Antimicrobial photodynamic therapy using indocyanine green and biocompatible copolymers for pneumonia treatment: a murine study

Antimicrobial photodynamic therapy using indocyanine green and biocompatible copolymers for pneumonia treatment: a murine study | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 15 October 2025 V1 Latest version Share on Antimicrobial photodynamic therapy using indocyanine green and biocompatible copolymers for pneumonia treatment: a murine study Authors : Lorraine Gabriele Fiuza 0009-0006-5067-8129 [email protected] , Isabelle Almeida de Lima , Michelle Barreto Requena 0000-0002-8690-3053 , Vadila Guerra , Layla Pires , Cristina Kurachi 0000-0001-7175-5337 , Natalia Mayumi Inada , and Vanderlei Salvador Bagnato Authors Info & Affiliations https://doi.org/10.22541/au.176051195.52799577/v1 204 views 108 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Pneumonia remains one of the leading global public health challenges, with rising antimicrobial resistance severely limiting the effectiveness of conventional therapies. Antimicrobial Photodynamic Therapy (aPDT) has emerged as a promising alternative to combat lower respiratory tract infections. Nonetheless, its in vivo efficacy has been hampered by lung surfactant (LS), which sequesters photosensitizers (PS) and prevents them from reaching bacterial targets, thereby reducing therapeutic outcomes. To address this barrier, recent studies have demonstrated that indocyanine green (ICG) complexed with the copolymer Gantrez AN-139 maintains activity against Streptococcus pneumoniae in vitro , even in the presence of LS. Building upon these findings, the present study evaluated the effectiveness of this formulation in a murine pneumonia model. Among the tested protocols, nebulization of 200 µM ICG with 4% (w/w) Gantrez AN-139 followed by external near-infrared illumination (808 nm, 120 J/cm²) achieved nearly 3 log₁₀ bacterial reduction and improved survival (80% at 21 days). These Introduction Pathogens such as fungi, viruses, and bacteria cause respiratory tract infections. Pneumonia is the most prevalent of these infections and is a leading cause of global mortality and morbidity, resulting S. pneumoniae accounted for the largest proportions of lower respiratory infection (LRI) episodes and deaths, with an estimated 97.9 million (92.1–104.0 million) cases and 505,000 (454,000–555,000) deaths globally. 1–3 Bacterial pneumonia is the second leading cause of death across all age groups, particularly affecting the elderly and children under five. It causes alveoli inflammation, leading to fluid accumulation and highly affecting the respiratory function. The most common common bacterial cause in both adults and children S. pneumoniae . 1,3–9 Given the crisis in conventional treatments and the rise of antimicrobial resistance, there is a growing need for new methods to combat bacterial agents. In this context, Antimicrobial Photodynamic Therapy (aPDT) has emerged as a promising treatment option 10,11 . This technique relies on a photodynamic reaction involving the simultaneous interaction of three elements: a PS, light, and oxygen. 12–15 This interaction generates reactive oxygen species, including the reactive singlet oxygen, inducing damage to the biomolecules in the vicinity, mostly affecting cell membranes and walls, resulting in microorganism inactivation. One of the key advantages of this method is its nonspecific biological target, unlike antibiotics, resulting in a low likelihood of resistance development. Moreover, aPDT offers other significant benefits, including dual selectivity—allowing both the PS and light to be precisely targeted to the treatment site—absence of cumulative toxic effects, and its non-invasive nature. 13,14,16 With this motivation, our research group has been exploring the application of aPDT since 2013 to combat pulmonary infections caused by S.pneumoniae . In pursuit of a minimally invasive treatment, we have proposed the pulmonary administration of the PS combined with external illumination, using indocyanine green (ICG) due to its ability to be activated in the infrared spectrum, a wavelength range that offers greater tissue penetration, allowing irradiation to be performed externally. 17–24 The overall conception of the technique is presented in Figure 1. However, although light delivery and PS administration have been proven in animal models, some barriers limit the photoinactivation of microorganisms in the alveoli, such as lung surfactant (LS). 