CORNERSTONE RESEARCH FINANCIAL AND ECONOMIC CONSULTING AND EXPERT TESTIMONY The Economics of Irreparable Harm in Pharmaceutical Patent Litigation Rahul Guha, Cornerstone ResearchMaria Salgado, Cornerstone Research TABLE OF CONTENTS The Hatch-Waxman Act greatly simplifies the process of obtaining FDA approval for a generic drug. Under the Act, a generic company need only file an Abbreviated New Drug
Ifes.edu.brJ Nanopart Res (2013) 15:1879DOI 10.1007/s11051-013-1879-8 Improved photodynamic action of nanoparticles loadedwith indium (III) phthalocyanine on MCF-7breast cancer cells Carlos Augusto Zanoni Souto • Kle´sia Pirola Madeira • Daniel Rettori •Mariana Ozello Baratti • Letı´cia Batista Azevedo Rangel • Daniel Razzo •Andre´ Romero da Silva Received: 1 April 2013 / Accepted: 16 July 2013Ó Springer Science+Business Media Dordrecht 2013 Indium (III) phthalocyanine (InPc) was (1.8–7.5 lmol/L), incubation time (1–2 h), and laser encapsulated into nanoparticles of PEGylated poly power (10–100 mW) were studied on the photody- (D,L-lactide-co-glycolide) (PLGA-PEG) to improve namic effect caused by the encapsulated and the free the photobiological activity of the photosensitizer. The InPc. Nanoparticles with a size distribution ranging efficacy of nanoparticles loaded with InPc and their from 61 to 243 nm and with InPc entrapment efficiency cellular uptake was investigated with MCF-7 breast of 72 ± 6 % were used in the experiments. Only the tumor cells, and compared with the free InPc. The photodynamic effect of encapsulated InPc was depen- dent on PS concentration and laser power. The InPc-loaded nanoparticles were more efficient in reducingMCF-7 cell viability than the free PS. For a light dose C. A. Z. Souto A. R. da Silva (&)Federal Institute of Espı´rito Santo, Campus Aracruz, of 7.5 J/cm2 and laser power of 100 mW, the effec- Avenida Moroba´, 248, Moroba´, Aracruz, tiveness of encapsulated InPc to reduce the viability ES 29192-733, Brazil was 34 ± 3 % while for free InPc was 60 ± 7 %.
Confocal microscopy showed that InPc-loaded nano- particles, as well as free InPc, were found throughout Biotechnology Program/RENORBIO, Health Sciences the cytosol. However, the nanoparticle aggregates and Center, Federal University of Espı´rito Santo, Vitoria, the aggregates of free PS were found in the cell ES 29040-090, Brazil periphery and outside of the cell. The nanoparticles aggregates were generated due to the particles con- Department of Exact Sciences and Earth, Federal centration used in the experiment because of the small University of Sa˜o Paulo, Diadema, SP 09972-270, Brazil loading of the InPc while the low solubility of InPccaused the formation of aggregates of free PS in the M. O. BarattiDepartment of Cellular Biology, University of Campinas, culture medium. The participation of singlet oxygen in Campinas, SP 13083-863, Brazil the photocytotoxic effect of InPc-loaded nanoparticleswas corroborated by electron paramagnetic resonance experiments, and the encapsulation of photosensitizers Department of Pharmaceutical Sciences,Federal University of Espı´rito Santo, Vitoria, reduced the photobleaching of InPc.
ES 29040-090, Brazil PLGA-PEG Indium phthalocyanine Nanoparticles MCF-7 cells Department of Physical Chemistry, Institute of Chemistry,University of Campinas, Campinas, SP 13083-970, Brazil Photodynamic therapy J Nanopart Res (2013) 15:1879 Since poly(lactide-co-glycolide) (PLGA) is the mostwidely used polymer in pharmaceutical products Photodynamic therapy (PDT) is an important thera- approved by the Food and Drug Administration peutic option for treating oncology (O'Connor et al.
(FDA) due to their biocompatibility and biodegrad- ; Sharma et al. ; Triesscheijn et al. and ability, this polymer has a long safety record (Blander non-oncology (Allison et al. ; Calzavara-Pinton and Medzhitov ; Mundargi et al. ). There- et al. ; Qiang et al. ) diseases. This modality fore, our group has been exploring PLGA to encap- combines a photosensitizer, light and oxygen mole- sulate photosensitizers (Silva et al. , ).
cules (Juzeniene et al. ; Ochsner ). After Several works have shown the benefits of PEGy- administration of the photosensitizer, the diseased lation across a broad range of polymer molecular tissue is illuminated with visible light. The irradiation architectures and macromolecular assemblies in leads to excitation of the PS to a singlet electronic state increasing the circulation half-life of polymeric nano- (S1), which can be deactivated to the triplet state (T1) particles that facilitate more opportunities for the by non-radioactive processes (intersystem crossing) passage of nanoparticles from the systemic circulation (Juzeniene et al. ). In this state, the PS can interact into the disordered and permeable regions of tumor with oxygen molecules or other biomolecules that are vasculature (Cruz et al. ; Gref et al. ; Jokerst present near the irradiated region, thus generating et al. ). In addition, polyethylene glycol (PEG) reactive species that can damage the neoplastic tissues has been clinically validated for many different (Bozzini et al. ; Juzeniene et al. Ochsner applications, and is currently listed as ‘‘Generally Recognized as Safe'' by the FDA. This makes the Administration of the lipophilic PS is a challenge in PEGylated polymer particularly attractive for use in PDT because of the poor solubility of the molecules in the encapsulation of a hydrophobic PS (Knop et al.
physiologically compatible solvent media (Korbelik et al. To overcome this problem, various Metallic phthalocyanines stand among the most delivery strategies have been studied so far to preserve promising photosensitizers due to their intense absorp- the hydrophobic photosensitizer in the aqueous envi- tion in the ‘‘photodynamic window'' (600–800 nm), ronment (Bechet et al. Konan et al. ; long triplet lifetimes, and large singlet oxygen quan- Korbelik et al. ). Indeed, the development of drug tum yields (Garcia et al. ; O'Connor et al. delivery systems such as liposomes, micelles, and Considering that several metallic phthalocyanines nanoparticles could improve the unfavorable biodis- have been used in clinical and preclinical trials in tribution of free PS (i.e., improvement of photosensi- oncology (Sekkat et al. ) and that the presence of tizer pharmacokinetic properties, better targeting of In(III) in the core of the photosensitizer structure diseased tissues due to particle size using the enhanced enhances the in vitro and in vivo photodynamic permeability, and retention effect, association to efficacy (Chen et al. ; Rosenfeld et al. ), we serum proteins and specific activation of the PS decided to study indium(III) phthalocyanine (InPc) as through localized delivery of the PS) as well as a photosensitizer. Unfortunately, the InPc has a strong avoidance of aggregation and loss of phototoxic hydrophobic character that promotes aggregation in activity/fluorescence which should result in a better high polarity media hindering their systemic admin- therapeutic outcome (Acharya and Sahoo ; Ma- istration and restricting clinical studies. However, the eda et al. Sekkat et al. ; Soares et al. encapsulation of InPc in nanoparticles can solve this Polymeric nanoparticles have been prominent among the other delivery systems because of their capacity to The aim of this study was to encapsulate InPc in release drugs at an experimentally predetermined rate PLGA-PEG nanoparticles to improve the photobio- over a prolonged period of time; maintain drug logical activity of the photosensitizer. The photody- concentration with therapeutically appropriate ranges namic efficacy of nanoparticles loaded with InPc on in circulation and within tissues; protect drugs from MCF-7 human breast tumor cells was evaluated and hepatic inactivation, enzymatic degradation and rapid compared with free InPc. The particles were charac- clearance in vivo, and due to their simple preparation terized with respect to surface morphology, size, zeta methods (Kamaly et al. ; Mundargi et al. potential, InPc loading, and entrapment efficiency.
