Research Article www.acsami.org
Design of Pluronic-Based Formulation for Enhanced RedaporfinPhotodynamic Therapy against Pigmented Melanoma Barbara Pucelik,† Luis G. Arnaut,§ Grazẏ na Stochel,† and Janusz M. Dąbrowski*,† †
Faculty of Chemistry, Jagiellonian University, 30-060 Kraków, Poland CQC, Chemistry Department, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal
§
S Supporting Information *
ABSTRACT: The therapeutic outcome of photodynamic therapy (PDT) with redaporfin (a fluorinated sulfonamide bacteriochlorin, F2BMet or LUZ11) was improved using Pluronic-based (P123, F127) formulations. Neither redaporfin encapsulated in Pluronic nor micelles alone exhibited cytotoxicity in a broad concentration range. Comprehensive in vitro studies against B16F10 melanoma cells showed that redaporfin-P123 micelles enhanced cellular uptake and increased oxidative stress compared with redaporfin-F127 or photosensitizer alone after short incubation times. ROSsensitive fluorescent probes showed that the increased oxidative stress is due, at least in part, to a more efficient formation of hydroxyl radicals, and causes strong light-dose dependent apoptosis and necrosis. Tissue distribution and pharmacokinetic studies in tumor-bearing mice show that the Pluronic P123 formulation of redaporfin increases its bioavailability as well as the tumor-to-muscle and tumor-to-skin ratios, in comparison with Cremophor EL and Pluronic F127 formulations. Redaporfin in P123 was most successful in the PDT of C57BL/6J mice bearing subcutaneously implanted B16F10 melanoma tumors. Vascular-targeted PDT combining 1.5 mg kg−1 redaporfin in P123 with a light dose of 74 J cm−2 led to 100% complete cures (i.e., no tumor regrowth over one year posttreatment). This remarkable result reveals that modification of redaporfin with Pluronic block copolymers overcomes the resistance of melanoma cells to PDT possibly via increased tumor selectivity and enhanced ROS generation. KEYWORDS: bacteriochlorins, cancer, delivery vehicle, melanoma, photodynamic therapy, targeting
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INTRODUCTION Malignant melanoma is a skin cancer characterized by high aggressiveness and significant resistance to various therapeutic approaches. Important advances were made in understanding the risk factors as well as in the epigenetic background of melanoma, but the prognosis of successful treatment and cures of metastatic melanoma remain rather disappointing.1 The inherent resistance of melanoma to traditional forms of chemotherapy and radiotherapy motivated the development of alternative immune-based strategies. Promising preclinical and clinical results geared attention to immunotherapy using adjuvants such as interleukin-2 (IL-2), humanized monoclonal antibodies directed against CTLA (Ipilimumab) and PD-1 (Pembrolizumab, Nivolumab), or high dose interferon alpha-2b (INTRON A), and immunotherapy is now the one of most auspicious FDA-approved treatment of melanoma.2,3 Although traditional treatments as well as innovative immunotherapy fostered significant clinical advances, the heterogeneity at the cellular and molecular levels together with highly metastatic character of malignant melanoma, limit to subsets of patients the response to immune checkpoint blockade, and the overall therapeutic benefit remains unsatisfactory. In view of the complex nature of this disease, combination therapies acting via inter-related, but different mechanisms, involving signaling © 2016 American Chemical Society
pathways of cancer as well as tumor specific properties are likely to offer additional benefits.4 Photodynamic therapy (PDT) has emerged as a promising, clinically approved strategy in oncology. PDT offers various advantages over alternative strategies, such as the specific targeting of tissues, and the possibility of combination with other therapies.5−8 Its multiple approaches to optimize therapeutic regimens and the targeting of tumor vasculature may be especially useful in the treatment of melanoma.9−13 Relevant parameters to optimize PDT are drug and light doses (in mg·kg−1 BW and J, respectively), the drug-to-light interval (DLI, in units of time) between the administration of the drug and the illumination of the target tissues, the radiant exposure (in J cm−2), and the irradiance (in W cm−2).7 However, high melanin levels may act as an optical shield and as antioxidants that might result in reduced PDT efficacy in the treatment of melanoma.12 PDT photosensitizers and protocols need to be carefully optimized in order to overcome these challenges. The treatment of melanoma requires very phototoxic photosensitizers that absorb light at long wavelengths (700−800 Received: June 11, 2016 Accepted: August 5, 2016 Published: August 5, 2016 22039
