**7. miRNAs as therapeutic targets in acute lymphoblastic leukemia**

Nowadays, advances in our understanding of the molecular carcinogenesis of the human cancers and the extensive research on generate and implement new combined and targeted therapies, and have allowed to know specific molecular therapeutic targets. However, there is still a continuous need for development of new therapeutic tools for applicability.

RNA molecules actually are the therapeutic targets promising in the molecular oncology. The ability of miRNAs to regulate important cellular processes, by concurrently regulating multiple targets, their inherent role in carcinogenesis as oncogenes or tumor suppressor genes, and the aberrant dysregulation of their

*Advances in Hematologic Malignancies*

independent of p53 [50].

prognosis [61].

longer over survival [64].

role for miR-125b in early hematopoiesis [46].

cells ectopically expressing miR-125b that showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. These mice developed B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm, suggesting an important

Patients group with high miR-21 expression was significantly associated with those aged <2 and > 10 years, lower platelets count, more incidence of central nervous system (CNS) infiltration, and poorer treatment outcome also; patients with high miR-21 showed a significantly poorer disease-free survival (DFS) and overall survival (OS) compared with those with low miR-21 expression group [47]. Also, miR-92a expression is significantly higher in ALL compared with peripheral blood mononuclear cells (PBMNCs) from healthy volunteers. Likewise, the expression levels of miR-99a, miR-100, and miR-128b correlated high-risk prognostic factors, including white blood cell (WBC) count, ALL subclassification (T-cell and B-cell ALL), the MLL-rearranged gene, and the BCR-ABL fusion gene, suggesting possible relation of miR-99a, miR-100, and miR-218b with prognosis [48, 49].  Wang et al. (2010) observed that miR-146a, miR-181a/c, and miR-221 were significantly associated with overall survival of the ALL patients. Expression level of miR-146a and miR-181a/c was associated with a poor outcome (i.e., poor prognosis/short-term survival), whereas that of miR-221 was associated with a good outcome (i.e., good prognosis/long-term survival) [54], while that of miR-423-5p is associated with a poorer survival in patients with ALL [55]. Otherwise, the reduced expression of miR-155, miR-181b, miR-182, miR-143, miR-210, and miR-335 is associated with poor outcome of pediatric ALL [56–60]. Also, the expression of miRNAs miR-18a, miR-532, miR-218, miR-625, miR-193a, miR-638, miR-550, and miR-633 is associated with early relapse in childhood ALL, suggesting possible relation of these miRNAs with

The high miR-16 expression is associated with hyperleukocytosis and poor cytogenetic groups. In B-cell ALL patients, the DFS was significantly shorter in patients with high miR-16 levels. While in T-cell ALL patients, for both DFS and overall survival, a significant trend was found with a survival shortening from the lowest to the highest miR-16 levels [62, 63]. Likewise, it was reported that the expression of miR-16 was upregulated in cases of T lymphoblastic lymphoma/leukemia (T-LBL/ ALL), and the high expression group of miR-16 was significantly correlated with

For instance, Gimenes-Teixeira et al. reported that T-ALL patients with high miR-221 expression had significantly lower 5-year overall survival (OS) rates compared with those with low miR-221 expression [65]. Oliveira et al. observed that lower levels of miR-29a were significantly associated with higher blast counts in the bone marrow and with increased disease-free survival in T-ALL patients [66].

**6. miRNAs in response to commonly used chemotherapy agents in**

Despite the great effort of current treatment strategies, drug resistance still remains a major cause of chemotherapy failure and relapse in pediatric patients.

**pediatric acute lymphoblastic leukemia**

**62**

expression levels in cancer, can represent a viable therapeutic strategy and a powerful intervention tool in **leukemia** [73]. For example, in leukemia cells isolated from individuals with BCR/ABL, TKI-resistant Philadelphia-chromosome-positive acute lymphoblastic leukemia (Ph + ALL) was observed an increase in levels of DNMT3A in association with downregulation of miR-217; these observations are clinically relevant; and inhibition of DNMT3A by forced expression of miRNA-217 may benefit in preventing drug resistance to TKI treatment in Philadelphiachromosome-positive ALL patients [74]. Another therapeutic strategy for BCR-ABL-positive ALL is miRNA-203, which has as direct target to BCR-ABL1 and ABL1, proteins with activity tyrosine kinase. This miRNA is silenced by genetic and epigenetic mechanisms in hematopoietic malignancies expressing either ABL1 or BCR-ABL1. However, the restoration of the miRNA-203 expression reduces ABL1 and BCR-ABL1 levels and inhibits cell proliferation [75]. miRNA-143 was identified as a regulator of MLL-AF4 expression and is epigenetically repressed by promoter hypermethylation in MLL-AF4-positive primary blasts and cell lines;upregulation of miRNA-143 expression by demethylation has therapeutic promise for MLL-AF4 B-cell ALL [76].

It is also important to consider that some miRNAs can behave as oncogenes in one cancer type and as tumor suppressive genes in others. It has been reported that miR-221 maintains a high expression in hepatic cancer and exerts an oncogenic function by targeting tumor suppressor PTEN, but this miRNA acts as a tumor suppressor in erythroblastic leukemia by inhibiting the KIT oncogene expression [77, 78]. Thus, identification of specific biological functions, type of cancer, and targets of miRNAs is a basic aspect when considering miRNA therapeutics.

## **8. Summary and future directions**

Various studies have demonstrated that the oncomiRs or tumor suppressor miRNAs expression may significantly have potential how diagnostic and/ or prognostic biomarkers, as well as monitoring the disease progression and in the response to treatment, and it may be a therapeutic target for treatment in ALL. Also, miRNAs expression levels may play an important role in the genesis and evolution of the ALL. Nevertheless, the biological effects and relevant target genes of many miRNAs that are deregulated and/or prognostically relevant in ALL need to be identified and characterized. Therefore, novel anti-ALL agents are needed to overcome chemotherapy resistance and reduce cytotoxicity. The mimics- and/or anti-miRNAs may be a good alternative. However, more experiments are required to evaluate the feasibility and safety of mimics- and/or anti-miRNAs in the clinical treatment.

