**Appendix B**

This is a sampling of The Achieving Styles Inventory™ Items, used in this study in a pre–post-test format:

#### **A Direct Items**

*A1 Intrinsic:*

For me, the most gratifying thing is to have solved a tough problem.

More than anything else, I like to take on a challenging task.

For me, the greatest satisfaction comes from breaking through to the solution of a new problem.

*A2 Competitive:*

For me, winning is the most important thing.

I am not happy if I don't come out on top of a competitive situation.

I select competitive situations because I do better when I compete.

*A3 Power:*

I want to be the leader.

I want to take charge when working with others.

Being the person in charge is exciting to me.


**1. Introduction**

More than 5.8 million people die from severe trauma worldwide each year and approximately 40% of which are caused by uncontrolled bleeding or its consequences [1]. Massive blood loss from incompressible injuries has been reported to cause hemorrhagic shock coagulopathy, multiple organ failure, life-threatening sepsis, and acidosis. Moreover, interventional diagnosis and surgical treatment are prone to hemorrhage or intraluminal hemorrhage, especially in sites close to the heart, parenchymal organs, vital blood vessels, etc. [2]. Therefore, research into hemostatic technologies and materials with superior performance is critical to reducing adverse side effects and even saving lives.

Recently, exploring fast and effective methods for controlling bleeding in different application environments has always been an important subject of multidisciplinary research [3]. Sutures and staplers are commonly used to close wounds, however, they have the risk of infection, blood exudation, hyperplasia, and keloid formation [4]. For quick hemostasis in superficial wounds, some conventional hemostatic materials, such as tourniquets [5], hemostatic gauze [6], bandages [7], etc., have been utilized extensively. Nevertheless, common materials struggle to meet the demand for quick and efficient hemostasis in cases of intracavitary hemorrhage or injuries involving essential tissues/organs. Additionally, as gauze and bandages are not biodegradable and can cause secondary injury, delayed healing, and additional discomfort, they need to be removed after hemostasis [8,9]. Therefore, various advanced hemostatic powders, hydrogels, sponges, adhesives, and hemostatic agents [10,11], have been extensively explored (Figure 1).

Adhesives can bind different tissues as well as blood vessels together, whereas hemostatic drugs are known to stop bleeding mechanically or by accelerating the coagulation cascade. Additionally, tissue sealants can stop blood dripping from blood vessels [12]. Nevertheless, some of these products are not universally applicable and have obvious disadvantages. For example, fibrinogen and thrombin-based injection solutions (fibrin sealants) may be washed away by the bloodstream and cannot be used effectively in severe

**Citation:** Han, W.; Wang, S. Advances in Hemostatic Hydrogels That Can Adhere to Wet Surfaces. *Gels* **2023**, *9*, 2. https://doi.org/ 10.3390/gels9010002

Academic Editor: Christian Demitri

Received: 23 November 2022 Revised: 11 December 2022 Accepted: 12 December 2022 Published: 22 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

or emergency arterial bleeding [13,14]. As emerging medical adhesives, cyanoacrylates may cause allergic reactions, and their rapid curing process is exothermic [15]. Furthermore, such products have not been used inside the body due to concerns about the potential toxicity of the degradation products. As a consequence, designing an ideal hemostasis technique for effective hemostasis in complicated clinical situations remains challenging. *Gels* **2023**, *8*, x FOR PEER REVIEW 2 of 23

**Figure 1.** The current methods of hemostasis used for external and internal bleeding management. Figure modified from [11] with permission. Copyright 2020. **Figure 1.** The current methods of hemostasis used for external and internal bleeding management. Figure modified from [11] with permission. Copyright 2020.

