Home » Synthesis and preliminary therapeutic evaluation of zinc nano particles against diabetes mellitus and -induced micro- (renal) and macrovascular (vascular endothelial and cardiovascular) abnormalities in rats

Synthesis and preliminary therapeutic evaluation of zinc nano particles against diabetes mellitus and -induced micro- (renal) and macrovascular (vascular endothelial and cardiovascular) abnormalities in rats

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Dr Maham Fatima1,Dr Anwar Ahmed Wains1,Dr Qurat Ul Ain Agha2

1.Nishtar Medical University,Multan

2.Cmh Institute Of Medical Sciences,Bhawalpur Key

https://doi.org/10/molbev/msr191(upcoming)

Abstract

The current study synthesized and investigated the effect of low-dose zinc nanoparticles (ZnNPs) against diabetes mellitus and -induced experimental micro- (nephropathy) and macro-vascular (cardio and endothelium) complications. Diabetes mellitus (DM)-induced vascular abnormalities were revealed by the reduction in acetylcholine-induced endothelium-dependent relaxation, the decrease in aortic and serum nitrite/nitrate concentration, increased CKMB, LDH, SGOT/SGPT, serum creatinine, and blood urea nitrogen, and the induction of proteinuria and oxidative stress. However, treatment with low-dose ZnNPs after streptozotocin administration reduced serum glucose concentration. Moreover, ZnNPs had shown a partial but significant prevention of cardio-vascular structural and functional abnormalities in diabetic rats. Increased bioavailability of NO in the endothelium and reduction in oxidative stress might be the possible mechanisms involved for the protective role of ZnNPs against diabetes-induced micro- and macrocomplications.

  1. Introduction Type 2 diabetes mellitus (DM) is one of the most important health emergencies in the 21st century. The population suffering from DM is estimated for 463 million worldwide and is rapidly growing. Predictions say that the number may reach 578 million by 2030, and 700 million by 2045 [1]. DM is associated with higher relative risk of cardiovascular disorders (CVD) which is estimated between 1.6 and 2.6 and slightly higher in women [2]. Cardiovascular disease in DM patients occurs approximately 15 years earlier than in healthy subjects. It is the main cause of mortality in this group which is different based on sex-dependence. CVD mortality in men with diabetes increases 1–3 times compared to diabetes-free individuals, while the same coefficient estimated in women varies from 2 to 5 times [3]. It is not clear what plays the major role in inducing the differences, but the most important factor seems to be the attenuation of estrogens protective influence against cardiovascular complications Nutrients 2021, 13, 2306. https://doi.org/10.3390/nu13072306 https://www.mdpi.com/journal/nutrients Nutrients 2021, 13, 2306 2 of 16 in DM women. It was proved that DM impairs endothelial response in women to a greater extent than among males [4]. DM diminishes antiproliferative effects of estrogen on the vascular smooth muscle cells by modifying expression, activity, and balance between the estrogen receptors (ER): ERα and ERβ. Domination of ERβ over ERα activity results in increased inflammatory profile, excessive reactive oxygen species (ROS) formation, and aggravated atherosclerotic plaque formation [5]. In that way, estrogens increase the nitric oxide (NO) bioavailability reducing NO inactivation and additionally may directly increase NO generation [6]. Furthermore, diabetic females are characterized by worse cardiovascular profile, including higher average HbA1c, LDL cholesterol, and BMI levels [4]. Catalan et al. proved that, despite the fact that men more often present carotid plaques in general, in a subgroup of newly diagnosed diabetic women, carotid atherosclerosis was more prevalent [7]. Cardiovascular risk in DM patients is highly dependent on endothelial dysfunction as well as micro- and macrovascular complications coexisting in DM. Among numerous factors, nitric oxide (NO) bioavailability and its metabolic pathway abnormalities seem to be the most crucial. Asymmetric dimethylarginine (ADMA) is a pivotal element leading to the NO synthesis disturbances by competitive inhibiting the nitric oxide synthase (NOS). Significant evidence indicates that not only ADMA concentration per se decides on endothelial function but also the decreased Arginine level and the Arginine/ADMA ratio seems to be additional and potentially more sensitive markers of endothelial dysfunction. The Arginine/ADMA ratio as an indicator of reduced global bioavailability of Arginine and NO production was postulated to be an independent CVD risk factor and to correspond with the severity of hypertension, congestive heart failure, and atherosclerosis [8,9]. Up to date, most of the studies have focused on the NO metabolism abnormalities being seen in the plasma compartment of diabetic subjects. Hardly any of them aims to evaluate the erythrocytes’ role in this process. As the red blood cells (RBC) remain in constant contact with the endothelium and enable nitric oxide transport into distant hypoxic areas, their role in regulating the nitric oxide bioavailability
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    might be underestimated. For many years erythrocytes have been considered to just eliminate the NO, which easily reacts with hemoglobin. However, recent studies pointed out that erythrocytes contain NOS and are able to produce NO. Subsequently they release it into the vasculature, regulating the blood flow in distal hypoxic regions [10]. It demonstrates that the conventional theory needs to be verified and the specific role of erythrocytes in the nitric oxide metabolism and its influence on endothelial dysfunction requires detailed evaluation. Hence, the main goal of this study was to evaluate the nitric oxide metabolism abnormalities in plasma and erythrocytes of diabetic subjects with a close assessment of endothelial function using different tools. Additionally, it was intended to define which of the nitric oxide metabolites reflects better endothelial dysfunction at the early stages of diabetes mellitus. 2. Materials and Methods 2.1. Ethical Approval All experiments were conducted and approved in accordance with the guidelines of the Bioethics Committee at the Wroclaw Medical University (KB155/2019) from 28 February 2019 and adhered to the principles of the Declaration of Helsinki (Seventh Revision, 64th World Medical Association meeting, Fortaleza, 2013). All of the individuals agreed to participate in the study by signing a written informed consent. 2.2. Patients A total of 100 patients were investigated in the study. The inclusion criteria comprised diabetes mellitus diagnosed according to the American and Polish Diabetes Associations criteria, treated with oral metformin at age of 35–80 years. Subjects with the presence of diabetic complications, including microangiopathy, macrovascular diseases, past history of stroke, or myocardial infarction, as well as taking anticoagulant or antiplatelet treatment, Nutrients 2021, 13, 2306 3 of 16 were excluded from this study. In order to exclude the potential variables affecting the differences between groups, we excluded subjects with concomitant hypertension. Finally, a total of 80 consecutive subjects met properly the inclusion and exclusion criteria and were enrolled to the study, including 35 patients with diabetes mellitus (female: 12, male: 23) and 45 healthy individuals qualified to the control group, respectively. The control group was recruited from outpatient clinics in pursuance of demographic characteristics (age, sex, region), meeting the inclusion and exclusion criteria properly. All of them had previously undergone the screening for the presence of the glucose metabolism alterations, including diabetes, impaired fasting glycemia, impaired glucose tolerance, and insulin resistance. Subjects with any of these disturbances were excluded from the control group. All of the participants underwent a standard detailed physical examination (Scheme 1). 