J Nanopart Res (2013) 15:1879 The effects of incubation time, InPc concentration, method, the dispersion of an immiscible organic and laser power were studied on the photocytotoxicity solvent in an aqueous phase with high pressure vapor, of encapsulated or free InPc. The degree of internal- such as dichloromethane, is necessary. However, InPc ization of InPc-loaded nanoparticles and of free InPc is not soluble in dichloromethane. Thus, experiments into cells was evaluated by confocal microscopy and were performed to determine the best solvent to afterward quantified by fluorescence. The formation of solubilize InPc for preparing the PLGA-PEG nano- singlet oxygen by the encapsulated and free photo- particles loaded with InPc. An absorbance measure- sensitizer was evaluated by electron paramagnetic ment of the free InPc solution (1.0–5.0 lmol/L) was resonance (EPR), and the influence of photobleaching obtained in 1-methyl-2-pirrolidone (MP), ethyl acetate on these results was studied.
(EA), dimethylformamide (DMF), and dimethylsulf-oxide (DMSO). Graphics of absorbance versus con-centration were obtained using the InPc absorbance values at the maximum absorbance wavelength ineach solvent (682 nm for MP, 683 nm for DMF, 686 nm for DMSO, and 683 nm for EA) to determinethe absorptivity coefficient. The solvent with the highest absorptivity coefficient was chosen as the best PLGA ? 5 kDa PEG) was purchased from Evonik solvent for InPc.
Rohm GmbH (Darmstadt, Hessen, Germany). Indiu- For nanoparticle preparation, 50 mg of PLGA- PEG were dissolved in 7.0 mL of dichloromethane poly(vinyl alcohol) (PVA) (M and 0.30 mg of InPc was dissolved in 3 mL of MP.
w 13–23 kDa, 89 % The percentage of 30 % (v:v) of MP in the organic phase was adequate to prevent InPc aggregation (TritonÒ X-100), trypsin, ethylenediaminetetraacetic when the InPc solution was mixed to PLGA-PEG acid (EDTA), trypan blue, 2-amino-2-(hydroxy- solution. The organic phase (InPc solution ? PLGA- methyl)-1,3-propanediol (TrizmaÒ Base), sodium PEG solution) was added slowly to 50 mL of an pyruvate, penicillin, gentamicin, amphotericin B, aqueous solution of PVA (1.5 % m/v) and ethanol (5 % v/v), which was homogenized for 15 min at (phalloidin-TRITC), and propidium iodide were pur- 24,000 rpm (UltraTurrax T25, IKA, Wilmington, chased from Sigma Chemical Company (St. Louis, NC, USA). Since PLGA-PEG is characterized by a MO, USA). Dimethylsulfoxide (DMSO), ethanol, low glass transition temperature (Tg) (30 °C) (Loch- sodium chloride, potassium chloride, dibasic sodium mann et al. ), the jacketed glass was connected phosphate, monobasic potassium phosphate and para- to an ultrathermostatic bath that maintained the formaldehyde were obtained from Vetec Quı´mica internal circulating water at 15 °C. The initial and Fina Ltda (Duque de Caxias, RJ, Brazil). RPMI 1640 final temperatures of the solution were 20 ± 1 and medium and fetal bovine serum were purchased from 26 ± 2 °C, respectively. The resulting emulsion was Cultilab (Campinas, SP, Brazil). ProLongÒ Gold with maintained in magnetic agitation for 24 h for 40,6 diamidine-2-phenylindol (DAPI) was obtained evaporation of organic solvents. The particles were from Invitrogen (Sa˜o Paulo, SP, Brazil). The water recovered by centrifugation at 64,0009g for 22 min used throughout the experiment was first bi-distilled at 19 °C (Beckman J2-21, Beckman Instruments, and then deionized (Millipore). All other chemicals Fullerton, CA, USA), and washed 3 times with water were of analytical grade and were used without further to remove excess PVA and non-incorporated InPc.
(mannitol: particle mass ratio of 1:1) were frozenin liquid nitrogen and freeze-dried at 28 lmHg Nanoparticle preparation and—45 °C in a LioBras lyophilizer, model LIOTOPL101 (Sa˜o Carlos, SP, Brazil) for 2 days. Three The particles were prepared using the emulsion/ independent formulations were prepared using this evaporation technique (Jeffery et al. For this J Nanopart Res (2013) 15:1879 Particle morphology and mean size represent the mean ± standard deviation (SD) forthree independent preparations of nanoparticles.
The morphology of nanoparticles was ascertained byscanning electron microscopy (SEM; JSM-6360 LV, Cultivation of cancer cells JEOL, Tokyo, Japan). Approximately 2 mg of lyoph-ilized particles were dispersed in deionized water and MCF-7 human breast tumor cells (Rio de Janeiro Cell a droplet of this aqueous suspension was placed Bank, Duque de Caxias, RJ) were cultured in an RPMI directly onto a metallic stub. Samples air-dried over 1640 medium supplemented with 10 % fetal bovine the stub were coated with Au using a MED 020 Bal- serum, 1.0 mmol/L sodium pyruvate, 2.0 mmol/L Tec coater (Balzers, Liechtenstein). The mean particle L-glutamine, 14 U.I./mL penicillin, 10 lg/mL genta- size was determined by dynamic light scattering using micin, and 3.5 lg/mL amphotericin, being incubated a NPA152 Zetatrac from Microtrac (York, PA, US).
at 37 °C in a humidified environment having 5.0 %CO2 (Sanyo, Bensenville, Il). After confluence wasreached, the cells were washed twice with a phos- Determination of InPc content in the nanoparticles phate-buffered saline (PBS) solution (1.8 mmol/LKH An amount of lyophilized InPc-loaded nanoparticles 10.1 mmol/L Na2HPO4, 136.9 mmol/L NaCl, 2.7 mmol/L KCl) and harvested with trypsin (2.0 mg) without mannitol was dissolved in MP (0.25 % m/v)-EDTA (0.02 % m/v) solutions. They (2.0 mL) (a good solvent for both PLGA-PEG and the were seeded at a density of 1.5 9 105 cells/well in InPc). The InPc was quantified by UV–Vis spectroscopy 96-wells plates and allowed to grow for 48 h.
(Agilent Cary 50 Conc, Santa Clara, CA, USA) at682 nm using an analytical curve obtained with ten Photocytotoxic activity of the encapsulated different InPc concentrations. The PLGA-PEG polymer did not cause interference at the selected wavelength.
InPc incorporation efficiency was calculated using Eqs.
The photocytotoxic effect of encapsulated and free ) and ). The determinations were carried out in InPc on the viability of MCF-7 cells was determined triplicate and their mean values are reported.