### **Acknowledgements**

This work was supported by the Universidad Autónoma de Guerrero. Yazmín Gómez-Gómez (CVU: 236728) and Jorge Organista-Nava (CVU: 236745) were recipient of postdoctoral fellowships from CONACYT.

**65**

**Author details**

Yazmín Gómez-Gómez†

and Marco Antonio Leyva-Vázquez\*

provided the original work is properly cited.

† These authors contributed equally to this work

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to<EMAIL_ADDRESS>and<EMAIL_ADDRESS>
, Jorge Organista-Nava\*†

Molecular Biomedicine Laboratory, School of Chemical-Biological Sciences, Autonomous University of Guerrero, Chilpancingo, Guerrero, Mexico

, Berenice Illades-Aguiar

*miRNAs in Acute Lymphoblastic Leukemia: Diagnosis, Prognosis and Target Therapeutic*

*DOI: http://dx.doi.org/10.5772/intechopen.84318*

## **Conflict of interest**

The authors declare that there are no conflicts of interest.

*miRNAs in Acute Lymphoblastic Leukemia: Diagnosis, Prognosis and Target Therapeutic DOI: http://dx.doi.org/10.5772/intechopen.84318*


*<IP_ADDRESS> Total energy minimization*

The first step in atomistic simulations is to find a stable structure by the total energy minimization process (geometry optimization). The lowest energy configuration of the atoms in a system can reach by adjusting their coordinates. The net forces acting on the atoms are zero.


*5.3.1 Removal organic and inorganic pollutants by activated carbon-spent coffee grounds*

Activated carbon is a material in which carbon is forming disordered graphite plates, in whose peripheries there is a wide diversity of functional groups, which gives it unique physico-chemical properties. Additionally, this material usually has raised surface areas, generally greater than 1000 m2 /g, which develops through various oxidation reactions [58]. Given these characteristics, this material is typically used in numerous applications, excelling in the removal of organic and inorganic compounds present in the gas and liquid phase.

Waste coffee grounds were used to produce activated carbon by KOH under Ar atmosphere at three temperatures (700, 800, and 900°C), the adsorbent material was tested to adsorb CH4 and H2. The activated carbon at 900°C showed a CH4 adsorption capacity of 1.96 mmol/g at 273 K and 100 kPa. However, at 3000 kPa, the highest adsorption capacity was reported to be 4.2 mmol/g. The three adsorbents materials (700, 800, and 900°C) were also tried to adsorb H2 at 77 K and 100 kPa, achieving the highest adsorption capacity of 1.75 wt% [48]. Activated carbon from waste SCG as the precursor was physically activated by CO2 or steam at high temperatures (700–900°C) and chemically activated by ZnCl2, KOH, and H3PO4 at 450 and 600°C. Nevertheless, in this work, only the raw material, the activated carbons by ZnCl2 or steam, was tested to adsorb Bisphenol-A. The removal of Bisphenol-A was found to be 98, 12, and 0% for carbon activated by ZnCl2, raw material, and carbon activated by steam, respectively. These results were compared

**143**

*Revalorization of Coffee Waste*

(127 mg/g), respectively [51].

and 0.5 were used [55].

and 2.3 mmol/g, respectively [56].

*DOI: http://dx.doi.org/10.5772/intechopen.92303*

the low surface area reported for this material (4 m2

with a commercial activated carbon, which showed a Bisphenol-A removal of 93%. The poor adsorption performance of the SCG carbon activated by steam is due to

SCG was used as a precursor to prepare activated carbon by chemical activation with ZnCl2 at three impregnation ratios, at room temperature, and during 8, 12, and 24 h. The adsorption of Cu(II) was conducted using this activated carbon. The experimental data were fitted by using the Langmuir, Freundlich, and Elovich isotherms; the maximum adsorption capacity (Langmuir) was 285.71 mg/g, and the maximum Cu(II) removal reported was 18% at 100 rpm, and the pH solution value was not reported [51]. The production of SCG-based activated carbon was carried out at three impregnation ratios, g ZnCl2/g precursor (1:0.5, 1:1, and 1:2); the impregnated precursor was carbonized under N2 atmosphere at 800°C during 60 min, and this material was tested for H2S separation. The SCG activated carbon was used to study H2S dynamic breakthrough capacity passing a dilute flow of H2S-Air (1000 ppm, 80% humidity) through a fixed bed. The adsorbents activated at impregnation ratios of 1:2 and 1:1 showed the lowest (18.2 mg/g) and the highest breakthrough capacity

Chemical activation of SCG was performed with KOH using 2:1 and 4:1 impregnation ratio (KOH: precursor) to produce activated carbons. The carbonization process was carried out at 400 or 700°C under N2 flow for 2 h. These activated carbons were tested to adsorb CO2 at 0, 25, and 50°C and 0–10 bars. The highest adsorption capacity obtained was 6.8 mmol/g at 1 bar and 0°C, when the activated carbon was produced at 700°C and an impregnation ratio of 4:1. However, when the carbonization process was performed at 10 bars, the highest uptake achieved was 23.26 mmol/g [53].

SCG microporous activated carbon (using potassium hydroxide as activation agent) was synthesized and characterized. The precursor was pyrolyzed using 1:9, 1:18, and 1:36 mmol KOH:g SCG of impregnation ratios and under N2 flow at 800°C during 1 h. SCG activated carbons were used to adsorb phenol and methylene blue. The equilibrium was attained within 100 and 360 min for phenol and methylene blue, respectively. The maximal adsorption capacity, based on Langmuir isotherm, for phenol and methylene blue was 3008 and 1058 mmol/g, respectively [54].

