Perioperative fluid management, a critical aspect of major surgeries, is characterized by pronounced stress responses, altered capillary permeability, and significant fluid shifts. Recognized as a cornerstone of enhanced recovery protocols, effective perioperative fluid management is crucial for optimizing patient recovery and preventing postoperative complications, especially in high-risk patients. The scientific literature has extensively investigated various fluid infusion regimens, but recent publications indicate that not only the volume but also the type of fluid infused significantly influences surgical outcomes. Adequate fluid therapy prescription requires a thorough understanding of the physiological and biochemical principles that govern the body’s internal environment and the potential perioperative alterations that may arise. Recently published clinical trials have questioned the safety of synthetic colloids, widely used in the surgical field. A new clinical scenario has arisen in which crystalloids could play a pivotal role in perioperative fluid therapy. This review aims to offer evidence-based clinical principles for prescribing fluid therapy tailored to the patient’s physiology during the perioperative period. The approach combines these principles with current recommendations for enhanced recovery programs for surgical patients, grounded in physiological and biochemical principles. Intravenous fluid infusion is a ubiquitous therapy in perioperative patient management . The misCrystalloid fluids have traditionally been used to prevent perioperative hypovolemia. Not until this millennium was it realized that fluid overload is a common problem and that the “therapeutic window” for crystalloid fluids is actually quite narrow. In fact, fluid therapy protocols must carefully consider the optimal amount of fluid to be infused in different clinical situations . DespiteTherefore, one of the primary objectives of perioperative fluid therapy is to maintain adequate circulating blood volume while preventing both hypervolemia and hypovolemia, and AKI, as these conditions are all known to contribute to unfavorable postoperative outcomes . See FigAnother pivotal function of perioperative fluid therapy is to preserve the homeostasis of plasma and prevent disruptions to the internal environment that could adversely impact vital organs . InfusedThe aim of this article is to provide an overview of the history, basic physiology, and currently recommended use of crystalloid fluids during the perioperative period.The infusion of electrolyte-based intravenous fluids has become a standard practice in healthcare settings. The origins of modern fluid therapy can be traced back to the cholera pandemic that brought havoc to India in 1831. As a result of trade movements, the disease quickly spread to Europe, including the British Isles, resulting in over 23,000 individuals dying in Britain from an undiagnosed illness .During that time, a young Irish physician named William Brooke O’Shaughnessy journeyed to England to aid in combating the pandemic. He described the disease caused by the Vibrio Cholerae bacterium as “sudden, deadly, overwhelming, the living death”. The existing remedies at the time, such as bloodletting, leech therapy, and powerful cathartics, had proven ineffective . HoweverTwo months after the publication of O’Shaughnessy’s paper, another British doctor named Thomas Latta, inspired by O’Shaughnessy’s work, performed the first intravenous fluid resuscitation in May 1832. Latta used a hypotonic aqueous mixture of sodium, chloride, and bicarbonate, saving the lives of 8 of the 25 treated patients .Intravenous therapy has been the subject of controversy since its inception. Some clinicians hailed it as a “miraculous and supernatural agent”, while others in the medical profession condemned the idea of violating the sanctity of the human body. The latter view seems to have prevailed, as O’Shaughnessy’s rehydration therapy was ignored during the six cholera pandemics of the 19th century. Of course, the lack of safe materials capable of ensuring infection-free intravenous infusions was also a likely culprit .Over the next 80 years, multiple scientific breakthroughs were made, largely driven by an improved understanding of cardiovascular pathophysiology. Around 1876, Sydney Ringer developed a physiological calcium-rich solution for intravenous perfusion based on his discovery that the infusion of such calcium-rich fluids into the hearts of experimental frogs enhanced their contractility. These ground-breaking findings fueled the advancement of intravenous infusion therapy . In 1934The first mention of a solution similar to 0.9% saline dates back to an article by WS Lazarus-Barlow published in 1896. Based on experiments by Dr. Hamburger, Lazarus-Barlow found that a saline concentration of 0.92% was normal in mammals. Hamburger had demonstrated the isotonicity of 0.9% saline with human plasma in a series of in vitro studies, leading to the misconception that 0.9% saline was physiologically saline .In recent years, authors have drawn on past experience and on current evidence to search for intravenously infused fluids that closely resemble the electrolytic composition of plasma and can keep patients’ physiological parameters and internal environment as close as possible to normal levels. In this regard, balanced solutions have garnered significant interest and have largely replaced 0.9% saline as the standard of care in the operating room . HoweverA solution is the outcome of a homogeneous mixture of multiple substances, with the solvent, typically water in biological contexts, being the most abundant component. Substances dissolved in water are referred to as solutes. Water can form solutions with various solutes . The quaThe osmotic effect of a solution is determined by the number of dissolved particles it contains, regardless of their weight, electrical charge, or valency, as one mole of any element consists of the same number of molecules (the Avogadro constant) . One osmConsequently, it is possible for a hyperosmotic solution to be hypotonic. One example is ethanol, which greatly increases osmolality but easily enters cells and, therefore, does not redistribute water. Many amino acids, such as glycine, enter cells with only minor difficulty. Another example is a 20% glucose solution, which, despite being hyperosmotic due to its glucose content, acts as “free water” in vivo after the glucose has been metabolized. −, H+, phosphates, albumin, Na+, K+, and others, as they enable the preservation of complex and efficient cell functions related to the acid–base balance [Acute changes in blood pH induce regulatory effects on protein and enzyme structure and function, leading to changes in cell function such as glycolysis, gluconeogenesis, mitosis, and DNA synthesis, among others. Hence, it is important to understand the concurrence of elements governing pH maintenance within physiologic boundaries, such as HCO3 balance .The traditional model, known as the Henderson-Hasselbach equation ;Stewart’s theory, based on the Law of Conservation of Matter, the Law of Mass Action (stating that any incompletely dissociated substance reaches a state of dissociation equilibrium), and the Principle of Electroneutrality .Two chemical models have been developed to elucidate the ionic behavior of the acid–base balance:2 pressure (PaCO2) [In the traditional approach using the Henderson-Hasselbalch equation, bicarbonate independently determines pH. In Stewart’s approach, however, bicarbonate is only one of several dependent ions and is thus conceived as a marker, not a mechanism. According to the latter model, three independent variables can govern the acid–base behavior of bodily fluids if they exert control over the physical medium of these fluids. These variables are the total weak acid concentration (ATOT), the strong ion difference (SID), and the partial CO (PaCO2) . See FigAs the influence of fluid