2.2. Patients A total of 100 patients were investigated in the study. The inclusion criteria comprised diabetes mellitus diagnosed according to the American and Polish Diabetes Associations criteria, treated with oral metformin at age of 35–80 years. Subjects with the presence of diabetic complications, including microangiopathy, macrovascular diseases, past history of stroke, or myocardial infarction, as well as taking anticoagulant or antiplatelet treatment, were excluded from this study. In order to exclude the potential variables affecting the differences between groups, we excluded subjects with concomitant hypertension. Finally, a total of 80 consecutive subjects met properly the inclusion and exclusion criteria and were enrolled to the study, including 35 patients with diabetes mellitus (female: 12, male: 23) and 45 healthy individuals qualified to the control group, respectively. The control group was recruited from outpatient clinics in pursuance of demographic characteristics (age, sex, region), meeting the inclusion and exclusion criteria properly. All of them had previously undergone the screening for the presence of the glucose metabolism alterations, including diabetes, impaired fasting glycemia, impaired glucose tolerance, and insulin resistance. Subjects with any of these disturbances were excluded from the control group. All of the participants underwent a standard detailed physical examination (Scheme 1). Scheme 1. Scheme 1. A flow chart presenting the study protocol. A flow chart presenting the study protocol. 2.3. Blood Sample Collection Forty-four milliliters of peripheral blood was collected using the Sarstedt S-Monovette® system (Sarstedt AG & Co., Nümbrecht, Germany). The sample (1.6 mg EDTA/mL blood) tubes within 30 min after the collection, were centrifuged at 1000× g for 15 min at 4 ◦C and stored at −80 ◦C until further analysis. Nutrients 2021, 13, 2306 4 of 16 2.4. Erythrocytes Preparation The erythrocytes were removed from the plasma within the 10 min following blood drowning. Erythrocytes samples were thawed on ice. Subsequently, a 10 µL of internal standards solution and a 1200 µL of cold extraction solution containing methanol, acetonitrile, and water (5:3:2) were added and vortexed (for 15 min, 1200 rpm at 4 ◦C). Samples were centrifuged (for 15 min, 22,000 rpm at 4 ◦C) and the supernatants were transferred into new microtubes. Samples were then dried at 55 ◦C and afterwards dissolved in 100 µL of water and vortexed (for 5 min, 1200 rpm at 25 ◦C). Subsequently, 50 µL of borate buffer (0.025 M Na2B407 × 10H2O, 1.77 mM NaOH, pH = 9.2), 400 µL of acetonitrile, and 10 µL of 10% benzoyl chloride (BCl) in acetonitrile were added and vortexed again (for 10 min, 1200 rpm, at 25 ◦C). After derivatization samples were dried at 55 ◦C using the SpeedVac evaporator. Dried samples were reconstituted in 50 µL of 3% of methanol in water and centrifuged (for 10 min, 15,000 rpm, at 4 ◦C). Clear supernatant was transferred into chromatographic polypropylene vial with attached 200 µL insert. 2.5. Plasma Preparation Plasma concentrations of metabolites involved in nitric oxide (NO) synthesis were measured according to method described by Fleszar
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    et al. [11]. Briefly, a 100 µL of plasma, 50 µL of borate buffer, 10 µL of internal standard solution (100 µM D7Arginine, 20 µM D7- ADMA, 25 µM D6-DMA, 100 µM D6-ornithine, and 50 µM D4-citrulline) were transferred into the 2 mL polypropylene tubes and mixed (for 1 min, 1200 rpm, at 25 ◦C). Then, 400 µL of acetonitrile and 10 µL of 10% BCl in acetonitrile were added and mixed (for 10 min, 1200 rpm, at 25 ◦C). Subsequently, samples were centrifuged (for 7 min, at 4 ◦C, 22,000 RCF) and 100 µL of clear supernatant was diluted four times with water, transferred to chromatographic glass vials, and analyzed. 2.6. Plasma and Erythrocytes Samples Analysis The LC-MS/MS analysis was performed using the Acquity UPLC system (Waters, Milford, MA, USA) equipped with cooled autosampler. The sample temperature in an autosampler was 6 ◦C and the injection volume was 2 µL. The Waters BEH Shield C18 column (1.7 µm, 2.1 × 10 mm) was thermostatted in a column oven at 60 ◦C. The flow rate was 0.350 mL/min, and a total run time was 8 min. Eluents: A: water with 0.1% formic acid (FA), B: methanol with 0.1% FA. The following gradient was used: 0.0 min–3% B, 2.5 min–14% B, 4.6 min–60% B, 4.8 min–90% B, 6.1 min–3% B. MS analysis was performed using the SYNAPT G2 Si mass spectrometer (Waters, Milford, MA, USA) equipped with electrospray ionization source (ESI) in a positive ionization mode. The spray voltage, source temperature and the de-solvation temperature were set at 0.5 kV, 140 ◦C, and 450 ◦C, respectively. Data acquisition was performed using the MassLynx software (Waters) for the following ions (m/z): 150.0919, 156.1295, 237.1239, 243.1339, 263.1090, 267.1382, 279.1457, 286.1897, 307.1770, and 314.2209 for DMA, D6-DMA, ornithine, D6-ornithine, citrulline, D4-cytrulline, Arginine, D7-Arginine, ADMA, symmetric dimethylarginine (SDMA), and D7-ADMA, respectively. Standard calibration curves were prepared using the following concentration ranges: 3 to 150 µM for ornithine, 5 to 250 µM for Arginine, 0.05 to 2.5 µM for ADMA and SDMA, 1 to 50 µM for citrulline, and 0.14 to 7 µM for DMA. 2.7. Laser Doppler All the individuals underwent the laser Doppler flowmetry (LDF) measurements using the Perimed PeriFlux System 5000, Sweden, with a PeriFlux heating unit, performed strictly according to the manufacturer’s instructions. The protocol was adapted from our previous study [12]. First, all the individuals were advised to rest for 15 min in horizontal position in a quiet laboratory at the room temperature 22 ◦C. Afterward, their forearm was fixed by a vacuum pillow and the laser probe was placed on a skin, where no superficial veins were visible. The baseline flow and values were Nutrients 2021, 13, 2306 5 of 16 obtained within the first 10 min with a probe heated to 33 ◦C. It was subsequently heated to 43 ◦C by the PeriFlux heating unit for the next 30 min of protocol. According to the previous studies [13–16], there are at least two independent mechanisms that are involved in the skin microvascular vasodilatation in response to local heating. First—a peak observed in laser Doppler flowmetry is caused by the fast-responding vasodilator axon reflex activated by heat-sensitive receptors on afferent nerves mediated by antidromic release of a vasodilatory neurotransmitters (calcitonin gene-related peptide (CGRP), substance P) [17]. After initial brisk increase, the plateau phase is observed, which is followed (after 10–20 min) by slowly responding vasodilator system based on nitric oxide production during prolonged local heating. In order to evaluate the change in the cutaneous blood flow, the maximum heating index (MHI) was counted. It is the ratio between areas under the curve of 10 min blood flow during maximum heating (late NO-dependent phase) to 10 min baseline flow (before heating). Using the MHI better reflects nitric-oxide dependent endothelial function, reducing the overlap influence of autonomic system. 2.8. EndoPAT The profile of endothelial function was assessed using the EndoPAT 2000 device (Itamar Medical, Caesarea, Israel). The EndoPAT detects plethysmographic pressure changes in the fingertips, caused by the arterial pulse, where special sensors are placed and translates it to a peripheral arterial tone (PAT). After 6 min of basal register, the occlusion by sphygmomanometer cuff inflated 40 mmHg over systolic pressure was made. Occlusion measurement was done with additional 5 min of post occlusion measurement (hyperaemic period). The PAT values obtained from contralateral limb serve as a control to exclude the individual systemic changes in blood flow. The achieved data were calculated using a computerized automated algorithm provided with the device. The results were compared using the reactive hyperaemia index (RHI) and its logarithm (lnRHI), which corresponded with the endothelium-mediated changes in vascular tone after occlusion and reflected the arterial endothelial function. Additionally, the augmentation index (AI) was assessed by analyzing the two pressure peaks signals from ascending aorta generated during the cardiac cycle. The first peak was derived from the pulse wave generated by the left ventricle, where the second was the reflection from arterial walls which superimpose to the first ventricular wave. AI is defined by the difference in these pressures, expressed as a percentage of the measured pulse pressure [18]. Its value depends on the vascular stiffness and aorta elasticity. Since it was shown that the augmentation index is heart rate dependent, a more reliable indicator is the augmentation index normalized for a heart rate of 75 bpm (AI-75) [19]. The whole protocol was conducted in pursuance of the manufacturer’s instructions. According to the Bonetti et al. study, the RHI cut-off value was set at RHI < 1,67 referring endothelial dysfunction [20]. All experiments were performed in an air-conditioned, quiet