Adhesives can bind different tissues as well as blood vessels together, whereas hemostatic drugs are known to stop bleeding mechanically or by accelerating the coagulation cascade. Additionally, tissue sealants can stop blood dripping from blood vessels [12]. Nevertheless, some of these products are not universally applicable and have obvious disadvantages. For example, fibrinogen and thrombin-based injection solutions (fibrin sealants) may be washed away by the bloodstream and cannot be used effectively in severe or emergency arterial bleeding [13,14]. As emerging medical adhesives, cyanoacrylates may cause allergic reactions, and their rapid curing process is exothermic [15]. Furthermore, such products have not been used inside the body due to concerns about the potential toxicity of the degradation products. As a consequence, designing an ideal hemostasis technique for effective hemostasis in complicated clinical situations remains challenging. Hydrogel-based biomaterials show many advantages over traditional hemostasis methods [16,17]. A hydrogel is a 3D cross-linked hydrophilic polymer with a structure similar to that of the natural extracellular matrix (ECM) [18]. They are widely used in biomedicine, and due to their injectability and fluidity, hydrogels are crucial for achieving quick and lasting hemostasis for a variety of irregular wounds and intraluminal injuries [19]. In addition, the hydrogel-based biomaterial is safe for use in vivo due to its exceptional biocompatibility and biodegradability [20,21]. An ideal polymer hydrogel for hemostatic applications should have the following properties: (i) the hydrogel should be able to effectively promote wound healing and immediately block the bleeding, [22] (ii) the hemostatic hydrogel should have superior mechanical qualities that enable it to quickly adhere and completely seal the damage, especially in humid and dynamic settings, and [23] (iii) the hydrogels must have quick coagulation to prevent the migration of hemostatic hydrogels from the bleeding site [24].

Hydrogel-based biomaterials show many advantages over traditional hemostasis methods [16,17]. A hydrogel is a 3D cross-linked hydrophilic polymer with a structure similar to that of the natural extracellular matrix (ECM) [18]. They are widely used in biomedicine, and due to their injectability and fluidity, hydrogels are crucial for achieving quick and lasting hemostasis for a variety of irregular wounds and intraluminal injuries High pressures and blood flow rates may remove or destroy general hydrogels due to their limited underwater adhesion and weak adhesion mechanisms [25]. Therefore, it is crucial to enable hemostatic hydrogels to adhere to tissues and maintain their integrity in humid environments. Moreover, an ideal wet-adhesive hydrogel should completely cover the wound area, minimize surgical scope or complications, carry therapeutic cells and drugs, and release them to the wound site [26]. This review introduces the properties of

[19]. In addition, the hydrogel-based biomaterial is safe for use in vivo due to its exceptional biocompatibility and biodegradability [20,21]. An ideal polymer hydrogel for hemostatic applications should have the following properties: (i) the hydrogel should be

the hemostatic hydrogel should have superior mechanical qualities that enable it to quickly adhere and completely seal the damage, especially in humid and dynamic settings, and [23] (iii) the hydrogels must have quick coagulation to prevent the migration of

High pressures and blood flow rates may remove or destroy general hydrogels due to their limited underwater adhesion and weak adhesion mechanisms [25]. Therefore, it is crucial to enable hemostatic hydrogels to adhere to tissues and maintain their integrity in humid environments. Moreover, an ideal wet-adhesive hydrogel should completely

hemostatic hydrogels from the bleeding site [24].

hemostatic hydrogels that can adhere to wet surfaces, briefly discusses the basic principles of hemostasis, and then focuses on the adhesion mechanism of hydrogels on wet surfaces. This review is anticipated to promote further research progress on wet viscous hemostatic hydrogel and create more possibilities for rapid and effective emergency hemostasis.

### **2. Properties of Wet Adhesive Hemostatic Hydrogel**

The complicated dynamic equilibrium environment of the human body should be adapted when hydrogels are employed to stop bleeding. The ideal adhesive hemostatic hydrogel should possess the properties of strong wetting adhesion, hemostasis, antibacterial, and biocompatibility.


**1. Introduction**

Diuretic drugs aim to regulate the volume and composition of body fluids by increasing the rate of urine flow and sodium excretion. They are widely used in clinics for the treatment of edematous disorders, such as those associated with congestive heart failure, as well as liver or renal failure and hypertension [1,2]. Furosemide (FUR, Scheme 1), 4-chloro-2-[(2-furanylmethyl)-amino]-5-sulfamoylbenzoic acid, is classified as a high ceiling loop diuretic drug. Its mechanism of action is related to the inhibition of the sodium-potassium-2chloride co-transporter (Na+-K+-2Cl−) located in the thick ascending limb of the loop of Henle in the renal tubule.