therapy on acid–base balance involves the metabolic component, the respiratory component is beyond the scope of this review.The approximate amount of ATOT is calculated using the following equation :2.5 × AlAn increase in ATOT is linked to acidosis, while a decrease in ATOT leads to alkalosis. A decrease in ATOT is caused by hypoalbuminemia. The calculation formula for ATOT emphasizes the significant impact of albumin. In critically ill patients, hypoalbuminemia is primarily caused by fluid overload, endothelial leak, and nutritional deficiencies . While i+, K+, Ca2+, Mg2+, and Cl−, exclusively exist in fully ionized form within body fluids. At physiological pH, this phenomenon primarily applies to anions with pKa values ≤4, especially in quantitative terms. Examples of such anions include sulfate, lactate, and beta-hydroxybutyrate. Stewart referred to compounds that display this characteristic as strong ions, which originate from substances that completely dissociate in water [+, K+, Ca2+, Mg2+, and Cl−. Notably, body fluids exhibit an abundance of strong cations, quantified by Stewart as the SID. In simple terms, this so-called apparent SID (SIDa), where the contribution of weak acids is not considered, can be calculated through subtracting the concentration of strong anions from that of strong cations [Certain elements, namely Nain water . The mai cations , and it For normal plasma, SIDa can be calculated using the following formula: The in vitro SID of all fluid (SID in a bag) is zero according to the law of electroneutrality. However, when balanced crystalloids are mixed with plasma, the buffers are eliminated. Due to this gap, the in vivo SID becomes higher than zero. It can be hypothesized that administering a crystalloid with a similar in vivo SID to that of normal plasma  will not alter the plasma SID. However, when crystalloid solutions are infused, they typically lead to the dilution of weak acids, especially albumin, which may increase the patient’s susceptibility to metabolic alkalosis . ConsideHowever, for the determination of the global metabolic component of the acid–base equilibrium using the Stewart model, the strong ion gap (SIG) must calculated, and for this, the apparent strong ion difference apparent (SIDa) and the effective strong ion difference (SIDe) must be known first . SIDe is determined by the formula:ases SIG . No stanases SIG . + and Cl− ions at supraphysiological levels (154 mmol/L). Its osmolarity and osmolality closely resemble those of plasma [Crystalloids are aqueous solutions that consist of inorganic cations and organic and inorganic anions . Saline,f plasma .2. The most commonly used buffers in crystalloids include the following:(a)L-Lactate is metabolized primarily in the liver within approximately one hour. However, one major disadvantage of lactate becomes particularly obvious in cases of peripheral tissue hypoxia, as the accumulation of l-lactate can cause significant interference, making it difficult to accurately measure systemic perfusion. It is not recommended for use in patients with diabetes ,53(b)Acetate is typically metabolized within 15 min, al.though the exact duration may vary among patients. After entering the bloodstream, it undergoes swift absorption by cells and is subsequently metabolized into acetyl-coenzyme within those cells. This metabolic transformation is possible within 15 min in all body cells that engage in aerobic oxygen metabolism, although the exact duration may vary among patients. The most elevated metabolic rates are observed in the heart, liver, skeletal muscles, and kidneys. It does not pose a risk of buildup. Acetate is associated with the lowest oxygen consumption and the highest carbon dioxide production. In large quantities, such as in dialysis applications, it may lead to vasodilation and reduced cardiac contractility. However, the amount of acetate in balanced fluids is generally low, mitigating these concerns ,54.(c)Gluconate undergoes partial but rapid metabolism in the liver, with around 80% being cleared through the kidneys. It is associated with the highest metabolic consumption of oxygen and the highest production of carbon dioxide . Glucona(d)Malate: While malate is metabolized at the muscular level , there iBalanced crystalloids are solutions designed to minimize disruptions in the acid–base balance caused by fluid infusion. In these solutions, chloride anions are replaced by sodium bicarbonate or other buffers. Initially, balanced crystalloids achieved this through replacing chloride with bicarbonate anions. However, due to the instability of bicarbonate-containing solutions in gas-permeable bags, modern balanced crystalloids now incorporate buffers, which are often rapidly metabolized or excreted . The metSurgery induces comprehensive lifestyle changes, demanding higher performance levels from patients, not only due to social or personal reasons but also because of underlying conditions and the physiological stress caused by anesthesia and surgery themselves . In respNeuroendocrine changes resulting from stress manifest through a dual effector system: the hypothalamic–pituitary–adrenal (HPA) axis and the sympathetic–adrenal–medullary (SAM) axis. The alterations caused by the HPA axis are the ones exerting the strongest impact on the internal environment. The HPA axis triggers the secretion of vasopressin (ADH) in synergy with the corticotropin-releasing hormone, which acts on the pituitary gland and promotes the release of corticotropin (ACTH) into systemic circulation. ACTH stimulates the production and release of cortisol and aldosterone, affecting various physiological processes . AdditioDue to the aforementioned hormonal changes, surgery leads to water and salt retention in the body. Moreover, the anesthesia-associated reduction in the arterial pressure unloads the baroreceptors, which increases the sympathetic nerve activity in the kidneys and also results in strong water and sodium retention ,63. Comp2/FiO2 at 24 h and prolonged assisted ventilation [Sodium and chloride are two essential ions that play a crucial role in maintaining the internal environment. Sodium is a major determinant of plasma tonicity and is consequently essential for proper osmoregulation. Despite this, the human kidney lacks an active sodium excretion mechanism . The hortilation . Furthertilation .The optimal functioning of organs, particularly the kidneys, relies on maintaining appropriate chloride plasma concentrations. Although there is no clear limit for diagnosing hyperchloremia, the most commonly accepted is Cl > 110 mEq/L. Hyperchloremia caused by underlying diseases or fluid therapy is common in ICUs, within 25–45%. Hyperchloremia often occurs together with acidosis. In this case, the pH is lowered by decreased SID and not only by hyperchloremia. Hyperchloremia can also appear with alkalosis; this situation occurs in the presence of greater hypernatremia and therefore with increased SID. This fact highlights the presumably greater importance of relative chloremia compared to absolute chloremia, although it is a controversial issue . These cIn normal conditions, approximately 60–65% of an adult’s body weight consists of water, with this percentage gradually decreasing during childhood until the age of ten, when it becomes comparable to that of adults. Neonates, on the other hand, may have water levels as high as 75–80%. In women, the percentage of water is slightly lower, around 55%. Water is dynamically distributed among three bodily compartments: two-thirds are allocated to the intracellular compartment, while the remaining third is distributed to the extracellular space, which can be further divided into the interstitial space (75–80%), the intravascular space (5–6%), and the transcellular space, housing specific types of fluid, such as pleural or pericardial fluid, crucial for organ integrity [The intracellular compartment is separated from the extracellular fluid by a semipermeable cell