InPc loading ð%Þ razolium bromide (MTT) assay. The culture medium mass of photosensitizer in particles was removed and 200 lL fresh RPMI medium, mass of particles without fetal bovine serum and containing either InPc-loaded nanoparticles or free InPc, were added toeach well of a 96-wells plate so that the irradiation of a Entrapment efficiency ð%Þ well does not reach the neighboring wells containing photosensitizer solution. The irradiation of the wells Theoretical InPc loading was done in the laminar flow cabinet. For theexperiments performed with free InPc, the phthalocy-anine was solubilized in MP solution (0.18 % volumeMP: volume RPMI). The final InPc extracellular Zeta potential measurement of nanoparticles concentration was 7.5 lmol/L. Then, the MCF-7 cells(1.5 9 105 cells/well) were incubated for 1–2 h at The zeta potential of nanoparticles was measured using 37 °C. After the desired incubation time, the wells dynamic light scattering technology (NPA152 Zeta- were washed with PBS solution and a fresh culture trac, Microtrac Instruments, York, US) joined with the medium was added. Each well was irradiated with a interaction of random Brownian motion with driven light dose of 7.5 J/cm2 using a laser diode INOVA electric field motion of particle suspensions. Typically, 665 nm of Laserline (Amparo, SP, Brazil). This laser 3 mg of the lyophilized nanoparticles were dispersed has a current selector, allowing us to work with in 10 mL of deionized water, followed by sonication different powers. A laser power of 60 mW was used for a period of 1 min. The measure was done two times for the experiments performed to determine the for each preparation. The zeta potential values J Nanopart Res (2013) 15:1879 encapsulated InPc on cell viability. Immediately after saline solution. Then 2.0 mL of culture medium irradiation, the culture medium was removed from the containing InPc-loaded nanoparticles or free InPc well and 15 lL of MTT solution (5.0 mg/mL) were (7.5 lmol/L) were added to each well. After 2 h of incubated for 4 h with cells. After this period, 70 lL of incubation, cells were washed three times with the sodium dodecyl sulfate solution (10 % m:v) in HCl culture medium to eliminate excess particles or free InPc 0.01 mol/L were incubated with cells to solubilize the that was not entrapped by the cells and harvested with formazan crystals. After 12 h, absorbance of the trypsin-EDTA solution. The suspension was centrifuged formazan was measured in 570 nm in each well using at 5009g for 5 min and the cells were resuspended with an ELISA plate reader (Bioclin MR-96-A, Belo 500 lL of TritonÒ X-100 solution (0.5 % m:v) in a Horizonte, MG, Brazil) to determine cellular viability.
sodium hydroxide solution (0.2 mol/L) to permeabilize The absorbance of treated cells and the control the cellular membrane and expose the encapsulated and (untreated cell) were used for determining the per- free InPc. Subsequently, 1.5 mL of MP were added to centage of cell viability (Eq. ).
solubilize the encapsulated and free InPc. The InPc in thecell extracts was determined spectrofluorimetrically, absorbance of treated cells % cell viability ¼ based on a previously constructed analytical curve using absorbance of the control a PerkinHelmer LS 55 fluorescence spectrometer (Wal-tham, MA, USA). The values presented are the The photocytotoxicity of InPc-loaded nanoparticles mean ± SD of three independent replicates. Statistical and of the free InPc was also evaluated by varying: analyses were carried out using the Student's t test with a (i) InPc concentrations (1.8–7.5 lmol/L) and (ii) laser significance level of p 0.05.
power (10–100 mW). All experiments were carried outin a dark room to prevent the influence of surrounding Cellular localization of InPc using confocal radiation on the photocytotoxic effect. Dark control toxicity was examined through wells containing onlycells incubated with RPMI, or cells incubated with MCF-7 cells (7.5 9 105 cells/mL) were seeded in RPMI and InPc-loaded nanoparticles or with free InPc, plastic Petri dishes (16 9 10 mm) containing a spher- or cells incubated with RPMI and 0.18 % MP, or cells ical slide ([ = 13 mm) immersed in RPMI 1640 incubated with RPMI and InPc-free nanoparticles. Light medium complemented with 10 % fetal bovine serum.
control toxicity was also examined through wells The Petri dishes were then incubated at 37 °C and 5 % containing only cells incubated with RPMI and irradi- of CO2 for 48 h. The culture medium was removed ated by the laser diode 665 nm (light), or cells incubated and 500 lL of RPMI medium containing InPc-loaded with RPMI and 0.18 % MP and irradiated by the laser, nanoparticles or free InPc (7.5 lmol/L) were added to or cells incubated with RPMI and InPc-free nanoparticle each dish. After 2 h of incubation, the cells were and irradiated by laser diode. The equivalent concen- rinsed twice with a phosphate-buffered saline solution tration of PLGA-PEG nanoparticles loaded with InPc to eliminate the encapsulated or free InPc which was was used in the experiment performed with free InPc not attached to the cellular culture. Then, the cells nanoparticle. The results presented are the mean ± SD were fixed with 4 % (m/v) paraformaldehyde solution of three independent replicates. Statistical analyses were for 20 min at room temperature, rinsed three times carried out using the Student's t test with a significance with PBS, and the slide was removed from the Petri level of p 0.05.
dish. A TritonÒ X-100 solution (0.2 % m:v) inTrizma-buffered saline (TBS) solution (150 mmol/L Quantification of internalized InPc of NaCl and 50 mmol/L of TrizmaÒ base) was addedon the slide surface for 5 min to permeabilize the cells.
The encapsulated and free InPc internalized in MCF-7 The permeabilization was blocked by placing a cells was quantified through the method described by solution of bovine albumin (3 % m:v) in PBS on the Win et al. (Win and Feng MCF-7 cells were slide surface for 30 min at room temperature. Subse- grown in a 12-wells plate at 37 °C for 48 h using an quently, 100 lL of phalloidin-TRITC were placed RPMI 1640 medium. After this period, the wells with onto the slide for 30 min to visualize the cell cells were washed twice with a phosphate-buffered cytoskeleton. The slides were washed twice with J Nanopart Res (2013) 15:1879 PBS to eliminate residual phalloidin-TRICT and is detectable using EPR measurements (Lion et al 100 lL of a ProLongÒ Gold solution, containing ). Typically, TEMP solutions (50 mmol/L) in 40,6-diamidine-2-phenylindol (DAPI) were added to RPMI 1640 medium, containing InPc-loaded nano- stain the cell nucleus at room temperature. All particles or free InPc (7.5 lmol/L), were added to a experiments were carried out in a dark room to cylindrical glass cell and afterward irradiated with a prevent photodegradation of the probes. The slides light dose of 3.0–7.5 J/cm2 and a laser power of were examined under a Zeiss Confocal LSM 510 100 mW at room temperature (25 °C). The same microscope (Carl Zeiss microimaging, Inc., Thorn- irradiation system (a laser diode 665 nm) was used in wood, NY) equipped with Argon (kex = 543 nm) and this experiment. Because of the hydrophobicity of Helium–Neon (kex = 633 nm) lasers. Phalloidin- InPc, the free InPc stock solution was prepared in MP TRICT was excited at 543 nm and its fluorescence solvent. Thus, the EPR experiment with free InPc was was selected with a BP 560–615 filter that passes performed in culture medium containing 0.18 % (v:v) radiation with wavelengths ranging from 560 to of MP. EPR spectra were recorded with a JES-FR30 615 nm. The InPc was excited at 633 nm and its JEOL spectrometer using a rectangular cavity after the fluorescent emission was selected using an LP650 incident light dose. Experiments were also performed filter that passes radiation with wavelengths higher in the presence of 5.0 lmol/L of NaN3 (singlet oxygen than 650 nm. The DAPI was excited using a mercury suppressor) and 0.24 mmol/L of TweenÒ 20 (non- lamp and no filter was used. Optical cross-sections ionic surfactant). For assessing if singlet oxygen is were obtained with a gradual increase of 1.57 and generated by encapsulated InPc or by InPc molecules 0.79 lm in depth to evaluate the InPc-loaded nano- that were released from nanoparticles into the culture particles and free InPc distribution in the cells, medium, the nanoparticles were incubated for 30 min in the medium, after which, the medium was centri- InPc-loaded nanoparticles were incubated for 2 h fuged (24,0009g, 30 min) and the supernatant was with only culture medium to evaluate if the nanopar- irradiated in the presence of TEMP using a light dose ticle could aggregate in the culture medium during the of 7.5 J/cm2. Measurements were carried out at room incubation time with MCF-7 cells. The experiment temperature with the following instrument settings: was also performed in water to evaluate if the culture microwave power: 4 mW; microwave frequency: medium could cause the aggregation of the particles.