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    room with a constant air temperature of 20 ◦C. 2.9. Statistical Analysis The data are presented as the mean ± SD. The differences between two continuous parameters were assessed using a Mann–Whitney U-test or a Student’s t-test, following the Shapiro–Wilk test and Levene’s test as appropriate. Additionally, Spearman’s rank correlation coefficient was performed. All calculations were made with Statsoft® Statistica 13.3 software, Krakow, Poland and Graph Pad PRISM® 8.4 San Diego, CA, USA. 3. Results 3.1. Baseline Characteristics The study diabetic subjects and healthy controls were matched with respect to the age and sex distribution. There were, however, significant differences in the waist to hip ratio (WHR), weight, body mass index, lipid and glucose metabolism (Table 1). Additionally, higher levels of high-sensitive CRP and uric acid with decreased magnesium and sodium Nutrients 2021, 13, 2306 6 of 16 concentration were noted in the diabetic subjects. As compared to healthy individuals, patients suffering from DM had significantly higher HOMA-IR and lower QUICKI indexes. Table 1. Demographic and biochemical characteristics between studied groups including cardiovascular risk stratification parameters. Results are presented as mean ± SD. Parameter Diabetes Group n = 35 (Mean ± SD ) Control Group n = 45 (Mean ± SD ) p Value Age (year) 59.80 ± 9.00 55.42 ± 10.75 NS Women (%) 12 (34%) 19 (42%) NS Weight (kg) 88.37 ± 14.49 78.16 ± 14.43 < 0.05 vs. control Figure 1. Assessment of endothelial function by EndoPAT 2000 and Laser Doppler Flowmetry. Abbreviations: RHI: reactive hyperaemia index (EndoPAT 2000); AI (%): augmentation index (EndoPAT 2000), AI-75 (%): augmentation index normalized for a heart rate of 75 beats/min (EndoPAT 2000), MHI: maximum hyperaemia index (Laser Doppler Flowmetry). Correlation of endothelial function with NO and biochemical metabolites in DM patients. *- p < 0.05 vs. control. 3.3. Parameters of the Nitric Oxide Bioavailability in Erythrocytes and Plasma Three of the six evaluated metabolites of the nitric oxide pathway were found to be significantly different in the plasma compartment (Arginine, DMA, citrulline). One was found to be altered in the erythrocytes (citrulline), when compared to the control group. The citrulline level was decreased in both compartments among DM patients, which was more noticeable in the erythrocytes. Simultaneously, the Arginine level was reduced in plasma, with no differences in the erythrocyte levels between groups. On the contrary, the dimethylamine (DMA) concentration was increased in plasma which was consecutively not observed in red blood cells (RBCs). All of the alterated nitric oxide metabolites were found at higher concentrations in plasma in both groups. In order to define the involvement of particular pathways in the alterations of the nitric oxide biotransformation the product to substrate ratios both, in the plasma and erythrocyte compartment in particular reactions were assessed. The study revealed significantly increased ornithine/Arginine and decreased ADMA/DMA ratio in the plasma of DM subjects. Additionally, reduced Arginine/ADMA ratio was noted in plasma with preserved proportion in erythrocytes. By comparing these ratios, higher NO bioavailability in the RBC of diabetic subjects was also identified. Futhermore, Arginine and citrulline compartmental concentration ratio were proved to be statistically different between the groups (Table 3 and Figures 2–4). Nutrients 2021, 13, 2306 8 of 16 ADMA/DMA 0.37 ± 0.23 0.47 ± 0.24 < 0.05 vs. control. Figure 2. (a–f) Nitric oxide metabolites concentration in different compartments. *- p < 0.05 vs. control. Nutrients 2021, 13, 2306 9 of 16 (e) (f) Figure 2. (a–f) Nitric oxide metabolites concentration in different compartments. *- p < 0.05 vs. control. (a) (b) Figure 3. Figure 3. (a, ( b a ) Citrulline and Arginine compartment ratios. *- ,b) Citrulline and Arginine compartment ratios. *- p < 0.05 vs. control. p < 0.05 vs. control. Nutrients 2021, 13, x FOR PEER REVIEW 10 of 17 Figure 4. Nitric oxide (NO) bioavailability ratio. *- p < 0.05 vs. control. 3.4. Assessment of the Relationship between Biochemical Results and Endothelial Function The analysis of biochemical results and endothelial function revealed a moderate correlation between AI and eGFR and BNP. A similar correlation was found between endothelial function measured using EndoPAT and ADMA concentration, ADMA/Arginine ratio, and NO bioavailability in the RBC compartment with no such dependence in the plasma of DM patients (Figure 5). Figure 4. Nitric oxide (NO) bioavailability ratio. *- p < 0.05 vs. control. Table 3. The Nitric oxide pathway metabolites ratios. Plasma Ratios DM Group (Mean ± SD) Control Group (Mean ± SD) p Value RBC Ratios DM Group (Mean ± SD) Control Group (Mean ± SD) p Value Plasma/RBC Ratios DM Group (Mean ± SD) Control Group (Mean ± SD) p Value Arginine/ ADMA 94.6 ± 40.3 116.5 ± 50.4 < 0.05 vs. control Contrary to the RBC compartment, substantial abnormalities in the NO synthesis in the plasma of DM patients were confirmed. First of all, a significantly reduced Arginine concentration was shown. It may be caused by shunting the L-Arginine from the eNOS to the arginase pathway with its enhanced arginase degradation, as an increased ornithine to Arginine ratio in plasma was found [18,22,23]. It is noteworthy that the results are in accordance with the studies by Shemyakin et al., proving that the downregulation of arginase improves endothelial function among patients with DM and may become a promising therapeutic target [24]. Additionally, enhanced Arginine transport from plasma to erythrocytes were found as Arginine compartment ratios were significantly different. Increased transport via system y+ in human erythrocytes had already been confirmed in different diseases affecting nitric oxide
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    transformation, such as renal failure and chronic heart diseases [25,26]. It may indicate that the same upregulation of y+ transport occurred in the DM group as a compensatory response to maintain the NO production. Furthermore, low level of Arginine leads to the NOS uncoupling and results in excessive reactive oxygen species (ROS) formation, which also stems from NADPH oxidase Figure 6. Nitric oxide metabolism alteration in different compartments. Abbreviations: ADMA: asymmetric dimethylarginine; DMA: dimethylamine; NOS: nitric oxide synthase; NO: nitric oxide RBC: erythrocyte SDMA: symmetric dimethylarginine. *- p < 0.05 vs. control. Contrary to the RBC compartment, substantial abnormalities in the NO synthesis in the plasma of DM patients were confirmed. First of all, a significantly reduced Arginine concentration was shown. It may be caused by shunting the L-Arginine from the eNOS to the arginase pathway with its enhanced arginase degradation, as an increased ornithine to Arginine ratio in plasma was found [18,22,23]. It is noteworthy that the results are in accordance with the studies by Shemyakin et al., proving that the downregulation of arginase improves endothelial function among patients with DM and may become a promising therapeutic target [24]. Additionally, enhanced Arginine transport from plasma to erythrocytes were found as Arginine compartment ratios were significantly different. Increased transport via system y+ in human erythrocytes had already been confirmed in different diseases affecting nitric oxide transformation, such as renal failure and chronic heart diseases [25,26]. It Nutrients 2021, 13, 2306 12 of 16 may indicate that the same upregulation of y+ transport occurred in the DM group as a compensatory response to maintain the NO production. Furthermore, low level of Arginine leads to the NOS uncoupling and results in excessive reactive oxygen species (ROS) formation, which also stems from NADPH oxidase (NOX) and mitochondrial complexes reactions [27]. As a result, the NO bioavailability is reduced in different ways—by decreased production because of substrate depletion and increased elimination through reaction with reactive oxygen species. Moreover, significantly reduced Arginine to ADMA ratio in the DM group was also revealed, pointing at reduced NOS action and a subsequently diminished NO production. As no difference in ADMA concentration per se was found nor an increased DMA level, it collectively points at higher ADMA turnover in DM patients. Current literature is inconclusive in the assessment of dimethylarginine. Dimethylaminohydrolase (DDAH) activity in diabetics, which is related with ADMA concentration. Numerous studies suggest that the high glucose level accompanied by increased concentration of proinflammatory cytokines, such as tumour necrosis factor (TNF), results in decreased DDAH activity and consequent increased ADMA level [28–32]. Our study cohort was composed of early-stage diabetic patients treated with metformin without the micro- and macrovascular complications. The low HbA1c levels reflect good glucose control, which may progressively result in preserved DDAH activity. This observation is consistent with the Xiong et al. study, proving