Ablation of Hypoxic Tumors with Dose-Equivalent Photothermal, but Not Photodynamic, Therapy Using a Nanostructured Porphyrin Assembly ^ ) Cheng S
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ARTICLE Ablation of Hypoxic Tumors with Dose-Equivalent Photothermal, but Not Photodynamic, Therapy Using a Nanostructured Porphyrin Assembly ^ ) Cheng S. Jin,†,‡,§ Jonathan F. Lovell,§, Juan Chen,† and Gang Zheng†,‡,§, ,* †Ontario Cancer Institute, Campbell Family Cancer Research Institute and Techna Institute, University Health Network (UHN), Toronto, Canada M5G 2M9, ‡Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Canada M5S 3M2, §Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada M5S 1A1, ^Department of Biomedical Engineering, University at Buffalo, State University of New York, Buffalo, New York 14260-2050, United States, and Department) of Medical Biophysics, University of Toronto, Toronto, Canada M5G 1L7 ABSTRACT Tumor hypoxia is increasingly being recognized as a characteristic feature of solid tumors and significantly complicates many treatments based on radio-, chemo-, and phototherapies. While photodynamic therapy (PDT) is based on photosensitizer interactions with diffused oxygen, photothermal therapy (PTT) has emerged as a new phototherapy that is predicted to be independent of oxygen levels within tumors. It has been challenging to mean- ingfully compare these two modalities due to differences in contrast agents and irradiation parameters, and no comparative in vivo studies have been performed until now. Here, by making use of recently developed nanostructured self-quenched porphysome nanoparticles, we were able to directly compare PDT and PTT using matched light doses and matched porphyrin photosensitizer doses (with the photosensitizer being effective for either PTT or PDT based on the existence of nanostructure or not). Therefore, we demonstrated the nanostructure-driven conversion from the PDT singlet oxygen generating mechanism of porphyrin to a completely thermal mechanism, ideal for PTT enhancement. Using a novel hypoxia tumor model, we determined that nanostructured porphyrin PTT enhancers are advantageous to overcome hypoxic conditions to achieve effective ablation of solid tumors. KEYWORDS: tumor hypoxia . nanoparticle . photothermal therapy . photodynamic therapy . porphyrin . porphysome umor hypoxia, the condition in which with porphyrin or porphyrin analogues of- Ttumor cells persist in a low oxygen ten used as photosensitizers, among which environment, is a common feature of Photofrin remains the gold standard PDT solid tumors. It poses a major therapeutic agent in clinical practice.14 Thus, PDT re- problem, as hypoxia-induced cellular changes quires synchronizing interactions among can result in more clinically aggressive light, photosensitizers, and oxygen to be phenotypes,1À5 and moreover, the reduced effective.15 However, the oxygen-dependent ff partial O2 pressure in tumor tissue creates an nature of PDT limits its e ectiveness in hypoxic obstacle for numerous cancer therapies, in- tumors, and when partial oxygen pressure ffi cluding ionizing radiotherapy,6,7 certain types (pO2)isbelow40mmHg,PDTecacy 10,16,17 of chemotherapy,7À9 and photodynamic ther- decreases. In addition, both photosensi- tizer-mediated oxygen consumption and apy (PDT).10,11 * Address correspondence to blood vessel damage during PDT further po- [email protected]. PDT is a form of phototherapy that uses photosensitizers that are exposed selec- tentiate tumor hypoxia, impeding therapeutic 10,12,18,19 Received for review December 19, 2012 tively to light, whereupon they produce outcomes. Thus, an oxygen-indepen- and accepted February 10, 2013. dent phototherapy such as photothermal ther- highly reactive oxygen species through Published online 12,13 apy (PTT) could be a useful alternative for either type I or type II reactions, result- 10.1021/nn3058642 ing in toxicity to targeted tissues. Classical treating hypoxic tumors. Upon selective light PDT mostly follows the type II mechanism treatment, instead of production of reactive C XXXX American Chemical Society JIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX A www.acsnano.org ARTICLE oxygen species, PTT agents absorb light and dissipate tumor model to investigate the advantages of PTT for the absorbed energy through nonradiative decay treatment of hypoxic tumors. The study rationale and (heating), which induces a temperature increase in design are schematically illustrated in Figure 1. the local treatment environment resulting in irreversi- RESULTS ble cell damage.20,21 Many light-absorbing species have been investi- Acute Hyperoxia and Hypoxia. We developed a multi- gated as effective PTT agents. Most of the PTT agents pronged approach to generate tumor hypoxia and developed to date are based on inorganic