According to the Biopharmaceutics Classification System (BCS), FUR belongs to class IV drug, defined by low solubility and low permeability values [3]. Indeed, furosemide is almost insoluble in water [4], which results in significant intraindividual variations in absorption and very poor oral bioavailability [5]. Despite this relevant drawback, FUR has shown great efficacy, hence it is highly used in therapeutics worldwide, including chronic treatments. Thereby, the development of improved oral formulations of furosemide, which aim to achieve higher bioavailability, are certainly relevant for the pharmaceutical industry.

The design of multicomponent pharmaceutical solids is actually one of the latest research strategies in the development of new drug alternatives in the pharmaceutical

**Citation:** Acebedo-Martínez, F.J.; Alarcón-Payer, C.; Rodríguez-Domingo, L.; Domínguez-Martín, A.; Gómez-Morales, J.; Choquesillo-Lazarte, D. Furosemide/Non-Steroidal Anti-Inflammatory Drug–Drug Pharmaceutical Solids: Novel Opportunities in Drug Formulation. *Crystals* **2021**, *11*, 1339. https://doi.org/10.3390/ cryst11111339

Academic Editor: Aidar T. Gubaidullin

Received: 18 October 2021 Accepted: 31 October 2021 Published: 2 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

industry [6]. They can be defined as crystalline materials in which at least one component is an active pharmaceutical ingredient (API). The other components, incorporated in the crystal lattice—so-called coformers—must be found in a stoichiometric ratio and considered pharmaceutically acceptable—i.e., included in the Generally Recognized as Safe (GRAS) list within the FDA's "Substances Added to Food" Inventory. APIs and coformers recognize themselves by different kind of non-covalent intermolecular interactions, so-called supramolecular synthons, mainly H-bonds, which organization has a profound impact on the intimate 3D structure of the solid and therefore on its macroscopic physicochemical properties. The development of this novel strategy is rather interesting because allows industry to save money compared to the traditional drug development scheme, still guarantying the possibility of generating intellectual property rights [7]. In this context, there has been reported several studies devoted to studying pharmaceutical cocrystals and salts of furosemide [8–13].

**Scheme 1.** Chemical formula of furosemide (FUR), ethenzamide (ETZ), and piroxicam (PRX).

One of the most recent approaches in the development of multicomponent pharmaceutical solids is the concurrent administration of two or more APIs, leading to drug–drug or co-drug pharmaceutical solids [14]. APIs within the formulation might have similar or different mechanisms of actions, but always looking for a synergic effect, either targeting one metabolic pathway at different levels or different pathways related to a particular disease.

Along with diuretics, nonsteroidal anti-inflammatory drugs (NSAIDs) are also widely prescribed worldwide. Interestingly, the combination of diuretics—particularly furosemide—and NSAIDs is rather common, especially among the elderly. However, although not contraindicated, there is clinical evidence on the moderate interaction between these two kinds of drugs. The use of NSAIDs may decrease natriuretic response to loop diuretics, thus reducing their efficacy and resulting in adverse effects on patients with different edematous states. In addition, some NSAIDs may also show adverse nephrotoxic effects, which may be exacerbated by diuretic therapy [15–17]. In these cases, dose adjustments or special monitoring of the renal function and blood pressure are required for safety's sake. Unfortunately, the insights of such interactions are still poorly understood because they do not seem to follow the same mechanism for all combination of drugs, some of them being associated with the suppression of plasma renin activity or impaired synthesis of vasodilator prostaglandins.

Since the concurrent prescription of FUR and NSAIDs is quite common, it is worthwhile exploring the formulation of drug–drug multicomponent pharmaceutical solids involving such a combination, seeking new therapeutical alternatives that would improve the bioavailability of the APIs and/or reduce the abovementioned drug–drug interactions. In this work, the synthesis and physicochemical characterization of two different drug– drug pharmaceutical solids, including the loop diuretic furosemide and one NSAID drug: ethenzamide (ETZ, 2-ethoxybenzamide) or piroxicam (PRX, 4-hydroxyl-2- methyl-N-2 pyridinyl-2H-1,2,-benzothiazine-3-carboxamide 1,1-dioxide) are reported (Scheme 1). To the best of our knowledge, there are no conclusive studies on the interaction between furosemide and ethenzamide, while one study was reported on piroxicam–furosemide drug interaction in the late 1980s [18].