9.41 GHz; field modulation frequency: 100 kHz; field The same concentration of nanoparticle (1.28 mg/mL) modulation amplitude: 1 G; time constant: 0.30 s; scan incubated with MCF-7 cells was used in this exper- time: 120 s; number of scans: 1; field center: 3375 G; iment. The average size of nanoparticle and the field width: 15 G. All experiments were carried out in a percentage of particles with a certain average size dark room to prevent the influence of surrounding were measured in determined period during the incubation time (2 h) using the light dynamic scatter-ing. The same procedure was performed in the culture Free and encapsulated InPc photobleaching medium to measure the size and percentage of freeInPc aggregates. In this case, the concentration of free Stock solutions of free InPc were prepared in MP due InPc was the same used in the experiment of irradi- to the low solubility of the photosensitizer in water. To ation of the cells (7.5 lmol/L). After 2.0 h of incuba- obtain the desired concentration of InPc in the tion, a TweenÒ 20 solution (0.24 mmol/L) was added photobleaching assays (5.0 lmol/L), aliquots of the to the nanoparticle or the free InPc solutions and the stock solution were added to a photooxidation size and percentage of particles were measured again.
medium composed of PBS solution and TweenÒ 20(0.24 mmol/L). The concentration of TweenÒ 20 was EPR measurements for detection of singlet oxygen (0.04 mmol/L) to reduce the formation of free InPc Generation of singlet oxygen was detected by EPR aggregates. The final percentage of MP in the photo- spectroscopy using TEMP. The reaction between 1O2 oxidation medium was the same percentage used in the and TEMP generates a stable nitroxide radical 2,2,6,6- experiments performed with MCF-7 cells (0.18 % tetramethyl-4-piperidone-N-oxide (TEMPONE) that v/v). The photooxidation medium containing free InPc J Nanopart Res (2013) 15:1879 solution were added to a quartz cuvette and irradiated Results and discussion with a light dose of 0.5–5.0 J/cm2 at room temperatureusing a laser diode 665 nm. The experiments were Preparation and characterization of InPc-loaded performed using several laser powers (1–60 mW).
The same conditions were used to irradiate the freeInPc solutions (5.0 lmol/L) prepared using MP from Figure disclosed that MP was more efficient to dilution of stock solution. In this experiment, the solubilize the InPc compared with other studied irradiated solution contained only InPc and MP. The solvents since the absorbance of free InPc was the InPc absorbance was monitored from 550 to 800 nm highest in MP. The absorptivity coefficient for InPc after each incident light dose. Then, a graphic of in MP [(1.8 ± 0.1) 9 105 L/mol cm] was 15 times relative absorbance intensity versus light dose was higher than the smallest absorptivity obtained in obtained at 682 nm for free InPc solution prepared in AE [(1.2 ± 0.1) 9 104 L/mol cm] and increased MP (without PBS solution and TweenÒ 20), and at according to the following order of solvents: AE [(1.2 690 nm for the free InPc solution diluted in PBS ± 0.1) 9 104 L/mol cm] DMSO [(2.2 ± 0.4) 9 solution containing TweenÒ 20. The relative absor- 104 L/mol cm] DMF [(4.5 ± 0.7) 9 104 L/mol cm] bance intensity was obtained by dividing the InPc MP [(1.8 ± 0.1) 9 105 L/mol cm]. Therefore, we absorbance intensity after light dose by absorbance decided to use MP to dissolve InPc to prepare the InPc- intensity before irradiation. This procedure was loaded PLGA-PEG nanoparticles.
repeated for each laser power. Suspension of InPc- Since InPc is not soluble in dichloromethane, we loaded nanoparticles (with InPc concentration of evaluated whether the mixture of InPc solution in MP 5.0 lmol/L) in PBS solution with 0.24 mmol/L of with dichloromethane could cause changes in the InPc TweenÒ 20 was also irradiated with the same incident spectrum. Figure shows that the ratio of 30 % (v:v) light doses and laser powers used in the photobleach- of MP with 70 % (v:v) of dichloromethane did not ing assays of the free InPc. After the light dose, the change the InPc spectrum after 15 min of mixture absorbance spectrum of encapsulated InPc was mea- (time used for preparing the nanoparticles). The sured from 550 to 800 nm and the graphic of relative PLGA-PEG also did not change the InPc spectrum absorbance intensity at 690 nm versus light doses was when the polymer was dissolved in the mixture of MP obtained. In order to conclude whether the encapsu- and dichloromethane, and it did not show absorbance lation of InPc decreases the photobleaching of the from 300 to 800 nm (Fig. inset). The result photosensitizer, the suspension was centrifuged at revealed that this mixture of MP with dichloromethane 20,4009g for 20 min and the InPc-loaded nanoparti- (30 and 70 % respectively) is efficient for maintaining cles were recovered and dissolved in MP for three the monomeric state of InPc molecules evidenced by a particular situations: before the irradiation of the sharp Q band, typical of a monomeric phthalocyanine suspension, after the incident light dose of 5 J/cm2 complex (Stillman and Nyokong ). This is with a laser power of 60 mW and after the same light important because the aggregation decreases the dose but with a laser power of 100 mW. Subsequently, photodynamic efficacy of photosensitizer. Therefore, the absorbance spectrum of InPc in MP was measured this mixture was used to prepare the PLGA-PEG and compared before and after incident light dose. All nanoparticles loaded with InPc.
experiments were carried out in a dark room to prevent The SEM image of nanoparticles (Fig. shows the influence of surrounding radiation on InPc that particle shapes are spherical and are relatively homogenous in size. Dynamic light scattering results Free InPc spectra were obtained in MP or in PBS show that the average particle size was (127 ± 8) nm, solution containing TweenÒ 20 for evaluation of the and the particle sizes distribution ranged from 61 to monomeric state of the InPc before the irradiation 243 nm, with 90 % of the nanoparticles smaller than period carried out in the photobleaching experiments.
200 nm (images not shown). Particles smaller than Absorbance measurements were also performed with 200 nm stay in the bloodstream longer due to the and without TweenÒ 20 to evaluate the presence of reduced recognition of these particles by the mono- nanoparticle aggregates during the experiment with nuclear phagocytic system (Bourdon et al. ; Hans the encapsulated InPc.
and Lowman ; Konan et al. They also
J Nanopart Res (2013) 15:1879 the cells (results not shown). Therefore, InPc-freenanoparticles, light and MP did not influence theresults obtained for cell viability of MCF-7 cellsincubated with InPc-loaded particles or the free InPc.
Effect of incubation time on phototoxicity A significant decrease in cell viability was observed aftera light dose of 7.5 J/cm2 when the MCF-7 cells wereincubated with InPc-loaded PLGA-PEG nanoparticlesfor 1 h since the viability decreased from (100 ± 5) %(number of control cells) to (44 ± 4) % (cells ? nano/InPc ? light) (Fig. ). A similar decrease in cellviability was obtained when cells were incubated withInPc-loaded nanoparticles for 1.5 and 2 h and irradiatedby the same light dose since viability was reduced to(49 ± 9) % and to (47 ± 10) %, respectively, suggest-ing the photodynamic effect was not changed after 1 h ofincubation (Fig. (100 ± 10) % (number of control cells) to (69 ± 6) %(cells ? free InPc ? light) only after 1.5 h of incubationand subsequent irradiation (Fig. ). The increase inincubation time of 1.5–2 h did not have any significanteffect on cell viability since viability was maintained at Fig. 1 a Absorbance intensity versus InPc concentration in (69 ± 4) %, suggesting the photodynamic effect was not several solvents. The absorbance values were determined at the changed after the incubation time of 1.5 h (Fig. ).