membrane. Fluid shifts between these compartments occur by virtue of osmosis, simple diffusion, and transmembrane channels. Changes in tonicity within either compartment allow water to flow freely between them. Regarding simple diffusion, the cell membrane permits the passage of a few solutes, which can move effortlessly down a concentration gradient . On the The movement of solutes or water across capillary walls predominantly occurs through filtration and simple diffusion. Volume kinetic studies suggest that the passage of crystalloid fluid across the capillary wall occur 4–5 times faster than the urine flow in the conscious state ,79, and The glycocalyx, a dense network of glycoproteins and associated proteoglycans attached to the luminal side of endothelial cells, forms a primary barrier preventing macromolecules from entering the sub-glycocalyx space immediately below, which is mostly devoid of proteins. The forces governing plasma exchange between the vascular system and the interstitium are the differences in hydrostatic and osmotic pressures between the interstitial space and the subglycocalyx region, covering the intercellular pores . MoreoveThe lymphatic system plays a vital role in the regulation of fluid balance, immune defense, and overall health. It is composed of lymphatic vessels, lymph nodes, lymphoid organs, and lymphoid tissues, forming a transportation network for lymph. Lymph is a clear fluid that carries nutrients, waste products, and immune cells. Lymphatic vessels run parallel to blood vessels throughout the body, ranging in size from microscopic capillaries to larger collecting ducts . The lymThe primary function of the lymphatic system is to maintain fluid balance in the body. Excess fluid, proteins, and cellular waste products that escape from the blood capillaries into the interstitial space are collected by the lymphatic capillaries. This fluid, now known as lymph, is transported through the lymphatic vessels, filtered in lymph nodes, and eventually returned to the bloodstream. Through preventing the accumulation of excess fluid, the lymphatic system promotes tissue health and prevents swelling, also known as edema .Normal lymphatic pump function is determined by the intrinsic properties of lymphatic muscle and the regulation of pumping by lymphatic preload, afterload, spontaneous contraction rate, contractility, and neural influences. Lymphatic contractile dysfunction, barrier dysfunction, and valve defects are common themes among pathologies that directly or indirectly involve the lymphatic system .The movement of interstitial fluid into the lymphatic system is facilitated by two types of tissue forces: extrinsic and intrinsic forces . ExtrinsVolume kinetic analysis of crystalloid fluid suggests that the infused volume distributes between three body fluid compartments: a central volume (the plasma) and one fast-exchange and one slow-exchange interstitial compartment. An infusion of <500 mL distributes only in the central space while larger volumes equilibrate between the plasma and the two interstitial spaces. The entrance of fluid to the slow-exchange space is slow and creates a typical biphasic plasma volume (PV) expansion curve which gives crystalloid fluid a fairly effective PV expansion during infusion (approximately 50% of the infused volume). The distribution phase lasts only for 25–30 min, which means that the strong PV expansion is of short duration ,101. HowThe data were derived from a volume kinetic analysis of 217 infusion experiments where 1.5 L (mean) of Ringer´s solution was infused over 30 min in 106 non-dehydrated volunteers and 111 patients undergoing surgery. The slowly equilibrating interstitial fluid pool accumulates more volume during general anesthesia due to the impairment of the return flow to the plasma, which creates a “lock-out” effect. Preliminary data suggest that the remote pool shifts volume to the plasma during blood loss (unpublished).Fluids prescribed for therapy should be considered medications in their own right. Therefore, fluid therapy should be based on the careful selection of a specific product administered at the appropriate dose, tailored to address a specific indication over a defined period. As with any medication, it is crucial to identify any adverse events associated with fluid therapy .When prescribing fluids, it is essential to consider factors such as electrolytic composition, the route of administration, osmolality, tonicity, and the presence of buffers. Additionally, the patient’s clinical status, the potential for renal and/or liver failure, acid–base balance, capillary leaks, water balance, albumin levels, and other relevant parameters should be evaluated to guide a physiologically appropriate prescription .Furthermore, fluid selection varies depending on the purpose of the therapy, which includes four indications for fluid administration: resuscitation, replacement, nutrition, or maintenance, either individually or in combination . See FigThe primary objective of resuscitation fluid therapy is to save patients’ lives and prevent organ failure . It addr2 and lactic acid [In certain situations, optimizing preload can be beneficial in achieving an optimal volumetric state and striking a balance between oxygen supply and demand. Ensuring the prevention of hypoperfusion is of utmost importance in the intraoperative setting, and early detection and treatment are crucial to minimize the risk of organ failure . It is wtic acid ,110. ManCrystalloid fluid has an indisputable positive effect on a frequent cause of shock such as hemorrhage, as the restored intravascular volume normalizes the body’s physiology to the pre-bleeding situation. The background is more complex during anesthesia where crystalloid fluid is indicated even if no bleeding occurs. A short explanation for this indication is that general anesthesia reduces the driving pressure for circulation, which is called the mean circulatory filling pressure (MCFP), by some 25% . Both veReplacement fluids are administered to address fluid deficits that cannot be adequately replenished through oral intake. These deficits can arise from various sources, including drains, stomata, fistulas, hyperthermia, open wounds, and conditions such as polyuria . However, data on replacement fluids remain limited. Several recent guidelines recommend matching the composition and the quantity of fluids and electrolytes as closely as possible to the lost fluids. Typically, replacement fluids are isotonic balanced solutions. In cases where there is a significant loss of fluids via the gastrointestinal tract, solutions with a high chloride content, such as saline solution (NaCl 0.9%), may be useful as replacement fluids .Nutritional fluid therapy represents a crucial indication for the administration of intravenous fluids as it plays a significant role in meeting the patient’s nutritional requirements. However, the administration of intravenous fluids for nutritional purposes can lead to fluid overload .Maintenance fluid therapy primarily meets the patient’s baseline needs. During the intraoperative period, maintenance fluid therapy compensates for traditionally overestimated insensible and other losses, including diuresis. Postoperatively, maintenance fluid therapy provides free water and meets the patient’s daily electrolyte and glucose needs .A fluid therapy protocol consists of two main components: the selection of an ideal fluid and the determination of the appropriate volume for infusion. The administration of fluids often deviates from a linear course due to the dynamic nature of surgical patients. Therefore, the prescription strategy should be adaptable, aiming to detect clinical changes and perioperative complications as early as possible . Two disThe initial phase of any fluid therapy protocol is the rescue phase, which occurs when the patient is in a state of shock. During this phase, the primary objective is to restore the balance between oxygen supply and