*2.1. Hydro-Densitometry—Two Compartmental, Indirect*

The indirect method of underwater weighing, termed hydro-densitometry, dates back to ~300 BC and uses assumed densities [2] and prediction formulae for determination of two-compartmental FFM and FM [2,3]. Historically, measures of body density established via underwater weighing have represented the 'criterion' for both accurate and reliable measurement of body composition and as a means for other methods to be validated against [4]. Employing the 'Archimedes principle' of measuring body volume, this technique is based upon the understanding that when an object is submerged underwater, the measurement of the 'buoyant force' is deemed to be equal to the weight of the water that it displaces [5]. When carried out by a qualified technician, the method can be accurate and reliable. However, limitations include (1) assumption of specific tissue densities, which may differ in elite athletic populations; (2) residual lung volume can be a source of error, given an individual must exhale all of their air whilst remaining static for a stable value; (3) some individuals may become claustrophobic whilst underwater or not be comfortable in water; (4) the equipment is now uncommon and costly, whilst needing to be maintained, sterilised and cleaned regularly; (5) the technique cannot measure distribution of FFM or FM; (6) air can be contained within an individual's swimsuit, skin, head, and/or body hair or internally such as in their digestive tract, all of which can be included in false estimations [6]; (7) air temperature, barometric pressure, nitrogen analyser and force sensor calibrations all contain sources of error, which could contribute to the overall measurement error [7]; and (8) is time consuming and potentially uncomfortable. Despite the fact that hydro-densitometry has long been considered a criterion assessment of body composition, the emergence of accurate surrogate techniques are now seen as more suitable alternates in applied sport [8] and in laboratory-based sport science research [9–11].

#### *2.2. Air Displacement Plethysmography—Two Compartmental, Indirect*

Air displacement plethysmography (ADP) is an alternative to hydrostatic weighing and has more practicality in applied sport, where instead of water, air is utilised to measure body density. Some consider ADP to overcome several of the aforementioned limitations of hydro-densitometry [6]. Machines such as the BOD POD (COSMED, Rome, ITA), use Poisson's Law to calculate air displacement and thus volumetric calculation. Isothermal air is then measured via inbuilt systems or generated via a prediction formula and combined to calculate a corrected body volume and body composition via numerous predictive equations [12]. Correlations of body density to assess validity when compared to hydrodensitometry have been found to be high [4,13,14]. Additionally, ADP has a high level of reliability (CV = 1.7 ± 1.1%) [4]. Despite these benefits, ADP is (1) insufficiently sensitive to detect in-competition changes in elite athletic body composition [15]; (2) sensitive to clothing, body hair, air movement, moisture, pressure and temperature changes [12,21–23]; and (3) expensive to purchase for those in the applied sports setting. When compared with hydro-densitometry, ADP overestimates BF% by 'at least 1.28%' [6]. When estimating adiposity in comparison to DXA, which will be covered later in this review, ADP diverges at the extremes of the BMI spectrum [24], which may be a cause for concern when used with certain athletic populations. Additionally, much like the use of hydro-densitometry, ADP is considered to be limited by its lack of ability to differentiate FM distribution, which may be of interest to practitioners. As such, this technique is not commonly used in applied sports practice and is primarily utilised in laboratory-based sport science research.

