Raman Spectroscopy and Histopathology of Rat Bladder Cancer treated by Doxorubicin and Cisplatin using reduced graphene oxide as carrier

INTRODUCTION
Raman spectroscopy is capable of identifying biomolecular changes associated with cancer progression; thus, this technique can be used as non-invasive tool to help diagnosing bladder cancer. A literature review has followed the evolution of studies on this topic, from the first experiment conducted in vitro to the application of this technique in vivo near 2014. It identified how diagnostic algorithms were developed and provided molecular information correlated to the etiology of the biochemical continuum of cancer progression [1]. According to histopathologists, Raman spectroscopy has the potential to help identifying and classifying biochemical changes associated with carcinogenesis. Raman demonstrating also a rapid, reproducible and non-destructive tissue during the analyses of a specific composition [2].
Bladder samples were examined through Raman spectroscopy; analysis results have shown healthy tissues, carcinoma in situ (CIS), as well as low- (LGC), moderate- (MGC) and high-grade carcinoma (HGC), after routine cystoscopy [3]. The mean spectra observed for each group has demonstrated subtle differences; principal component analysis was used to divide the five pathology groups presented by the analyzed specimens as much as possible. Stone et al [3] have explained these differences as increasing malignancy grades and likely the hard task to fully differentiate these groups based on only histopathology.
Raman spectroscopy was capable of distinguishing non-tumor from tumor bladder tissues. The compact fiber probe-based imaging Raman (CFPBI) system was enabled to Cordero et al [4] to correctly classify these tissues with high sensitivity and high specificity. The Raman endoscopic system was applied to 107 tissue samples collected from patients with bladder cancer during transurethral tumor resection; spectra were analyzed based on multivariate statistical methods. Multivariate algorithm enabled differentiating non-malignant from malignant tissues at sensitivity and specificity of approximately 79%. The authors have suggested that, despite the small number of samples, data have shown great potential to identify bladder wall lesions during endoscopy [5,6].
Laser-based confocal Raman micro-spectroscopy and principal component analysis (PCA)/support vector machines were used to build diagnostic algorithms capable of determining histological diagnosis based on the leave-one-out cross validation of its Raman spectrum. Nucleic acid peak intensity of Raman spectra in bladder cancer cells and protein contents in healthy bladder cells have significantly increased. PCA and support vector machines (SVM) were effective tools capable of differentiating bladder cancer from healthy bladder tissues. Excellent specificity, sensitivity, positive and negative predictive values of ~73% for bladder cancer diagnosis were observed through leave-one-out cross validation [7]. Wang et al. [7] have suggested that PCA/SVM-based Raman spectroscopy applications have promising potential to be used in bladder cancer diagnosis.
A preliminary study applied low-resolution fiber-optic Raman sensing (LRFORS) system to assess the potentially of Raman spectroscopy to diagnose diverse bladder pathologies ex vivo. Spectra from 32 bladder specimens were classified into 3 groups based on histopathological analysis, such as: healthy bladder tissues, low-grade and high-grade bladder tumors. Results have shown the potential of this technique - which uses LRFORS system - to diagnose bladder cancer in vivo [8,9].
The most recent technique comprised the combination between OCT (optical coherence tomography) and RS (Raman spectroscopy) to improve the diagnosis of, as well as to differentiate, different bladder cancer stages and grades ex vivo by associating complementary data supplied by these two techniques.  RS was performed upon the identification of degenerated tissue via OCT in order to define the molecular profile of it through point observations at suspected sites. OCT enabled clearly differentiating healthy from malignant tissues through tomogram inspection (71%) of tumor staging, from pTa to pT2, as well as over texture analysis and k-nearest neighbor categorization. RS provided 93% accuracy at the time to differentiate low- from high-grade lesions through PCA analysis, which was followed by k-nearest neighbor categorization. Data have evidenced the potentiality of a multi-modal strategy associated with OCT to be used for fast pre-selection and staging cancerous lesions, as well as of using RS to enhance the differentiation between low- and high-grade bladder cancer in a l3bel-free, non-destructive and non-invasive manner [10]. Most recently, a literature review has addressed the application of RS in breast cancer cases; it discussed its potential to analyze either tissues ex-vivo or liquid biopsy samples. Its potential to be used for applications in vivo in research about cancer, as well as for translational and clinical applications [11].
Therefore,  the aim of the current research was to apply an exhaustive histopathological analysis based on RS to rats chemically induced to NMIBC in order to investigate its potential to be used for tissue featuring purposes [5, 12-14].

