Targeted and synergic glioblastoma treatment: multifunctional

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Targeted and synergic glioblastoma treatment: multifunctional nanoparticles delivering verteporfin as adjuvant therapy for temozolomide chemotherapy Diogo S. Pellosi, Leonardo B. de Paula, Maryanne T. de Melo, and Antonio C. Tedesco Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01001 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Molecular Pharmaceutics

Targeted and synergic glioblastoma treatment: multifunctional nanoparticles delivering verteporfin as adjuvant therapy for temozolomide chemotherapy Diogo S. Pellosi#,†, Leonardo B. Paula†, Maryanne T. de Melo†, Antonio C. Tedesco†* #Laboratory

of Hybrid Materials, Department of Chemistry, Federal University of São Paulo,

Diadema, Brazil. †

Center of Nanotechnology and Tissue Engineering, Photobiology and Photomedicine Research

Group, Department of Chemistry FFCLRP— São Paulo University, Ribeirão Preto, Brazil.

ACS Paragon Plus Environment

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ABSTRACT Despite advances in cancer therapies, glioblastoma multiforme e treatment remains inefficient due to the brain-blood barrier (BBB) inhibitory activity and to the low Temozolomide (TMZ) chemotherapeutic selectivity. To improve therapeutic outcomes, in this work we propose two strategies: (i) photodynamic therapy (PDT) as adjuvant treatment and (ii) engineering of multifunctional theranostic/targeted nanoparticles (m-NPs) that integrate biotin as a targeting moiety with rhodamine-B as a theranostic agent in Pluronic P85/F127 copolymers. These smart m-NPs can surmount the BBB and co-encapsulate multiple cargoes under optimized conditions. Overall, the present study conducts a rational m-NP design, characterization, and optimizes the formulation conditions. Confocal microscopy studies on T98-G, U87-MG, and U343 glioblastoma cells and on NIH-3T3 normal fibroblast cells show that the m-NPs and the encapsulated drugs are selectively taken up by tumor cells presenting a broad intracellular distribution. The formulations display no toxicity in the absence of light and are not toxic to healthy cells, but they exert a robust synergic action in cancer cells in the case of concomitant PDT/TMZ treatment, especially at low TMZ concentrations and higher light doses, as demonstrated by nonlinear dose–effect curves based on Chou-Talalay method. The results evidenced different mechanisms of action related to the disjoint cell cycle phases at the optimal PDT/TMZ ratio. This effect favors synergism between the PDT and the chemotherapy with TMZ, enhances the antiproliferative effect, and overcomes cross-resistance mechanisms. These results point out that m-NP-based PDT adjuvant therapy is a promising strategy to improve TMZ-based glioblastoma multiforme e treatments. KEYWORDS: Nanotechnology, Theranostic, Temozolomide, Verteporfin, Photodynamic therapy.


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INTRODUCTION In the last 20 years, nanotechnology has been applied in several cancer treatments. Nanotechnology has helped to overcome current chemotherapy issues and has contributed to targeted and less aggressive treatment. Unfortunately, these advances have not improved the therapeutic outcomes of brain tumors. Among these tumors is glioblastoma multiforme (GBM), which is one of the most aggressive cancers and culminates in patient death a few months after diagnosis.1 GMB cells are resistant to apoptosis, and the blood-brain barrier (BBB) prevents drug delivery at the tumor level, which all account for the poor prognosis. The low BBB permeability stems from the low pinocytosis activity in the tight brain endothelial cell junctions and from the presence of specific cellular efflux systems like the ATP binding cassette, which limit drug and macromolecule transport to the brain tissue.2,3 The standard GBM treatment initiates with surgical tumor resection, which is followed by radiotherapy for six weeks and systemic administration of temozolomide (TMZ), the most important chemotherapeutic drug for GBM.4 However, survival benefits are still limited due to the BBB. High TMZ doses have been administered to surmount the BBB, but this strategy has led to multiple drug resistance (MDR) and severe side effects.5 Innovative approaches, such as the co-administration of two or more drugs with different antitumor mechanisms, are promising tools to potentiate therapeutic outcomes (synergism) and to overcome tumor resistance mechanisms even at low drug doses. This strategy can minimize possible side effects, which is especially required in the case of treatments of central nervous system diseases.6 In this context, the combination of TMZ with adjuvant therapies based on visible light phototherapy, including photodynamic therapy (PDT),7,8 has provided encouraging results. PDT is an anticancer treatment that uses light-activated photosensitizers (PS). In the


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presence of molecular oxygen, PSs generate various reactive oxygen species (ROS) in situ, particularly singlet oxygen (1O2), which induces cell death. PDT constitutes a valuable adjuvant therapy for TMZ in GBM treatment because it causes minimum toxicity to healthy tissues (due to PS light-driven activation), reduces the long-term morbidity of even inoperable cancers, and avoids the development of cross-resistance mechanisms.9,10 Photodynamic-based treatments should be ideally applied after surgical resection while brain cavity is still open in order to facilitate the light application in the desired area.11,12 Among the many available PS molecules, we highlight verteporfin (VP), which is an FDAapproved PS for the clinical treatment of age-related macular degeneration (Visudyne®). VP has also been demonstrated to be a highly effective antitumor agent against numerous cancers13,14 and it is currently under clinical trial for the treatment of primary breast cancer (NCT02872064). VP/TMZ co-administration is a promising strategy to potentiate GBM therapy, but both drugs are poorly soluble in biological fluids, which hinders their application.14 Therefore, it is crucial that a drug delivery system that can overcome these issues and release both drugs at the targeted tissue be engineered. Nanotechnology development provides new strategies for spatiotemporal controlled release of drug combinations: nanotechnology allows the drugs to be integrated into a single nanoparticle (NP). Overwhelming attention is now being devoted to polymeric carriers with tailored properties as an attempt to achieve efficient drug transport in the body.15,16 Pluronic® block copolymers are representatives of such materials. In water, pluronics self-assemble as core–shell NPs that entrap poorly water-soluble drugs, protect these drugs against degradation and renal clearance, and increase the selectivity of these drugs for tumors through the enhanced permeability and retention (EPR) effect. Pluronics also have the extraordinary ability to