#### *2.3. Bioelectrical Impedance Analysis and Spectroscopy—Multi-Compartmental, Doubly Indirect*

Bioelectrical impedance methods are commonly used within general populations for assessment of body composition due to the speed of procedure, minimal expertise required to administer the test, portability and cost in comparison to other approaches. Indeed, it is not uncommon to see such units in gymnasiums and sports clubs. The methods of bioelectrical impedance are categorised by the number of frequencies used for analysis [25]. Techniques which are single frequency are commonly referred to as bioelectrical impedance analysis (BIA) devices (i.e., hand to hand), while multiple frequency methods are described as bioelectrical impedance spectroscopy (BIS) devices (i.e., hand and foot contact) [25]. BIS methods are considered superior to BIA methods as the calculation of body fluid volumes are based on Cole modelling and mixture theories as opposed to the simple regression equations used by BIA (for a more in-depth review see [25]). Despite this, the majority of previous research using bioelectrical impedance cites BIA methods. For both techniques, the currents are generated and measured using electrodes or metal contacts, which send a small voltage through the body to indirectly assess TBW volume. Based on the resistance to current flow observed within the body, FFM has more water and is less resistant than FM, which contains less water and is therefore more resistant. Bioelectrical impedance was previously validated for measuring TBW volume and may also distinguish between intracellular and extracellular fluid compartments [26,27]. Nevertheless, this method has several limitations including (1) outputs that can be affected by temperature and hydration status [28]; (2) sensitivity to conductive surface of electrodes and electrode placement [29]; (3) makes assumptions on the composition of the body in formula and calculations, irrespective of population group [30]; and (4) the limbs contribute a large proportion to whole-body impedance, despite the relatively low contribution to overall BM [31].

Within athletic populations, there are a limited amount of studies that have assessed the validation of bioelectrical impedance methods to measure FM and FFM, with conflicting findings when compared to DXA. Some investigations reported an underestimation of FM and overestimation of FFM [32–36], with others that reported overestimations of FM [37]. There is also large variability between devices [38], alongside large differences in the equations used to estimate body composition, making comparisons highly complex [25]. However, using bioelectrical impedance as a way of assessing change (provided methods are standardised) may be a useful tool [38] and is re-emerging in an applied sport context.


**2. Study on powerful antioxidants**

#### **2.1 Study on resveratrol**

The reducing agent treatment efficacy was observed by evaluation of anion permeability for chloride, lipid peroxidation and hemolysis in RBCs. Anion permeability for chloride is an indicator of membrane protein damage and is evaluated in RBCs by the specific absorption of methemoglobin (CM) at 590 and 635 nm after treatment of heparinized blood with NaNO2. The measurement of the membrane lipid degradation is obtained by the determination of MDA. The lipid peroxidation susceptibility is observed after the oxidative stress induced by AAPH. The hemolysis assays are conducted on blood samples in phosphate buffer saline. To evaluate in human RBC the *in vitro* effects of AAPH and the antioxidant activity of polyphenols from red wine (resveratrol, catechin and naturally red wine), authors start the research evaluating RBC anion permeability for chloride. **Figure 1** shows the values of membrane anion permeability for chloride in both groups (controls and AAPH-treated RBCs). The statistical analysis of data shows that AAPH *in vitro* treatment lowers the parameter in comparison to all controls. Furthermore, the reducing agent treatment (T2-T4) also significantly recovers the activity up to values higher than AAPH treatment. The recovery of anion permeability for chloride is always lower than control data. Anion permeability for chloride values were slightly higher after red wine and resveratrol treatments (as reducing agents) in both experimental groups (controls and AAPHtreated RBCs), corresponding to a preserved architecture and anion permeability for chloride of the human erythrocyte membranes. To confirm the efficacy of oxidant and reducing treatments on human RBC *in vitro* authors evaluated lipid peroxidation, hemolysis and RBC morphology. In **Figure 2**, MDA levels are described in membranes from several varieties of treated and untreated RBC with AAPH, under the action of reducing agents or without treatment. On the contrary, as described in **Figure 2**, AAPH-induced increases of MDA levels are partially recovered by natural reducing agents at levels almost comparable to control the group. C2-C4 results always slow down MDA levels in RBC membranes, but catechin is less effective. Red wine, among them, is almost as powerful as resveratrol alone in both groups (controls and AAPHtreated RBCs). As shown in **Figure 3** time courses of RBC hemolyses are described at several incubation times (60, 120, 180 and 240 min) either under the action of natural oxidative pathways or by treatment with radical donor AAPH (60 mM). This experiment shows also the natural reducing agents partially recover the effect of both oxidative and reducing agent treatments. As described in all experiments, AAPH-