MATERIALS AND METHODS
Reduced graphene oxide synthesis
rGO (reduced graphene oxide was obtained and featured, as previously published [15,16]. Doxorubicin (DOX) and Cisplatin (CIS) were provided from LC Laboratories (Woburn, EUA) and LibbsFarmacêutica LTDA (Embu, São Paulo, Brasil), respectively.

rGO disperssion in Pluronic® F68
Dispersion of rGO was obtained by adding a proper quantity of rGo to 1% of Pluronic F68 in such away to find the final solution of 2 mg/mL of rGO based on ultrasonication bath application for 20 min.
CIS and DOX adhesion to rGO    
In total, 0.2 mg of DOX was added to 0.2 mL of physiological solution. This mix was added with 1 mL of rGO dispersion (2 mg/mL). Subsequently, 1.0 mL of rGO dispersion (2mg/mL) was added to 0.2 mL of CIS (0.05 mg) and shacked for 2h. 

Experiment conducted in vivo with rats
Thirty female of seven weeks old Fischer 344 rats were purchased in the Multidisciplinary Center for Biological Investigation (CEMIB) at University of Campinas (UNICAMP). Animal studies were accredited by the institutional Ethics Committee on Animal Research (CEUA/UNICAMP, protocol no. 3569-1). Rats were anesthetized with 10% ketamine (60 mg/kg, i.m.; Vibra®, Roseira, SP, Brazil) and 2% xylazine (5 mg/kg, i.m.; Vibra®, Roseira, SP, Brazil) before they were subjected to intravesical catheterization performed with 22-gauge angiocatheter. Animals remained anesthetized for approximately 45 min after catheterization in order to eliminate spontaneous micturition. Control group – Group 1: five control animals were treated with 0.30 ml of 0.9% physiological saline, intraperitoneally (i.p.) administered once a week, for 14 successive weeks. NMIBC induction was carried out in 25 rats that intravesically received n-methyl-n-nitrosourea (MNU; 1.5 mg/kg, dissolved in 0.30 ml of 1 M sodium citrate, pH 6.0) every other week, for two months [14]. Two weeks after the last MNU dose, all rats were subjected to ultrasound examination in order to assess the incidence of tumors. Then, MNU-treated rats were further separated into five groups (n=5 per group): the Cancer+rGO group (Group 2) was treated with 2.0 mg/mL of rGO, i.p. administered once a week, for one month and a half successive weeks; the Cancer + rGO + CIS group (Group 3) was treated with  0.2 ml of rGO added with 0.05 mg cisplatin (CIS), i.p. administered and complexed in rGO dispersion (2 mg/mL) once a week, for one week and a half successive weeks; the Cancer+rGO+ DOXO group (Group 4) was treated with  0.2 ml of rGO  added with 0.2 mg doxorubicin (DOXO), i.p. administered and complexed in rGO dispersion (2mg/mL) once a week, for one month and half successive weeks; the Cancer+rGO+CIS+ DOXO group (Group 5) was subjected to the same procedure as Groups 3, 4 and 5.
After treatment was over, all animals were euthanized and all the their urinary bladders were collected and analyzed by histopathological and Raman spectroscopy analyses.

Histopathological Analysis
Urinary bladder samples were collected from all rats and fixed in Bouin solution for 12 hs. Post-fixation process, tissues were washed in 70% ethanol and subjected to dehydration in an rising up series of alcohols. Subsequently, tissue fragments were diaphanized in xylene for 2 h and included in plastic polymers (Paraplast Plus, ST. Louis, MO, USA). Afterword, the materials were cut (thickness = 5 μm ) in Leica RM 2165 rotary microtome (Leica, Munich, Germany),  stained with hematoxylin-eosin and photographed in Zeiss Axiophot light microscope (Zeiss, Munich, Germany). Studied groups were characterized based on the consensus staging suggested by the World Health Organization/International Society of Urological Pathology [17,18].
Histopathological results were confronted to a proportion test. The divergence between the two proportions was subjected to proportion test at 1% type-I error. 

Raman spectroscopy measurement
Measurements    
The experiment was carried out at the Electrical Engineering and Computing School, Department of Semiconductors, Instruments and Photonics, in Unicamp. Urinary bladder fragments were frozen in liquid nitrogen and stored in frezeer at -80°C. Raman Scattering Spectroscopy (in Via Renishaw microscope, England) with infrared laser excitation  ʎ = 785 nm and 20x magnification lens was used to analyze changes in the investigated samples and to find the Raman spectra. Samples were subjected to the same analysis conditions, the observation field was selected at random, as well as the representative of the sample spectrum.