InPc maximum absorbance wavelength for each solvent Neither encapsulated InPc nor free InPc caused cytotoxic (682 nm for MP, 683 nm for DMF, 686 nm for DMSO and683 nm for EA). The solvent with the highest absorptivity effects (without light) to MCF-7 cells under the same coefficient was chosen as the best solvent for InPc. b Absorbance conditions (Fig. ). Statistical analysis shown that the spectra of InPc (5.0 lmol/L) dissolved in MP and in the mixture encapsulated InPc was more efficient than free InPc in of 30 % (v:v) of MP with 70 % of dichloromethane. The PLGA-PEG also did not change the InPc spectrum (inset) when thepolymer was dissolved in the mixture of MP and dichlorometh-ane, and it did not show absorbance from 300 nm to 800 nm interact more efficiently with cellular membranes(Win and Feng The PLGA-PEG nanoparticlesexhibited an InPc entrapment efficiency of around(72 ± 6) %, a loading value of around (0.43 ±0.04) % and a zeta potential of -33.9 ± 3 mV.
Viability of MCF-7 cells in action involvingonly InPc-free nanoparticles, light and MP The photocytotoxic effect on the viability of MCF-7cells after incubation and irradiation with InPc-freenanoparticles was not observed. This is consistent with Fig. 2 SEM images of InPc-loaded PLGA-PEG nanoparticles, PLGA-PEG biocompatibility. The light emitted by the prepared by the emulsion/evaporation method using PVA as laser and MP (0.18 % v:v) were also not cytotoxic to
J Nanopart Res (2013) 15:1879 causing cellular death since less cell viability was for a concentration of 1.8 lmol/L, subsequently to obtained using InPc-loaded nanoparticles with 1 h (63 ± 3) % (number of cells ? nano/InPc ? light) [(44 ± 4) %], 1.5 h [(49 ± 9) %], or 2 h [(47 ± for a concentration of 3.8 lmol/L and then to 10) %] of incubation and 7.5 lmol/L of InPc whether (47 ± 6) % (number of cells ? nano/InPc ? light) compared with the viability obtained using free InPc for when the InPc concentration was 7.5 lmol/L (Fig. 1 h [(92 ± 9) %], 1.5 h [(69 ± 6) %], or 2 h [(69 ± However, free InPc only reduced cell viability from 4) %] of incubation (Fig. ). It seems that encapsulated (100 ± 6) % (number control of cells) to (71 ± 8) % InPc was internalized faster than free InPc since the (number of cells ? free InPc ? light) when the InPc photocytotoxic effect was observed after an incubation concentration was 7.5 lmol/L (Fig. ). The increase time of 1 h for InPc encapsulated and after 1.5 h for free of free InPc concentration to 30 lmol/L (results not InPc. Since the reduction in cellular viability between 1.5 shown) did not change significantly the results and 2 h was not significantly different for MCF-7 cells obtained using the concentration of 7.5 lmol/L. The incubated with InPc-loaded nanoparticles, or with free ability of free zinc phthalocyanine (ZnPc) in reducing InPc, after irradiation with a light dose of 7.5 J/cm2 and the viability of A549 cells was also not changed after laser power of 60 mW, an incubation time of 2 h was concentration 17 lmol/L and the average viability used for subsequent experiments.
was maintained at 62 % (Soares et al. ). FreeZnPc was not cytotoxic even using the concentration Effect of InPc concentrations on phototoxicity at 69 lmol/L (Soares et al. ). Probably theaggregate state of free InPc in the culture medium The increase in encapsulated InPc concentration from decreased the photodynamic efficiency of free InPc to 1.8 to 7.5 lmol/L increased the photocytotoxicity of reduce the cell viability (as will be shown later). The the nanoparticulate formulation since cell viability encapsulated InPc in PLGA-PEG nanoparticles or the decreased from (100 ± 5) % (number of control cell) free InPc was not cytotoxic for MCF-7 cells in the to (82 ± 3) % (number of cells ? nano/InPc ? light) dark. The results shown that the InPc-loaded nano-particles were more photocytotoxic than the free InPcsince the cell viability was reduced from (82 ± 6) %(number of cells ? free InPc ? light) to (63 ± 3) %(number of cells ? nano/InPc ? light) when the InPcconcentration was 3.8 lmol/L, and from (71 ± 8) %(number of cells ? free InPc ? light) to (47 ± 6) %(number of cells ? nano/InPc ? light) using the con-centration 7.5 lmol/L (Fig. This is in agreementwith results obtained by other researchers who encap-sulated photosensitizers into polymeric nanoparticles(Konan et al. Zeisser-Labouebe et al. These results also revealed that free InPc photocyto-toxicity was not dependent on the range of studied InPcconcentration. Since the encapsulated and free InPc Fig. 3 Percent of cell viability versus incubation time on exhibit significant photocytotoxic effects at concentra- cytotoxicity (without light) and photocytotoxicity (with light) of tions of 7.5 lmol/L in experiments with light, this InPc-loaded nanoparticles and free InPc. InPc concentration inInPc-loaded nanoparticles and in the aqueous cell culture media concentration was used in subsequent assays.
was 7.5 lmol/L. The MCF-7 cells were incubated with InPc-loaded nanoparticles and free InPc for different times (from 1 to Effect of laser power on InPc phototoxicity 2 h) and then irradiated with a light dose of 7.5 J/cm2 (light infigure legend) using a 665 nm laser diode with a power of 60mW. The InPc-free nanoparticles, the source light and MP were The effectiveness of encapsulated InPc (Fig. ) in not cytotoxic for cells (results not shown). Each data point is the causing cellular death was influenced by laser power mean ± SD of three values. (*) The differences of ‘‘cell'' vs.
since viability was reduced from (100 ± 9) % (num- ‘‘cell ? nano/InPc ? light'', or ‘‘cell'' vs. ‘‘cell ? free InPc ? ber control of cells) to (64 ± 3) % (number of light'' or (**) ‘‘cell ? free InPc ? light'' vs. ‘‘cell ? nano/InPc ?light'' were significant at p B 0.05 cells ? nano/InPc ? light) and then to (34 ± 3) %
J Nanopart Res (2013) 15:1879 Fig. 5 Percent of cell viability versus laser power on cytotox- Fig. 4 Percent of cell viability versus InPc concentrations in icity (without light) and photocytotoxicity (with light) of InPc- InPc-loaded nanoparticles and in aqueous cell culture media on loaded nanoparticles and free InPc. InPc concentrations in InPc- the cytotoxicity (without light) and photocytotoxicity (with loaded nanoparticles and in aqueous cell culture media was light) of InPc-loaded nanoparticles and free InPc. MCF-7 cells 7.5 lmol/L. MCF-7 cells were incubated with InPc-loaded were incubated with InPc-loaded nanoparticles or free InPc for nanoparticles or free InPc for 2 h and then irradiated using a 2 h and then irradiated with a light dose of 7.5 J/cm2 (light, in 665 nm diode laser with different powers (10–100 mW) (light, figure legend) using a 665 nm diode laser with a power of in figure legend). The InPc-free nanoparticles and MP were not 60 mW. The InPc-free nanoparticles, source light and MP were cytotoxic for cells (results not shown). Each data point is the not cytotoxic for cells (results not shown). Each data point is the mean ± SD of three values. (*)The differences of ‘‘cell'' vs.
mean ± SD of three values. (*) The differences of ‘‘cell'' vs.
