Simulation, In Vitro, and In Vivo Cytotoxicity Assessments of Methotrexate-Loaded pH-Responsive Nanocarriers
Authors: Saman Sargazi
Farshid Zargari
Mohammad Reza Hajinezhad
Mahira Zeeshan
Sadanand Pandey
Mahmood Barani
Mehrdad Khatami
Abbas Rahdar
Journal: Polymers (2021)
Discipline: CHEMISTRY
Keywords: in-vitro; in-vivo; methotrexate; niosome; pH-responsive
Affiliations: Medical Mycology and Bacteriology Research Center, Kerman University of Medical Sciences, Kerman 7616913555, Iran., Basic Veterinary Science Department, Veterinary Faculty, University of Zabol, Zabol 9861335856, Iran., Cellular and Molecular Research Center, Research Institute of Cellular and Molecular Sciences in Infectious Diseases, Zahedan 9816743463, Iran., Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan., Department of Physics, University of Zabol, Zabol 9861335856, Iran., Department of Chemistry, College of Natural Science, Yeungnam University, 280 Daehak-Ro, Gyeongsan 38541, Korea., Noncommunicable Diseases Research Center, Bam University of Medical Sciences, Bam 76617-71967, Iran., Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-111, Iran., Pharmacology Research Center, Zahedan University of Medical Sciences, Zahedan 9816743463, Iran., Department of Chemistry, Faculty of Science, University of Sistan and Baluchestan, Zahedan 98135674, Iran.

Abstract:  In this study, pH-responsive niosomal methotrexate (MTX) modified with ergosterol was prepared for potential anticancer application. The prepared formulation had a size of 176.7 ¬± 3.4 nm, zeta potential of -31.5 ¬± 2.6 mV, EE% of 76.9 ¬± 2.5%, and a pH-responsive behavior in two different pHs (5.4 and 7.4). In-silico evaluations showed that MTX intended to make a strong hydrogen bond with Span 60 compartments involving N2 and O4 atoms in glutamic acid and N7 atom in pteridine ring moieties, respectively. The cytotoxic effects of free and pH-MTX/Nio were assessed against MCF7 and HUVECs. Compared with free MTX, we found significantly lower IC50s when MCF7 cells were treated with niosomal MTX (84.03 vs. 9.464 ¬µg/mL after 48 h, respectively). Moreover, lower cell killing activity was observed for this formulation in normal cells. The pH-MTX/Nio exhibited a set of morphological changes in MCF7 cells observed during cell death. In-vivo results demonstrated that intraperitoneal administration of free MTX (2 mg/kg) after six weeks caused a significant increase in serum blood urea nitrogen (BUN), serum creatinine, and serum malondialdehyde (MDA) levels of rats compared to the normal control rats. Treatment with 2 and 4 mg/kg doses of pH-MTX/Nio significantly increased serum BUN, serum creatinine, and serum lipid peroxidation. Still, the safety profile of such formulations in healthy cells/tissues should be further investigated. 

Retracted: No

PubPeer comments:
 **The references [9], [10]. [13] and [17] are irrelevant to the subject of this article and or to the short sentence!**  , 
 #1 There is relevance to all four references provided in support of the sentence.  Recently, nanotechnology revolutionized medicine and drug delivery and advanced drug therapeutics to a new level, is a common sentence on the use of nanotechnology in health care. As a result, references 9,10, and 13 all discuss cosmetics and microemulsions (F127 and AOT microemulsions), in which F127 and AOT are surfactants. In medicine and health sciences, cosmetics and microemulsions (oil-in-water and water-in-oil) stabilized by different surfactants such as F127 pluronic and AOT are therapeutic agents. According to references in the literature review, these references concern nanocarriers, such as niosomes, liposomes, microemulsions, etc. A research paper in reference number 17 discusses carbon nanotubes as adsorbents for humic acid from aqueous solutions (removal of pollutants from aqueous solutions in medicine and health sciences), which is relevant to health discourse in health and medicine. These references are related to that public sentence for the reasons outlined above , 
 Table 1 is identical to table 1 of [Barani et al, ACS Omega, 2021] . The free MTX curve in Figure 7 is more similar than expected (specially given that these are very different molecule) to the free DFX curve shown in Fig 5 of [Barani et al, ACS Omega, 2021] . Table 3 in both papers has signs   instead of commas in numbers. Furthermore, although the results are supposed to represent different kinetic models, the equations are linear in all cases. The text mentions (in both cases) an  exponent but there is not one in the equations. , 
 #3 Sorry for our late response. unfortunately, Pubpeer is filtered in Iran and we must use VPN to get access to it. Table 1 is identical to table 1 of Barani et al, ACS Omega, 2021. Response: We use the molecular modeling especially molecular dynamic simulation in these works to portrait the drug-niosomes bilayer at atomic level. However, lack of sufficient hardware facilities leads us to simulate a part of niosomes interacting with a number of drugs in the simulation box. The model of niosomes bilayers that we set up in both paper is the same and it contains a 35:35:30 molar ratio of soan60:tween60:ergostrole. However, the structural analysis of the bilayer, including bilayer APL and bilayer thickness have the same values (even with separate analysis) in both paper which prove the reproducibility of our data. The free MTX curve in Figure 7 is more similar than expected (especially given that these are very different molecules) to the free DFX curve shown in Fig 5 of Barani et al, ACS Omega, 2021. Response: For the release behavior of both studies, we used the same dialysis bag with the same pore size. By the way, two studies showed different results. For example, after 1 hr MTX released 22% and DFX 25 % and so on up to MTX 96.1% and DFX 95.2% that is the end of the analysis. Table 3 in both papers has signs   instead of commas in numbers. Response: The u201c u201d is because of the different language of authors and publishers and is not a big deal that is u201c.u201d Furthermore, although the results are supposed to represent different kinetic models, the equations are linear in all cases. The text mentions (in both cases) an  exponent but there is no one in the equations. Response: u201cnu201d is the Diffusion or release exponent that is estimated from linear regression of log ( Mt M ) versus log t. For release data, we obtain all data from the estimated linear regression of each kinetic. Based on KORSMEYER-PEPPAS EQUATION we obtained u2018nu2019 . Korsmeyer u2013 peppas equation is: F = (Mt M ) = Km tn Where: F = Fraction of drug released at time u2018tu2019 Mt = Amount of drug released at time u2018tu2019 M = Total amount of drug in dosage form Km = Kinetic constant n = Diffusion or release exponent t = Time in hours *If you have further questions, please reach me at mahmoodbarani7@gmail.com. ,