RESULTS AND DISCUSSIONS
Macroscopic and Histopathological Analyses
Macroscopic changes were not observed in the peritoneum of the Control or Cancer group (Fig. 1A). However, animals in the Cancer + rGO group presented dark agglomerates in the peritoneum (Fig. 1B). It may have happened due to slow rGO compound absorption by mesenteric vessels when it was intraperitoneally (i.p.) administered.
This perspective has shown that  GO and rGO administered via i.p. remained for more than a month in animals’ body when PEG was used as polymer in carbon nanotubes. Although this finding leads to concern about toxicity, no toxicity of graphene-based materials (GBMs) has been proven, so far [19]. Guo and Mei [19] have also reported that animals did not present damage in the main organs, although dark spots on the liver and spleen were observed - they subsided over time. The observed aggregate can lead to thrombus formation and, later on, it can lead to animals’ death. In addition,  GO-PEG i.p. administration has led to the presence of nanomaterial in animals’ bodies for three months, and it evidenced that GBMs’ behavior in vivo and toxicology  depend on the administration route [20].
Dark agglomerates were not observed in the peritoneum of the herein investigated animals when CIS and/or DOXO were dispersed with rGO (Fig. 1C); this finding has indicated higher absorption of these compounds by mesenteric vessels. In addition, carbon nanotubes in contact with biological fluids have adsorbed biomolecules and formed their “corona” by reducing the surface energy of carbon nanotubes and by promoting its dispersion – this factor played important role in carbon nanotubes identification process [21]. 
With respect to histopathological results, the Control group did not show histological changes in bladder tissue (Figs. 2a, 2b; Table 1). Healthy bladder urothelium was composed of basal, intermediate and surface cell layers (umbrella cells) (Figs. 2a, 2b). On the other hand, all animals in the Cancer group have shown 100% malignant lesions, such as urothelial carcinoma with lamina propria invasion (pT1) (Figs. 2c, 2d; Table 1). 
Urothelial papillary carcinoma (pTa) (Figs. 2e, 2f) and pT1 were the most frequent neoplastic lesions observed in the Cancer+rGO group – they affected 60% and 40% of rats, respectively (Table 1). Similarly, the Cancer+rGO+CIS group has shown 100% malignant lesions, such as pTa (Figs. 2g, 2h) and pT1 – these lesions affected 80% and 20% of rats, respectively (Table 1).
Rats in the rGO+DOXO group have shown malignant lesions, such as flat carcinoma in situ (pTis) (Figs. 2i, 2j) and pTa. These lesions affected 60% and 40% of rats, respectively (Table 1).
Animals treated with rGO+CIS+DOXO have clearly shown better histopathological recovery from cancer than those subjected to the other treatments – this group has shown decreased neoplastic bladder  lesion progression in 40% of animals (Table 1). Benign lesions, such as flat hyperplasia (Figs. 2k, 2l), were found in 40% of rats in this group (Table 1). The most frequent neoplastic lesion found in this group was pTis, which affected 20% of rats (Table 1).