nanomater- hyperoxia in vivo. In general, partial oxygen pressure 22À24 ials including gold nanoparticles (e.g., nanocages, (pO2) of the tumor could be modified by exposing mice 25,26 27,28 29,30 nanorods, nanoshells, nanospheres ) and to different inhalation concentrations of O2. To inves- À carbon nanotubes.31 34 They are efficient in photon- tigate PDT and PTT in tumor hyperoxia and hypoxia to-thermal conversion due to the surface plasmon conditions, mice were provided inhaled oxygen con- resonance (SPR) oscillation and high absorption cross centrations of 100 and 7%, respectively, prior to and section of NIR light.35,36 Many chromophores have also during laser irradiation (Figure 2a). To further generate been developed as PTT agents since the 1980s,37 such acute hypoxia conditions, a tourniquet was clamped À as endogenous chromophores in tissue38 40 externally onto one leg bearing a xenograft tumor in conjunction 41,42 added dyes such as indocyanine green, naphthal- with 7% O2 to ensure low oxygen levels at the tumor. ocyanine,43,44 and transition-metal-coordinated Mice under hyperoxia conditions behaved normally porphyrins.37,45 However, PTT capability of monomeric during inspiration of 100% oxygen for 30 min. Their chromophores is limited by their relatively low light arterial partial oxygen pressure was 513.2 ( 67.1 À À absorption (ε =104À106 M 1 cm 1) compared to gold mmHg (Figure S1 in Supporting Information), and nanoparticles with extinction coefficients orders of hemoglobin was highly saturated with O2 measured À À magnitude higher (ε =109À1011 M 1 cm 1).36,44,46 by photoacoustic (PA) imaging to be 99.9 ( 0% Porphyrins have also been conjugated to gold nano- (Figure 2b and Figure S2 in Supporting Information). particles or carbon nanotubes for enhanced light en- Mice in the hypoxia group breathing 7% O2 exhibited a ergy conversion and combination of photodynamic faster breathing rate and reduced movement. Partial À and photothermal therapy,47 49 but only recently, oxygen pressure in artery blood circulation was re- porphyrin-based nanoparticles, termed porphysomes, duced to 37.0 ( 4.3 mmHg (Figure S1 in Supporting have been developed to be the first organic PTT agents Information). In addition, real-time PA imaging showed with comparable optical absoprtions to gold nanopar- that the arterial oxyhemoglobin saturation rate of the ticles for high photothermal efficiency.50 The liposomal mice decreased rapidly in the first 10 s upon exposure nanostructure of porphyrin lipids provides additional to 7% oxygen, reaching 75% after 1 min. Low oxygen advantages over inorganic PTT agents such as biocom- conditions were maintained, and the arterial oxyhe- patibility and biodegradability.51 moglobin saturation rate was kept around 74.7 ( 3.4% Porphysomes are self-assembled from porphyrin during the following 30 min using hypoxic gas expo- lipid into liposome-like nanoparticles (∼100 nm sure together with the leg tourniquet (Figure 2b and diameter). The porphyrin packing density per particle Figure S2 in Supporting Information). We further eval- is high (>80 000 per particle), so they absorb light with uated hypoxic conditions in the tumor by HIF-1R extremely high efficiency. As the packing density also staining assay. HIF-1R is a heterodimeric transcription induced highly self-quenching porphyrin excited factor that plays a critical role in the cellular response to states, the absorbed energy is released as heat, provid- hypoxia.52 As shown in Figure 2c, the nuclei of tumor ing exceptional properties as PTT agents. Therefore, cells of the hypoxia group stained dark brown, indicat- unlike monomeric porphyrins, the unique nanoassem- ing the nuclear accumulation of HIF-1R under hypoxic bly of porphyrin lipids results in the conversion of the conditions, while the nuclei of hyperoxic tumor cells porphyrin photosensitizers from a singlet oxygen gen- remained blue, indicating HIF-1R was not detected. erating mechanism, useful for PDT, to a thermal me- Both the in vivo measurement of partial oxygen pres- chanism, ideal for PTT enhancement. This creates a sure arterial oxyhemoglobin saturation rate and ex vivo unique opportunity for the first time to compare, HIF-1R staining confirmed that, by combining con- in vivo, the conversion of porphyrins from PDT photo- trolled inhalation gases (100 or 7% oxygen) together sensitizers to PTT transducers, by comparing porphyrin with tourniquet-based blood restriction, biologically monomers to porphyrin nanoparticles. relevant acute hyperoxic and hypoxic tumor condi- In this study, we evaluated the nanostructure-driven tions could be generated. conversion of the mechanism of PDT activation of Drug Dark Toxicity. To keep the drug accumulation porphyrin to PTT activation by in vivo studies, comparing consistent for treatment in hyperoxic and hypoxic the PDT