demand as quickly as possible . PatientThe goal of the optimization phase is to maintain normovolemia, particularly during the intraoperative period. Although intraoperative hypovolemia may not be initially associated with hypoperfusion, studies have linked it to poorer outcomes in surgical patients . During The stabilization phase differs from the previous two stages by the absence of shock or an imminent threat of shock. At this point, the focus shifts toward providing organ support, and the patient enters a stable steady state. During this phase, fluid therapy is primarily required for ongoing maintenance to compensate for normal fluid losses, such as renal, gastrointestinal, and insensible losses . ReplaceIn the maintenance phase, fluids should only be administered to fulfill daily fluid requirements, considering other sources of fluid and electrolytes. If a patient already receives adequate water, glucose, and electrolytes through alternative means such as enteral or parenteral nutrition, medication solutions, and the like, specific intravenous maintenance fluids should be discontinued . It is eThe term “de-resuscitation” was initially proposed in 2012 and officially coined in 2014. It specifically refers to late goal-directed fluid removal and late conservative fluid management . Late goAssessing preload responsiveness can still be valuable in determining the initiation and cessation of fluid removal . If no sSince the late 1990s, there has been a resurgence in the recognition and exploration of surgical stress, driven by increasing concerns regarding its impact on patient recovery and surgical outcomes. This renewed focus has led to a better understanding of the physiological response to stress and its underlying mechanisms. As a result, a comprehensive set of measures has been devised and compiled aimed at adopting a less metabolically demanding approach during the perioperative period .One prominent manifestation of this approach is the implementation and widespread adoption of Enhanced Recovery After Surgery (ERAS) protocols, which place significant emphasis on perioperative fluid therapy. These protocols have demonstrated remarkable efficacy in reducing surgical morbidity and mortality and hospital stays and overall healthcare costs .In the past three decades, numerous studies have investigated the use of balanced crystalloids compared to 0.9% saline for resuscitation fluids. These studies encompassed controlled trials involving healthy volunteers, observational studies, and clinical trials conducted in surgical settings and with adult critical patients.The SPLIT trial   involvedBuilding upon these studies, two large-scale clinical trials have recently been completed involving nearly 30,000 adults. The SMART  trial  and the p = 0.04). In patients with sepsis or septic shock, the 30-day in-hospital mortality was lower in the balanced crystalloid group compared with the 0.9% saline group . Using balanced crystalloids in patients at higher risk of severe renal damage or death resulted in absolute risk reductions of 3.7% and 4.2%, respectively.The SMART trial  recruiteThe SALT-ED trial  enrolledAlthough the SMART and SALT-ED trials have limitations, such as being single-center studies with a low total volume of fluids infused, they provide valuable insights. Additionally, the SOLAR trial   trial  and the An updated meta-analysis  incorporIn conclusion, the evidence from these studies supports the use of balanced crystalloids as resuscitation fluids during the perioperative period. They have shown potential benefits, including reduced risks of severe renal dysfunction, in-hospital mortality, and major adverse kidney events when compared with 0.9% saline. However, further research is needed to address limitations and provide more robust evidence in specific patient populations and clinical settings.Prolonged preoperative fasting is associated with various harmful effects on surgical patients, including anxiety, dehydration, nausea, vomiting, and impaired insulin resistance during the postoperative phase . IntensiThe preoperative administration of a carbohydrate-rich drink (200–300 mL), primarily consisting of maltodextrins, the night before surgery and up to 2 h preoperatively does not increase gastric volume or pose a risk of aspiration and has been found to enhance postoperative recovery . The rouThere are three main strategies for the administration of fluid during surgery.-Fluid balance approach: The anesthetist summarizes measured and perceived losses of fluid during the surgery and replaces the volumes according to the known plasma volume (PV) expansion effects of crystalloid and colloid infusion fluids. This individualized method is the most common worldwide and is appropriate for surgeries of short duration (we suggest <1 h) .-Outcome-based approach: Here, researchers have compared the incidence of postoperative complications in groups of patients randomized to receive one of two pre-determined fluid regimens, such as liberal/restrictive or crystalloid/colloid. A fluid strategy according to this approach is appropriate for intermediate-length surgery (1–3 h) without major hemorrhage.Over the past two decades, numerous studies have compared various intraoperative infusion protocols, using multiple definitions of liberal and restrictive regimens. Typically, in major abdominal surgery, a regimen that administers an intraoperative infusion exceeding 7 mL/kg/h is categorized as liberal, while an approach characterized by an infusion rate not surpassing 5 mL/kg/h is considered a restrictive strategy ,142.-Goal-directed fluid therapy should be applied for major surgery with an expected larger hemorrhage, such as the Whipple operation or abdominal cancer surgery. The fluid administration is then guided according to invasive hemodynamic measurements whereby the stroke volume is maintained at the flat portion of the Frank–Starling curve. The interested reader is referred to special literature on this topic . HistoriAdhesion to the perioperative protocols for goal-directed fluid therapy can result in volume overload if applied in intensive care. Here, fluid boluses should be provided when signs of poor organ perfusion  or hemodynamic instability appear.The need for PV expansion during surgery is mainly due to the previously mentioned reduction in the MCFP. In addition, the excreted urine, bled volume, and insensible fluid losses need to be replaced. The goal is to maintain constancy in the internal environment despite the complex fluid balance situation that is at hand. Traditionally, insensible losses during surgery have been overestimated . This, cFluid creep refers to the amount of fluid (and sodium) administered to dilute intravenously administered drugs, infuse prediluted medications, facilitate catheter flushing, or aid in the advancement of medication through a catheter. A retrospective study conducted in a Belgian ICU involving nearly 15,000 patients concluded that fluid creep accounted for one-third of a critically ill patient’s fluid intake during their ICU stay . HoweverOral intake should be encouraged as soon as possible, and the administration of intravenous fluids should be limited . HoweverA cross-sectional observational study on fluid therapy by Uña et al. revealed that fluid therapy regimens prescribed in clinical practice often fail to meet patients’ daily requirements, frequently providing excessive amounts of sodium and chloride and insufficient amounts of potassium, calcium, and magnesium . Such imPerioperative fluid therapy plays a crucial role in determining postoperative outcomes. Crystalloids are the preferred choice of fluids during the perioperative period. An in-depth understanding of these agents and the biochemical and physiological factors governing their in vivo performance is essential for their safe prescription to surgical patients, allowing for the preservation of the dynamics, composition, and homeostasis of the internal environment.