Raman spectroscopy.
The current study took into consideration bladders with lesions that were more representative of the analyzed groups of animals, as well as the treatment dispersions they were subjected to, which led to the spectra (Fig. 3), based on which, it was possible generating the table with the observed peaks (Table 2). According to reports, healthy bladder tissues have shown bands at 875; 1,080; 1,289; 1,303; 1,446 and 1,657 cm-1, which are considered high and can be attributed to lipids and proteins [8]. On the other hand, bands observed in bladder cancer at 1,330; 1,462 and 1,662 cm-1were associated with guanine, proteins and tryptophan, respectively [6]. Peaks observed for healthy tissues are often displaced (between 1 and 5 cm-1) or missing in tumor tissues. It is worth emphasizing that detecting non-aromatic amino acids was a defying task, since they generate weak vibrational signals due to their poor polarity; but, aromatic amino acids present significant vibrational peaks due to the incidence of benzene ring. Nucleic acid peaks were observed through vibration in DNA bases, which are masked in healthy tissues. Cells proliferate without control; DNA amount significantly increases during malignant transformations - this process is followed by changes in phosphate, deoxyribose or bases [8]. The incidence of nucleic acids (~ 1,340 cm-1) observed in the cancer group resulted from malignancy of the neoplastic process, which is basically a cellular process presenting high protein and lipid levels [22]. Protein visualization through Raman spectrum provides information about amino acid chains and that plays crucial role in interactions between protein structure and function [8].
Only peaks different from those presented in the control and cancer groups were taken into consideration at the time to observe nanocomposites’ interaction in NMIBC treatment. The following peaks were observed in the rGO group: 930; 1,285; 1,770 and 2,958 cm-1. Carbohydrate peaks at 930 cm-1 according to studies that the accumulation of the compound occurs during maturation and disappear with the loss of differentiation during neoplasia [23]. The 1,285 cm-1 band is associated with cytosine [24]; nucleic acid-associated peaks and DNA structure suggest the initiating of DNA fragmentation during apoptosis. The 1,770 cm-1 band is associated with vibration strength at C = O bond of δ lactone. 
Peak in the rGo+CIS was observed at 1667 cm-1, which referred to protein band, C = C band stretching, amide I helix structure and structural mode of tumor proteins. According to Chen et al [8] studies, bands representing proteins vary in cancer tissues, and it suggests weak interactions between chemical amino acid bonds in cancer cells. It happens because hydrogen bonds can be damaged and lead to protein structure loss or to alterations in the microenvironment of amino acid residues, in case of increased setting and unsetting of α-helix from β sheets [8].
The rGO+DOXO group presented the following peaks: 858; 1,259; 1,325; 1,667 and 2,953 cm-1. The peak at 858 cm-1 refers to tyrosine and collagen; increased DNA and decreased collagen levels are expected, since the core / cytoplasm ratio increases from healthy tissues to neoplastic ones. Cells become more abundant and the extracellular matrix, which presents abundant collagen amounts, reduces until the tumor gets bigger, and collagen I and III in the bladder become more abundant [3].
This process means that cancer cells produce and release matrix metalloproteinase in order to degrade the protein matrix, in the case of collagen, a fact that enables metastasis because the proliferation of cancer cells can mask the collagen signal in the matrix [8]. 
In special, the phenyl peak at 1,005 cm-1 and the protein C–H deformation and adenine, guanine nucleic acid peak at 1,342 cm-1 presented sharp intensity diminishes for all but one cell at 48 h time points, doing these excellent biomarkers of cell death [24]. In 1,640 cm-1 protein band stretching of the band C = C α-amide structure of the amide I structural mode of tumor proteins [25].The 2,953 cm-1 band, which refers to asymmetric CH3 elongation, has shown that Raman spectroscopy was capable of differentiating CH3 from CH and CH2CH3, which have the same features in the spectrum.
Changes in the optical properties of tissues, such as increased urothelium thickness, as well as morphological changes (increased cell density), can mitigate excited light penetration, and decrease the amount of photons emitted in the stromal region of neoplastic tissues in comparison to that of healthy tissues [26].
The following peaks were observed in the rGo+CIS+DOXO group: 1,242; 1,328 1,457 and 2,951 cm-1. Peak 1,242 cm-1 represents amide III (β sheet and random distribution were attributed to amide III vibrations, in both of protein structure such as elastin and collagen [27]. The 1,328 cm-1 band has shown typical phospholipids [21]; “healthy” tissues were rich in lipids, which were uniformly distributed, whereas tumor tissues were more concentrated and evident. The 1,457 cm-1 band corresponds to deoxyribose and at 2,951 cm-1appeared the asymmetric stretch of CH3.
Bands ranging from 1,230 to 1,300 cm-1 refer to lipids and proteins [6] that derive from the lamina propria and muscular layer in healthy bladder tissues. According to reports, lipid peaks were mainly generated due to cell membrane vibration, C-H and C-C bonds of lipids and C = C unsaturated fatty acids. Peaks associated with amino compound III were mainly produced by C-N elongation vibration and N-H binding vibration, which indicated presence of protein in the β-sheet structure [9].
These changes are associated with disease progression, since bladder tumors are often papillary and quite vascular, and they do not penetrate necrotic sites [3]. Identifying chemical changes in the Raman spectrum of healthy and pathological tissues enables better understanding the effects of treatments, as well as allows optimizing pathological information. Moreover, cell death does not take place as a slow biomolecule degradation process [28], but as a singular fast event2. Raman spectroscopy allowed the first approximation between biochemical bases to help better understanding the pathological process of carcinogenesis [3].
The techniques herein adopted to feature the rGO sample were very useful, since they were capable of providing data that confirmed the nature of this compound. Using a set of techniques was essential and necessary to help finding reliable and safer results. The rGO tests conducted in vivo did not show significant toxicity; rGO sheets in the group subjected to the combination between CIS and DOXO chemotherapy complexes had positive effects on NMIBC therapy in comparison to the groups treated with isolated chemotherapeutic agents. Raman tests have confirmed structural and biochemical changes in rats’ urinary bladder due to pathological process and to their exposure to chemotherapeutic agents.

CONCLUSIONS
At the end of the activities developed and in light of the objectives outlined in the conception of this study, it can be concluded that:
All of the techniques employed in the characterization of the rGO sample proved to be very useful, providing data that confirmed the nature of the compound. The use of a set of techniques has proven to be of importance and necessary to obtain reliable and safer results; in the in vivo tests rGO did not present significant toxicity and the group with combination of the CIS and DOXO chemotherapy complexes the rGO sheets presented positive effects against the NMIBC therapy when compared to the groups with isolated chemotherapeutic agents; Raman tests confirm structural and biochemical changes in the urinary bladder due to the pathological process and exposure to chemotherapeutic agents.

ACKNOWLEDGEMENTS
Support from FAPESP, CNPq, NanoBioss (MCTI), INOMAT (MCTI/CNPq) and Prof. Dr. Hudson Zanin/FEEC/Unicamp-Brazil for the Raman equipment .

CONFLICT OF INTEREST
The authors declare no conflicts of interest.