The influence of the impregnation ratio of H2SO4 over SCG granular activated carbon to treat leachate was studied. Six samples of leachate with the following chemical and biological parameters were treated: COD (1010–1815 mg/L), BOD5 (184–338 mg/L), NH4-N (2208–2780 mg/L), iron (4.25–4.73 mg/L), and PO4-P (220–284 mg/L). However, in this research, only the removal percentage of iron and PO4-P was found to be 77 and 84%, respectively, when impregnation ratios of 2.5

SCG obtained from a trademark coffee (Nespresso®) was used as a precursor to produce activated carbon. KOH was used as an activating agent at four impregnation ratios. The chemical activation process was carried out at 873 K, and physical activation with CO2 was carried out at 973 K. These materials were used to capture CO2, which is a byproduct of the combustion process. However, in this study, a pure CO2 flow was tested at 298 K and 101 kPa. The CO2 adsorption capacities of the adsorbents activated by using chemical activation and physical activation were 3

SCG carbon activated with phosphoric acid and zinc chloride was used to adsorb Pb(II) and Cd(II). The precursor was mixed with ZnCl2 or H3PO4 at chemical agent/ coffee residue mass ratios of 25, 50, 75, and 100% at 85°C for 7 h. When the precursor was activated with H3PO4 (50% impregnation ratio), the maximal adsorption capacities, based on Langmuir isotherm, were as follows: 89.28 mg Pb(II)/g and 46.95 mg Cd(II)/g. With ZnCl2 (75% impregnation ratio) as the activation agent, the maximal adsorption capacities were 63.29 mg Pb(II)/g and 37.04 mg Cd(II)/g [57].

/g) [46].

#### *Revalorization of Coffee Waste DOI: http://dx.doi.org/10.5772/intechopen.92303*

*Coffee - Production and Research*

**Total pore volume (cm3 /g)**

> 0.765 (ZnCl2) 0.618 (HPO3)

**Pore width (nm)**

1039 0.481 4.7 500 ZnCl2

1082 0.51 3.0 600 and 700 KOH or

3.44 (ZnCl2) 2.44 (HPO3)

*Reports of chemical activation conditions to produce activated carbon from coffee wastes.*

831 0.44 400, 450,

**Pyrolysis temperature (°C)**

1040.3 0.635 — 700–900 KOH Ar — [48]

1280 0.77 3 600 ZnCl2 N2 26 [50]

1121 0.954 1–3 800 ZnCl2 N2 — [52] 2785 1.36 1.051 400 and 700 KOH N2 11–16% [53] 1778 0.657 — 800 KOH N2 — [54]

and 500

146.1 0.0705 1.6 600 H2SO4 N2 42.77–

**Activation agent**

Steam

CO2

H3PO4

600 ZnCl2 or

**Flow Yield**

N2 40%

ZnCl2 Air 15.99

**(%)**

20%

22.95%

51.85%

N2 23–29% [56]

N2 — [57]

**Ref.**

[49]

[51]

[55]

**Surface area (m2 /g)**

aqueous solutions.

889 (ZnCl2) 1003 (HPO3)

**Table 1.**

*grounds*

According to the data shown in **Table 1**, SCG can be considered an excellent precursor to produce activated carbon. The large surface area achieved in SCG activated carbon could be used to remove inorganic and organic compounds from

Activated carbon is a material in which carbon is forming disordered graphite plates, in whose peripheries there is a wide diversity of functional groups, which gives it unique physico-chemical properties. Additionally, this material usually has

ous oxidation reactions [58]. Given these characteristics, this material is typically used in numerous applications, excelling in the removal of organic and inorganic

Waste coffee grounds were used to produce activated carbon by KOH under Ar atmosphere at three temperatures (700, 800, and 900°C), the adsorbent material was tested to adsorb CH4 and H2. The activated carbon at 900°C showed a CH4 adsorption capacity of 1.96 mmol/g at 273 K and 100 kPa. However, at 3000 kPa, the highest adsorption capacity was reported to be 4.2 mmol/g. The three adsorbents materials (700, 800, and 900°C) were also tried to adsorb H2 at 77 K and 100 kPa, achieving the highest adsorption capacity of 1.75 wt% [48]. Activated carbon from waste SCG as the precursor was physically activated by CO2 or steam at high temperatures (700–900°C) and chemically activated by ZnCl2, KOH, and H3PO4 at 450 and 600°C. Nevertheless, in this work, only the raw material, the activated carbons by ZnCl2 or steam, was tested to adsorb Bisphenol-A. The removal of Bisphenol-A was found to be 98, 12, and 0% for carbon activated by ZnCl2, raw material, and carbon activated by steam, respectively. These results were compared

/g, which develops through vari-

*5.3.1 Removal organic and inorganic pollutants by activated carbon-spent coffee*

raised surface areas, generally greater than 1000 m2

compounds present in the gas and liquid phase.

**142**

with a commercial activated carbon, which showed a Bisphenol-A removal of 93%. The poor adsorption performance of the SCG carbon activated by steam is due to the low surface area reported for this material (4 m2 /g) [46].

SCG was used as a precursor to prepare activated carbon by chemical activation with ZnCl2 at three impregnation ratios, at room temperature, and during 8, 12, and 24 h. The adsorption of Cu(II) was conducted using this activated carbon. The experimental data were fitted by using the Langmuir, Freundlich, and Elovich isotherms; the maximum adsorption capacity (Langmuir) was 285.71 mg/g, and the maximum Cu(II) removal reported was 18% at 100 rpm, and the pH solution value was not reported [51].

The production of SCG-based activated carbon was carried out at three impregnation ratios, g ZnCl2/g precursor (1:0.5, 1:1, and 1:2); the impregnated precursor was carbonized under N2 atmosphere at 800°C during 60 min, and this material was tested for H2S separation. The SCG activated carbon was used to study H2S dynamic breakthrough capacity passing a dilute flow of H2S-Air (1000 ppm, 80% humidity) through a fixed bed. The adsorbents activated at impregnation ratios of 1:2 and 1:1 showed the lowest (18.2 mg/g) and the highest breakthrough capacity (127 mg/g), respectively [51].