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hypersensitize multidrug-resistant cells by inhibiting the glycoprotein P-mediated drug efﬂux, the main mechanism of drug transport in the BBB.17,18 Our group has developed pluronic P123/F127 mixed micelles that are stable in biologically relevant media and which can load VP and the antiangiogenic drug Sorafenib® for combined cancer treatment.13,19 Despite their good formulation characteristics, pluronic NPs are poorly taken up by cancer cells due to their surface hydrophilicity. To overcome this drawback, we have recently developed biotin-decorated pluronic P123/F127 micelles for tumor targeting purposes. This smart NP enhances niclosamide and VP internalization rates and anticancer activities in different cancer cell lines.20,21 With this idea in mind, here we propose the development of multifunctional pluronic P85/F127 NPs (designated m-NPs hereafter). We have chosen biotinylated-F127 because it is biocompatible, and cancer cells, even glioblastoma cells, have previously been reported to take up biotinylated NPs.22,23 Pluronic P85 is the most effective pluronic when it comes to hypersensitizing glycoprotein-P and surmounting the BBB.18,24 In addition, in this work we have functionalized P85 with the fluorescent dye rhodamine-B for theranostic purposes. In other words, herein we use a rational design for m-NP construction that enables diagnosis and targeted combined therapy to be integrated into a single nanoplatform (Figure 1). Through a precise and controlled delivery of the optimal drug ratio (VP/TMZ ratio) at the desired site, we expect that this design will elicit a synergic antitumor effect on glioblastoma multiforme cells. To this end, we first optimize the m-NP formulations to obtain the best drug loading conditions and long-term stability. Then, we conduct in vitro fluorescence microscopy studies on U87-MG, T98-G, and U343 glioblastoma cells and on NIH-3T3 normal fibroblast cells, as control, to understand the role that the m-NPs play in drug uptake by cancer cells and in drug intracellular


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distribution. Cytotoxicity assays helped us to assess the influence of different conditions. Finally, we discuss combined therapy-induced cell death mechanism on the basis of cell cycle assays. EXPERIMENTAL SECTION Materials. The photosensitizer Verteporfin (VP), the chemotherapeutic drug Temozolomide, pluronic F127 (EO100–PO65–EO100, MW = 12,600 g mol-1), 4-dimethylaminopyridine (DMAP), N,N’-dicyclohexylcarbodiimide (DCC), rhodamine B, pyrene, and 1,6-diphenyl-1,3,5hexatriene (DPH) were purchased from Sigma (Sigma-Aldrich co., St. Louis, MO, USA). Pluronic P85 (EO26–PO40–EO26, MW = 4600 g mol-1) was acquired from BASF chemicals (Mount Olive, NJ, USA). The cryoprotector trehalose was obtained from Calbiochem. The fluorescent cell probes Alexa Fluor® 488 Phalloidin and DAPI® (4′,6-diamidino-2phenylindole) were supplied by Thermo Fisher Scientific Inc. The Guava Nexin reagent was purchased from Millipore (Bedford, MA). Ethanol was acquired from J.T. Becker® as analytical grade. All the chemicals were of analytical reagent grade and were used without previous purification. Rhodamine B-tagged pluronic P85 synthesis. The procedure we used to synthesize rhodamine B-tagged pluronic P85 (designated rhodaminated-P85) was adapted from the synthesis of biotinylated-F127.20 Briefly, 1.5 g of F127 was dissolved in 40 mL of CH2Cl2 followed by addition of 0.22 g of rhodamine B. After that, 0.002 g of DMAP was added to each reaction flask, and the solution was cooled to 0 °C. Then, 0.04 g of DCC was added dropwise via a dropping funnel over 20 min, and the reaction was carried out at room temperature for 48 h, under stirring. The reaction mixture was extracted with sodium bicarbonate solution 10%. After this step, the organic phase was frozen overnight, and the insoluble substances were removed by


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filtration. The organic solution was precipitated twice in cold diethyl ether. The functionalized polymer was filtered and dried overnight under vacuum. Rhodaminated-P85: reaction yield: 77%. 1H-NMR (500 MHz, DMSO-d6, TMS), δ (ppm): 1.000-1.206 (s, -CH3 b), 1.229 (t, N-CH3, 12H), 3.255-3.561 (m, -O-CH2-CH2- a), 3.670 (d, N-CH2, 8H), 6.991 (m, H2-H7-H4-H5, 4H), 7.104 (d, H8-H1, 2H), 7.463 (dd, H6’, 1H), 7.792 (m, H4’-H5’, 2H) and 8.344 (dd, H3’, 1H). 13CNMR (75 MHz, DMSO-d6, TMS), δ (ppm): 12.2 (ethyl CH3), 14.0-17.0 (Pluronic CH3), 46.5 (ethyl CH2), 64.9 (C=O), 69.0-74.0 (Pluronic CH2, CH2-O, CH), 97.3 (C4-C5), 115.1 (C2-C7), 130.3 (C1-C8), 131.0-134.1 (C1’-6’), 154.7 (C3-C6), 159.2 (C9), 165.5 (C=O) High concentrations of rhodaminated-P85 was used to get carbon signals of rhodamine B-tagged molecule. NMR spectra are presented in supporting information (Figure S1). Multifunctional nanoparticle preparation. Unloaded and VP-, TMZ-, and VP/TMZ-loaded multifunctional pluronic P85/F127 nanoparticles (multifunctional pluronic P85/F127 nanoparticles are designated m-NPs) were prepared by the thin-film hydration methodology.13 Briefly, 100 mg of a P85/F127 mixture were dissolved in 1 mL of ethanol in a round-bottom flask. Different mass ratios between P85/F127 were evaluated in order to obtain the best formulation conditions. For functionalized micelles formulations, biotinylated-F127 was added in a way that ensures a final concentration of 20.0 μmol.mL-1 of biotin in the NP surface. Rhodaminated-P85 mass was added in order to give a rhodamine B concentration of 1.0 µg.mL-1 in the final formulations. To obtain drug-loaded m-NPs, different amounts of VP and/or TMZ were dissolved in ethanol and added to the P85/F127 solution. Then, the solvent was removed by rotary evaporation (Rotavapor R-215, Buchi) at 40 °C for about 20 min. The residual solvent was eliminated in a vacuum desiccator for 24 h. After that, the dried film was hydrated with 10 mL of filtered