DOI: 10.1021/acsami.6b07031 ACS Appl. Mater. Interfaces 2016, 8, 22039−22055
Research Article
ACS Applied Materials & Interfaces
delivery of photosensitizers such as hydrophobic porphyrins, chlorins or phthalocyanines.33−35 Such photosensitizers readily aggregate in water, hampering the administration of the drug in vivo, and diminishing their PDT efficacy.36,37 For instance, Park reported that conjugation of the chlorin e6 with Pluronic F127 enhanced tumor-specific distribution and, after PDT, inhibited CT26 tumor growth in athymic nude mice.38 Dolphin reported a better delivery to lipoproteins and a higher PDT efficacy of benzoporphyrin derivative monoacid ring A (BPD-MA) toward M1 rhabdomyosarcoma when BPD-MA was formulated with Pluronic P123 rather than with other lipid-based formulations.39 It was also demonstrated that Pluronic P123 is able to solubilize ring-B BPD as monomers in aqueous media, which are difficult to solubilize in liposome formulations.24,36 Pellosi et al. reported P123/F127 formulations of both ring-A and ring-B BPDs in an attempt to enhance photophysical properties (e.g., singlet oxygen quantum yields) and stability in aqueous solutions, and showed that such formulations did not compromise in vitro PDT efficacy against HeLa and A549 cancer cells.40 In summary, it can be expected that Pluronicsbased formulations may overcome challenges associated with the delivery of hydrophobic photosensitizers, such as poor solubility, cellular internalization, and tumor targeting. This work explores the incorporation of redaporfin (also referred by the code names F2BMet and LUZ11) in Pluronic micelles. Redaporfin exhibits high PDT efficacy in the treatment of mice with subcutaneously implanted CT26 colon carcinoma41−44 and light-pigmented S91 melanoma tumors,10 and is currently in Phase II clinical trials for head and neck cancer. Redaporfin is characterized by an intense absorption of light ca. 750 nm and by the ability to generate ROS via Type II (energy transfer) and Type I (electron transfer) processes with efficient formation of singlet oxygen, superoxide ion and hydroxyl radicals, respectively.8,41−44 Redaporfin is practically insoluble in water and has a noctanol:water partition coefficient POW = 80. The formulation used in clinical trials is a redaporfin concentrated solution in a 1:5 mixture of CrEL and ethanol, which is further diluted in saline before administration. The possible hypersensitivity reactions are countered by premedication. Cremophor micellar formulations were shown to be most effective for PDT with short DLI,43,44 and redaporfin-PDT was also shown to be most efficient with DLI = 15 min. It is very challenging to increase the redaporfin concentration in the tumor just 15 min after the intravenous (i.v.) drug administration above the level already attained with CrEL formulations. The design of new micellar formulations for redaporfin was motivated by the clinical value of improving tumor targeting at very short DLI. This work shows that it is possible to obtain safer and more efficient redaporfin-PDT treatments using a Pluronic P123 formulation with appropriate micellar size, high loading of nonaggregated redaporfin molecules, capable of increasing the photostability of redaporfin while increasing also the generation of hydroxyl radicals, with enhanced redaporfin release at low pH and rapid internalization by cells. This formulation significantly increases the concentration of redaporfin in the tumor at DLI = 15 min and leads to an increase in redaporfin-PDT efficacy that is manifested in the complete cure of mice with subcutaneously implanted B16F10 melanoma tumors. The animals have been followed for over one year after the treatment, and there are no signs of tumor regrowth. This unprecedented success in the treatment of a very challenging tumor model is related to precise engineering of the treatment protocol together with that