Chemical activation of SCG was performed with KOH using 2:1 and 4:1 impregnation ratio (KOH: precursor) to produce activated carbons. The carbonization process was carried out at 400 or 700°C under N2 flow for 2 h. These activated carbons were tested to adsorb CO2 at 0, 25, and 50°C and 0–10 bars. The highest adsorption capacity obtained was 6.8 mmol/g at 1 bar and 0°C, when the activated carbon was produced at 700°C and an impregnation ratio of 4:1. However, when the carbonization process was performed at 10 bars, the highest uptake achieved was 23.26 mmol/g [53].

SCG microporous activated carbon (using potassium hydroxide as activation agent) was synthesized and characterized. The precursor was pyrolyzed using 1:9, 1:18, and 1:36 mmol KOH:g SCG of impregnation ratios and under N2 flow at 800°C during 1 h. SCG activated carbons were used to adsorb phenol and methylene blue. The equilibrium was attained within 100 and 360 min for phenol and methylene blue, respectively. The maximal adsorption capacity, based on Langmuir isotherm, for phenol and methylene blue was 3008 and 1058 mmol/g, respectively [54].

The influence of the impregnation ratio of H2SO4 over SCG granular activated carbon to treat leachate was studied. Six samples of leachate with the following chemical and biological parameters were treated: COD (1010–1815 mg/L), BOD5 (184–338 mg/L), NH4-N (2208–2780 mg/L), iron (4.25–4.73 mg/L), and PO4-P (220–284 mg/L). However, in this research, only the removal percentage of iron and PO4-P was found to be 77 and 84%, respectively, when impregnation ratios of 2.5 and 0.5 were used [55].

SCG obtained from a trademark coffee (Nespresso®) was used as a precursor to produce activated carbon. KOH was used as an activating agent at four impregnation ratios. The chemical activation process was carried out at 873 K, and physical activation with CO2 was carried out at 973 K. These materials were used to capture CO2, which is a byproduct of the combustion process. However, in this study, a pure CO2 flow was tested at 298 K and 101 kPa. The CO2 adsorption capacities of the adsorbents activated by using chemical activation and physical activation were 3 and 2.3 mmol/g, respectively [56].

SCG carbon activated with phosphoric acid and zinc chloride was used to adsorb Pb(II) and Cd(II). The precursor was mixed with ZnCl2 or H3PO4 at chemical agent/ coffee residue mass ratios of 25, 50, 75, and 100% at 85°C for 7 h. When the precursor was activated with H3PO4 (50% impregnation ratio), the maximal adsorption capacities, based on Langmuir isotherm, were as follows: 89.28 mg Pb(II)/g and 46.95 mg Cd(II)/g. With ZnCl2 (75% impregnation ratio) as the activation agent, the maximal adsorption capacities were 63.29 mg Pb(II)/g and 37.04 mg Cd(II)/g [57].

The use of activated carbon derived from SCG was recently reported, and the activation procedure was carried out with ZnCl2. To optimize the activated carbon production, an experimental design was performed; the independent factors were temperature (450 and 600°C), activation time (40 and 120 min), and impregnation ratio (0.5 and 1.5 g ZnCl2/g SCG), and the experimental responses were surface area, yield, and hardness. The optimal conditions were impregnation ratios of 1.5, 600°C, and 40 min. At these conditions, the experimental responses were surface area 1279.96 m2 /g, yield 26%, and hardness 76.77%. The activated carbon produced at these conditions was used to adsorb phenol from aqueous solutions, based on Langmuir isotherm, the maximum adsorption capacity was 160.52 mg/g, and the equilibrium was attained less than 150 min [50].


**2.4. Extraction of cellular protein**

312 Chromatography – The Most Versatile Method of Chemical Analysis

**2.2. Cultivation of P. aeruginosa MTCC 2453**

and stored at -70oC for further study.

**2. Materials and methods**

**2.1. Chemicals and reagents**

Previous researchers [10] adopted genetic engineering techniques and other bacterial species for purification of azurin. This study is concerned of enhanced azurin synthesis from different strains of *P. aeruginosa* with lucid homogeneity by customized methods. The growth of different *P. aeruginosa* MTCC strains 1934, 741, 2453, and 1942 for the synthesis of azurin were scrutinized for enhanced azurin synthesis. The enhanced azurin synthesis from *P. aeruginosa*  strains was improved by the CuSO4 and KNO3 containing media under facultative anaerobic condition. The purification of azurin had been performed by ion-exchange and gel-filtration chromatography. High yield was reported in *P. aeruginosa* 2453 strain than other strains.

Growth medium constituents were of analytical grade obtained from Hi-Media laboratories, India. The buffer ingredients were purchased from Merck Chemicals Ltd, India. Sephadex G-25, G-75, diethyl amino ethyl cellulose (DEAE) cellulose and carboxy methyl (CM) cellulose were all obtained from Sigma-Aldrich, USA. The 3, 5-dimethoxy, 4-hydroxy cinnamic acid otherwise called as sinnapinic acid a matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) was also acquired from Sigma-Aldrich. Protein concentrations were measured by Lowry's method with bovine serum albumin as standard. Standard dialysis bag with 3 kDa cutoff was purchased from Sigma-Aldrich. Powder of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), propidium iodide (PI) and dimethylsulfoxide (DMSO) solution were procured from Sigma Aldrich, India. Cell culture media and other constituents of media are purchased from Hi-media Laboratories

Ltd, India. Fetal bovine serum was obtained from Invitrogen Life Technologies, USA.