the correlation of ADMA level with the macroangiopathy occurrence but not with the disease duration [33]. Additionally, DM patients were treated with metformin, which is a structural analog of ADMA reducing its concentration, possibly in the DDAH-dependent manner [34–37]. Furthermore, metformin reduces the advanced glycation end-products (AGEs) concentration what additionally restores the NO bioavailability [38]. Significantly lower plasma values of the citrulline in DM patients resulting from reduced eNOS activity caused by depletion of Arginine and increased inhibition were also found. Additionally, the use of metformin reduces citrulline levels [39,40]. According to Breier, M. et al. studies, short-term therapy with metformin reduces citrulline concentration by mean of 24% which is in compliance with results of this study—a 26% depletion on average of citrulline concentration between groups [39]. The mechanism of metformindependent citrulline reduction is not clear. Some authors seek the mechanism in urea cycle activity changes, inhibition of mitochondrial complex I, increased urinary excretion by kidneys, or decreased gut absorption associated with gastrointestinal side effects of metformin [39,41–43]. Reduced citrulline concentration in the erythrocyte compartment, more pronounced in the DM group was also noted. In this study, the endothelial function using the two independent methods was also evaluated. First, the vascular response was assessed using the peripheral arterial tonometry with a post-occlusive reactive hyperaemia (PORH) measured as a reactive hyperaemia index (RHI). Second, with the laser Doppler flowmetry (LDF). A diminished endothelial function was found only by using the second method. The pathophysiology of the vascular relaxation in response to numerous stimuli (heating or occlusion) is different and may explain to some extent, why only local thermal hyperemia (LTH) indicates endothelial dysfunction in the diabetic subjects. The sensory nerves response and endothelium-derived hyperpolarizing factors have been described as the major points affecting both, the initial peak and the following increased blood flow after occlusion. Putative elements affecting PORH include the cytochrome epoxygenase metabolites and the large-conductance calcium-activated potassium channels (BKCa) in the vascular smooth muscle cells and in sensory nerves [44–46]. Hence, compared to other mechanisms regulating vascular response, the role of nitric oxide pathway in the post-occlusion response turned out not to be so crucial [47,48]. On the contrary, in the LDF response to heating stimuli, nitric oxide is
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    responsible for vascular response in approximately 70% [49]. The first phase is mediated by a local sensory nerve axon reflex and the plateau depends mostly on the NO [45,49]. The maximum Nutrients 2021, 13, 2306 13 of 16 hyperemia index (MHI) was used to eliminate the neuronal influence. It reflects the nitric oxide-dependent plateau phase and expresses the endothelial function. Applying MHI results in a higher sensitivity and explains the discrepancies between the LTH and PORH outcomes. The results of this research are in line with Faisel and Heier studies, who confirmed endothelial dysfunction in diabetes mellitus type 1 group using LDF with intact vascular reaction in peripheral arterial tonometry [50,51]. The results showed that the augmentation index (AI) in diabetics was statistically higher, strictly corresponding with the increased vascular stiffness and the reduced flexibility of the aorta among diabetic patients Additionally, the AI-75 positively correlated with BNP concentration (r = 0.55) and negatively with the eGFR value (r = −0.43). It reflects that arterial stiffness in DM patients appears at the early stage of the disease and induces the organ-mediated complications from the beginning. The nitric oxide metabolic pathway was also compared by correlating the products’ concentrations with the endothelial function results. The analysis confirmed that the endothelial function in DM patients correlated with NO bioavailability and NOS activity more in the RBC compartment than in plasma (r = 0.38 and r = 0.46). Furthermore, similar dependency was found within the erythrocyte ADMA concentration (r = −0.58) (Figure 2). Hence, it may be postulated that erythrocytes play an important role in compensating the endothelial dysfunction occurring in DM. Further studies are needed to evaluate their specific position in the NO metabolism and to define novel therapeutic targets to prevent the DM micro- and macrovascular complications. 5. Conclusions Patients at an early stage of DM revealed endothelial dysfunction, which could be diagnosed earlier using the laser Doppler flowmetry. This group of subjects showed significant differences in the nitric oxide metabolism, which was more pronounced in the plasma compartment. Physical methods Among the physical synthesis of zinc nanomaterials, we can distinguish such methods as physical vapor deposition, arc plasma method, thermal evaporation, ultrasonic irradiation or laser ablation [56], [63], [64], [65], [66], [67] (Fig. 2). ZnO NPs can be synthesized by laser ablation of zinc metal (bulk material) in a solution – this method was reported to offer several benefits such as technical simplicity and chemical pureness of the yield. The synthesis efficiency and the characteristics of produced nano-zinc particles depend upon many process parameters, including ablation time, fluence and wavelength of the laser [68]. The laser ablation conditions may lead to narrow distribution of size, shape and physical parameters of nanoparticles [67], [69], [70], [71]. Kim et al. [67] synthesized ZnO nanoparticles by laser ablation in neat water; the influence of different conditions such as different ablation times (10–40 min), fluences (50–130 mJ/pulse) and wavelengths (1064, 532, and 355 nm) on the nanoparticle size and shape were also tested. The results of the experiment showed that the size of the ZnO nanoparticles was not affected by the ablation time and fluence – they had a similar band gap and exciton emission wavelength at 1064 nm. However, the mentioned parameters had a significant effect on the density of the ZnO nanoparticles because the band gap and exciton emission wavelength of Z355 were different from those of Z1064 and Z532, which was attributed to quantum confinement effects. Another physical method is thermal evaporation. It is a process in which condensed or powdered source material is vaporized at elevating temperature and then the resultant vapor phase condenses under certain conditions such as temperature, pressure, atmosphere or substrate to form the desired product [72]. Wang et al. [65] showed that it is possible to obtain ZnO nanoparticles in a different shape or size by the method described above. They reviewed various growth morphologies such as nanorods, nanobelts, nanocombs and nanorings. Zou et al. [63] synthesized ZnO nanotubes by heating zinc powder at 600–700°°C at the gas pressure of 20 Pa; ZnO NPs with diameters of 50–200 nm were obtained. The thermal evaporation method used there was found to have some advantages – it is simple, low cost, and catalyst-free. Besides, it can be used for fabrication of ZnO nanotubes on an industrial scale for photocatalytic degradation, because of their high photocatalytic activity. Zheng Wei Pan et al. [73] used thermal evaporation of oxide powder without the presence of catalyst to produce nanobelt structures of ZnO. Furthermore, they showed that the belt-like geometrical morphology is a common structure obtained by thermal evaporation – they also synthetised nanobelts of In2O3, CdO and SnO2. 2.2. Chemical methods Chemical methods are classified according to the physical state, i.e. solid phase, liquid phase (so-called wet chemical methods) or vapor phase applied for the synthesis of zinc nanocomposites [62]. All of them are shown in Fig. 2. The wet chemical methods are the ones most commonly used, and they can be a valuable alternative to gas phase synthesis, with known commercial applications [74]. Water-based chemical methods offer numerous advantages such as being environmentally friendly, using cheap and easy to handle reagents as well as uncomplicated equipment, and they simultaneously require only low energy input. Moreover, they make the easy
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    tailoring of synthesis parameters throughout the whole process possible, which is helpful in gaining control of the composition, shape and size of the resulting nanomaterials [54]. Among the wet-chemical methods we can distinguish microemulsion [57], sol-gel synthesis [58], precipitation [75], hydrothermal and solvothermal method [55], [76].