‘‘cell ? nano/InPc ? light'', or ‘‘cell'' vs. ‘‘cell ? free InPc? ‘‘cell ? nano/InPc ? light'', or ‘‘cell'' vs. ‘‘cell ? free InPc ? light'' or (**) ‘‘cell ? free InPc ? light'' vs. ‘‘cell ? nano/InPc ? light'' or (**) ‘‘cell ? free InPc ? light'' vs. ‘‘cell ? nano/InPc ? light'' were significant at p B 0.05 light'' were significant at p B 0.05 (number of cells ? nano/InPc ? light) when cells cells ? free InPc ? light) to (34 ± 3) % (number of were incubated with InPc-loaded nanoparticles and cells ? nano/InPc ? light) when laser power was irradiated with a laser power of 47 and 100 mW, respectively. The same dependence was not observedfor free InPc when laser power was increased from 47 Localization and uptake of the encapsulated to 100 mW since viability was only reduced from and free InPc into the MCF-7 cells (100 ± 9) % (number control of cells) to 60 ± 7 %when laser power was 100 mW (Fig. ). Neither InPc- Figure is the combination of four micrographies that loaded nanoparticles nor free InPc caused the MCF-7 relate InPc green fluorescence, DAPI blue fluores- cell death when a laser power of 10 mW was used. The cence bound to the nucleus, phalloidin red fluores- energy density emitted by laser was probably not cence bound to the cytoskeleton, and a sum of these efficient to observe the photodynamic effect when the micrographies. InPc-loaded nanoparticles (Fig. a) as laser power of 10 mW was used to excite the well as free InPc (Fig. b) were localized throughout the cytosol and in the perinuclear region, since the 10–47 mW was used to excite the free InPc. Cells micrographs very clearly show the fluorescence emit- not incubated with free or encapsulated InPc were ted by InPc during excitation with a helium–neon irradiated using different powers and no change was laser. Micrographies also disclosed the presence of observed in the cell viability, indicating that the laser intense fluorescence of encapsulated InPc in the energy did not fried the MCF-7 cells. Results show micro-region of the cytosol near the peripheral surface clearly that the encapsulated InPc was more effective of the cell, suggesting the presence of aggregates of in causing the death of MCF-7 cells than free InPc PLGA-PEG particles loaded with InPc inside and in since the cell viability was reduced from (90 ± 9) % the periphery of MCF-7 cells (Fig. a). The PLGA- (number of cells ? free InPc ? light) to (64 ± 3) % PEG nanoparticles possess a highly hydrophilic sur- (number of cells ? nano/InPc ? light) when laser face coated with a PEG polymer. This characteristic power was 47 mW, and from (60 ± 7) % (number of could favor the interaction of nanoparticles with the
J Nanopart Res (2013) 15:1879 Fig. 6 Confocal micrographs of MCF-7 cells incubated for 2 h (c) Phalloidin red fluorescence (argon laser kex = 543 nm) with 7.5 lmol/L of a InPc encapsulated into nanoparticles or bound to F-actin for staining the cytoskeleton and (d) a sum of b free InPc. (a) InPc green fluorescence (helium–neon laser, the three earlier micrographies. Similar results were obtained kex = 633 nm); (b) DAPI blue fluorescence (mercury lamp, with the other nanoparticle formulations and free InPc (results kex = 365–372 nm) bound to DNA for staining the nucleus; hydrophilic external membrane of the cell, causing a (Fig. a). Experiments were also performed with water concentration of PLGA-PEG nanoparticles in the instead culture medium to evaluate if the aggregates peripheral area of the cell. However, intense fluores- were generated by interaction of nanoparticles with the cence was also detected in the region away from the culture medium (Fig. The results disclosed that surface of the cell. Therefore, it was evaluated if the the population of large aggregates of particles aggregation could be caused due to the interaction [(20 ± 2) %] was bigger in water than that obtained between culture medium and nanoparticles. The in the culture medium [(6 ± 1) %] after 2 h of average size was measured at determined period incubation, as well as the size was also larger since during the incubation of nanoparticles only with the average size of aggregates was (5950 ± 66) nm culture medium for 2 h. One population of nanopar- while in the culture medium the average size was (1579 ± 16) nm (Fig. ). The presence of non-ionic (132 ± 2) nm and other population [(2 ± 1) %] with surfactant reduced the population and the size of the an average size of (383 ± 13) nm were observed after aggregates since the average size was reduced from 0.33 h of incubation, suggesting that the nanoparticle (5950 ± 66) to (778 ± 17) nm and from (1579 ± 16) aggregates were generated in the culture medium to (1155 ± 17) nm when TweenÒ 20 (0.24 mmol/L) (Fig. a). Large particles were not observed for was added in water and in medium culture, respec- incubation time smaller than 0.33 h.
tively. This result allows hypothesizing that the culture The nanoparticle aggregates increased during the medium helped to reduce the aggregates of InPc- incubation time since the population of large particles loaded nanoparticles. Probably, the aggregates of increased from 0 to (6 ± 1) % and the population of nanoparticles observed in the medium culture and in the confocal micrographies were generated due to the (94 ± 1) % after 2 h of incubation with the culture nanoparticles concentration used in the experiments medium, while the average size of large particles because of the small loading of the InPc into the increased from 0 to (1579 ± 16) nm and the small nanoparticles. Unfortunately, the better values for particles increased from (128 ± 10) to (151 ± 12) nm loading were not possible because of the diffusion of
J Nanopart Res (2013) 15:1879 Studies have shown that hydrophobic photosensitizerstend to be localized in cytoplasmatic membrane (Moor; Ochsner ). However, the intense fluores-cence of free InPc was also detected in regions awayfrom of the cell. The free InPc solution in the culturemedium was also monitored by dynamic light scatter-ing to evaluate the presence of aggregates. Theoreti-cally, free InPc aggregates should not be detected if thesolution was homogenous, but when free InPc solutionprepared in MP was diluted in the culture medium, theaggregates were detected in initial of the experiment(Fig. c). The average size of small aggregatesincreased from (209 ± 10) to (283 ± 13) nm after1.67 h of incubation. One population [(1.9 ± 1) %] oflarge aggregates were detected after 2.0 h. However,the large aggregates were eliminated when TweenÒ 20(0.24 mmol/L) was added in medium culture suggest-ing that the large aggregates were reduced to smallersize. These results suggest that the molecules of freeInPc aggregated in culture medium due to the hydro-phobicity of InPc (as will be shown later). Therefore,the intense fluorescence detected outside of the cellwas emitted by aggregates of free InPc.
Fluorescence from InPc-loaded nanoparticles or free InPc within the nucleus was not observed. Thus,there is no risk of photocarcinogenesis for survivingcells (Berg et al. ).
Cells incubated with nanoparticles of PLGA-PEG without entrapped InPc did not present fluorescence(not observed for nanoparticles loaded with InPc is onlydue to the photosensitizer. The fluorescence intensityof internalized InPc-loaded nanoparticles was higherthan that of the free InPc, suggesting that a greater Fig. 7 Average size and percentage of InPc-loaded PLGA- amount of encapsulated InPc was internalized in the PEG nanoparticles incubated with a the culture medium andb water. Nanoparticles were incubated from 0 to 2 h and the cells in comparison with free InPc. This hypothesis average size and percentage of particles were measured in a was evaluated by a quantification of the amount of predetermined time. c Average size and percentage of aggre- encapsulated and free InPc internalized in the cells.
gates of the free InPc incubated with culture medium was also Although the results have corroborated the confocal measured. After 2.0 h of incubation, a TweenÒ 20 solution(0.24 mmol/L) was added to the nanoparticle or the free InPc micrographs (not shown), it was not possible to solutions and the size and percentage of particles were measured consider the quantification of the amount of encapsu- again. Each data point is the mean ± SD of three values lated and the free InPc into the cells since thenanoparticle aggregates and free InPc aggregates were the InPc for the aqueous phase due to the interaction not eliminated when the cells were rinsed with with ethanol and PVA (results will be published).
phosphate-buffered saline solution. Probably the Similarly, an intense fluorescence of free InPc results reflected the amount of encapsulated and free aggregates was localized in the peripheral region of InPc localized inside and outside of the cell. Studies MCF-7 cells (Fig. suggesting that free InPc have shown that the encapsulation of photosensitizers aggregates could be localized on the surface of cells.