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distilled water, the sample was sonicated for 5 min, and the formulation was filtered through 0.22-µm filters to remove non-incorporated drug(s) or large polymer aggregates. The process recovery yield was calculated from an aliquot of the m-NP dispersion that was freeze-dried and had its solid residue weighed. The results are expressed as the ratio of the actual weight to the theoretical polymer weight x 100. Formulation optimization was assessed based on m-NP size, polydispersity index, and drug (VP and/or TMZ) encapsulation efficiency. VP and TMZ encapsulation efficiency. The TMZ- and/or VP-encapsulation efficiencies were evaluated by dissolving a known amount of freeze-dried m-NPs (10 mg) in 1 mL of DMSO and by analyzing the resulting solution by UV-Vis spectrophotometry on a GE Ultrospec 7000 (GE healthcare). Before the analyses, the samples were filtered through a 0.22-µm filter. The recovered VP was quantified on the basis of its molar absorptivity (34,000 L.mol-1.cm-1 at 692 nm) in DMSO.13 The recovered TMZ was calculated by using a standard calibration curve plotted for TMZ DMSO solutions at known concentrations (from 1.0 x 10-6 to 1.4 x 10-4 mol.L1);

the absorbance values were read between 250 and 400 nm. Statistical analysis of regression

by least squares, Analysis of Variance (ANOVA), and Test of Lack of Adjustment were applied. Potential VP interference in TMZ absorption was preliminarily assessed by spiking a TMZ solution in DMSO with different amounts of VP. The results are reported as the mean of three separate measurements of three different samples (n = 9) ±SD. Particle size and zeta potential. The hydrodynamic diameter (DH), the polydispersity index (PI), and the zeta potential values of the m-NP formulations were determined with a Zetasizer Nano ZS (Malvern Instruments Ltd. Worcestershire, UK). The freeze-dried formulations were dispersed in Milli-Q water, and the measurements were performed at 37.0 °C, at an angle of 90°.


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The results are reported as the mean of three separate measurements on three different micelle batches (n = 9) ±SD. Formulation stability in aqueous medium and under lyophilization. The stability of the VP/TMZ-loaded m-NPs was evaluated in bovine serum albumin (BSA) 1% in phosphate buffer saline (PBS) solution, incubated in a temperature-controlled bath (37 °C), and stirred at 100 rpm for 72 h. At predetermined time intervals, each sample was previously filtered through a 0.22-μm filter and analyzed as described previously. Non-rhodaminated m-NPs were employed in these measurements to avoid spectral interferences. All the results are reported as the mean of three separate measurements (n = 3) ±SD. To obtain solid formulations, trehalose (2% of total weight of pluronic) was added to a freshly prepared m-NP aqueous solution, which was then frozen at 70 °C and lyophilized for 24 h in a Lyph-Lock 4.5 freeze-dryer (Labconco, Kansas City, USA). The solid product was subsequently stored under two controlled conditions: at 25 °C and at 4 °C, in the dark. Lyophilized samples without trehalose were also prepared as control. To verify the stability of the lyophilized formulations, the samples were periodically rehydrated, and the drug content, size, and zeta potential values were evaluated as described above. All the results are reported as the mean of three separate measurements (n = 3) ±SD. In vitro release studies. For release studies in PBS or in cell culture medium, the optimized formulation (P85/F127 m-NPs 80:20 m/m – [VP] = 5.0 µg.mL-1 and [TMZ] = 700 µg.mL-1) was dispersed in 1 mL of PBS buffer ou DMEM with 10% FBS and placed in a dialysis bag (MWCO=3500 Da, Spectra/Por®). The sample was plunged in 5 mL of PBS containing 5% v/v of polysorbate 80 (sink condition) and kept at 37 ºC up to 72 h. At selected time intervals, 1 mL of release medium was withdraw and replaced with an equal volume of fresh medium. TMZ and VP quantitative analyses were carried out by UV-Vis as described above. It is worthy to mention


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that as TMZ hydrolyzes to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) in aqueous buffered solutions, MTIC is the compound tracked by UV-vis analysis (λanal = 315 nm). As control, release profile of non-combined drugs formulations diluted in PBS or DMEM with 10% FBS medium are reported. Results are expressed as release % over time (n=3) ±SD. Accelerated stability studies: Lumisizer. The accelerated stability of the dispersions was analyzed with the LUMiSizer 611 equipment (LUM GmbH, Berlin, Germany). The evaluated samples were empty and drug-loaded (P85/F127 80:20 m/m) m-NPs (1.0% w/v). The analyses were performed as follows: 400 μL of the samples without prior dilution were placed in rectangular cuvettes suitable for the type of sample with maximum radial position of 129.5 mm (10-mm optical path). The samples were measured at 25 °C, which provided a transmission profile at 880 nm of each sample every 65 s, for 4.56 h (at 3168 rpm). Such characteristics were equal to one-year accelerated storage stability. The shelf life was obtained as follows: shelf life (time in seconds) = (tmr x RCF)

(1)

where tmr is the experiment duration, in seconds, and RCF is the relative centrifugal force (table value according to Stokes' Law). To convert the shelf life from seconds to months, the time calculated in seconds was divided by 3,600 (number of seconds in 1 h) x 24 (number of hours in one day) x 30 (number of days in one month). The results were obtained with the software SEPView V.5.1 (LUMGmbH, Berlin, Germany). NP morphology. Scanning electron microscopy: The morphology of the m-NPs in the solid form (lyophilized), formulated with trehalose or not, was obtained by scanning electron microscopy (SEM) in a Carl Zeiss Evo 50 microscope (Carl-Zeiss, Cambridge, UK) with 20-kV electron beam acceleration voltage. The samples were sputtered with gold on a Bal-Tec model:


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SCD 050 (Fürstentum, Liechtenstein) for 240 s, and the images were obtained with the ImageJ program. To verify the stability of formulation in solid state we can use powder or powder state (after lyophilization), the samples were analyzed immediately after lyophilization (t = 0) and 12 months after the lyophilization process. Atomic force microscopy: A drop (5.0 μL) of the m-NP (1% w/V) formulations diluted 100x was deposited and spread on a mica surface. Micrographic images were obtained at room temperature, without the need of sample coverage; the Scanning Probe Microscope model SPM-9600 (Shimadzu) and the SPM Online software were employed. The images were obtained in the intermittent contact mode; a cantilever with length of 124 μm, operating with resonance frequency in the range of 324–369 KHz, stiffness ranging from 34 to 51 N.m-1, and constant force was used. The dimensional results were processed with the program software itself. At least ten images of each sample were analyzed to ensure reproducible results. Cell cultures. The human brain glioblastoma multiforme e cells T98-G, U87-MG (grade IV GBM), and U343 (grade III GBM) and the mouse embryo NIH-3T3 fibroblast cell line were purchased from the American Type Tissue Culture Collection (ATCC, Rockville, Maryland). The cells were grown in DMEM with Glutamax supplemented with fetal serum bovine (FBS) 10%, streptomycin 100 U.mL-1, and penicillin 100 μg.mL-1 (all from Sigma) and maintained at 37 °C in humidified atmosphere containing 5% CO2 m-NP uptake by cells. All the cell lines were grown in 24-well plates (5×104 cells/well) for 24 h and incubated for different times (1, 2, 4, or 12 h) with m-NPs diluted in a medium added with FBS 3% without phenol red to avoid spectral interference. Subsequently, at the optimum incubation time (4 h), the cells were incubated again under different conditions: with m-NPs containing biotin on their surface, with m-NPs without biotin on their surface, or with medium containing excess free biotin (1 mmol.mL-1) compared to theoretical biotin in the surface of m-


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NP (20.0 μmol.mL-1) calculated from the amount of biotinylated-F1127 added in the formulation process. This procedure named competitive assay aims to saturate biotin receptors on cell surface prior to m-NP addition. In both cases, free VP dispersed in the cell culture medium from a concentrated DMSO stock solution was analyzed as the control. At the end of the incubation time, the cells were washed twice with PBS and dispersed with propanol, to lyse the cells and to solubilize the fluorophores. After incubation for 30 min under stirring, the fluorescence signal of the samples was analyzed with a Tecan Safire2 Microplate reader (Tecan, Switzerland). The samples were excited at 430 nm for VP and at 520 nm for rhodaminated-P85. The fluorescence intensity was read at 690 and 560 nm for VP and rhodaminated-P85 detection, respectively. Confocal microscopy. For subcellular localization, 2×104 cells/well were carefully added under glass coverslips (13 mm) previously attached to the bottom of a 24-well plate. The cells remained in culture for 24 h before being incubated with m-NPs diluted in the medium added with FBS 3% for 4 h. After this time, the cells were washed twice with PBS and fixed with paraformaldehyde 4% for 20 min. Then, the cells were washed twice with PBS containing glycine 100 mmol.L-1, permeabilized with Triton X-100 0.1% diluted in PBS for 7 min, and stained with Alexa Fluor® 488 Phalloidin (Thermo Fisher Scientific Inc.) diluted in PBS (1:800) for 15 min. After this second incubation step, the cells were washed twice with PBS, and the coverslips containing the adhered cells were removed from the wells and mounted onto glass slides with the ProLong Gold antifade reagent containing DAPI (4’,6-diamidino-2phenylindole) and mounting medium (Thermo Fisher Scientic, USA). The slides were analyzed with a Leica Platform TCS SP8 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany), and the images were elaborated with the ImageJ software.


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Cytotoxicity assay in the absence of light. All the cell lines were seeded (5 × 103 cells/well) in 96-well plates (24 h of growth at 37 °C and with 5% CO2) and incubated with VP-loaded mNPs (without TMZ), empty n-NPs, or free VP (controls) for 4 h, in the dark. After incubation, the cells were washed with PBS and incubated again for 24 h, in the dark. After this time, the cells were detached from the plates with trypsin, which was neutralized by addition of mediumenriched with FBS (10% v/v). The cells were centrifuged and suspended in ViaCount reagent, and cell viability was analyzed by flow cytometry (Guava easyCyte 8HT; Millipore, Boston, MA, USA). For this analysis, 5 x 104 events/sample were acquired, and the data were analyzed with the Guava CytoSoft 4.2.1 Software Environment - Guava ViaCount module. Rhodaminated-P85 was not used for the flow cytometry experiments to avoid possible interferences with the cell viability stain. The viability of the treated cells was expressed as a percentage of the absorbance of the control cells, which in turn were taken as 100% viability. Combined treatment: PDT (VP) and chemotherapy (TMZ). The cells were cultured as described in section 2.9 and incubated with VP, TMZ, VP/TMZ-loaded m-NPs, or empty mNPs, as control, for 4 h, in the dark. The VP concentration was kept at 5.0 μg.mL-1, and three different TMZ concentrations were analyzed: 700 μg.mL-1 (condition close to TMZ IC50 in glioblastoma cells25), 500 μg.mL-1, and 300 μg.mL-1. The cells were then washed twice with PBS and irradiated with a Brilliant B Nd-YAG Q-switched laser (Quantel) at 690 nm for VPinduced photodynamic action; irradiation time was 2 minutes for all samples, the laser energy was adjusted in each case in order to obtain three different light doses (0.3, 0.7, and 1.0 J.cm-2). Cell viability was measured by flow cytometry 24 or 72 h after exposure to light, as described above for the cytotoxicity assays in the absence of light.