Matas recommended the venoclysis route, with the intravenous infusion continued guttatim for as long as circulatory doubt existed.3 However, case descriptions of intravenous hydration included patients developing congestion of the lungs and heart when fluid was rapidly administered, hence Matas’ recommendation of a “drop by drop” rate of infusion.5 These early descriptions were observed in critically ill patients, and the understanding of ischemia-reperfusion syndrome may not have been known.5The administration of intravenous fluids, crystalloids and colloids, has been an integral part of care for most surgical procedures, yet the replacement volume needed to maintain intravascular normovolemia continues to be a daily issue for most anesthesiologists.12 Historically, the development of postpneumonectomy pulmonary edema as originally described by Zeldin, Normandin, and colleagues13 demonstrated higher complication rates when associated with excessive intravenous fluid therapy. Consequently, Zeldin et al and others recommended restricting the administration of intravenous fluids during pulmonary surgery.17 Other researchers, however, could not associate postpneumonectomy pulmonary edema with excessive intravenous fluid administration.18 In contrast, restricting fluid therapy risks inadequate organ perfusion with worse postoperative outcomes.19 Although the goal is to maintain intravascular normovolemia,21 the classic measures of intraoperative systemic hemodynamics and urine output do not provide satisfactory measures of intravascular normovolemia.22 Furthermore, investigations with the use of complex monitoring instruments to measure perioperative intravascular volume status have not answered this need,22 hence the continued use of guidelines rather than individualization measures to maintain intravascular normovolemia during the surgical procedure.17What is the right fluid balance for pulmonary resection procedures or for surgical procedures in general? The study of perioperative fluid therapy has increased in recent years as studies show that a fluid administration strategy can influence postoperative outcomes.20 and Bundgaard-Nielsen, Secher, and Kehlet21 proposed using volumetric dose-response curves to associate perioperative fluid administration with the incidence of postoperative morbidities. We agree with that concept. The authors proposed that the volumetric curves would be shaped in the form of a U, with the limbs of the U representing increased morbidities and the optimum fluid administration range located at the bottom of the U.21 The approach in our study was to examine the dose-response relationships to previously reported adverse events to determine the optimum intraoperative fluid administration rates23 and then compare these findings to other lung resection studies that reported their analyses with conventional summation statistics.27Bellamy23 rather than the hypothesized U-curves.21 J-curves are not a new concept, as they have been used in the medical literature to optimize blood pressure therapy31; to examine relationships between alcohol intake and noncardiogenic strokes,33 between plasma low-density lipoprotein concentrations and recurrent coronary events,34 and between pediatric serum lead levels and intellectual development37; and to investigate the role of body mass index on the incidences of cancer, hip fractures, and mortality.41In our study, the associations of the intraoperative crystalloid administration rate (mL/kg/hr) to previously reported adverse events were in the shapes of J-curves (see 23 However, when using a different statistical fitting tool, knotted splines on this same association, a different fit is obtained (42 However, these observations need further investigation.In our study, postoperative hospital length of stay was fitted to intraoperative crystalloid administration rate (We can also examine the range of postoperative hospital length of stay with another statistical tool, the outlier plot (The use of dose-response curves allows investigators to visualize optimal intraoperative crystalloid infusion rates and use these results to develop intraoperative fluid management guidelines for this patient population. This approach provides clinical information that cannot be determined when data are solely presented as summations. This method provides an additional tool to improve our understanding of perioperative medicine.


Unlike sonochemistry insemi-infinite fluids, resonance and so constructive interference ofsound pressure can be established in thin layers. Constructive interferenceenables substantial amplification of sound pressure at solid fluidinterfaces. Fluid properties of sound velocity and attenuation, oscillatorinput frequency, and thin fluid layer thickness couple to establishedresonance in underdamped conditions. In thin layer sonochemistry (TLS),thin layers are established where ultrasonic wavelength and oscillator–interfaceseparation are comparable, about a centimeter in water. Solution ofa one dimensional wave equation identifies explicit relationshipsbetween the system parameters required to establish resonance andconstructive interference in a thin layer.Sonochemistry ina thin fluid layer has advantages of no visiblecavitation, no turbulence, negligible temperature changes (≲1°C), low power transducers, and transmissibility (sound pressureamplification) of ≳10 Ultrasound is exploited to impact chemicalreactions and interfacial rates in syntheses of materials and nanomaterials,electrocatalysis, electrosynthesis, kinetics, analysis, deposition,and plating.23Sonochemistryis typically undertaken by irradiating a bulk fluidwith ultrasound. Ultrasound is generated by a transducer at frequenciesabove human hearing, ≳ 20 kHz. In bulk fluids, ultrasound generateslocalized high temperatures (5000 K) and pressures .23 Pressureand temperature transients in the bulk fluid arise from the localizedenergy release that generates cavitation. Cavitation is a chaoticprocess because as the voids collapse, energy is dispersed in indiscriminatedirections. In classical sonochemistry (CS) undertaken in bulk fluids,substantial energy dissipates as heat in the fluid. CS requires highpower transducers because transduction of input sound pressure tothe fluid is inefficient.In bulk fluids, sound pressure irradiation manifests in cavitation.Ultrasound is input from the transducer as a sinusoidal oscillationof sound pressure. The fluid undergoes compression and rarefactionunder the pressure oscillations, see 24 assketched in Sonochemistry in a thin layer of fluid exploitsconstructive interferenceof the sound pressure oscillation,27 The rates of slow interfacial (heterogeneous) electron transferreactions are increased with no impact on mass transport. From voltammetryin an aqueous electrolyte, oxide layers on platinum electrodes areremoved on extended sonication. Notably, during these experiments,where resonance is established,24 no cavitationis observed, perhaps because constructive interference focuses directionalorder across the fluid so that the localized, chaotic void collapsesfound in bulk fluids are avoided. The temperature of the fluid doesnot increase to within ≤1 °C, consistent with efficientenergy transduction. A low power transducer such as a quartz crystaloscillator (QCO) is sufficient to establish constructive interferencein a thin layer. QCOs consume little power and energy, as QCOs aredriven by voltage with little current demand. QCOs are of high efficiency,commonly >90%. It is noted that construction of a TLS cell thateffectivelyexploits construction interference is sensitive to the oscillatorfrequency ω, thickness L, and fluid propertiesrequired to achieve resonance.24 Theseexperiments are undertaken voltammetrically, but the observationsextend to interfacial chemical