    2.2.1. Microemulsion
    Microemulsion is defined as thermodynamically stable dispersion of two immiscible liquids – usually water and hydrocarbon – and these types of fluid droplets are stabilized by the presence of surfactant molecules [77], [78]. Two types of microemulsions: direct (oil dispersed in water, O/W) and reversed (water dispersed in oil, W/O) are commonly used [79], [80]. The microemulsion method has been used to synthesize different types of materials including colloidal metals [81], ultrafine AgBr [82], nanocrystalline Fe2O3 [83], TiO2 [84] and zinc oxide nanocomposites. For the synthesis of ZnO nanoparticles, the reversed microemulsion with various types of zinc precursors, surfactants and other reagents is typically used [85], [86]. Different kinds of additives (i.e. polyethylene glycols) are applied to control the size, shape and optical properties of zinc nanomaterials [87]. Apart from the size, other important issues such as the particle size distribution and the degree of agglomeration should be considered during an experiment. Normally, the method of preparing ZnO particles through microemulsion [87] involves salt of zinc being incorporated in aqueous core of a micelle and it is precipitated to get precursor particles. One of the most commonly used surfactants which easily forms water-in-oil microemulsions is anionic sodium bis-2ethylhexylsulfosuccinate (AOT). It consists of two branched alkyl tails, has a negatively charged SO3− head group and an Na+ counterion [88]. Another popular surfactant is Triton X-100 [87]. Van der Rul et al. [54] prepared microemulsion with zinc chloride (ZnCl2) as a precursor and AOT as a surfactant. ZnO nanoparticles were formed in the aqueous cores of the microemulsion upon colliding the nanodroplets. The results of the DLS analysis showed that the ZnO NPs are approximately 12 nm with a narrow size distribution. However, extraction with acetone resulted in aggregation of nanoparticles. Singhal et al. [89] synthetized zinc oxide nanoparticles using ethanol-in-oil microemulsion. What is more, they used zinc-substituted surfactant for the first time. Oxalic acid (fine powder) was added to precipitate zinc oxalate. Finally, zinc oxide was produced after calcination of the oxalate and the obtained nanoparticles were about 12 nm in size. A similar approach was applied in another experiment conducted by Elen et al. [57]. They also used zincsubstituted surfactant, which played role both of a metal source and microemulsion stabilizer. This technique produced equiaxial nanoparticles with the sizes in the range of 10–20 nm. Unfortunately, calcined zinc powder showed large and dense aggregates as the addition of solid oxalic acid destabilizes the microemulsion and it results in large and irregularly shaped particles. As it was mentioned above, various types of additives can be applied to control the size, shape and optical properties of zinc nanomaterials. Li et al. [87] tested the effect of different concentrations of PEG-400 on the morphology (size and shape) of ZnO NPs. When the lowest and the highest concentration of polyethylene glycol (0% and 50% respectively) were used, zinc oxide nanoparticles were smallest (about 50 nm), and they were aggregated. Also the shape of nanostructures varied depending on the PEG-400 concentration. Those results show the crucial role of additives – their different dosage has an effect on the morphology and size of ZnO samples. The outcomes obtained by Li et al. correspond with the results achieved by Hou and colleagues [91] – they found that size and shape of zinc nanoparticles are related to both concentration and type of polyethylene glycol (PEG-200, 400, 600 and 1000).
    2.2.2. Sol-gel method
    The sol-gel process involves preparation of a colloidal solution (sol) that is converted to gel and solid materials. The procedure consists of hydrolization, condensation and polymerization reactions. Typical precursors are metal alkoxides (M(OR)x, where M = metal, i.e. Zn) or corresponding chlorides, in an aqueous or organic medium (usually alcohol). In case of zinc nanomaterials, the most commonly used precursor is zinc acetate hydrate in alcohol. There are several important factors, such as the nature of the alkyl group and the solvent, the concentration of each species in the solvent, the temperature, or the molar ratio of water to alkoxide which are known to affect the growth of ZnO nanoparticles [92]. The sol-gel method was first developed by Spanhel et al. [93] and later refined by Meulenkamp [94]. Both groups used zinc acetate solution as a precursor and they
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    obtained ZnO nanoparticles in the size range from 2 to 7 nm. Meulenkamp [94] also noted that the ratio of water and zinc acetate influenced the particle size. Several research groups [93], [94], [95], [96], [97], [98] proposed a new approach to this method, namely the use of certain additives such as surfactants or others. Vafaee and Ghamsari [95] used triethanolamine (TEA) as a surfactant and zinc acetate dihydrate as a precursor; three different ratios of both reagents were chosen. The results of this study show that what was created were ZnO NPs with the size of 3–4 nm and spherical shape. Their optical properties were also tested – according to the results from absorption spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR) analyses, it was proved that zinc oxide nanoparticles prepared with TEA have higher photoluminescence spectra comparing to those resulting from the traditional sol-gel method. Another useful surfactant can be ethylene diamine (EDA). Jiang et al. [96] found that ZnO NPs can be synthesized by the sol–gel method with the addition of EDA to the zinc acetate dihydrate and oxalic acid dihydrate reaction system. They obtained rod-like nanoparticles 20–320 nm in diameter. In the case of the lowest molar ratio of the surfactant and precursor (R = 0,1) the diameter of NPs was 280–300 nm; increase in molar ratio resulted in decrease of the NPs size and shape. When R was increased to 1, no rod-like nanoparticles were obtained. The experiment results showed that the morphology of nanoparticles can be controlled by changing the amount of EDA and that ethylene diamine plays an important role in the formation of ZnO nanorods. Ristic et al. [58] reported a novel method where tetramethylammonium hydroxide (TMAH) solution was added to the alcoholic solution of zinc 2-ethylhexanoate. TMAH is a strong organic alkali [98] and it can be also used to synthesize other types of nanoparticles, i.e. TiO2 nanocrystalline [99]. The researchers obtained nanocrystalline zinc oxide powders, and the results of X-ray diffraction (XRD) showed an average value of 25–35 nm for the basal diameter of the supposed crystallites whereas their height was 35–45 nm. TEM showed that the size of ZnO NPs varied between 20 and 50 nm; this indicates that particle and crystallite sizes in ZnO powders were approximately equal. Furthermore, it was found that the morphology of nanoparticles did not change significantly for different amounts of precursor. The sol–gel method has certain advantages over other chemical ways of preparing metal oxide nanoparticles – it offers faster nucleation and growth and can be used for large-scale industrial production of nanopowders. The disadvantage of this process is the high cost of the precursors of metal nanomaterials.