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The effects of PDT (24 and 72 h after exposure to light), chemotherapy (24 and 72 h of incubation with TMZ), and combined PDT/chemotherapy were evaluated. Chou-Talalay method for assessing drug synergism was implemented to determine the combination index (CI), dose reduction index (DRI), and fraction affected (Fa).26,27 CI theorem of Chou–Talalay analyses was calculated using CompuSyn software. We analyzed the CI based on the nonlinear dose–effect curves (effect-based approaches).27 To analyze the dose–effect parameters (% of cell death) of each compound alone (VP and TMZ) as well as in combination (VP/TMZ) and calculate the CI value, the parameters can be automatically determined from the median-effect equation.26 Comprehensive procedures of automated dose–effect dynamic analysis via mathematical induction for quantization of synergism in drug combination studies are given in the user’s manual for CompuSyn software. Brieﬂy, the CI-isobologram equation was used, where CI values of 1.1 are antagonistic. Cell cycle analyses. The cell cycle kinetics and the subG1 cell population analyses were performed under the conditions optimized in the previous assays: VP/TMZ-loaded m-NPs (1% w/V, TMZ concentration = 300 μg.mL-1, and VP concentration = 5.0 μg.mL-1), samples irradiated with light dose of 1 J.cm-2, and analysis after 72 h of treatment, as mentioned in topic 2.10. Cells exposed to similar conditions but without PDT and/or chemotherapy were also evaluated as controls. After treatment, the medium was removed and stored, while the cells were trypsinized and added to the previously stored medium and centrifuged at 1000 rpm and 4 °C for 5 min. The supernatant was removed, and the cell pellet was washed with PBS. The cells were centrifuged again. After centrifugation, PBS was removed, and the precipitate was suspended in 0.2 mL of 70% ethanol (4 °C) and maintained at -20 °C until reading in the flow cytometer. Before the analyses, the stored samples were centrifuged again (at 1000 rpm and 4


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°C for 5 min) and washed with 0.2 mL of PBS to remove ethanol. The pellet was suspended in 0.2 mL of propidium iodide solution (propidium iodide 30 μM, Nonidet P40 0.2%, and RNase A 10 μg/mL – Sigma-Aldrich, USA). After 20 min of storage in the dark, the material was analyzed on the flow cytometer (Guava EasyCyte 8HT) by using the Guava CytoSoft 4.2.1 Software Environment - Guava cell cycle module. A total of 5,000 events were acquired for each sample. Statistical analyses. The GraphPad Prism 6® software for biostatistics (GraphPad Software, San Diego, CA) was used to analyze the data, which are expressed as the mean ± standard deviation (SD) of at least three independent experiments. The difference between groups was evaluated by one-way ANOVA with Tukey's Post Hoc test. Significance was set at p < 0.05. RESULTS AND DISCUSSION Pluronic P85/F127 m-NPs: synthesis, assembly properties, and loading efficiency. The mNPs were first prepared from the synthesis of rhodaminated-P85 and biotinylated-F127. The rhodaminated-P85 1H NMR spectrum (Figure S1a) displays the main peaks corresponding to the PPO and PEO segments of the copolymer at 1.0–1.3 and 0.1–3.5 ppm, respectively. The rhodamine B signals appear in the aromatic hydrogen region (above 7 ppm). 13C NMR (Figure S1b) results also confirm binding of the fluorophore to P85. Biotinylated-F127 used in this work was previously synthesized and characterized with a view to enhancing targeting and uptake of this copolymer by cancer cells.20 Modified polymers above the critical micellar concentration (CMC) and the critical micellar temperature (CMT) (see below) can self-assemble, to form mixed multifunctional nanoparticles (m-NPs) where the drugs are co-encapsulated with


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functional molecules exposed to external media within the m-NP core, as represented in Figure 1a.

Figure 1. a) Schematic representation of pluronic P85/F127 m-NP preparation (80:20 w/w– total concentration = 1% w/V): b) UV-Vis spectrum of Verteporfin encapsulated in m-NP ([VP] = 5.0 µg.mL-1 without rhodamine-B to avoid spectral interference), c) Hydrodynamic diameter distribution, d) SEM image of the lyophilized formulation (scale bar = 200 nm), and e) atomic force microscopy tridimensional analysis. To obtain a nanocarrier system with higher hydrophobic drug encapsulation efficiency and stability in aqueous medium, we evaluated different functional P85/F127 ratios by using verteporfin (VP) as a hydrophobic drug model (see Table S1 in supporting information). It is worthy to mention that in these preliminary tests we used high concentrations of VP (10.0 μg.mL-1) to assess the capability of P85/F127 micelles in solubilize very hydrophobic drugs. At higher F127 (hydrophilic copolymer) concentrations, the VP encapsulation efficiency is low due self-aggregation state28 and two NP populations with different size distributions arise. All this


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demonstrates that P85 and F127 copolymers does not present an effective self-assembly aggregation at high F127 proportions. However, as the P85 proportion in the mixture increases, especially above 80% in weight, higher encapsulation efficiencies for VP in the monomeric form are observed, and there is one single m-NP size distribution (DH = 30.0 nm, P.I. = 0.18, and zeta potential = -5.19 mV) (see also Figures 1b and 1c). Therefore, hydrophobic P85 stabilizes the copolymer mixture and improves the VP encapsulation efficiency. Stability studies in BSA 1% solution demonstrated that after three-day incubation at 37 °C, the m-NPs with a P85/F127 ratio of 90:10 have phase separation and significant encapsulated VP loss (Figure S2). In other words, F127 at low concentration cannot avoid protein adsorption onto the NP surface.20,29 However, the m-NPs with a P85/F127 ratio of 80:20 are stable for at least three days in the same medium and maintain their original properties, showing that this is the best formulation condition. Further experiments conducted at this ratio showed that the low CMC (0.0045% - Figure S3) and CMT (17.1 °C - Figure S4) values ensure self-assembly of the copolymers as core-shell NPs under the working conditions (1% w/V and 37.0 °C), favoring drug encapsulation and transport in body fluids.30 It is important to highlight that TMZ is spontaneously hydrolyzed in aqueous buffer solutions at physiological pH to the highly unstable compound 5-(3-methyltriazen-1-yl)imidazole-4carboxamide (MTIC). This process is undesirable at formulation conditions since MTIC rapidly degrades.7 Figure S5 shows the absorbance of TMZ in DMSO and in m-NP formulation. Both spectra are similar with λmax ~330 nm demonstrating that even after formulation process TMZ was not hydrolyzed since it remains encapsulated inside m-NP core being protected from the outer water. The absorbance profile of TMZ did not suffer significant changes for at least 3 days