systems beyond electrochemistry, whereconstructive interference can be exploited in a thin layer to increaseinterfacial reaction rates. The paper provides a theoretical basisfor establishing constructive interference in a thin layer. Modeloutcomes are consistent with the experimental observations.Observed experimentally,where the reflective boundary is an electrode,several novel experimental outcomes are noted.31 Constructive interference hasbeen noted for high power transducers in bulk fluids.32 However, in thin fluid layers where constructive interference canbe exploited, sonochemistry is modeled very little. One paper reportsmodels and experiments for ultrasound in thin layers with cavitationwhere resonance is not established.30 Thesensitivity in construction of a thin layer sonoelectrochemical experimentthat eliminates cavitation is noted.24Quantitative models for various aspects of sonochemistry in bulkfluids have been developed.u is derived for sonochemistryin a thin layer. From the solutions to a wave equation, advantagesof TLS over CS are identified. To establish constructive interference,the system must be underdamped to achieve resonance. Resonance ischaracterized by u, dependent on ω, L, and properties of thefluid and solid.25 The model is derived for conditionswhere no sound pressure is lost into the solid across the solid–fluidinterface. There is a single input frequency ω. The model includessound attenuation in the fluid, which sets maximum sound pressuredelivered to the interface. Amplification at the interface is reportedas transmissibility, the ratio of maximum sound pressure in the fluidto sound pressure input by the transducer. Transmissibility is foundfrom u inthe thin layer. The mathematics establish why a thin layer is requiredto achieve resonance; identify the constraints for the coupling betweenω, L, and fluid properties; provide the naturalfrequencies of the system ωn; andestablish 2ωn must be only slightlyhigher than the attenuation frequency k. Cumulativeenergy input to the interface is discussed. In heterogeneous chemicaland electrochemical systems, TLS advantages over CS include lowerpower transducers (oscillators) for sound input and no obvious cavitationand temperature excursions. The model is consistent with TLS experimentsdemonstrated with an electrode as the reflective interface.27Here, a one-dimensional model of sound pressure x = L propagates asa wave toward the interfacial boundary at x = 0,where the sound pressure wave is reflected off the interface. Thereflected wave is in phase with the incoming sound pressure wave toachieve resonance. Resonances allows constructive interference, sothe amplitude of the wave builds additively over time. The increasedamplitude manifests as increased transmissibility of the sound pressurein the thin fluid layer. Transmissibility measures the amplificationof sound pressure in the fluid enabled by constructive interference.Fluid properties of acoustic velocity and attenuation rate limit themaximum amplitude. Reflectance at the interface is set by the acousticimpedance of the fluid and the interface.26 Acoustic impedance Z is the product of acoustic velocity and density in thephase, Z = ρc.The model system is sketched in x (m) and time is t (s). Oscillator frequency f (s–1) yields radial frequency 2π f = ω (rads–1).Distance in one dimensionis reported as x = L. Sound pressure u (Pa) is input to the fluid at the face of the transducer at x = L with time dependenttransducer output pressure h(t)(Pa). Typically, the transducer outputs a sinusoidally oscillationat frequency f, or equivalently, angular frequencyω. In the ultrasonic range, f ≳ 20 kHz.The transducer frequency and the acoustic velocity of the fluid c (m s–1) set the wavelength λ (m)of the sound wave in the fluid.c = 1482 m s–1 at 20 °C. Wavelength λof 1.00 cm is establishedfor f of 148.2 kHz. For the typical sonochemicalrange of 20 to 100 kHz, λ falls between 7.4 and 1.48 cm, andλ is on the order of centimeters. Medical imaging frequenciesare in the GHz range with wavelengths on the order of micrometers.Energyand power are introduced by the transducer at T measures the amplificationin the fluid as maximum sound pressure generated in the fluid relativeto the sound pressure input at x = L. Where sound is amplified in the fluid, T >1 ischaracteristic of constructive interference.Transmissibility k (rad s–1). Fora natural frequency of the system ωn, amplification is only possible for underdamped systems, where k < 2ωn. The modelfinds ωn and the relationship betweenωn and L. The temporalattenuation rate k is proportional to the spatialattenuation coefficient α (m–1).35 α is well approximated from the fluid propertiesof dynamic viscosity η (Pa s) and density ρ (kg m–3). Kinematic viscosity (m2 s–1) is ηρ–1.–4 Pa s and ρ is 1000 kg m–3 such that α = 1.823 × 10–16 (m–1 s2) f2. At22 kHz, α is 87.2 nm–1 and k is 8.18 × 10–4 rad s–1.Amplification marks resonance and so constructive interference. Amplificationis measured as transmissibility T that is found from u evaluated in themodel.Transmissibilityis limited by the rate sound pressure attenuatesin the fluid, u characterizes the response of the thin layer sonochemical systemthrough the one dimensional wave equation (utt = c2uxx) modified for sound attenuationat rate ut. ut is the first derivative in time. utt and uxx are second derivatives.x < L and t ≥ 0. The systemis specified with two boundary and two temporal conditions. The boundaryat x = 0 is completely reflective, and there is noflux of sound pressure into the phase at x < 0.h(t) at x = L.t = 0, initial conditionsare that sound pressure in the fluid is zero and the time rate ofchange of sound pressure is zero.Propagationof sound pressure x. The surfaces of the transducer and the reflective interfaceshave large, flat cross sections and the surfaces are parallel. Theattenuation coefficient k is a constant, x and time t independent. Fluid propertiesare homogeneous and invariant across 0 < x < L. No heat is dissipated in the fluid. The interface at x = 0 is totally reflective  = sin(ωt)  10.ux = a, where a is the fraction transmitted (lost) into the solid. Wheresound pressure is lost into the solid, the magnitude of the constructiveinterference is diminished. In practical systems, the sound pressuretransmitted into the solid may dissipate as heat. Experimentally,Duda observed small losses of silver from silver electrodes and disintegrationof graphite pencil electrodes on sonication at 20 kHz.27 Fluid temperatures measured with more reflective electrodes areunchanged.The most substantiallimitation of the model is that the interface is 100% reflective.In practice, ultrasound is lost to the solid. Reflectivity of theinterface varies with combinations of solid and fluid, as detailedin ref . For exah(t) suffices to characterize the oscillator,although unanticipated complexities of multiple frequencies may arise.Dependent on parameters that include L, λ,ω, k, and reflectivity, construction of thethin layer cell is somewhat delicate.24In practice, the transducer may output multiple frequenciesatvarying amplitudes, and the faces of the transducer and reflectivesurface may not be strictly parallel. The single sinusoidal input 27Despite the limitations of the model assumptions, the outcomesof the model in underdamped conditions are consistent with the experimentalresults for sonoelectrochemistry undertaken in a thin layer configurationabsent cavitation.The problemis re-expressed ash(t) is specified as a simple sine wave.The input from the oscillator Supporting Information.25Theone-dimensional partial differential equation is solved asdetailed in the v, as developed in v is expressed with variousparameters as the sum of two infinite series with index n.t → ∞. The second term characterizes compounding ofthe sound pressure oscillations over time coupled with damping dueto attenuation. All parameters subscripted with n evolve with the series index n, where n is an integer, n ≥ 0.The solution for the sound pressure isfound in n(x) is thetime-independent, cosine function that describes the standing wavesestablished in the fluid. This arises from the assumption that v is representedas a series that is the product of separable time vn(t) and space-dependentϕn(x) terms, eq SI.2, ϕn is a frequency that relatesthe natural frequency of the thin layer ωn to the temporal attenuation coefficient k. From k < 2ωn, so amplification requires βn >0. Constructive interference is best established as βn → 0. At βn = 0, the system is critically damped and constructive interferenceis not established. For a given fluid, βn is fixed.