nm), where melanin is transparent and the energy of light is still sufficient to generate reactive oxygen species (ROS) such as singlet oxygen or superoxide ion.9,11−13 Moreover, PDT should avoid defects in apoptotic pathways and the efflux of photosensitizer molecules by ATP binding cassette transporters. Although much recent work focused on developing several new strategies to improve the performance of PDT, the specificities of melanoma have not been satisfactorily overcome.13 Nanotechnology offers countless opportunities for improving the properties of drugs or photosensitizers, as well as diagnostic, therapeutic, and theranostic agents in general.14−18 Tunable nanosized formulations such as polymeric micelles, nanoparticles, or emulsions can be used to optimize drug pharmacokinetics and pharmacodynamics.19−21 Interesting examples of such materials are Pluronic copolymers, which are surfactant molecules containing two hydrophilic poly(ethylene oxide) (PEO) and one hydrophobic poly(propylene oxide) (PPO) regions arranged in a PEO−PPO−PEO triblock structure. Pluronics are capable of self-assembly in water as nanosized core−shell micelles above their critical micelle concentration (CMC). Generally the PPO segment is confined in the hydrophobic micelle core in which the methyl groups are thought to interact with solubilized drug via van der Waals interactions, while the two PEO blocks face out toward the aqueous media forming the micelle corona (shell) that confers water solubility through hydrogen bonding between ether oxygen and water molecules.22−25 Although the PPO units have the tendency to concentrate in the center and the PEO units are more exposed to the aqueous media, water molecules may also present in the “core”, because PPO is not particularly apolar. The appeal of Pluronics lies in the fine-tuning of its physicochemical properties by modifying PEO and PPO.26 This tuning provides tailored biological interactions with greater potential to overcome the problems of drug delivery of hydrophobic drugs and to improve their therapeutic indexes.25−27 Pluronics enable the incorporation of poorly soluble drugs, diminish drug extravasation into normal tissues, and increase drug circulation times possibly promoting passive targeting to solid tumors via enhanced permeability and retention (EPR) effect after intravenous injection.22 It is recognized that some of these block copolymers enhance drug release from the liposomes, increase therapeutic efficacy of Doxil, and increase inhibition of the growth of drug-resistant tumors.28 One of the first Pluronics-based formulation that entered clinical trials was the mixture of the doxorubicin with Pluronics L61 and F127 micelles, and this is currently in Phase III clinical trials for the treatment of tumors resistant to doxorubicin.25−29 Pluronics can also mediate the loading of photoactive compounds such as methylene blue (MB) and preserve its photochemical properties enabling optical labeling as well as PDT.30 Recently, Kulbacka and coauthors showed that the administration of the clinically approved photosensitizer Photofrin in P123/F127 micelles increased in vitro cytotoxic effects in SKOV-3 and MCF-7/WT cells.31 The enhancement of antitumor activity and the favorable safety profile of Pluronic F127/P123 mixed micelles led to the investigation of Pluronic formulations to overcome tumor resistance to paclitaxel. The main commercially available paclitaxel formulation, Taxol, contains a high amount of Cremophor EL (CrEL) that has been associated with anaphylactoid hypersensitivity reactions, and Pluronic formulations may be better tolerated.32 Pluronic micelles have also been considered promising vehicles for the 22040
DOI: 10.1021/acsami.6b07031 ACS Appl. Mater. Interfaces 2016, 8, 22039−22055
Research Article
ACS Applied Materials & Interfaces