A freeze dried culture of *P. aeruginosa* strains MTCC2453,741,1934,1942 was obtained from the Microbial type culture collection center, Chandigarh, India and was grown in a medium containing 7g yeast extract, l0g peptone, 20g of KNO3, 6.4g of KH2PO4, 3.6g of Na2HPO4 (anhydrous), 2.5g of NaCl, 5µg/ml of CuSO4 per liter. The initial pH was adjusted to 6.5 with NaOH. The strains were maintained on nutrient broth with 50% glycerol concentration

Inoculums' of *P. aeruginosa* strains was prepared by inoculating a loopful of colonies in individual 100 ml conical flasks with the exact constituents of the above prescribed media and incubated at 370C, in stirring mode at 100/rev for 21 hours. These inoculums was used to seed the bulk 500ml x 4 sterile medium in 2000 liter conical flasks (separate conical flask used for all

All P*. aeruginosa* strains MTCC2453, 741, 1934, and 1942 are inoculated separately in a sterile medium. Impact of copper sulphate and potassium nitrate in azurin synthesis were

four strains) which was also kept at stirred mode (100/rev) for 21 hours at 370C [11].

**2.3. Impact of copper sulphate and potassium nitrate on culture medium**

After 21 hrs incubation, cells were harvested by centrifugation method at 13200 g for 15-20 minutes by using ultra centrifuge (Eppendorf, Hamburg, Germany). Cell pellets was collected and suspended in the appropriate volume of 0.02M potassium phosphate buffer pH 7 with protease inhibitor and kept in the ice basket for sonication. Cells were sheared by Ultra sonicator (Cole Parmer, USA) of approximately 100 ml batches of cell suspension. All batches were sonicated for 1-2 minutes at 100W. After sonication the samples was stirred vigorously and centrifuged at 10000g for 20 minutes which removes cell wall debris. The green-brown crude supernatant was stored. Resuspended the precipitate in same buffer, stirred it vigorously and centrifuged as before and the supernatant were stored with the previous extracts [11, 12].


*Edited by Phil Ainslie*

Written by international experts, this publication provides the reader with the present knowledge and future research directions of diagnostic and therapeutic ultrasound and spectroscopy. Focused topics include Duplex ultrasound, transcranial color Duplex, MRA guided Doppler ultrasonography and near-infrared spectroscopy. New directions in the use and application of transcranial and color Duplex ultrasound are provided, as well as the use of ultrasound and arterial stiffness for measuring human vascular health and circulatory control. Novel use of ultrasound for the detection of intra-cardiac and intra-pulmonary shunts is also described along with its utility for the assessment of gastric regulation and emptying.

ISBN 978-953-51-0522-0

Applied Aspects of Ultrasonography in Humans

Photo by DariaRen / iStock


**2. Capillaroscopy procedures**

NFC is commonly performed in nailfold cuticles, as the capillary loops become more parallel to the skin surface in this area and can be observed in their full length in the last row [2]. Usually, eight fingers are examined: the 2nd, 3rd, 4th, and 5th [1, 19]. The thumbs are excluded because, in these fingers, capillaries are poorly observed and microtrauma is more frequent due to thumb's opponency. At least four images should be taken from each finger, in order to maximize nailfold area visualization (**Figure 1**). Less than eight nailfold reduces the sensitivity to detect capillary changes [20]. So, in a regular exam, at least 32 pictures are taken from each hand when using a videocapillaroscope.

The patient should stay at least 20 minutes in a climatized room (20–22°C), to reduce RP attacks, and a clinical examination should be performed in his or her hands, in order to avoid traumatic injuries and to detect sclerodactyly, edema, pitting scars, active arthritis, skin lesions, onychophagia, or other possible changes [19, 21]. As several physiological and external factors can affect NFC image quality, patients are asked to avoid cutting cuticle or even the nails in the previous week, prevent nail varnish removal, and avoid smoking or drinking caffeine-containing beverages in the preceding hours [7]. To improve the amount of light reaching the nails and then ameliorate image quality, an immersing oil is used between the skin and the lens.

Different devices can be used for microcirculation visualization:

**173**

include the following:

(**Figure 4A**).

*The Impact of Nailfold Capillaroscopy in the Approach of Microcirculation*

option and requires specialized training [7].

**3. Nailfold capillaroscopy assessment and classification**

abnormal shapes, or visibility of the subpapillary venous plexus.

the number of loops in the distal row in a 1 mm2

• Videocapillaroscope (**Figure 1**): it has an excellent image quality and reproducibility and variable magnification from 200–600×. It has a portable system enabling its use in patients with severe joint contractures, and it carries specific software for capturing and analyzing the images, yet it is the most expensive

*Nailfold capillaroscopy should be performed in 2nd, 3rd, 4th, and 5th fingers, dividing each nailfold cuticle into 4 areas and taking images from each of them. (A) Clinical examination of the fingers should be performed before the exam begins. (B) Performing videocapillaroscopy using Videocap biomicroscope, version 3.0. (C, D).*

NFC procedure includes the assessment of morphologic and some functional parameters: skin transparency, capillary density and orientation, venous plexus visualization, presence of neoangiogenesis or microhemorrhages, capillary loop diameter, length and morphology, and characteristics of blood flow [1, 21]. The number of capillaries has been validated as the most important criterion for the patient follow-up [22].

NFC studies lead to the detection of microvascular abnormalities. Some of them, rarer, have a clear pathological significance and can disclose early an AICTDthey are called the "major abnormalities" [1].  Other abnormalities, more frequent, have an uncertain pathological meaning and represent an overlap between the scope of normality and microangiopathy-they are called the "minor abnormalities." Those are principally tortuosity,

Capillaroscopic parameters are usually evaluated through qualitative, semiquantitative, and quantitative analysis. The qualitative assessment implies pattern recognition and describes the global microvascular array and architecture, shape and distribution of the capillaries, and abnormalities of single loops. It readily allows a distinction between a normal capillaroscopy exam and an abnormal one [20]. The semiquantitative analysis is focused on major NFC changes. The quantitative evaluation estimates capillary density, avascular areas, diameter of enlarged capillaries, and the frequency of each abnormality. The quantification of capillaries includes

Normal capillaries are hairpin shaped and present a homogeneous distribution, in a "comb-like" structure [1] (**Figure 3**). Capillary density varies from 9 to 14 capillaries per millimeter in adults and at least 6 in children [1, 7, 23]. Abnormal shapes

• **Tortuous capillaries**: arterial and venous limbs are curled but do not cross [23]

section of the nailfold (**Figure 2**).