βAn characterizes theamplitude of the cosine term of h(0) and rn, where rn is defined in eq SI.13.eq SI.15An = −2h(0)rnL–3. An is independent of L.Bn characterizestheamplitude of the sine term, dependent on rn, the initial oscillation at the oscillator face h(0) and the rate of change (the frequency) of the oscillationat the oscillator face h′(0). h(t). Equation SI.17 definesβnBn = −rnL–3[2h′(0)+ kh(0)]. Bn is also independent of L. As βn decreases, Bn increases.Rn(t) characterizes the compoundingof the sound energy by constructiveinterference, with acknowledged limits due to attenuation. This isdetailed in the SI, sections SI.1.4 and SI.1.5. Because the integrals for Rn(t) do not yield to immediate solution, Rn(t) is evaluatednumerically.T.The model was evaluated numerically using Pythonand Julia. COMSOLis an alternative and would be needed for more complex geometriesand conditions. The approach through solution of PDEs does provideseveral explicit numerical relationships between system parametersthat maximize x = L. Transmissibility is shown as a function of ωt in k ≤ 0.10 rad s–1. In common solvents, k ≲ 0.01 rad s–1 for T of the order of 106.24Transmissibility is evaluatedfrom the maximum sound pressure andthe sound pressure input at Each oscillation builds energy at the interface.Kinetic ratesof interfacial reactions are increased by energy deposited at theinterface by high frequency sound pressure oscillations.u. When constructive interference isestablished, the systemis in resonance and T > 1. Transmissibility isamplification,the ratio of the maximum sound pressure achieved in the fluid to soundpressure input by the oscillator. Several observations about TLS aredrawn from the solution for u in u and T and how TLSwithout cavitation compares to CS in practical terms.The model yields Severalinferences about sonochemistry in thin layers and undersemi-infinite conditions are drawn for the solution for sound pressure.The solution is discussed. Distinctions between TLS and CS are presented.x) identifies distinctions in CS and TLS. The general form of the solution for eq SI.4 is equivalently expressed as x is restricted to a range0 to L, so ϕn(x) is bounded and soundpressure waves are limited in amplitude. Under appropriate conditionsfor λ, the system comes into resonance and sound pressure isamplified  = 0, and resonance can not be established.In part, the form of ϕ(mplified 4 by consmplified 2. In then = 6 in L/λ = 0 propagates into thefluid. If the solid reflective interface is at L/λ= (2n + 1)/4, sound pressure is reflected at theinterface straight back toward the oscillator and the incoming andreflected waves add to generate constructive interference across thethin layer. The waves add as the condition ux(L/λ = (2n + 1)/4) = 0 and the reflected sound pressure wave overlays the incomingwave. Where the interface is ill-positioned at L/λ= (2n)/4, sound pressure is negligible and ux < 0. The little soundpressure reflected at the interface near L/λof n/2 is of the opposite sign of the incoming wave,and the incoming and reflected waves cancel to yield destructive interference.For the semi-infinite systems common in CS, where k is underdamped, there is no reflective boundary to generate constructiveinterference and no amplification. In real systems, the sound pressurewould dissipate at distances sufficiently far from the oscillatorbecause underdamped conditions k < 2ωn are only achieved as n →∞, as L → ∞.The model identifies constructive interference is established forunderdamped conditions.27 Moussatov, Granger, and Dubus30 developeda two-dimensional model for a thin fluid layer at a cylindrical horntransducer with cavitation, where L) “much smaller”than the acoustic wavelength (2πc/ω).Both linear components along the principle axis and radial componentsabout the horn tip contribute to amplification.Within the one-dimensionalmodel developed here, fluid properties are uniform and input soundpressure is sinusoidal of low intensity. No cavitation is found eitherfor this model in underdamped conditions or for TLS experiments whereresonance and constructive interference are established.L/λ = 0.05 mark L much smaller than λ, where u >0 and 0 < ux < 1. If a reflectiveboundary were established at L/λ = 0.05, somefraction of the reflected wave would add to the incoming wave, but ux ≠ 0 conveys that soundpressure also travels beyond L/λ = 0.05. Inreal systems, these conditions are not sufficient to establish cavitationor identify constructive interference as a major component of amplificationin thin layers with cavitation. However, amplification is clearlydemonstrated in experiments and models for thin layer systems with30 and without27 cavitation.It is not clearwhether constructive interference is critical to amplification ina thin layer with cavitation. Considered in the simple one-dimensionalframework in kt/2] by βn is captured in n(x) and An are independent of k and βn. But, Bn increases as βn decreases, where Bn ∝ βn–1. The coefficient of the sine term under the first summation on theright-hand side is proportional to βn–1 exp[−kt/2]. From section SI.1.4, Rn(t) convolutesexp[−kt/2] and βn, but the general pattern remains as a ratio βn–1 exp[−kt/2]. The ratio βn–1 exp[−kt/2] characterizes both summations.If time-independent βn does notapproach zero, then exp[−kt/2] will tend todamp u as t increases. Thus, to achieve resonance and amplification,βn → 0 is needed. One conditionto achieve resonance is an underdamped system where 2ωn > k, consistent with βn → 0.The counterbalance ofthe attenuation damping exp. For the second series for βn = 0, Rn(t) scales as βn–1 (Rn(t) increases without bound, where βn = 0 and v tends to infinity. The critically damped conditionswhere βn = 0 do not lead to theconstructive interference absent cavitation in the thin layer. Whethercritically damped conditions lead to cavitation is not determinedfrom the model. as βn–1 A.18. Rn in a thin layeris constrained by the two summations. u varies across the thin layer with h(t), the sinusoidal input from the oscillator at x = L. But, the impact of h(t) on u diminishes parabolically as x approacheszero. At the interface (x = 0), v = u. On input at x = L, h(t) induces sound wave oscillations in the fluid.The induced oscillations establish sound pressure at the interface(x = 0). Where resonance is achieved, T > 1 and sound pressure is increased at x = 0.From n to the distance between the oscillator andthe interface L and the sound velocity in the fluid.Multiple roots are provided by n where all valuesof n provide valid roots. In practice, large n may not be practicable. Resonance is achieved where L → ∞, ωn approaches zero for finite n.The principle outcome for solution of cω–1. To achieve constructive interferencein a thin layer, conditions require λ = 4L(2n + 1)−1  –1 will provide resonance. Given practical limitation on n, λL–1 falls between 4 andabout 4/7 to 4/9.The wavelength of ultrasound, λ = 2πn + 1)−1 13. The wn approaching zero,greatest resonance is