17


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in aqueous protein-rich media (see below) when encapsulated in m-NP (data not shown), reinforcing that polymeric chains protect its structure from water molecules We assessed the optimal conditions to obtain m-NPs with good encapsulation efficiencies for the VP/TMZ combination. Table 1 summarizes the physicochemical characteristics and the drugloading parameters of the m-NPs with a P85/F127 ratio of 80:20 loaded with VP, TMZ, or VP/TMZ. We kept the VP concentration constant at 5.0 µg.mL-1, which is relevant for optimized photodynamic action in cancer cells.20,29 Table 1. Composition and properties of pluronic P85/F127 m-NPs (80:20 – 1% w/V) loaded with verteporfin (VP) and temozolomide (TMZ). The results are reported as the average of three separate measurements on three different batches (n = 9) ±SD. VP

TMZ

(µg/mL)

(µg/mL)

--

--

5

--

VPa (EE %)b

TMZa (EE%)b

DHc (nm ± DP)

PId

Zeta Potential (mV ± DP)

--

--

27.3 ±1.8

0.156

-3.71 ±1.22

--

30.0 ±1.8

0.180

-5.19 ±0.78

28.4 ±3.0

0.153

-3.34 ±0.27

23.4 ±2.1

0.091

-2.11 ±0.77

23.9 ±4.9

0.106

-4.66 ±1.35

26.1 ±1.7

0.112

-3.28 ±0.90

4.56 ±0.2 (91.3 ±4.02)

--

300

--

--

500

--

--

700

--

--

1000

--

270 ±44.0 (90.0 ±5.01) 476 ± 51.2 (95.2 ±1.08) 640 ± 51.2 (91.4 ±3.17) 851 ± 104.9 (85.1 ±4.84)


18

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4.55 ±0.2

277 ±38.2

(91.0 ±3.74)

(92.3 ±3.21) 421 ± 44.0

5

300

5

500

4.30 ±0.2 (86.0 ±4.20)

5

700

4.49 ±0.6 (89.8 ±3.67)

5*

1000

3.82 ±0.9 (78.4 ±4.54)

a Encapsulation

(84.2 ±4.55) 617 ± 70.4 (88.1 ±4.09) 733 ± 141.4 (73.3 ±5.66)

32.9 ±2.6

0.197

-4.43 ±1.08

33.9 ±5.1

0.250

-6.75 ±2.96

30.2 ±4.9

0.179

-3.56 ±1.00

43.0 ±5.5

0.381

-2.20 ±1.49

expressed as μg of drug encapsulated in 10 mg of the m-NPs; bRatio between the

experimental and the theoretical loading x 100. c DH = hydrodynamic diameter. d PI = Polydispersity index. * The solution was slightly cloudy.

Given the DH of the empty and drug-loaded m-NPs, particle size ranges between 23 and 43 nm, and the PI value is satisfactory depending on drug concentration. All the formulations present slightly negative ζ-potential values, as in the case of polyethylene glycolated nanocarriers made of uncharged copolymers.31 Pluronic solutions may form a soft gel during film formation, which promotes high loading efficiency, as observed for TMZ entrapment up to 700 µg.mL-1. Loading of the VP/TMZ combination is feasible and controlled by the ratio between these two drugs. Considering that both VP and TMZ have low water solubility, we can hypothesize that their coentrapment elicits mutual interference in the NP core.32 In fact, lipophilic drugs accommodate inside the PPO core of pluronics and, when these domains become saturated, the encapsulation efficiency decreases, and the particle size and the PI value increase, as observed for the higher TMZ concentration. The slight increase in particle size after entrapment of both VP and TMZ may be due to increased PPO core size, which probably results from the presence of entangled drugs. Nevertheless, the micelles are still small enough for tumor-specific accumulation via the EPR


19


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effect.33 AFM studies confirmed the nanometric size and spherical shape of the m-NP formulations (Figure 1d). Three-dimensional analyses demonstrated relatively homogeneous mNP distribution with medium particle size of 44 nm (Figure S6), which agrees with the values obtained by DLS analyses (Table 1). VP- and TMZ-loaded Pluronic P85/F127 m-NP formulation stability. When therapeutic application is concerned, formulation stability is an important issue. The reasonably low m-NP CMC value indicates good stability upon dilution, which is critical for the potential m-NP application as drug carrier. However, studies in biologically relevant media, like protein solutions, are crucial to mimic possible effects in body conditions.34 Figure 2 represents the stability assay of the formulations developed herein along 72 h in aqueous PBS buffer + BSA 1%. The formulation with higher TMZ content is not stable in protein-rich medium (Figure 2a), which shows that TMZ in excess destabilizes the m-NP packaging, to culminate in copolymer phase separation and significant TMZ loss. We believe that TMZ is preferentially loaded into m-NP core and, when core capacity of loading is exceeded due high amounts of TMZ, new drug molecules tends to go to shell layer of the copolymers destabilizing it and realizing encapsulated drugs, especially that portion loaded in the shell part. Higher release of TMZ compared to VP (Figure 2a) prove this idea. On the other hand, the formulation with TMZ content of 700 µg.mL-1 has small particle size, PI, and drug loss (Figure 2b). The same behavior arises for lower TMZ concentrations in the mixture; i.e., 500 and 300 µg.mL-1 (data not shown). Minimal losses are due to drug release from the m-NPs, which depends on drug hydrophobicity. Therefore, the optimal formulation condition is VP 5.0 µg.mL-1 and TMZ 700 µg.mL-1, which are considered satisfactory loading values for a therapeutically relevant system.