*DOI: http://dx.doi.org/10.5772/intechopen.90525*

**Figure 1.**

*The Impact of Nailfold Capillaroscopy in the Approach of Microcirculation DOI: http://dx.doi.org/10.5772/intechopen.90525*

#### **Figure 1.**

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

(EULAR) study groups [15–17].

**2. Capillaroscopy procedures**

when using a videocapillaroscope.

(CSURI), to predict the appearance of new scleroderma ulcers and/or persistence of nonhealing lesions, within 3 months from NFC exam [14]. It has a good sensitivity, specificity, and positive predictive value, even in different devices. Its reliability has been successively demonstrated by European League Against Rheumatism

Indications for NFC do not resume to RP or other vascular acrosyndromes approach. It should be performed to any patient with microcirculation involvement from a systemic disease that includes AICTD, like systemic sclerosis, idiopathic inflammatory myositis, mixed connective tissue disease, and systemic lupus erythematosus, among others, but also other systemic diseases associated to microangiopathy, like vasculitides, diabetes, and arterial hypertension. As it also plays an important role in diagnosis, prognosis, and treatment monitoring of some diseases, capillaroscopy can be considered to act as a promising microcirculation biomarker [18].

NFC is commonly performed in nailfold cuticles, as the capillary loops become more parallel to the skin surface in this area and can be observed in their full length in the last row [2]. Usually, eight fingers are examined: the 2nd, 3rd, 4th, and 5th [1, 19]. The thumbs are excluded because, in these fingers, capillaries are poorly observed and microtrauma is more frequent due to thumb's opponency. At least four images should be taken from each finger, in order to maximize nailfold area visualization (**Figure 1**). Less than eight nailfold reduces the sensitivity to detect capillary changes [20]. So, in a regular exam, at least 32 pictures are taken from each hand

The patient should stay at least 20 minutes in a climatized room (20–22°C), to reduce RP attacks, and a clinical examination should be performed in his or her hands, in order to avoid traumatic injuries and to detect sclerodactyly, edema, pitting scars, active arthritis, skin lesions, onychophagia, or other possible changes [19, 21]. As several physiological and external factors can affect NFC image quality, patients are asked to avoid cutting cuticle or even the nails in the previous week, prevent nail varnish removal, and avoid smoking or drinking caffeine-containing beverages in the preceding hours [7]. To improve the amount of light reaching the nails and then ameliorate image quality, an immersing oil is used between the skin

• Ophthalmoscopes and traditional microscopes: they consist in low cost options and are widely available, which can be used with minimal training; the disadvantages include not only low magnifications (10–20×), but also it is hard

• Dermatoscopes: they have an intermediate cost; they are portable devices and easily available and have acceptable resolution and sensitivity for NFC abnormalities; however, they also have magnification restraint (20–40×) and can

• Stereomicroscopes: they also have an intermediate cost, they are easy to use, their magnification lens vary from 10–200×, but they are time-consuming and difficult to use in patients with joint contractures and they need an additional camera and fiber optic light source to capture the images, and a specialized

Different devices can be used for microcirculation visualization:

only detect gross NFC changes, and images are not reproducible.

handling and it has a poor reproducibility.

training is even required.

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and the lens.

*Nailfold capillaroscopy should be performed in 2nd, 3rd, 4th, and 5th fingers, dividing each nailfold cuticle into 4 areas and taking images from each of them. (A) Clinical examination of the fingers should be performed before the exam begins. (B) Performing videocapillaroscopy using Videocap biomicroscope, version 3.0. (C, D).*

• Videocapillaroscope (**Figure 1**): it has an excellent image quality and reproducibility and variable magnification from 200–600×. It has a portable system enabling its use in patients with severe joint contractures, and it carries specific software for capturing and analyzing the images, yet it is the most expensive option and requires specialized training [7].

NFC procedure includes the assessment of morphologic and some functional parameters: skin transparency, capillary density and orientation, venous plexus visualization, presence of neoangiogenesis or microhemorrhages, capillary loop diameter, length and morphology, and characteristics of blood flow [1, 21]. The number of capillaries has been validated as the most important criterion for the patient follow-up [22].

### **3. Nailfold capillaroscopy assessment and classification**

NFC studies lead to the detection of microvascular abnormalities. Some of them, rarer, have a clear pathological significance and can disclose early an AICTDthey are called the "major abnormalities" [1].  Other abnormalities, more frequent, have an uncertain pathological meaning and represent an overlap between the scope of normality and microangiopathy-they are called the "minor abnormalities." Those are principally tortuosity, abnormal shapes, or visibility of the subpapillary venous plexus.

Capillaroscopic parameters are usually evaluated through qualitative, semiquantitative, and quantitative analysis. The qualitative assessment implies pattern recognition and describes the global microvascular array and architecture, shape and distribution of the capillaries, and abnormalities of single loops. It readily allows a distinction between a normal capillaroscopy exam and an abnormal one [20]. The semiquantitative analysis is focused on major NFC changes. The quantitative evaluation estimates capillary density, avascular areas, diameter of enlarged capillaries, and the frequency of each abnormality. The quantification of capillaries includes the number of loops in the distal row in a 1 mm2 section of the nailfold (**Figure 2**).

Normal capillaries are hairpin shaped and present a homogeneous distribution, in a "comb-like" structure [1] (**Figure 3**). Capillary density varies from 9 to 14 capillaries per millimeter in adults and at least 6 in children [1, 7, 23]. Abnormal shapes include the following:

• **Tortuous capillaries**: arterial and venous limbs are curled but do not cross [23] (**Figure 4A**).

#### **Figure 2.**

*Capillary density. Panel A: 1 capillary/mm2 ; panel B: 6 capillaries/mm2 . (arrows) images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

#### **Figure 3.**

*Examples of images of nailfold capillaroscopy captured in healthy subjects and considered as normal. Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

#### **Figure 4.**

*Examples of capillaroscopy abnormalities: Tortuosity (arrow), crossing (X), meandering (black circle), bushy capillaries (six-pointed star), subpapillary plexus visibility (plus sign), and dilated loop (black square). Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

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*The Impact of Nailfold Capillaroscopy in the Approach of Microcirculation*

venous limbs or different capillaries [7] (**Figure 5**).