22 Figure 1. Proposed Antimicrobial Photodynamic Therapy scheme for pneumonia treatment: After infection is confirmed, the patient receives a photosensitizer via nebulization. Then, they are exposed to an external near-infrared light source, which penetrates biological tissues to activate the photosensitizer within the lungs, eliminating the infectious agent. Developed by the author. Created using Biorender. LS is a complex mixture of lipids and proteins secreted by type-II lung epithelial cells. It is essential for reducing alveoli surface tension, facilitating gas exchange and preventing alveolar collapse. 25–28 Additionally, LS plays a critical role in the lung’s immune system. Previous in vitro studies have demonstrated that surfactant components, particularly phospholipids, exhibit a high affinity for various PS molecules. 22 This interaction results in PS trapping, preventing their effective engagement with microbial targets and consequently reducing the efficiency of aPDT. 22 For decades, polymers have been widely used to enhance the solubility and bioavailability of therapeutic agents. More recently, these techniques have advanced the delivery and bioavailability of PS (photosensitizers) for antimicrobial photodynamic therapy (aPDT). 29–31 This includes using microneedles, transdermal patches, drug delivery systems in biofilms, and nanomaterials, thereby increasing the overall efficacy of the treatment. 32–36 To address the challenge posed by the lung surfactant impairing aPDT response, Lima et al . 23 employed Gantrez AN-139 as a carrier for ICG, and around 6.31 Log reduction in S. pneumoniae load in vitro was achieved even in the presence of two LS commercial compounds. Moreover, their study demonstrated the biocompatibility of this copolymer, at the used concentration with pulmonary cells, highlighting its potential use for ICG delivery to the lungs 23 . Building on these findings, the present study validated this protocol in a murine model, paving the way for innovative treatments targeting pneumonia. These results are particularly significant in the context of rising antimicrobial resistance, providing a foundation for future research involving larger animal models and eventual first-in-human studies. Results Gantrez-ICG aerossol: the Gantrez-ICG formulation was characterized based on the volume median diameter (VMD) and particle dispersion. The VMD values for ICG were close to 9 µm, showing little variation, while the dispersion values were below 0.3 µm, indicating low particle size variability. On the other hand, for solutions containing 4% (w/v) Gantrez, it was necessary to adjust the initially intended concentrations (400, 200, and 100 µM) because the polymer significantly increased the solution’s viscosity, preventing the particles from being uniformly nebulized and detected by the Spraytec system. Consequently, the tests were carried out with lower-than-intended concentrations. Under these conditions, the dispersion values exceeded 1.0 µM, indicating a high variability in particle size. The results are summarized in Table 1. Table 1. Size of particles Distilled water 8.90 0.27 0.47 0.21 ICG 400Um 9.34 ICG 200uM 9.39 ICG 50uM 9.41 0.29 ICG 10uM 9.39 0.35 ICG 7uM ICG GA 10uM 8.82 9.13 0.49 5.38 ICG GA 7uM 10.42 3.29 Bacteria and animal model Single treatment: This study was approved by the Animal Ethics Committee of the São Carlos Institute of Physics, University of São Paulo (Approval No. 6505060323). All experiments were conducted in accordance with ARRIVE guidelines and National Council for the Control of Animal Experimentation (CONCEA). 72 female BALB/c mice, aged 8 weeks and weighing approximately 25g, were used, considering 3-5 animal per group. The animals received the Gantrez-ICG formulation via intratracheal instillation or nebulization. The irradiation (fluence of 40, 60, 120, and 200 J/cm2) was performed with a laser-based device at 808 nm beginning simultaneously to the drug administration and for the needed irradiation time to achieve the desired delivered fluence. The bacteria quantification of the Bronchoalveolar Lavage (BAL) and lung macerate post-aPDT with different fluences is shown in Figure 2. Figure 2. Recovered bacterial load (CFU/Mouse) after a single aPDT treatment using the instillation (A) or nebulization delivery (B) and different delivered fluences. The Gantrez-ICG formulation and aPDT at low fluences levels of 40 and 60 J/cm 2 did not significantly reduce the bacteria load for any of the drug delivery methods evaluated. On the other hand, for fluences over >120 J/cm 2 , there was a significant CFU/Mouse reduction of approximately 2.45 and 2.60 log 10 for intratracheal instillation and nebulization, respectively. No statistically significant differences were observed between the bacteria recovery methods (BAL and tissue macerate). Dual treatment : to improve the aPDT efficacy against S. pneumoniae, a dual treatment protocol was tested. The Gantrez-ICG was delivered