20

100

80

240

b)

43%

210

9%

150 40 91%

20

60 50 40 30 20 10 0

57%

81%

0 0

24

50

100

40

80

30

60

20

40

10

20

180

19%

48

Size (nm)

60

Size (nm)

a)

0

0

72

0

24

100

d)

80

100 80

Drug release (%)

Drug release (%)

72

Time (hours)

Time (hours)

c)

48

60 40 20 0

60 40 20 0

0

10

20

30

40

50

60

70

80

0

10

Time (hours)

20

30

40

50

60

70

80

Time (hours)

Figure 2. Stability of VP/TMZ-loaded pluronic P85/F127 m-NPs (80:20 m/m – total concentration = 1% w/V) with trehalose in BSA solution (1%) in PBS buffer. [VP] = 5.0 µg.mL1

and a) [TMZ] = 1000 µg.mL-1, b) [TMZ] = 700 µg.mL-1. T = 37.0 °C. Release profile of TMZ*

and VP from m-NP formulations in c) PBS or d) DMEM with FBS 10%. The external medium used for dialysis was PBS buffer with polysorbate 80 (5% v/v) at pH 7.4; T = 37 °C. [VP] = 5.0 µg.mL-1 and [TMZ] = 700 µg.mL-1. Black squares = VP, red triangles = TMZ. Data are reported as the mean of three independent experiments (n = 3) ±SD. *Monitoring MTIC spectrum at λanal = 315 nm, as described in the Experimental Section.


21

Relative concentration (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Relative concentration (%)

Page 21 of 62


Page 22 of 62

Release profile of dual-drug loaded m-NP dispersed in PBS and DMEM medium with 10% of FBS at pH 7.4 and 37.0 °C were evaluated by dialysis method (Figure 2c and 2d). Polysorbate 80 in the external medium ensured both sink conditions and prevention of drug aggregation. These studies allow understand how different media affect m-NP stability and its drug-release profile even in proximity of tumor tissues (DMEM enriched with FBS 10% v/v studies). TMZ presented a sustained released pattern while VP remained substantially entrapped in the m-NP core. It is worth noting that unlike conventional drug delivery systems, photosensitizes as VP does not necessarily require to be released from nanoparticles to induce a photodynamic activity.13 Release of drugs from formulations containing TMZ or VP (single-drug formulations) presented similar behavior from dual-loaded m-NP (data not shown).. This results highlights that the entrapment of both drugs at proposed condition (i.e., [VP] = 5.0 µg.mL-1 and [TMZ] = 700 µg.mL-1) does not affect m-NP stability. In addition, the results shows that one drug does not affect the other one concerning about their release, indicating different locations for drugs inside pluronic NP. The non-observation of a strong burst release and the similar release rates observed in both media highlights the stability of the formulation even in protein-rich media, which is quite favorable for in vivo applications. Bearing the storage shelf life in mind is also essential when developing new NP formulations. In this context, the LumiSizer® technique is a valuable tool because it provides up to 2300 times faster NP destabilization analysis than conventional tests. Lumisizer monitors time and spaceresolved extinction profiles of the sample reading the transmission of light during the centrifugation. The progression of the obtained transmission profiles contains the complete information on the stability kinetics of the formulations identifying any changes in the sample due to cream formation, sedimentation, flocculation, coalescence or separation of phases.35


22

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Figure 3 depicts one-year stability simulation studies showing that the formulations are not stable for long periods: the first profile (red) is distant from the last profile (green). The quantitative data obtained by integration of the transmission profiles (Figure S7 and Table S2 in supporting information) allow the m-NP formulation shelf life to be estimated. The presence of TMZ reduces the kinetic stability due high drug content entrapped into pluronic chains. The estimated shelf life of the VP/TMZ-loaded m-NPs is about three months when formulations are stored in the liquid phase, which leads to significant encapsulated drug loss due to NP flocculation, sedimentation, or coalescence. Therefore, new formulation and storage methodologies must be considered.

Figure 3. Accelerated stability study: evolution of the transmission profiles of blank and VP/TMZ-loaded pluronic P85/F127 m-NPs (80:20 w/w – total concentration = 1% w/V) with trehalose in PBS. [VP] = 5.0 µg.mL-1 and a) [TMZ] = 700 µg.mL-1. The centrifugation rate was 3168 rpm for 4.56 hours. T = 37.0 °C. The abscissa shows the position (nm), and the ordinate shows light transmission.


23


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The freeze-drying process (lyophilization) is known to be a robust methodology to preserve the original properties of different formulations.36 We applied trehalose as cryoprotector during lyophilization. The m-NP formulations containing trehalose produce a fluffy material that is readily reconstituted in aqueous medium after manual shaking, whereas formulations without trehalose undergo slower rehydration. High recovery yield and the absence of significant changes in the VP visible spectrum (absence of self-aggregation signals – data not shown) highlight that no NP dissociation occurs. Scanning electron microscopy images (Figure 1c and Figure S8) show a regular porous structure for trehalose-lyophilized formulations, which is a favorable characteristic for the development of new products based on pharmaceutical nanotechnology.37 Formulations without a cryoprotector present a more irregular solid distribution, which hinders sample hydration. These structures also explain differences in the long-term stability of solid formulations. After the one-year storage study (Figure S9), we verified that NP size and drug content do not change significantly, so the formulations containing trehalose have high kinetic stability at both temperatures. Therefore, under the proposed conditions, the VP/TMZ-loaded m-NPs have large drug loading capacity for VP and TMZ as well as high kinetic stability in biologically relevant media and during storage upon lyophilization. These constitute suitable characteristics for the VP/TMZ-loaded m-NPs to function as nanovector for cancer theranostic applications. Uptake by cancer cells: pluronic P85/F127 m-NP theranostic application. Selective drug delivery to tumor cells is a major goal in modern cancer therapy, especially in the case of brain tumors such as glioblastoma multiforme .38 We quantified the availability of the biotin targeting moiety on the micelle surface by measuring the HABA/avidin complex absorbance at 500 nm (Figure S10). The theoretical amount of available biotin is 20.0 μmol.mL-1 if we bear in mind