J Nanopart Res (2013) 15:1879 photosensitizer in the cell, a fact that will depend on Hydrophobic photosensitizers tend to aggregate in the photosensitizer, the carrier used in encapsulation cell culture media due to the susceptibility of the and the cell line studied (Nishiyama et al. ; Konan hydrophobic skeleton to avoid contact with water et al. The encapsulation of InPc and the low molecules (Rosenthal The aggregation state solubility of free InPc in the culture medium explain hinders the efficacy of InPc by decreasing its bio- the greater efficiency of encapsulated InPc in reducing availability and limiting its capacity to absorb light MCF-7 cell viability than free InPc.
(Bechet et al. Juzeniene et al. The Optical cross-sections were obtained from the top of the cells, with a gradual increase of 1.57 and0.79 lm in depth, respectively, for cells incubatedwith InPc-loaded nanoparticles or with free InPc toevaluate if encapsulated or free InPc were distributedwithin cells and/or near the cell surface. Figure confirmed the presence of InPc-loaded nanoparticlesinside MCF-7 cells since the fluorescence persisted inall optical sections (Fig. However, the distribu-tion of InPc-loaded nanoparticles in the cells was nothomogeneous since the fluorescence intensity fromnanoparticles was not uniform within these opticalsections. Similar results were observed for the distri-bution of free InPc into the MCF-7 cell (Fig. b). But,the intensity of green fluorescence from free InPc Fig. 9 Free and encapsulated InPc absorbance and (inset) within the optical sections was smaller than that fluorescence spectra at a concentration of 7.5 lmol/L in RPMI1640 medium. Free InPc absorbance spectrum was also obtained emitted by InPc-loaded nanoparticle, corroborating in the same concentration but in MP. InPc was excited at 620 nm with results obtained in Fig.
in both solutions Fig. 8 Confocal micrographs of optical cross-sections taken nanoparticles or b free InPc (7.5 lmol/L) for 2 h. InPc was with a gradual increase of 1.57 and 0.79 lm in depth from the excited with a helium–neon laser (kex = 633 nm) top of the cell (top row) after incubation with a InPc-loaded J Nanopart Res (2013) 15:1879 aggregation of free InPc in an RPMI 1640 medium Generation of singlet oxygen by encapsulated was also evaluated by absorbance and fluorescence measurements (Fig. The absorbance spectrum offree InPc in an RPMI 1640 medium, compared with The capacity of encapsulated and free InPc in generating that obtained in MP, suggested that the InPc is singlet oxygen was monitored by EPR measurements aggregated in the culture medium since the wave- using TEMP as a singlet oxygen trapping agent (Lion length of maximum absorbance of free InPc was red et al. ; Shutova et al ). The irradiation of the shifted by 20 nm in the RPMI medium, with a culture medium containing nanoparticles loaded with concomitant decrease in absorbance intensity and a InPc led to the generation of an EPR signal (Fig. a).
broadening of the Q band (400–800 nm) (Fig. The triplet EPR signal is characteristic of a stable corroborating with results obtained in Fig. c. The nitroxide radical (TEMPONE) resulting from the reac- molar absorptivity of the photosensitizer decreases tion between singlet oxygen and TEMP (Lion et al.
significantly when large self-associated supra-struc- Xu et al. ). The EPR signals increased tures are formed, causing a decrease in absorbance gradually with the increase in incident light from 3 to intensity and, consequently, a reduction in the free 7.5 J/cm2, suggesting that singlet oxygen was gener- InPc capability to absorb incident radiation (Reddi and ated. This result is important because it shows the ability Jori ). These large aggregates can also scatter of encapsulated InPc to interact with molecular oxygen incident radiation favoring a reduction in the absorp- and that PLGA-PEG does not act as an impermeable tion of free InPc. The InPc fluorescence also decreased barrier. Before irradiation of TEMP, a small signal (Fig. , inset) in the RPMI medium, in comparison was detected due to the presence of TEMPONE with the spectrum intensity in MP. The aggregates have a reduced tendency for crossing plasmatic The EPR signal did not change with an increase in membranes and consequently have lower cellular the light dose when the culture medium was irradiated uptake. Thus, lower effectiveness of free InPc in in the presence of free InPc (Fig. b). Experiments causing MCF-7 cell death can be associated to the were performed in the presence of a non-ionic lipophilic character of InPc, which favors the aggre- surfactant to evaluate the importance of aggregation gation state in the aqueous culture medium. This on generating singlet oxygen. The free InPc generated reduces the capability of InPc to absorb incident singlet oxygen in the presence of TweenÒ 20 radiation and decreases its cellular uptake, and its (Fig. suggesting that the photosensitizer aggre- subsequent ability to produce singlet oxygen.
gation reduced the efficacy of free InPc in generating The absorbance and fluorescence spectra of the singlet oxygen, and consequently the effectiveness in encapsulated InPc, compared with that obtained in decreasing the viability of MCF-7 cells. This result MP, also suggested the presence of aggregates of corroborates with the decrease in absorbance and photosensitizer in the culture medium since the fluorescence intensity of the free InPc spectrum.
absorbance and fluorescence intensities decreased A similar experiment performed with InPc-loaded and Q band suffered a broadening. However, the nanoparticles and TweenÒ 20 caused an increase in absorbance and fluorescence intensities of encapsu- EPR signal intensity (Fig. suggesting the mole- lated InPc were higher than that obtained to the free cules adsorbed in the surface of nanoparticles could be InPc. Especially, for absorbance spectrum of encap- released into the culture medium in the presence of a sulated InPc, the wavelengths considered in the surfactant. Besides, InPc molecules adsorbed on the conclusion were between 600–800 nm since wave- nanoparticles surface could be responsible for the lengths smaller than 600 nm presented higher scatter- photodynamic effect of the encapsulated photosensi- ing, causing an exponential profile in the spectrum. It tizer on the death of MCF-7 cells. To evaluate this is known that scattering is more intense in regions of hypothesis, we incubated the InPc-loaded nanoparti- smaller wavelength since the scattering intensity (SI) cles with TEMP solution for 30 min. Before irradia- is inversely proportional to the wavelength raised to suspension was centrifuged and the fourth power (SI a 1/k4) (Li et al. ). These nanoparticles were discarded. The results revealed results suggest that the aggregation of the encapsu- that InPc-loaded nanoparticles were important for the lated InPc was smaller than that the free InPc.
generation of singlet oxygen since the EPR signal was J Nanopart Res (2013) 15:1879 Fig. 10 EPR spectra of TEMPONE generated by photooxida- For assessing whether singlet oxygen is generated by encapsu- tion of 2,2,6,6-tetramethylpiperidone (23 mmol/L) at room lated InPc or by InPc molecules that were released from temperature (25 °C) in the presence of a encapsulated and b free nanoparticles into the culture medium, the nanoparticles were InPc (7.5 lmol/L), in an RPMI 1640 culture medium. The incubated for 30 min in the medium and after this time, the generation of TEMPONE signal by c free and d encapsulated medium was centrifuged before irradiation. Light source: laser InPc was also monitored in the presence of 0.24 mmol/L of diode 665 nm. Light doses of 7.5 J/cm2 and laser power of 100 TweenÒ 20 and 5.0 l mol/L of azide (singlet oxygen quencher).