• **Ramified capillaries:** there are abnormal connections between arterial and

• **Neoangiogenesis:** neoformations can be heterogeneous and may comprise shape heterogeneity, four or more capillaries within a single dermal papilla, elongated loops, branching, and interconnected capillaries [1] (**Figure 5**).

• **Bizarre loops**: capillaries with an atypical morphology, distinct from other

• **Dilated or enlarged loops**: there is not a universally accepted definition for loop enlargement, but it is usually considered if the limbs are enlarged about four times the normal width or if the width diameter is >20 μm. Nevertheless, a recent study from Cutolo et al suggests that dilations below 30 μm may be

• **Megacapillary or giant capillary**: capillaries with homogeneously enlarged

• **Hemorrhages**: microbleeding appears as dark masses adjacent to distal row, due to hemosiderin deposits. They result from disruption of the capillary wall,

• **Avascular areas**: lack of two or more successive capillaries. Loss of capillaries is associated with tissue hypoxia and subsequent digital ulcers and ischemia

arrangement of vessels with a greater caliber than the capillaries. Enlargement and congestion of venules and capillaries related to persistent opening of arteriovenous anastomoses, thus enabling a greater visibility [1] (**Figure 4**).

• **Subpapillary venous plexus visibility**: observation of large and linked

• **Capillary blood flow**: with powerful magnifications, when the blood flow is slow, it is possible to see the packs of red blood cells moving as a capillary

• **Capillary array and architecture disarrangement**: when shapes, length, and diameter vary in continuous loops, it leads to a complete distortion of a normal

Major NFC patterns divide into scleroderma and nonscleroderma pattern. Scleroderma pattern was first described by Maricq et al, through a combination of widening of the capillary loop, loss of capillaries, and disorganization of the nailfold capillary bed [1]. This pattern is frequently seen in scleroderma spectrum disorders, like systemic sclerosis, dermatomyositis, and mixed connective tissue disease and in RP without a clear diagnosis. Later, Cutolo et al further classified the scleroderma pattern into "early," "active," and "late" stages. "Early" scleroderma pattern presents with few giant capillaries and hemorrhages, relatively well-preserved capillary

• Elongated capillaries: capillary loops longer than 300 μm [7] (**Figure 7**).

loops with a branch diameter above 50 μm [1, 23] (**Figure 6**).

Other capillaroscopic parameters include the following:

either spontaneous or traumatic [1, 7] (**Figure 8**).

*DOI: http://dx.doi.org/10.5772/intechopen.90525*

described categories [23].

[7, 23] (**Figure 2**).

sludge [1] (**Figure 9**).

capillary pattern [1] (**Figure 10**).

considered nonspecific [23] (**Figure 6**).

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

• Crossing capillaries: arterial and venous limbs cross at one point [23]

*Examples of capillaroscopy abnormalities: Tortuosity (arrow), crossing (X), meandering (black circle), bushy capillaries (six-pointed star), subpapillary plexus visibility (plus sign), and dilated loop (black square). Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold*

*Examples of images of nailfold capillaroscopy captured in healthy subjects and considered as normal. Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy*

*; panel B: 6 capillaries/mm2*

*Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of*

*. (arrows) images were taken using*

• **Meandering loops**: the limbs cross upon themselves or with each other several

• Bushy capillaries: the limb branches present themselves in small and multiple

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**Figure 4.**

**Figure 3.**

*Clinic of Hospital Curry Cabral.*

**Figure 2.**

*Hospital Curry Cabral.*

*Capillary density. Panel A: 1 capillary/mm2*

(**Figure 4B**).

times [23] (**Figure 4A**).

*Capillaroscopy Clinic of Hospital Curry Cabral.*

buds [7] (**Figure 4C**).

Other capillaroscopic parameters include the following:

Major NFC patterns divide into scleroderma and nonscleroderma pattern. Scleroderma pattern was first described by Maricq et al, through a combination of widening of the capillary loop, loss of capillaries, and disorganization of the nailfold capillary bed [1]. This pattern is frequently seen in scleroderma spectrum disorders, like systemic sclerosis, dermatomyositis, and mixed connective tissue disease and in RP without a clear diagnosis. Later, Cutolo et al further classified the scleroderma pattern into "early," "active," and "late" stages. "Early" scleroderma pattern presents with few giant capillaries and hemorrhages, relatively well-preserved capillary

#### **Figure 5.**

*Images from three different phases of scleroderma pattern, belonging to three different patients: Early, active, and late (from left to right). Capillary density decreases from early to late pattern. Capillaroscopic findings include giant capillaries (black spot), hemorrhages (\*\*), and neoangiogenesis (star). Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

#### **Figure 6.**

*Capillary limb dilatation: Dilated (diameter* ≥ *30 μm) and giant (diameter* ≥ *50 μm) capillaries. Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

#### **Figure 7.**

*Elongated capillaries (capillary loops* ≥*300 μm). Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

distribution, and no loss of capillaries. "Active" scleroderma pattern reveals frequent giant capillaries and hemorrhages, moderate loss of capillaries, mild disorganization of capillary bed, and absent or mild ramified capillaries (i.e., neoangiogenesis). "Late" scleroderma pattern shows an irregular enlargement of the capillaries, few or absent giant capillaries and hemorrhages, severe loss of capillaries with avascular areas (the *plages désertes*), and ramified or bushy capillaries (**Figure 5**).