simultaneously with the light and one hour later, a second similar treatment was carried out. Results for cohorts treated with two treatments of 40 J/cm 2 (total 80 J/cm 2 ) and of 120 J/cm 2 (total 240 J/cm 2 ) are shown in Figure 3. Figure 3. Recovered bacterial load (CFU/Mouse) after a dual aPDT treatment using the instillation (A) or nebulization delivery (B) and different delivered fluences. The dual treatment did not significantly improve the treatment outcome. With bacteria reductions (CFU/Mouse) of 0.74 and ~ 2.83 log 10 , these results were similar to those achieved with a single treatment and 40 J/cm² and 120 J/cm². Antimicrobial activity of Gantrez-AN 139: Considering the intrinsic antimicrobial properties of the Gantrez polymer, 37,38 These effects were also evaluated under light exposure. No bacterial reduction was observed in figure 4, confirming that the data in Figures 2 and 3 are indeed a result of the photodynamic action caused by the ICG activated by light. Figure 4. Antimicrobial effect of the Gantrez AN-139 polymer alone, administered with a light dose of 120 J/cm². Creator by the author. Based on these results, we conducted experiments to evaluate the effects of aPDT on survival in a murine pneumonia model (n=5 per group). The animals were daily monitored for 21 days after aPDT treatment. Within a week after the treatment, the survival rate from the control group was approximately 40% compared to 80% of the aPDT group. These rates were maintained for the remaining evaluation period. (Figure 5). Figure 5. The Kaplan-Meier curve illustrates the survival of untreated control mice and mice treated with PDT (120 J/cm² light + 200 μmol/L ICG + 4%(w/v) Gantrez) over 21 days following infection with 10⁷ S. pneumoniae cells. The treatment was performed one day after bacterial inoculation. In the control group, 60% of the animals (3 out of 5) died within six days of infection. In contrast, only one animal in the aPDT group died during the 21-day observation period. Discussions These findings align with a series of previously published data from our group on the efficacy of PDT for bacterial decontamination and further expand its application to PDT with external light-mediated illumination in cases of pneumonia. 17,18,20–22 Studies conducted by Lima et al. 23 demonstrated that a fluence of 40 J/cm² combined with 0.2% (w/v) Gantrez-ICG effectively inactivated bacteria (6.31 Logs CFU/mL) and confirmed that, under these conditions, the Polymer, at the used concentrations, even in the under 808 nm irradiation, exhibits no toxicity. Building on these findings, which highlight the potential of aPDT to eliminate S. pneumoniae , the in vivo results presented in this study provide additional evidence and demonstrate the effectiveness of using external near-infrared light illumination to treat pneumonia. 21 The present study evaluated different aPDT protocols combined with 4% (w/v) Gantrez AN-139 formulations associated with ICG, to improve PS delivery to the lungs and enable external light-mediated aPDT. Figure 2 presents the results obtained with the formulation used in vitro with different fluences. At 40 J/cm², the expected bacterial reductions, based on previous in vitro studies, were not achieved, consistent with the in vitro study as described in the literature. 23 Although the formulation demonstrated highly effective performance in vitro , it faces different and more complex conditions in the in vivo model, including challenges during nebulization and the drug’s way through the animal’s upper respiratory system up to the lungs. The Gantrez-ICG ratio used in the in vitro studies was maintained, but the concentration was optimized to 200 µM of ICG and 4% Gantrez. Additionally, the dynamics of the LS production and the bacteria conditions in an infection that affects the aPDT response, are not reproduced in a simple in vitro condition. Adjustments to the fluence and nebulization time did not lead to the expected increase in efficacy at 60 J/cm². Significant changes in the results were only achieved when the fluence reached levels of 120 J/cm² and 200 J/cm². A bacteria reduction (CFU/Mouse) of approximately 2.45 and 2.60 Log 10 in the BAL and lungs macerated, respectively, can be significant for a pneumonia treatment, demonstrating the protocol’s viability (Figure 2). However, the nebulization parameters could not be varied due to technical limitations associated with the used ultrasonic nebulizer. 39,40 The implementation of two aPDT sessions combined with nebulization did not show an improved efficiency when a 1-hour interval between sessions was used. Multiple sessions could be a potential option for enhancement, but this approach still requires further investigation of different protocols, including distinct intervals between sessions. This will undoubtedly be an experimental project to explore in the near future. Furthermore, both recovery techniques showed no statistically significant difference. 