mW were used in this experiment not observed after the discarding of nanoparticles Studies of free and encapsulated InPc before the light dose. The TweenÒ 20 probably reduced the nanoparticle aggregates, favoring photo-dynamic action of the greater amount of InPc-loaded Phthalocyanines have the tendency to suffer photo- nanoparticles, increasing the EPR signal.
bleaching (Bonnett and Martinez ). Thus, it is An experiment performed in the presence of azide possible that InPc photobleaching reduces the photo- (a quencher of singlet oxygen) did not show the triplet dynamic efficacy of free and encapsulated InPc in EPR pattern for the encapsulated or the free InPc, decreasing the MCF-7 cell viability or generating the corroborating that the photodynamic effect was EPR signal. For evaluating whether the encapsulated caused by a generation of singlet oxygen. Control and free InPc suffer photobleaching during the irra- irradiations of only the culture medium or the free InPc diation and whether the encapsulation decreases the in water were carried out and no changes were photobleaching of the photosensitizer, we monitored observed (results not shown).
the absorbance spectrum of free and encapsulated InPc J Nanopart Res (2013) 15:1879 Fig. 11 Free InPc photobleaching in a PBS solution and (inset)cin organic solvent. The decrease in relative absorbance of InPcsolution with concentration of 5.0 lmol/L was monitored at682 nm for the free InPc solubilized in MP, and at 690 nm forthe PBS solution containing the free InPc and 0.24 mmol/L ofTweenÒ 20. The solutions were irradiated with light doses from0.5 to 5.0 J/cm2 and laser power from 1 to 60 mW, using a laserdiode 665 nm. b Absorption spectra of free InPc (5.0 lmol/L) inMP and in PBS solution with TweenÒ 20, and of encapsulatedInPc (5.0 lmol/L) in PBS solution with and without TweenÒ 20.
The encapsulated InPc spectrum was also measured aftercentrifugation of the suspension for discarding the nanoparticlesloaded with InPc. c The photobleaching of encapsulated InPcwas also monitored in the PBS solution. (c, inset) Absorbancespectra of InPc-loaded nanoparticles (InPc concentration of5.0 lmol/L) dissolved in MP before and after irradiation with alight dose of 5 J/cm2 and a laser power of 60 and 100 mW. Afterthe light dose, the suspension of InPc-loaded nanoparticles inPBS solution was centrifuged at 20,4009g for 20 min, thesupernatant was discarded and the nanoparticles were dissolvedwith MP during light doses (Fig. ). It is known that the RPMI1640 medium is a mixture of enriched salts with aminoacids, vitamins, glucose and glutathione. We showedthat RPMI 1640 can reduce the photobleaching ofphotosensitizers because some constituents of theculture medium quenched the singlet oxygen (Silvaet al. Thus, we decided to study InPc photo-bleaching in a PBS solution to prevent the influence ofthe culture medium on the experiment results.
The results show that the relative absorbance intensity of free InPc (Fig. solubilized in PBSsolution and TweenÒ 20, decreases from 1.0 to 0.8with the increase in incident light from 0 to 5 J/cm2using a laser power of 60 mW. A smaller reduction inrelative absorbance intensity was obtained when thelaser power was 1 and 10 mW than when using apower of 60 mW. After a light dose of 5 J/cm2 therelative absorbance intensity was reduced from 0.94 to0.88 (Fig. when the laser power was increasedfrom 1 to 10 mW, and then to 0.80 when the laser aggregation state of the free InPc in PBS solution power was 60 mW. These results suggest that the free was corroborated by an InPc absorbance spectrum InPc was photobleached during the light doses. Free characterized by a large Q band with small absorbance InPc dissolved in MP (an organic solvent), irradiated intensity (Fig. Thus, the aggregation state of free with the same light doses and the same laser powers, InPc favors a decrease in InPc photobleaching.
revealed higher photobleaching (Fig. a, inset) than The results of the encapsulated InPc photobleach- what was observed for free InPc in PBS solution ing were inconclusive since relative absorbance (Fig. since after a light dose of 5 J/cm2 the intensity decreased and increased using the same light relative absorbance intensity of free InPc in organic doses and the same laser power as used for experi- solvent was reduced from 0.93 to 0.47 when the laser ments with free InPc (Fig. c). Probably, the light power was increased from 1 to 60 mW. The scattered by nanoparticles in PBS solution hampered J Nanopart Res (2013) 15:1879 the measurements of absorbance. These results could Therefore, encapsulated InPc suffers less photoble- suggest that the encapsulated InPc was not photo- aching than free InPc, which favors better photody- bleached. This hypothesis was evaluated measuring namic efficiency for encapsulated InPc in generating the InPc absorbance spectrum after light dose on the the EPR signal. The photobleaching of free InPc and nanoparticle solution, subsequent centrifugation of the its aggregation state in the culture medium probably nanoparticle suspension, discarding of the superna- reduces the efficacy of the free photosensitizer for tant, and dissolution of the InPc-loaded nanoparticles causing tumor cell death.
in MP. The intensity of the absorbance spectrumobtained by dissolving the InPc-loaded nanoparticlesin MP decreased after irradiation with a light dose of 5.0 J/cm2 and a laser power of 60 and of 100 mW(Fig. inset). This is an indication that encapsu- This study demonstrated that the encapsulation of InPc lated InPc was photobleached and that the oxygen can into PLGA-PEG nanoparticles improves the photobi- interact with the photosensitizer encapsulated in the ological and photodynamic activity of the photosen- nanoparticle. This outcome corroborates with EPR sitizer. The photocytotoxicity of encapsulated InPc results since encapsulation is not a barrier for photo- was observed after 1 h of incubation with MCF-7 bleaching or for generating singlet oxygen. Figure cells, and was depended on photosensitizer concen- (inset) disclosed that the decrease in InPc absorbance tration and laser power. The same result was not intensity was smaller for encapsulated InPc than observed for free InPc. InPc-loaded nanoparticles observed for free InPc using the same light dose and were more effective than free InPc in inducing MCF-7 laser power, since the relative absorbance of free InPc cell death. The increase in phototoxicity of InPc- after a light dose of 5 J/cm2 and a laser power of loaded nanoparticles was related to the low solubility 60 mW was reduced from 1 (integral absorbance of free InPc in the culture medium. The aggregation intensity) to 0.79 (a decrease of 20 % in InPc state and the photobleaching of free InPc reduce both absorbance intensity) (Fig. while the relative cellular uptake and its ability to produce singlet absorbance of encapsulated InPc was reduced from 1 oxygen. Encapsulation is not a barrier to InPc for to 0.93 (a decrease of 7 % in InPc absorbance generating singlet oxygen. Moreover, encapsulation intensity) (Fig. c, inset). Even a laser power of decreases the photobleaching of InPc, favoring the 100 mW reduced the relative absorbance of encapsu- augmentation of their photocytotoxicity.
lated InPc from 1 to 0.86 (a decrease of 14 % in InPc We thank the Conselho Nacional de absorbance intensity) (Fig. c, inset).
Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and the Absorbance measurement was also performed for Federal Institute of Espı´rito Santo for financial support, the encapsulated InPc in a PBS solution with and without Instituto Nacional de Cieˆncia e Tecnologia de Fotoˆnica surfactant before the light doses (Fig. The Aplicada a Biologia Celular (INFABIC) for the confocalmicroscopy analysis and Prof. Geovane Lopes de Sena from absorbance intensity of the encapsulated InPc spec- Federal University of Espı´rito Santo for fluorescence analysis.
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