#### **Figure 1.**

*RBC membrane anion permeability for chloride. Data are mean* � *standard error of the mean on 10 determinations. \*P < 0.05 according to one-way analysis of variance and Bonferroni* post-hoc *test coupled data at each time were performed. C = control sample,T = treated sample [8] C1 = control; C2 = control + red wine 5, 2 mM (reducing power equivalents of gallic acid); C3 = control + resveratrol 5 μM; C4 = control + Catechin 50 μM; T1 = AAPH 60 mM; T2 = AAPH 60 mM + red wine 5,2 mM (reducing power equivalents of gallic acid); T3 = AAPH 60 mM + resveratrol 5 μM; T4 = AAPH 60 mM+ Catechin 50 μM.*

#### **Figure 2.**

*Effects of lipid peroxidation and induced by 2.2*0 *-azobis-2 amidinopropan dihydrochloride (60 mM) (*t *= 60 min) on the malonyldialdehyde levels of human RBC membranes. Data are mean* � *standard error of the mean on 10 determinations. Column labels are according to \**P *< 0.05 and \*\**P *< 0.01 according to one-way analysis of variance and Bonferroni* post-hoc *test for coupled data at each time were performed (Figure 1).*

*Reducing Compounds Roles in Oxidative Stress Relieving of Human Red Blood Cells DOI: http://dx.doi.org/10.5772/intechopen.99977*

#### **Figure 3.**

*Effect of 2.2*0 *-azobis-2 amidinopropan dihydrochloride-induced hemolysis in sportive human RBCs at several times. Data are mean* � *standard error of the mean on 10 determinations. Column labels are according to incubation times a = (60 min); b = (120 min); c = (180 min); d = (240 min). \**P *< 0.05, \*\** P *< 0.01 and \*\*\** P *< 0,001 according to according to one-way analysis of variance and Bonferroni* post-hoc *test for coupled data at each time were performed (Figure 1). C = Control sample,T = Treated sample (Gallo et al., 2013) C1 = Control; C2 = Control + Red Wine 5, 2 mM (reducing power equivalents of gallic acid); C3 = Control +Resveratrol 5 μM; C4 = Control + Catechin 50 μM; T1 = AAPH 60 mM; T2 = AAPH 60 mM + Red Wine 5,2 mM (reducing power equivalents of gallic acid); T3 = AAPH 60 mM + Resveratrol 5 μM; T4 = AAPH 60 mM+ Catechin 50 μM. Of course C1 and T1 are a = (60 min). And so on, saying the others.*

induced hemolysis is more relevant than in other treated samples. As described in previous experiments (**Figures 1** and **2**) resveratrol and red wine polyphenol treatments are more efficient than catechin in preserving RBC membrane structural recovery at 60 and 120 min of incubation. In the present study, polyphenol (resveratrol and red wine) treatment is associated with an improvement in erythrocyte anion permeability for chloride-related with cell membrane derangements. The cell system exchange of Cl� for HCO3 � represents a target of membrane oxidative damage and is evaluated in RBCs by the specific CM at 590 and 635 nm after treatment of heparinized blood with NaNO2. According to our research, the in vitro action model of artificial agent AAPH on RBC plasma membranes shows that anion permeability for chloride (evaluated by CM [9]) decreases after treatment and is partially recovered after administration of either resveratrol or red wine polyphenols extracts. Our data confirm those on LDL peroxidation [10] with AAPH by MDA, hemolysis and RBC morphology study from our laboratory [8, 11]. Moreover in the present work all previous results are compared, so that a relationship among them is evident and correlate each other, so that the action of red wine polyphenol extracts are demonstrated as effective reducing agents both alone and in mixture. MDA, hemolysis and RBC morphological study confirmed the resveratrol and red wine efficacy on improvement of RBC membrane integrity against in vitro oxidative stress damages. The described damages could be linked to the erythrocyte membrane framework, so a lack of adequate lipid organization can significantly influence ion permeability. The alteration of membrane fluidity consequent to lipid oxidation represented by MDA level increase can reflect the change of the structure and function of membrane lipids and proteins. The permeability is another important property of erythrocyte membrane. Depending on the exchanger (Cl� \$ HCO3 �), that transports anions across the membrane, it is related to the framework of erythrocyte membrane. To sum up, the alteration of anion permeability can reflect the change of erythrocyte membrane protein framework. Moreover, our results could describe the overall effect of the three described alterations together.