39 Studies involving conventional methods and specific alternative therapies have explored different antimicrobials and delivery mechanisms, 41,42 distinct from the approach reported in this work. The study conducted by Kato et al . 42 investigated the efficacy of conventional antibiotics Garenoxacin (GRNX) and Levofloxacin (LVFX), demonstrating that GRNX exhibited superior antimicrobial activity compared to LVFX, with reductions of 2.02 log₁₀ for Streptococcus pneumoniae and 1.12 log₁₀ for Parvimonas micra . While these results are significant, the observed bacterial reduction values are lower than those achieved in this study using Antimicrobial Photodynamic Therapy (aPDT). The combination of aPDT with antibiotics, as described by Soares et al . 10 , could further enhance treatments, offering a synergistic approach against bacterial infections. The antimicrobial effect of Gantrez AN-139 is widely discussed in the literature. Figure 4 presents the results from tests conducted using only the polymer under the irradiation 120 J/cm². It is evident that no bacterial reductions occur under these conditions, confirming that the observed effect is attributable to aPDT effect. 37,38 Finally, the animal survival experiment (Figure 5), with a 21-day follow-up, provides even more conclusive evidence of the significant impact of aPDT with polymer-loaded ICG on survival rates. Among the animals treated as described in Section Survival rate , 80% survived, compared to only 40% in the control group. In the control group, 3 out of 5 animals died between the 5th and 6th days after infection. In contrast, in the treated group, only one animal died on the 8th day post-infection, the 7th day after treatment. Even without a bacteria eradication, the achieved reduction contributed to the animal response, probably the immune system, resulting in the pneumonia resolution. These results highlight the technique’s feasibility and strongly support continued investment in its development. Two factors appear to be relevant to the limitations observed so far. The first concerns the distribution of ICG particles associated with the polymer, which exhibited sizes over the one considered ideal. 43 To effectively reach the human lungs, particles are recommended to have an average diameter of 4 µm. Studies indicate that particles with a Volume Median Diameter (VMD) of approximately 9 µm would result in about 80% of the inhaled dose, with only half of that dose being deposited in the lungs. 43–47 However, the measured sizes of ICG particles exceed 9 µm under any condition in aqueous solution. Furthermore, the addition of the polymer slightly increases the average particle size and significantly increases dispersion. In the presence of the polymer, an average size of 10 µm and a dispersion value 3.2 µm were observed, indicating that only a small fraction of the particles reaches the lower respiratory tract. 46,47 This indicates that, in the current situation, a limited amount of the formulation being delivered to the site needed for the procedure. Most of the droplets are retained in the upper respiratory tract or in the intratracheal tube for the nebulization and intratracheal instillation methods, hindering their arrival at the infected target. A new aerosolization method could improve the distribution of the formulation as an aerosol and significantly increase drug delivery. 48 However, despite the limitations, it is surprising that the technique still shows effectiveness. Significant improvements can be achieved in aerosol dispersion if new nebulization methods are introduced. Parallel to this current study, we are developing techniques to produce aerosols with low dispersion and smaller mean particle sizes. This approach is expected to result in a deeper and more efficient delivery of the photosensitizer. A second reason for the limitations in the results is related to the photodynamic efficiency of ICG when associated with the polymer. Preliminary studies suggest that the binding of ICG to the polymer causes changes that need further investigation. We believe that using ICG with chemical modifications or introducing additives into the biopolymer composition could lead to further improvements in the results already achieved. Currently, ICG is strategically advantageous due to its absorption wavelength at the near-infrared range, where there is lower light attenuation, resulting in higher penetration at biological tissues. This is undoubtedly a promising new avenue that requires further development. A