    2.2.3. Precipitation
    The precipitation method has been successfully used to design different nanostructures of zinc oxide. Synthesis with this method involves a reaction of zinc salts, such as Zn(NO3)2, Zn(CH3COO)2·2H2O, ZnSO4·7H2O etc. with basic solutions containing LiOH, NH4OH and NaOH [75], [100], [101], [102], [103]. The synthesis starts with a reaction between zinc and hydroxide ions followed by the process of aggregation. Formation of a stable colloid suspension of ZnO nanoparticles is usually performed in an alcohol solution [103] since Zn(OH)2 was formed from aqueous solutions. Obtaining zinc oxide NPs with different morphologies is possible by controlling various parameters of the precipitation process such as solution concentration, pH, washing medium or calcination temperature [104]. Kumar et al. [105] used zinc sulphate heptahydrate and sodium hydroxide as precursors to formulate zinc oxide nanostructures. Results from analytical assays (XRD, SEM and UV–VIS spectroscopy) indicated that the morphology of synthetized NPs changes with calcination temperature. At 300°C and 500°C the samples were nanoflakes while increasing the temperature to 700 °C resulted in spherical nanoparticles. In the work of Wang and Muhammed [106] the precipitation method was used to obtain ZnO NPs with controlled morphology. They synthetized nanoparticles at the size of about 20 nm with zinc chloride as the precursor and ammonium carbamate as precipitating agent. The results from thermogravimetric analysis and SEM showed that formation of precipitates and morphology of nanoparticles depend on the concentration of zinc precursor. The formation of precipitates became more dominant with the rise of zinc concentrations, but on the other hand high precursor amount caused the formation of aggregates. Moreover, size and shape of ZnO NPs seem to be concentration-dependent too; using a solution of 1 M zinc chloride resulted in plate-like particles at 0,5 μm size while the precipitation with 0,1 M precursor led to about 20 nm rod-shaped NPs. In conclusion, higher Zn2 + concentration caused formation of larger and aggregated particles with different shapes. Pourrahimi et al. [107] presented results of using different precursors of zinc oxide such as zinc nitrate, chloride, sulphate and acetate; the reaction conditions were the same in all cases. The most narrowly sized particles with the average size of 25 nm were obtained from acetate precursor. Precipitation with chloride and sulphate precursors resulted in nanoparticles at 10–30 nm and 80–100 nm size respectively. In the case of nitrate precursor, the particles were
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    star-shaped with an average size of about 500 nm. According to these results, it can be concluded that a successful precipitation synthesis depends on the proper selection of zinc NPs precursor. 2.3. Biological methods Nanoparticles have been produced with physical and chemical methods for a long time, but recently the development of green chemistry seems to be an approach generating much interest Synthesis using biological systems is a promising alternative and provides many advantages in comparison to traditional chemical methods. This type of nanoparticle preparation is more eco-friendly, allows the researchers to control the synthesis of NP size and shape and does not use toxic and expensive organic solvents [108], [109], [110]. Biological methods require considering several process parameters such as selection of the organisms that are the most suitable (with regard to their enzyme activities and biochemical pathways) and optimal conditions for cell growth or enzyme activity (medium, temperature, pH, buffer). Optimization of these factors makes it possible to control the morphology of ZnO nanoparticles. Among biological methods, we can distinguish synthesis with bacteria, yeast, fungi and plant extracts [61], [112], [114], [115]. In the case of biosynthesis with plants, it is supposed that they are able to reduce metal ions by flavonoids, terpenoids and polysaccharides, and through reduction, such plants can produce nanoparticles [59]. Furthermore, some plants are known for their heavy metal accumulation and the ability to detoxify such substances. There are many mechanisms of metal tolerance in plants including cell wall binding, chelation of metals and active transport of metal ions into the vacuole [109]. This property has attracted the attention of researchers and can be used in biosynthesis of metal and metal oxide nanoparticles – it is possible to use Zn hyperaccumulating plants as a source of precursor to form zinc oxide NPs. The synthesis of zinc oxide NPs with plants has been carried out using Physalis alkekengi L. [114], Sedum alfredii Hance [115], Trifolium pretense flowers [61], Pongamia pinnata [116], Cassia Auriculata [117], [118], and Plectranthus amboinicus leaf extracts [119]. It was reported [114] that crystalline zinc oxide nanoparticles at 72.5 nm size were synthesized using Physalis alkekengi. This plant is able to grow in soils with high zinc level (from 50 to 5000 mg/kg). The bladder cherry plant can incorporate Zn into its aerial parts and accumulate it in the biomass. The results of the experiment by Qu et al. [114] indicate that P. alkekengi could be used for remediation of zinc-contaminated soils and for synthesis of nano zinc oxide. Another zinc hyperaccumulating plant is Sedum alfredii. Qu et al. [115] also reported the synthesis of hexagonal wurtzite ZnO nanoparticles from this plant. The results from TEM indicated that the formed nanoparticles were agglomerated and single ZnO nanoparticles were pseudospherical in shape with the size about 54 nm. Pongamia pinnata contains a wide range of biologically active compounds such as flavonoids, terpenoids, phenols etc., and due to these properties it was chosen for biosynthesis of zinc oxide NPs. Sundrarajan et al. [116] obtained spherical nanoparticles about 100 nm in size, with hexagonal structure and of high purity. The results from the use of the diffusion method showed the antibacterial activity of NPs against both Gram-positive and Gram-negative bacteria (S. aureus and E. coli respectively). Dobrucka and Dugaszewska [61] used Trifolium pretense water extract for the synthesis of ZnO NPs. The results from SEM and XRD (theta degrees 32, 34, 36, 47, 57, 63, 67, 68, 69, 73 and 77 correspond to the peaks identified as (1 0 0), (0 0 2), (1 0 1), (0 1 2), (1 1 0), (0 1 3), (2 0 0), (1 1 2), (0 0 4) and (2 0 2) respectively) analysis show that the obtained nanoparticles had hexagonal crystalline structure and size about 60–70 nm. The FT-IR spectra confirmed the presence of zinc oxide – the peak at 515 cm− 1 is
    related to Zn O bond [61]. What is more, the synthetized nanoparticles exhibited high antimicrobial activity against S. aureus, E. coli and P. aeruginosa. Fu and Fu [119] used leaf extract of Plectranthus amboinicus, a perennial plant from Lamiaceae family, for biosynthesis of ZnO nanoparticles. This plant had been previously used successfully to synthesize silver nanoparticles – Ajitha et al. [120] reported the use of P. amboinicus leaf extract as the reducing agent for synthesizing Ag NPs. The synthesized nanoparticles were spherical with the average size about 18 nm and exhibited good antimicrobial activity against E. coli and Penicillium spp. The results of an experiment conducted by Fu and Fu [119] prove that the extract of P. amboinicus can also be utilized in bioproduction of ZnO nanocomponents. The researchers obtained rod-shaped NPs sized 50–180 nm. Biological synthesis of ZnO nanostructures can be also carried out using organisms such as bacteria. It is believed that bacteria are better for the production of nanoparticles because of the ease of genetic manipulation and modification as well as owing to the possibility of extrapolating studies on one bacterium to others [121]. There are two main types of microbial synthesis: the nanoparticles are formed intracellularly or extracellularly. Intracellular production consists of transporting ions into the bacteria cell to form NPs in the presence of enzymes, coenzymes, ions and others. The extracellular synthesis of nanoparticles involves trapping the metal ions on the surface of the cells and interaction of zinc with the bioactive components released by microbial cells (e.g.
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    reductase, sugars, glycoproteins) leading to the formation of Zn/ZnO nanoparticles [122]. To produce zinc oxide NPs, probiotic bacteria like Lactobacillus sporogens or Lactobacillus plantarum are used [60], [112]. Lactic acid bacteria are non-toxic, easy to prepare (they are distributed in the majority of food products) and have negative electrokinetic potential in pH of synthesis, which easily attracts the cations and helps in biosynthesis of ZnO nanoparticles [123]. Biosynthesis with other types of bacteria such as Aeromonas hydrophila or Bacillus cereus is also known [124]. Selvarajan and Mohanasrinivasan [60], [125] synthesized pure and crystalline in nature spherical nanoparticles with size from 7 to 19 nm in diameter; they used ZnSO4°H2O as a precursor. FT-IR spectra confirmed the presence of zinc oxide – the peak observed at 528 cm− 1 corresponds to
    the stretching vibrations of Zn O bond. Prasad and Anal [112] used Lactobacillus sporogens and zinc chloride as a precursor for biosynthesis of ZnO nanoparticles. Data from XRD and TEM clearly illustrated the formation of hexagonal zinc oxide crystals sized 5–15 nm. The results from those two experiments indicate effective biosynthesis of zinc oxide nanomaterials with probiotic microorganisms. Jayaseelan et al. [124] described a lowcost and simple procedure for biosynthesis of ZnO NPs using bacteria Aeromonas hydrophila. X-ray diffraction analysis confirmed the crystalline nature of the nanoparticles which were spherical with an average size about 58 nm. The synthesized NPs showed significant antibacterial and antifungal activity – the maximum zone of inhibition was observed in the nanoparticles at the concentration of 25 μg/mL against Pseudomonas aeruginosa and Aspergillus flavus. To sum up, biological methods of synthesizing ZnO nanoparticles seem to be cheap, environmentally friendly and much safer than the chemical approach. The obtained nanoparticles have the expected hexagonal structure, are stable and of high purity. What is more, they exhibit antimicrobial activity against both Gram-positive and Gramnegative bacteria as well as antifungal properties. However, despite numerous research papers, the mechanism of biosynthesis is not yet fully clear and understood in its entirety, so further investigations are required.