Scheme 1. (a) Preparation of redaporfin-Pluronic-Based Formulation; (b) Effect of Pluronic on the Solubility and Aggregation of Redaporfin in Aqueous Solution; (c) Picture of Aggregated Redaporfin in PBS (left) and Redaporfin-P123 Solution (right)
these systems. Both processes are favored by the hydrophobic interactions in the micelle inner layers, which increase when PPO length increases or PEO length decreases.23 Pluronics with long PPO blocks and high hydrophobicity strongly modify the microviscosity of plasma membranes. It was demonstrated that a fine balance between hydrophilic (EO) and lipophilic (PO) components in the Pluronic molecule inhibits drug efflux systems in MDR cells. Overall, the best block copolymers are those with intermediate lengths of PPO block and relatively hydrophobic structure, such as Pluronic P123. Hydrophilic block copolymers with an extended PEO block do not incorporate well into lipid bilayers and inefficiently transport drugs into cells. Hence, the cellular membrane binding property of F127 may be limited by its larger hydrophilicity.22,23,25,38 Particle Size Measurements of Redaporfin-Pluronics Formulations. Dynamic light scattering (DLS) was employed to assess the diameters of redaporfin-Pluronics particles and evaluate the absence of redaporfin aggregation in PBS at concentrations relevant for PDT (i.e., up to 5 μM) for each poloxamer. As seen from Figure 1, the nanoparticles sizes of redaporfin-Pluronics formulations are similar to those of pure
of the materials developed for the treatment. The novelty of our study is also manifested in a high loading of nonaggregated redaporfin in a triblock copolymer and how this incorporation increases its stability, ability to generate ROS upon NIR irradiation, and release in the more acidic tumor environment.
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RESULTS AND DISCUSSION Physicochemical Characterization of Pluronic-Based Formulation of Redaporfin. Preparation of Redaporfin Formulations. The thin-film method was employed to prepare a water-soluble formulation of redaporfin36 using the strategy illustrated in Scheme 1. The polymeric micelles formed by hydrophobic interactions of the PPO chain incorporate redaporfin. This construct also protects the drug from its inactivation by unwanted interactions in biological media. The core−shell structure of Pluronic micelles exhibits a spherical shape of moderate uniform particle size27,45 surrounded by PEO long chains. The polymers selected for this study exhibit different morphism: P123 is a paste, whereas F127 is flake. They have similar molar mass percentage of PPO per unimer (about 65 units), but F127 has longer EO units (about 200 units) than P123 (39 units). The physicochemical properties of Pluronic copolymers mentioned above can be finely tuned by modifying the molar mass ratio between the PEO and PPO blocks (≈ 3:1 for F127 and 1:2 for P123), which also change their interactions with cells and membranes.25 The increase in the PPO length is related with the increase in the aggregation number, hydrophobic core size, and solubilization of hydrophobic molecules. Copolymers with a higher fraction of hydrophilic PEO block tend to form micelles having a lower aggregation number, smaller PPO core, and higher concentration of water in the core. As a result, an increase in the PEO content (as in F127) in the block copolymer usually lowers the solubilization of hydrophobic drugs. Moreover, Pluronics with different compositions can produce micelles having distinct structures, particularly different volumes of the core and corona. There is a fundamental relationship between the partitioning of the hydrophobic solute and micelle formation in
Figure 1. Particle size distribution by scattering intensity of Pluronic (10% w/v): P123 in PBS (bold black line), F127 in PBS (thin grey line), redaporfin-P123 (red line), redaporfin-F127 (dashed green line), and redaporfin aggregates in PBS (with 0.5% of DMSO) (blue line). 22041
DOI: 10.1021/acsami.6b07031 ACS Appl. Mater. Interfaces 2016, 8, 22039−22055
Research Article
ACS Applied Materials & Interfaces
Figure 2. TEM (left) and STEM (right) micrographs of P123 and F127 unloaded and loaded with redaporfin micelles.