third concern is the choice of the pneumonia murine model. When using the instillation method to induce the pneumonia, the animals will initially develop infected sites at the upper respiratory tract, and later infection of the lower tract. In this case, since the treatment targets the lower respiratory tract due to the location of the illumination, the collection of microorganisms for post-treatment counting may involve migration from the untreated upper tract to the lower tract. Additionally, the washing process may inadvertently include the upper tract. As a result, microbial counts may be artificially elevated, potentially obscuring the true effectiveness of the treatment and underestimating the potential for significantly improved outcomes. Nonetheless, this phenomenon may also better replicate the natural infection process associated with bacterial contamination in the respiratory tract. These findings highlight the possibility of achieving stronger microbial inactivation, underscoring the potential of externally delivered light-mediated aPDT as a promising approach for pneumonia treatment. Conclusions The results of this in vivo study suggest that the nebulization and tracheal instillation delivery of Gantrez-ICG, combined with external illumination, represents an innovative and promising strategy for treating pneumonia. This study provides critical proof of concept, showcasing the potential of this technique for future refinements that could pave the way for clinical application. These findings align with recent studies demonstrating the feasibility of combining antimicrobial photodynamic therapy (aPDT) with antibiotics. Such a synergistic approach offers a viable and effective solution to combat antibiotic-resistant bacterial pneumonia. The technique’s potential is significant, opening new avenues in the fight against bacterial resistance and marking an important step forward in the development of novel treatments for resistant bacterial infections 10,11 . Materials and Methods Preparation of the ICG-Gantrez formulation: a stock solution of 30% Gantrez AN-139™ (Ashland, USA) was prepared as described by literature. 49,50 In summary, polymer powder was diluted in Milli-Q water and kept at 95°C for 4-5h to induce gel formation through hydrolysis of the anhydride form of the copolymer into its corresponding acid. For the experiments, 4% (w/v) Gantrez working solution was prepared and mixed with different concentrations of ICG and centrifuged at 3500 rpm for 10 minutes to ensure complete homogenization and bubble removal. For tests using only the polymer, the volume allocated for ICG was replaced with Milli-Q water, following the same preparation procedure. Figura 6. Chemical structure of indocyanine green (ICG) and chemical structure of the Gantrez AN-139 polymer. Created by the author using Chemsketch software. Characterization of ICG-Gantrez aerossol : The particle size released by a nebulizer typically follows a normal distribution. The volume median diameter (VMD) represents the point where half of the particles are smaller than or equal to this value. The spread of the diameter distribution is measured by the Span, which is calculated by the RT Sizer software as 𝑆𝑝𝑎𝑛 = (𝐷90 − 𝐷10) / 𝑉𝑀𝐷, where D90 and D10 are the diameters below which 90% and 10% of the particles are found, respectively. The droplet size was determined using optical measurement with the Spraytec system (model RTS5134 from Malvern Instruments) and the RT Sizer software. The system allows the measurement of particles with a volume median diameter between 2.5 and 125 µm. The nebulizer was positioned in the optical path, and approximately 100 measurements were collected for each sample. For the ICG, solutions at 200 μg/mL were used. Bacteria and animal model: Streptococcus pneumoniae (ATCC® 49619™) was stored in Tryptic Soy Broth (TSB) with 15% glycerol at -80°C. A pre-inoculum was prepared by adding 200 µL of the bacteria to 10 mL of Brain Heart Infusion (BHI) and incubating it in a 5% CO 2 environment at 37°C until the optical density (OD 600nm) reached 0.2–0.8. The bacteria were then centrifuged, resuspended in Phosphate Buffered Saline (PBS), and prepared at a concentration of 5.0 x 10 7 CFU/mL. Mouse model: before the bacteria administration, cohorts of female Balb/c (n=3-5) were immunosuppressed with two doses of cyclophosphamide injected intraperitoneally. An initial dose of 3 mg was administered in 150 μL of saline, followed by a second dose of 2 mg in 100 μL 72 hours later. Twenty-four hours after the immunosuppression, the mice were infected via nasal instillation with 15 μL per nostril of S. pneumoniae at 1.0 x 10 7 CFU. Antimicrobial Photodynamic Therapy (aPDT) protocol For the proposed