  2. Physicochemical characterization of nanoparticles Various techniques are used to characterize nanoparticles and predict their ultimate properties including size, shape, surface charge and modifications, purity of the sample or antibacterial activity. Each of them often needs separate and precise tools which are at the same time simple and inexpensive. Some of the analytical methods used for NP characterization can be divided into static and dynamic approaches; their descriptions can be found below.
    3.1. Spectroscopy methods Dynamic light scattering (DLS) is commonly used for nanoparticle size determination. DLS measures Brown motion of NPs in suspension and relates its velocity to the size of nanoparticles according to the Stokes–Einstein equation. The result of DLS is reported as a mean particle size and the homogeneity of size distribution [126]. Van der Rul et al. [54] used this analysis to measure ZnO NPs size after microemulsion synthesis; they obtained nanoparticles at 12 nm with a narrow size distribution. Zeta potential (ζ – zeta, electrokinetic potential) as a physicochemical term of biocolloidal systems is an important tool for determining surface charge, understanding the state of the nanoparticle surface and predicting their long term stability [127]. Nanoparticles have a surface charge that attracts a thin layer of ions of the opposite charge to the nanoparticle surface; the electric potential at the boundary of the double layer is known as the zeta potential of the biocolloids [128]. Zeta potentials are typically obtained from the electrophoretic mobility (μe), according to the Smoluchowski equation (Fig. 3). Nanoparticles with zeta potential values between + 30 mV and − 30 mV typically have low degree of stability. Deviation from these values means higher stability of biocolloids (Fig. 3) [129].
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    Download : Download high-res image (110KB) Download : Download full-size image Fig. 3. Zeta potential of 150 mM AgCl: dispersion stability and Smoluchowski equation. One of the main problems during nanoparticle preparation and thus for the measurement of their size and zeta potential is aggregation of NPs. The ultrasonic process (i.e. sonication) is the standard approach for dispersing and deagglomerating nanomaterials [130]. In the work of Murdock et al. [131] different metal oxide nanoparticles were characterized by DLS before and after sonication. It was determined that ultrasonic processes can help better disperse NPs in the solution and gain more homogenous mixture. What is important, sonication did not seem to have a significant effect on the zeta potential of the nanoparticles. Unfortunately, it was observed that NPs are able to aggregate again after a period of time. Hexagonal wurtzite structure of ZnO and purity of the sample is usually determined by the X-ray diffraction method. XRD has a good potential for the analysis of nanostructures because the width and shape of reflections yield information about the substructure of the materials (i.e. sizes of crystallites) [132]. Reference theta degree values for ZnO are 31,84; 34,52; 36,33; 47,63; 56,71; 62,96; 68,13 and 69,18 for {1 0 0}; {0 0 2}; {1 0 1}; {1 0 2}; {1 1 0}; {1 0 3}; {1 1 2}; {2 0 1}, respectively [133], [134]. Elen and colleagues [57] attributed each peak gained with X-ray diffraction of calcinated zinc oxalate obtained from the microemulsion method to hexagonal wurtzite structure of ZnO NPs. Spectroscopy techniques also provide very useful information on the composition of nanomaterials – identification of the functional groups present in nanoparticles is a frequent requirement in nanotechnology research. Sharma et al. [135] applied the FTIR technique to characterize bio ZnO NPs. Peaks around 1632 and 3450 cm− 1 corresponded to the stretching vibrations of the OH groups; this was explained by the reabsorption of water molecules from ambient atmosphere. Another peak was observed between 1550 and 1490 cm− 1 and it was related to the vibration of C O bonds. FTIR analysis showed also a standard peak of zinc oxide around 464 cm− 1. In the study of Kumar and Rani [85], Fourier transformed infrared spectroscopy
    confirmed the presence of Zn O bond and adsorption of surfactant molecules at the surface of ZnO nanoparticles (υ = X cm− 1). For the optical characteristic of NPs, the UV–VIS spectroscopy is customarily used. The wavelength of bulk ZnO is 350–390 nm [136]. It has been found that the UV–VIS analysis of ZnO nanoparticles obtained by Kumar et
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    al. [85] showed an excitonic absorption peak at 214 nm which did not correspond to the band gap wavelength of bulk material. The authors suggested that this phenomenon may be due to strong aggregation of NPs. 3.2. Microscopy methods Microscopy approach is an essential tool in imaging and characterization of nanomaterials and is moreover the perfect complement of spectroscopic methods. Techniques such as fluorescent microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) are commonly used in nanotechnology [111], [113], [137], [138], [139]. Fluorescence microcopy makes it possible not only to measure the physical properties of nanoparticles but also to understand the interactions between the engineered nanomaterials and living matter such as cells or tissues. Shang et al. [137] compared the cellular uptake of different types of NPs (gold nanoclusters, CdSe/ZnS quantum dots and polystyrene nanoparticles) with various sizes and surface modification. All kinds of fluorescent labeled nanomaterials were tested with HeLa cells and mesenchymal stem cells (MSCs). The results from fluorescent confocal microcopy indicated the in situ internalization process of NPs in the size range of 3 to 100 nm by HeLa MS cells. Au NCs and CdSe/ZnS quantum dots at the size smaller than 10 nm were accumulated on the plasma membrane whereas polystyrene nanoparticles (diameter about 100 nm) were rapidly endocytosed without accumulating on the plasma membrane. Electron microscopy provides an accurate assessment of the size, shape, spatial resolution and differences in composition and structure of the NPs but has also a certain disadvantage – such techniques often require specific complicated sample preparation steps which can create artifacts (e.g. NP agglomeration during the drying process for electron microscopy) [138]. Kumar et al. [105] used the electron microscopy approach to check if the morphology of the synthesized ZnO NPs was changing with calcination temperature. The results shown in Fig. 4 indicate that at 300 °C and 500 °C the samples were nanoflakes while the temperature increase to 700 °C resulted in spherical nanoparticles.