24

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the experimental formulation condition. The actual amount of available biotin is 18.2 μmol.mL-1, which means that approximately 90% of biotin is available on the m-NP surface. We assessed the uptake of the prepared formulations by the U87-MG, T98-G, and U343 glioblastoma cells and by the normal cell line NIH-3T3, as control, as well as their subcellular distribution. Because VP and rhodaminated-P85 present fluorescence emission, we were able to monitor both drugs and the m-NPs as a theranostic system. m-NP time-dependent uptake experiments (Figure S11) demonstrated that all the formulations generally reach a plateau after 4 h of incubation with the cells. We assessed the biotin targeting ability at this optimized time (Figure 4). The uptake of biotinylated m-NPs and VP-loaded m-NPS by cells is significantly higher (threefold higher) than the cell uptake of non-biotinylated m-NPs for all the cancer cell lines. Healthy NIH-3T3 fibroblast cells present very limited internalization of the biotinylated formulations. These results highlight the importance of NP functionalization: biotin insertion increases the selectivity of drug release into tumor cells rather than into the healthy tissue. The competitive assay (excess of free biotin - Figure 4) emphasizes the role of biotin receptors, which favor m-NP uptake by vitamin receptor-mediated endocytosis.39


25


free VP

a)

Biotin -

Biotin +

Competitive assay

7000 6000

Intensity (a.u.)

5000 4000 3000 2000 1000

b)

0 30000 25000

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 62

20000 15000 10000 5000 0

3T3

T98 G

U343

U87 MG

Figure 4. Cell uptake of free and VP-loaded non-biotinylated and biotinylated pluronic P85/F127 m-NPs (80:20 w/w – total concentration = 1% w/V) by the NIH-3T3, U87-MG, T98G, and U343 cell lines. Fluorescence signal after 4 h of incubation for a) rhodaminated-P85 (1.0 µg.mL-1) and b) VP (5.0 µg.mL-1). Data are reported as the means of three independent experiments ±SD. Confocal microscopy images (Figure 5) reveal a cytoplasmic distribution for both VP (red signal) and rhodaminated micelles (yellow signal), but no m-NPs or VP are found in the nucleus. Cytoplasmic distribution of micelles and VP were similar for 3T3, U343 and T98G cells, but not in the U87MG cells. Specific interactions inside cellular environment can drive this different behavior, but this is not the focus of present work. A previous study demonstrated that a


26

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fluorescent Pluronic P105 has broad-range distribution in the intracellular organelles40, corroborating with our results. The study also indicated that biotin drives NP uptake but not its intracellular location. It is well known that mitochondria is the VP preferential site of action11, but the encapsulation of VP in the m-NPs led to significantly larger cytoplasmic VP distribution (not specific to an intracellular organelle). This suggests that a potentiated photodynamic outcome is possible due to the VP multiple sites of action. As the proposed m-NPs do not enter the nucleus, which is the TMZ site of actio41, TMZ release from the nanocarrier after cell internalization and TMZ diffusion toward the nucleus are expected. Finally, higher selectivity, faster m-NP uptake by the cancer cells and favorable subcellular drug distribution are favorable premises for a targeted and potent synergic action during combined treatment.


27


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Figure 5. Confocal microscopy images of NIH-3T3, U87-MG, T98-G, U343 cells incubated with VP-loaded P85/F127 m-NPs (80:20 w/w – total concentration = 1% w/V) for 4 h. Green fluorescence: Alexa Fluor® 488 Phalloidin; Yellow fluorescence: Rhodaminated-P85 (rhodamine B = 1.0 µg.mL-1); Red fluorescence: VP. (5.0 µg.mL-1). Scale bar: 10 μm.


28

Page 29 of 62

Combined treatment: Synergic action for improved cytotoxicity. We assessed the m-NP cytotoxicity during 4-h incubation with total m-NP concentration of 1% w/V. Under these conditions, empty formulations do not induce any toxic response in any of the evaluated cell lines (data not shown). Cytotoxicity assays in the absence of light (Figure S12) demonstrated that VP-loaded m-NPs with VP up to 5.0 µg.mL-1 are not toxic in the dark (at least 90% of cell viability), which is a critical condition for PDT application.10 Next, we evaluated the TMZ cytotoxicity and the VP phototoxicity of TMZ and VP encapsulated alone (individual treatments) for different m-NP formulations (Figure 6). control (blank m-NPs) -1 -1 300 g.ml 500 g.mL

TMZ :

700 g.mL

-1

b)

100 80

**

**

** **

* *** ***

***

40 20

* *

80

* ***

60

0

100

*

*

*

control (blank m-NPs) -2 -2 0.3 J.cm 0.7 J.cm

Light dose:

Cell viability (%)

(a)

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0 J.cm

-2

* * **

*

*

*

* **

** ***

60

*** 40

20

0

NHI-3T3 3T3

T98-G

U343

U87-MG

3T3 NHI-3T3

T98 G T98-G

U343 U343

U87 MG U87-MG

Figure 6. Cell viability assays: a) TMZ cytotoxicity at different concentrations and b) VP (5.0 μg.mL-1) photocytotoxicity exposed to different red light doses (λexc = 690 nm). Each drug was individually loaded into the P85/F127 m-NP (80:20 w/w – total concentration = 1% w/V) formulation. The cells were irradiated after they had been exposed to the formulations for 4 h, and cell viability was measured by flow cytometry (5 x 104 events/sample) 72 h post-irradiation or post-incubation with TMZ. Data are reported as the means of at least three independent triplicate experiments ± SD. *p < 0.05, **p < 0.01, *** p < 0.01 vs controls (Tukey's test).


29


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Individual treatments do not induce cell death effectively. In general terms, normal NIH-3T3 cells present lower sensitivity to both individual treatments, which is attributed to their lower uptake of the m-NPs (Figure 4). Again, this highlights the m-NP selectivity toward cancer cells rather than the healthy tissue, as highly desired in the treatment of central nervous system pathologies.42 U87-MG and U343 cells are the most sensitive to TMZ (Figure 6a). TMZ is not a powerful drug and usually require high doses (

Targeted and synergic glioblastoma treatment: multifunctional

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