In healthy subjects, anatomic variations occur, and capillary abnormalities can be found. The estimated prevalence of these changes are meandering loops in 25%,

**177**

*The Impact of Nailfold Capillaroscopy in the Approach of Microcirculation*

*Examples of nailfold hemorrhages in patients with different diseases. Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry*

*Images from a single patient with Sjögren syndrome, presenting dilated loops (black square), meandering (black circle), crossing (X), reduced capillary flow with sludge (dashed arrow), and ingurgitated venous plexus (plus sign). (A: 5th finger of the right hand; B: 2nd finger of the left hand; C: 4th finger of the right hand.) images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold*

dilated capillaries in 12%, bushy loops in 7%, bizarre loops in 2%, and giant capil-

*Dermatomyositis-images from 3 different patients, revealing reduced capillary density, with a complete distortion of the vascular array, neoangiogenesis (star), giant capillaries (black dot), and hemorrhages (\*\*). Venous plexus is also ingurgitated (plus sign). Images were taken using Videocap biomicroscope, version 3.0,*

*magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

**4. Nailfold capillaroscopy in autoimmune connective tissue diseases**

AICTD complex pathogenesis usually includes microvascular changes, with occurrence of progressive structural and functional damage of the capillaries. Therefore, NFC became an important diagnostic and prognostic tool to use while

Systemic sclerosis has probably been the most studied disease with NFC. It is a severe AICTD in which the main pathological events are endothelial dysfunction,

laries in 0.3% of the healthy population [23]**.**

*Capillaroscopy Clinic of Hospital Curry Cabral.*

managing these disorders.

**4.1 Systemic sclerosis (SSc)**

*DOI: http://dx.doi.org/10.5772/intechopen.90525*

**Figure 8.**

*Cabral.*

**Figure 9.**

**Figure 10.**

*The Impact of Nailfold Capillaroscopy in the Approach of Microcirculation DOI: http://dx.doi.org/10.5772/intechopen.90525*

#### **Figure 8.**

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

distribution, and no loss of capillaries. "Active" scleroderma pattern reveals frequent giant capillaries and hemorrhages, moderate loss of capillaries, mild disorganization of capillary bed, and absent or mild ramified capillaries (i.e., neoangiogenesis). "Late" scleroderma pattern shows an irregular enlargement of the capillaries, few or absent giant capillaries and hemorrhages, severe loss of capillaries with avascular

*Elongated capillaries (capillary loops* ≥*300 μm). Images were taken using Videocap biomicroscope, version 3.0,*

*Images from three different phases of scleroderma pattern, belonging to three different patients: Early, active, and late (from left to right). Capillary density decreases from early to late pattern. Capillaroscopic findings include giant capillaries (black spot), hemorrhages (\*\*), and neoangiogenesis (star). Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of*

*Capillary limb dilatation: Dilated (diameter* ≥ *30 μm) and giant (diameter* ≥ *50 μm) capillaries. Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy*

In healthy subjects, anatomic variations occur, and capillary abnormalities can be found. The estimated prevalence of these changes are meandering loops in 25%,

areas (the *plages désertes*), and ramified or bushy capillaries (**Figure 5**).

*magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

**176**

**Figure 7.**

**Figure 5.**

**Figure 6.**

*Clinic of Hospital Curry Cabral.*

*Hospital Curry Cabral.*

*Examples of nailfold hemorrhages in patients with different diseases. Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

#### **Figure 9.**

*Images from a single patient with Sjögren syndrome, presenting dilated loops (black square), meandering (black circle), crossing (X), reduced capillary flow with sludge (dashed arrow), and ingurgitated venous plexus (plus sign). (A: 5th finger of the right hand; B: 2nd finger of the left hand; C: 4th finger of the right hand.) images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

#### **Figure 10.**

*Dermatomyositis-images from 3 different patients, revealing reduced capillary density, with a complete distortion of the vascular array, neoangiogenesis (star), giant capillaries (black dot), and hemorrhages (\*\*). Venous plexus is also ingurgitated (plus sign). Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

dilated capillaries in 12%, bushy loops in 7%, bizarre loops in 2%, and giant capillaries in 0.3% of the healthy population [23]**.**


**Conflict of interest**

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


**5. Conclusions**

In this paper, the effects of cast defects on the corrosion behavior and mechanism of the UNS C95810 in artificial seawater were studied. The conclusions are as follows:

**Author Contributions:** Conceptualization, X.Z., Y.Q. and Z.Z.; methodology, X.Z., Y.Q. and Z.Z.; software, X.Z.; validation, J.W.; formal analysis, X.Z. and Z.Z.; investigation, X.Z. and Z.Z.; resources, J.Z., L.Q., D.H., Y.Q. and Z.Z.; data curation, X.Z.; writing—original draft preparation, X.Z.; writing—review and editing, X.Z., Z.Z. and Y.Q.; visualization, X.Z. and J.W.; supervision, Z.Z. and Y.Q.; project administration, Z.Z. and Y.Q.; funding acquisition, Z.Z. and Y.Q. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Project of Equipment Pre-research Field Fund, grant number<PHONE_NUMBER>4 and Equipment Pre-research Sharing Technology Project, grant number<PHONE_NUMBER>6,41423060314.

**Acknowledgments:** Thanks to Zeng Kai for his help in the experimental part of this article.

**Conflicts of Interest:** The authors declare no conflict of interest.


*3.7. Cell Viability Assay*

The cytotoxic effect on HEP-G2, PC-3, and fibroblast cells after treatment with (a) 225Ac-rHDL and (b) 225Ac-DOTA-benzene-p-SCN was evaluated by performing an assay of mitochondrial dehydrogenase activity using the XTT kit (Roche Holding AG, Rotkreuz, Switzerland). Briefly, HEP-G2, PC-3, and fibroblast cells (1 × 104 cells/well) were seeded in 96-well microtiter plates. The medium was removed after overnight incubation and the cells were incubated for 1 h with each treatment (4 kBq/200 µL). After removing the treatments, the cells were kept at 37 ◦C, 5% carbon dioxide, and 85% relative humidity. Cell viability was assessed at 3, 24, 48, and 192 h by spectrophotometric measurements (microplate absorbance reader, EpochTM, BioTek) at 450 nm. Fibroblast cells that did not receive treatment were considered as the control group with 100% cell viability at the different time points.