    Download : Download high-res image (256KB) Download : Download full-size image Fig. 4. SEM images of ZnO samples calcined at three different temperatures. (a) 300 °C, (b) 500 °C, and (c) 700 °C. SEM can be also used for predicting antimicrobial activity of ZnO nanoparticles. In the work of Zhang et al. [41], the results from scanning electron microscopy indicated that the presence of nanoparticles in growing medium may cause damages to the E. coli cell membrane. Ristic et al. [58] synthetized zinc oxide NPs by sol-gel method; TEM showed that the size of nanoparticles varied between 20 and 50 nm. Comparison of DLS and electron microscopy made by Mahl et al. [139] showed differences in the size distribution data – the smaller particles were undetectable by dynamic light scattering and only electron microscopy and analytical disc centrifugation were able to give quantitative data on the size distribution. On the other hand, it is not possible to check the agglomeration level of NPs with electron microscopy, because aggregation may also have taken place during sample preparation stage i.e. during the drying of the sample. The research proved decreased NOS activity with aggravated ADMA degradation and Arginine depletion. It indicated that erythrocytes remain a buffer less affected by the DM deviations with higher NO bioavailability than in the plasma compartment. Additionally, it was revealed that endothelial dysfunction correlates to a greater extent with abnormalities of nitric oxide pathway noted in RBC than in plasma compartment. The disclosed findings show the importance of the RBC as a NO-buffer. Hence, the RBCs play a key-role in maintenance a healthy vascular status in those with early diabetes mellitus. Additional studies in that field should be performed in order to extend the knowledge regarding the RBC NObuffer, which may be used in the prevention or treatment of vascular complications in the diabetic group. 6. Limitations Several limitations of this study should be addressed. The first limitation regards the measured molecules. As NO easily reacts with hemoglobin it was not possible to measure it directly and our results are mainly based on by-products which are
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    more stable. Furthermore, different permeability of erythrocyte membrane may affect compartment distribution and study outcomes. The separation process might affect the analytes equilibrium, therefore additional experiments should be conducted to assess the potential significance of that process. In this study, subjects with newly onset of diabetes, without concomitant cardiovascular disorders, were investigated. Hence, the results of this study cannot be simply extrapolated to the whole spectrum of diabetic population. Author Contributions: Conceptualization, A.D. and D.G.; methodology, A.D., J.W., P.F., and E.S.-K.; software, D.G. and A.D.; validation, D.G., A.D., and J.G.; formal analysis, D.G. and A.D.; investigation, D.G. and J.G.; resources, A.D.; data curation, D.G.; writing—original draft preparation, D.G.; writing— review and editing, A.D.; visualization, D.G.; supervision, A.D.; project administration, J.G. and E.S.-K.; funding acquisition, J.G. and A.D. All authors have read and agreed to the published version of the manuscript. Nutrients 2021, 13, 2306 14 of 16 Funding: This research was funded by Wrocław Medical University according to the records in Simple system, grant number STM.A210.18.026 and STA210.17.057. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Bioethics Committee at the Wroclaw Medical University (KB-155/2019) from 28 February 2019. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The original data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations ADMA asymmetric dimethylarginine AGEs advanced glycation end-products AI augmentation index AI-75 [%] augmentation index normalized for a heart rate of 75 beats/min Arg L-Arginine BCl benzoyl chloride BMI body mass index BNP brain natriuretic peptide CVD cardiovascular disorders DDAH Dimethylaminohydrolase DM Diabetes mellitus DMA dimethylamine eGFR estimated glomerular filtration rate ER estrogen receptors ESC European Society of Cardiology HbA1c glycated hemoglobin HDL high-density lipoprotein hsCRP high-sensitivity C-reactive protein LDF laser Doppler flowmetry LDL low-density lipoprotein lnRHI reactive hyperaemia index logarithm LTH local thermal hyperemia MCH mean corpuscular hemoglobin MCHC mean corpuscular hemoglobin concentration MCV mean (red blood) cell volume MHI maximum heating index NO nitric oxide NOS nitric oxide synthase NOX NADPH oxidase NS result statistically non-significant PAT peripheral arterial tone PDW platelet distribution width PORH post-occlusive reactive hyperaemia RBC red blood cells RHI reactive hyperemia index ROS reactive oxygen species SDMA symmetric dimethylarginine TNF tumour necrosis factor TSH thyroid-stimulating hormone WBC white blood cells WHR waist–hip ratio Nutrients 2021, 13, 2306 15 of 16 References 1. International Diabetes Federation. International Diabetes IDF Diabetes Atlas, 9th ed.; International Diabetes Federation: Brussels, Belgium, 2019. 2. Sarwar, N.; Gao, P.; Kondapally Seshasai, S.R.; Gobin, R.; Kaptoge, S.; Di Angelantonio, E.; Ingelsson, E.; Lawlor, D.A.; Selvin, E.; Stampfer, M.; et al. Diabetes mellitus, fasting blood glucose concentration, and risk of vascular disease: A collaborative meta-analysis of 102 prospective studies. Lancet 2010, 375, 2215–2222. [PubMed] 3. Lee, W.; Cheung, A.; Cape, D.; Zinman, B. Impact of Diabetes on Coronary Artery A meta-analysis of prospective studies. Diabetes Care 2000, 23, 962–968. [CrossRef] 4. Ballotari, P.; Ranieri, S.C.; Luberto, F.; Caroli, S.; Greci, M.; Rossi, P.G.; Manicardi, V. Sex Differences in Cardiovascular Mortality in Diabetics and Nondiabetic Subjects: A Population-Based Study (Italy). Int. J. Endocrinol. 2015, 2015, 1–10. [CrossRef] [PubMed] 5. Dantas, A.P.V.; Fortes, Z.B.; de Carvalho, M.H.C. Vascular Disease in Diabetic Women: Why Do They Miss the Female Protection? Exp. Diabetes Res. 2012, 2012, 1–10. [CrossRef] 6. Novensa, L.; Selent, J.; Pastor, M.; Sandberg, K.; Heras, M.; Dantas, A.P. Equine Estrogens Impair Nitric Oxide Production and Endothelial Nitric Oxide Synthase Transcription in Human Endothelial Cells Compared with the Natural 17β-Estradiol. Hypertens 2010, 56, 405– 411. [CrossRef] [PubMed] 7. Catalan, M.; Herreras, Z.; Pinyol, M.; Sala-Vila, A.; Amor, A.; de Groot, E.; Gilabert, R.; Ros, E.; Ortega, E. Prevalence by sex of preclinical carotid atherosclerosis in newly diagnosed type 2 diabetes. Nutr. Metab. Cardiovasc. Dis. 2015, 25, 742–748. [CrossRef] [PubMed] 8. Yu, E.; Ruíz-Canela, M.; Hu, F.B.; Clish, C.B.; Corella, L.; Salas-Salvadó, J.; Hruby, A.; Fíto, M.; Liang, L.; Toledo, E.; et al. Plasma Arginine/Asymmetric Dimethylarginine Ratio and Incidence of Cardiovascular Events: A Case-Cohort Study. J. Clin. Endocrinol. Metab. 2017, 102, 1879–1888. [CrossRef] [PubMed] 9. Bode-Böger, S.; Scalera, F.; Ignarro, L. The l-arginine paradox: Importance of the larginine/asymmetrical dimethylarginine ratio. Pharmacol. Ther. 2007, 114, 295–306. [CrossRef] [PubMed] 10. CorteseKrott, M.M.; Kelm, M. Endothelial nitric oxide synthase in red blood cells: Key to a new erythrocrine function? Redox Biol. 2014, 2, 251–258. [CrossRef] 11. Fleszar, M.G.; Wi´sniewski, J.; Krzystek-Korpacka, M.; Misiak, B.; Frydecka, D.; Piechowicz, J.; Lorenc-Kukuła, K.; Gamian, A. Quantitative Analysis of l-Arginine, Dimethylated Arginine Derivatives, lCitrulline, and Dimethylamine in Human Serum Using Liquid Chromatography-Mass Spectrometric Method Mass to charge ratio MRM Multiple reaction monitoring NO Nitric oxide NOS Nitric oxide synthase OPA Ortho-phthaldialdehyde. Chromatographia 2018, 1, 911–921. 12. Doroszko, A.; Szahidewicz-Krupska, E.; Janus, A.; Jakubowski, M.; Turek, A.; Ilnicka, P.; Szuba, A.; Mazur, G.; Derkacz, A. Endothelial dysfunction in young healthy men is associated with aspirin resistance. Vasc. Pharmacol. 2015, 67–69, 30–37. [CrossRef] 13. Kellogg, D.L. In vivo mechanisms of cutaneous vasodilation and vasoconstriction in humans during thermoregulatory challenges. J. Appl. Physiol. 2006, 100, 1709–1718. [CrossRef] [PubMed] 14. Farrell, D.M.; Bishop, V.S. Permissive role for nitric oxide in active thermoregulatory vasodilation in rabbit ear. Am. J. Physiol. Circ. Physiol. 1995, 269, H1613–H1618. [CrossRef] 15. Shibasaki, M.; Wilson, T.E.; Cui, J.; Crandall, C.G. Acetylcholine released from cholinergic nerves contributes to cutaneous vasodilation during heat stress. J. Appl. Physiol. 2002, 93, 1947–1951. [CrossRef] 16. Shastry, S.; Dietz, N.M.; Halliwill, J.R.; Reed, A.S.; Joyner, M.J. Effects of nitric oxide synthase inhibition on cutaneous vasodilation during body heating in humans. J. Appl. Physiol. 1998, 85, 830–834. [CrossRef] 17. Minson, C.T.; Berry, L.T.; Joyner, M.J. Nitric oxide and neurally mediated
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