Pluronics in PBS, suggesting that redaporfin is solubilized and is not aggregated, at least in P123 micelles. Redaporfin-P123 micelles tend to have a smaller size than redaporfin-F127 micelles. Clearly, the presence of F127 increased the DH of the formulation because of the higher length of PEO segments. The comparison with the micellar samples without photosensitizer suggests that redaporfin incorporation slightly increases the hydrodynamic diameter of the particle, ca. 5 nm, reflecting the increase of the core size.24,36,40 The mean micelle size of redaporfin-P123 was less than 50 nm, which is smaller than the critical size required to avoid capture by the reticuloendothelial system (RES). The redaporfin-P123 size is in the lower half of the sizes of approved nanomedicines (12−125 nm), which allows them to take advantage of the EPR effect and benefit from deep tumor penetration.46,47 The shapes and the size changes of micelles before and after redaporfin encapsulation were confirmed by TEM and STEM measurements. Both TEM and STEM micrographs show that drug free P123 micelles tend to arrange in specific clusters, while redaporfin-P123 micelles are spread out rather evenly (Figure 2). Moreover, TEM/STEM micrographs indicated that redaporfin-P123 micelles exhibited spherical shape and smooth surface forming homogeneous nanostructures with a size in line with DLS data. It is also possible to note that F127 micelles loaded with redaporfin exhibit larger differences in size. There is one fraction of smaller structures (∼25 nm) and another one significantly bigger (∼200 nm), which is also consistent with DLS measurements. In view of the size of the particles, it is likely that redaporfinPluronics formulations facilitate redaporfin accumulation into tumor tissues combining the avoidance of the RES system with the EPR effect and low steric hindrance. Moreover, Pluronic P123 completely avoids redaporfin aggregation, while F127 may not have been as efficient. As previously mentioned, two main groups of particles were observed with Pluronic F127. This may be related to the formation of micellar clusters (>100 nm) or looser structures, revealing unstable and suboptimal formulation. The redaporfin loading efficiency was calculated
by determining the fraction of redaporfin content in the micelles compared to that used during micelle formulation. As summarized in Table 1, the efficiency of redaporfin loading was dependent on the type of poloxamer used. Table 1. Properties of the Redaporfin-Pluronic-Based Formulations: Drug Loading Content (DL), Drug Encapsulation Efficiency (EE), and Hydrodynamic Diameter of Prepared Micelles formulation P123 F127 F2BMet-P123 F2BMet-F127
DL (wt %)
9.88 4.11
EE (%)
84 57
particle diametera ± SD (nm)
PDIb
± ± ± ±
0.069 0.068 0.165 0.281
21.60 22.80 26.63 45.05
0.80 1.41 0.30 0.52
a Effective diameter measured by DLS at room temperature with 12 runs per measurements. bPolydispersity index measured by DLS.
Our results confirm that the nature of the poloxamer is an important determinant in the solubilization of lipophilic photosensitizers and that increasing the PPO content (69 × PO and 39 × EO for P123, 65, and 200 for F127,33 respectively) was effective in formulating redaporfin. Pluronic P123 gave the most stable structures, with the ability to form micelles and to provide a slightly hydrophobic environment to maintain the photosensitizer in monomeric form. The efficiency of redaporfin loading in P123 micelles was higher than that of F127:84% vs 57%. Thus, P123-based formulation stands as the most efficient in redaporfin loading and the most stable. Absorption Spectra. The solubility of redaporfin-P123 and redaporfin-F127 vs redaporfin was investigated using electronic absorption spectroscopy. Porphyrin derivatives have the tendency to form aggregates in aqueous solutions that are characterized by a decrease in absorption intensity, broader and red-shifted bands, especially for the lower energy electronic transitions. Figure 3 shows that in PBS with less than 0.5% DMSO, at a concentration relevant for the biological activity (5 22042
DOI: 10.1021/acsami.6b07031 ACS Appl. Mater. Interfaces 2016, 8, 22039−22055
Research Article
ACS Applied Materials & Interfaces
Figure 3. (a) Electronic absorption spectra of redaporfin-P123 in PBS, redaporfin in PBS (