treatment, two delivery methods were tested: nebulization and installation. Nebulization was delivered through a face mask coupled to an ultrasonic nebulizer. Instillation was carried out by attaching a catheter to the trachea, administering 10 µL of the formulation every 2 minutes of irradiation, totaling 40 µL, using the same doses of light as for nebulization, as shown in Table 2. The treatment utilized a light source consisting of 18 diode lasers emitting at 808 nm arranged in a matrix to ensure uniform illumination from all angles and behind the target area (Figure 3). The irradiance was of 120 mW/cm² at the surface of the treated area. All animals had their dorsal fur removed to minimize light scattering. The ICG-Gantrez formulation and light were delivered simultaneously. For the groups aPDT-40+40, aPDT-120+120, or dual-aPDT, the treatments were given 1h apart. Table 2. Treatment groups and aPDT protocols Groups Irradiation time (min) Fluence (J/cm²) Number of treatments ICG concentration (µM) Gantrez AN-139 concentration (%) Control (Infection) 0 0 0 0 0 Control (Gantrez AN-139 + light) 16 120 1 0 4 aPDT-40 6 40 1 200 4 aPDT-40+40 6 40 2 200 4 aPDT-60 8 60 1 200 4 aPDT-120 16 120 1 200 4 aPDT-120+120 16 120 2 200 4 aPDT-200 20 200 1 200 4 Figure 7. Schematic representation of the experimental setup used for external illumination of the animals. The red lines represent a uniform 808 nm light incident on the animal’s back and sides. The diffuse light reaches the lungs at approximately 25 to 30% of the incident fluence. Created using CAD Software. Analysis of treatment response Bacterial recovery of the lungs: two recovery methods were used, bronchoalveolar lavage (BAL) and tissue maceration. For the BAL, the mice were anesthetized with xylazine and ketamine 10 mg/kg and 100 mg/kg a 20 catheter was inserted into the trachea. The BAL was then collected by injecting 1000 uL of PBS into the catheter and aspirating the fluid back. The BAL was then centrifuged for 15 minutes at 3000 rpm. The supernatant was discarded, and the recovered cells resuspended in 300 μL of PBS. The lungs were then homogenized in 1.0 mL of PBS. Both the BAL and the homogenates were diluted and plated on blood agar plates and incubated at 37°C for 18 to 24 hours. Subsequently, colony-forming units (CFUs) were counted. Survival rate : the optimized aPDT protocol was repeated in a mice cohort (n=5) to assess whether the treatment could improve survival. The animals were monitored daily for up to 21 days. Humane endpoints included weight loss >20%, short breath, hunched posture, reduced movement and ruffled fur coat. Statistical analysis: a two-way ANOVA with Ordinary one-way ANOVA ad-hoc test (Prism, GraphPad 8.0) was conducted to assess the differences between treated and non-treated mice, as well as bacteria recovery methods (BAL vs maceration). Statistical significance was defined as p<0.05. Acknowledgments This work was supported by FAPESP (grant 2013/07276-1, 2022/03965-6, 2022/10860-6, 2023/04209-3), CNPq (grant 465360/2014-9; 381522/2024-5) and CAPES (grant 88887.999666/2024-00). Cooperation with Texas A&M University - Biomedical Engineering Depart under the contracts: CPRIT - grant RR220054; GURI - grant M230930 and CRI- 02-292034, are considerably appreciated. REFERENCES 1. Loscalzo. Joseph. HARRISON’S Pulmonary and Critical Care Medicine . (2010). 2. Bender, R. G. et al. 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Keywords antimicrobial photodynamic therapy biopolymers indocyanine green pneumonia Authors Affiliations Lorraine Gabriele Fiuza 0009-0006-5067-8129 [email protected] Universidade de Sao Paulo Instituto de Fisica de Sao Carlos View all articles by this author Isabelle Almeida de Lima Universidade de Sao Paulo Instituto de Fisica de Sao Carlos View all articles by this author Michelle Barreto Requena 0000-0002-8690-3053 Universidade de Sao Paulo Instituto de Fisica de Sao Carlos View all articles by this author Vadila Guerra Universidade Federal de Sao Carlos View all articles by this author Layla Pires Texas A&M University Department of Biomedical Engineering View all articles by this author Cristina Kurachi 0000-0001-7175-5337 Universidade de Sao Paulo Instituto de Fisica de Sao Carlos View all articles by this author Natalia Mayumi Inada Universidade de Sao Paulo Instituto de Fisica de Sao Carlos View all articles by this author Vanderlei Salvador Bagnato Universidade de Sao Paulo Instituto de Fisica de Sao Carlos View all articles by this author Metrics & Citations Metrics Article Usage 204 views 108 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Lorraine Gabriele Fiuza, Isabelle Almeida de Lima, Michelle Barreto Requena, et al. 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