo. Fluorescence microscopy showed that both the effective leakage of TCPP from the CAM blood vessels and its photothrombic efficiency were dependent on the size of the nanoparticle drug carrier. Indeed, the TCPP fluorescence contrast between the blood vessels and the surrounding tissue increased at the applied conditions, when the particle size decreased. This suggests that large nanoparticles are more rapidly eliminated from the bloodstream. In addition, after injection of a drug dose of 1mg/kg body weight and a drug-light application interval of 1 min, irradiation with a fluence of 10J/cm(2) showed that the extent of vascular damage gradually decreased when the particle size increased. The highest photothrombic efficiency was observed when using the TCPP-loaded nanoparticles batch with a mean diameter of 121 nm. Thus, in this range of applied conditions, for the treatment of for instance a disease like choroidal neovascularization (CNV) associated with age-related macular degeneration (AMD), these experiments suggest that the smallest nanoparticles may be considered as the optimal formulation since they exhibited the greatest extent of vascular thrombosis as well as the lowest extravasation

Improved photodynamic activity of porphyrin loaded into nanoparticles: an in vivo evaluation using chick embryos

Hydrophobic porphyrins are potentially interesting molecules for the photodynamic therapy (PDT) of solid cancers or ocular vascularization diseases. Their pharmaceutical development is, however, hampered by their lipophilicity, which renders formulation difficult especially when intravenous administration is needed. Encapsulation of a lipophilic derivative of porphyrin, the meso-tetra(p-hydroxyphenyl)porphyrin (p-THPP), into polymeric biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles proved to enhance its photodynamic activity against mammary tumour cells when compared to free drug. In order to further investigate these carriers, the efficacy of the encapsulated drug was assessed on the chick embryo chorioallantoic membrane (CAM) model. First, we identified a suitable solvent for the drug in terms of p-THPP solubility and tolerability by chick embryos. This solution was used as a reference. Then, the fluorescence pharmacokinetics and the photodynamic effects of the porphyrin on CAM vessels were evaluated after intravenous administration of either a p-THPP solution (free drug) or the drug loaded into nanoparticles. The results showed that: (i) the drug remained longer in the vascular compartment when incorporated into nanoparticles and (ii) vascular effects of p-THPP after light irradiation were enhanced with nanoparticle carriers. These results are discussed taking into account the extravasation of intravascular circulating photosensitizers and its influence on PDT performance

Treatment of pleural malignancies by photo-induction combined to systemic chemotherapy: Proof of concept on rodent lung tumors and feasibility study on porcine chest cavities

Background: Low-dose, Visudyne (R)-mediated photodynamic therapy (photo-induction) was shown to selectively enhance tumor vessel transport causing increased uptake of systemically administered chemotherapy in various tumor types grown on rodent lungs. The present experiments explore the efficacy of photo-induced vessel modulation combined to intravenous (IV) liposomal cisplatin (Lipoplatin (R)) on rodent lung tumors and the feasibility/toxicity of this approach in porcine chest cavities. Material and Methods: Three groups of Fischer rats underwent orthotopic sarcoma (n = 14), mesothelioma (n = 14), or adenocarcinoma (n = 12) implantation on the left lung. Half of the animals of each group had photoinduction (0.0625 mg/kg Visudyne (R), 10 J/cm(2)) followed by IV administration of Lipoplatin (R) w (5 mg/kg) and the other half received Lipoplatin (R) without photo-induction. Then, two groups of minipigs underwent intrapleural thoracoscopic (VATS) photo-induction (0.0625 mg/kg Visudyne (R); 30 J/cm(2) hilum; 10 J/cm(2) apex/diaphragm) with in situ light dosimetry in combination with IV Lipoplatin (R) administration (5 mg/kg). Protocol I (n = 6) received Lipoplatin (R) immediately after light delivery and Protocol II (n = 9) 90 minutes before light delivery. Three additional animals received Lipoplatin (R) and VATS pleural biopsies but no photo-induction (controls). Lipoplatin (R) concentrations were analyzed in blood and tissues before and at regular intervals after photo-induction using inductively coupled plasma mass spectrometry. Results: Photo-induction selectively increased Lipoplatin (R) uptake in all orthotopic tumors. It significantly increased the ratio of tumor to lung Lipoplatin (R) concentration in sarcoma (P = 0.0008) and adenocarcinoma (P = 0.01) but not in mesothelioma, compared to IV drug application alone. In minipigs, intrapleural photo-induction combined to systemic Lipoplatin (R) was well tolerated with no toxicity at 7 days for both treatment protocols. The pleural Lipoplatin (R) concentrations were not significantly different at 10 and 30 J/cm(2) locations but they were significantly higher in protocol I compared to II (2.37 +/- 0.7 vs. 1.37 +/- 0.7 ng/mg, P < 0.001). Conclusion: Visudyne (R)-mediated photo-induction selectively enhances the uptake of IV administered Lipoplatin (R) in rodent lung tumors. Intrapleural VATS photo-induction with identical treatment conditions combined to IV Lipoplatin chemotherapy is feasible and well tolerated in a porcine model. Lasers Surg. Med. 47:807-816, 2015. (C) 2015 Wiley Periodicals, Inc