reached where at least one experimentally accessibleωn is slightly greater than k/2. From n slightly greater than 3πρc2η–1 is needed.To satisfy βk on T is shownin k = 0 and the T grows without bounds. As k increases, T comes to steady state, where maximum T decreasesas k increases. The amplification of sound pressureis substantial at about 50-fold for k = 0.03 andabout 20-fold for k = 0.10. Values of k for common solvents are ≲0.01 rad s–1,with T ≳ 2 × 105. Where k > 0, the system approaches steady state by ωt of 100. Steady state is achieved rapidly as ω ishigh. For ω ≳20 kHz, time to steady state is less than5 ms. At steady state, each oscillation builds energy at the interface.At high frequencies, the accumulated energy can impact interfacialrates.The impact of kwater = 8.18 × 10–4 rad s–1 and Twater = 3.65 × 106. From the model, maximumtransmissibility and temporal sound pressure attenuation are inverselyproportional, Tk = kwaterTwater = 2986 rad s–1. For a given solvent, Tk is constant,24 and transmissibility is maximized where solvent-specific k is minimized. In common solvents, T ison the order of 106 with constructive interference andno cavitation.The amplification is dependent on the fluid properties.For waterat 25 °C, L = (2n + 1)λ/4 λ/4 13. For arference5. For n L, an oscillator at x = L inputs a sinusoidal signal h(t) that transmits across the layer toinduce oscillations. Resonance is established when sound pressure u reflected at theinterface (x = 0) adds constructively across thecell. Resonance and amplification require parameters of ωn, L, k,and c meet conditions specified by the model in h(t). The objective is tomaximize amplification, T > 1.Sonochemistryin a thin layer establishes constructive interference when the thinlayer system is in resonance. Without resonance, there is no amplification.In a thin layer of thickness Z2 – Z1)(Z2 + Z1)−1. For example, reflectance at the waterplatinum interface is high 95% but lower for the water graphite interface,38.5%. It is noted that construction of TLS systems is more delicatethan construction of CS systems.24Practicalconstraintsof how to construct a successful thin layer cell are presented inref . This inx = L and the interface at x = 0, the condition ux =0 sets the reflection of sound pressure at the interface that is neededto establish resonance. In CS, where effectively L is infinitely large, the boundary condition ux cannot be set. Attenuationwould damp oscillations across the fluid and conditions of 2ωn slightly greater than k and ωn = c(2n + 1)π(2L)−1 can not be simultaneously satisfiedas L → ∞. Commonly in CS, resonanceis not established in the bulk fluid and k dampsinput sound pressure over distance.Resonance can be establishedin a thin fluid layer but not in a semi-infinite homogeneous fluid.Amplification by constructive interference can be achieved in TLS.For TLS with the oscillator at n → 0. In critically damped systems, βn = 0 and constructive interference is lost.Whether critical damping leads to cavitation can not be determinedfrom the current model.In underdamped systems,constructive interference is established where βh(t) = sin(ωt), but higher amplitude generated by higher intensity oscillatorsare anticipated to further enhance amplification in the system providedthe constraints on ωn, L, k, and c are met. Increased impactsof oscillator intensity at electrodes in TL electrochemistry experimentshave been observed.27 Because the input energy is lowerin TLS, the hardware is simpler and electrical energy consumptionand cost are lower. The waste heat in a TLS is negligible as celltemperature changes ≤1 °C over extended time of sonication.27 No cavitation is observed.Resonance established in TLS requiresa lower energy oscillator than used in CS . The model is developedfor input 26Resonance is maximized at steadystate. Because ω is high,TLS systems rise to steady state rapidly. This is observed experimentally.x = 0) is power. For resonance at ωn, energy is delivered with each oscillation.Integration of the powerover time yields the energy delivered to the interface. For an oscillatorsuch as a QCO, the energy delivered is about 1 kJ mol–1 s–1. For a 20 kHz transducer, ≈0.05 J mol–1 reaches the interface with each oscillation. Sonicationover longer time increases energy at the interface. Evidence of accumulatedenergy is found for removal of oxide from platinum in water over about6–10 min.26The rate pressure is delivered to theinterface  =0 is replaced, which would perhaps require strictly numeric solutionas with COMSOL.Within the contextof the model, an important caveat is that theinterface is perfectly reflective and no energy is lost to the solidat the interface. The model accounts fluid properties but assumesa reflective interface with no flux of sound pressure across interfaceinto solid at By appropriate configuration to establish resonance,it may bepossible to control the energy at the interface to overcome the activationenergy of interfacial reactions. Energy input over time accumulateswith oscillations, provided the appropriate conditions are met toestablish constructive interference in a thin fluid layer.Why sonochemistry in a thin layer? Constructiveinterference amplifiessound pressure.k and c, properties of the oscillator ω, andseparationdistance L between the oscillator at x = L and the perfectly reflective solid fluid interfaceat x = 0. Constructive interference is not establishedunless the system is in resonance. At resonance, natural frequenciesωn are established by L, c, and ω −1. Fromthe model equations, constructive interference can not be establishedin bulk fluids of semi-infinite extent. Constructive interferenceis established in thin fluid layers for underdamped conditions whereultrasonic wavelength λ and L are comparable,4 ≤ λ/L ≲ 0.5. In criticallydamped systems, (βn = 0), constructiveinterference is not established.The model of the wave eq (25 is vetted against experimentsin TLS, where resonance and constructive interference are established,27 and there is no visible cavitation and no turbulence. Temperaturechanges ≲1 °C mark efficient energy transfer. Low poweroscillators, such as QCO, suffice to establish resonance in TLS systems.Although more delicate to set up than classical sonochemistry in thebulk,24 the advantages of substantial amplificationwithout cavitation and low energy transducers in TLS are substantial.The model


Herein, a comprehensive overview is provided of the recent preclinical experiences with radiolabelled imaging probes targeting angiogenesis.Angiogenesis plays a crucial role in tumour progression and metastatic spread; therefore, the development of specific vectors targeting angiogenesis has attracted the attention of several researchers. Since angiogenesis-associated aminopeptidase N (APN/CD13) is highly expressed on the surface of activated endothelial cells of new blood vessels and a wide range of tumour cells, it holds great promise for imaging and therapy in the field of cancer medicine. The selective binding capability of asparagine-glycine-arginine (NGR) motif containing molecules to APN/CD13 makes radiolabelled NGR peptides promising radiopharmaceuticals for the non-invasive, real-time imaging of APN/CD13 overexpressing malignancies at the molecular level. Preclinical small animal model systems are major keystones for the evaluation of the in vivo imaging behaviour of radiolabelled NGR derivatives. Based on existing literature data, several positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radioisotopes have been applied so far for the labelling of tumour vasculature homing NGR sequences such as Gallium-68 ( Comparable biodistribution patterns were observed in another study by Shao and co-workers, where the in vivo and in vitro comparative characterisation of NGR ([68Ga]Ga-NOTA-G3-NGR2) and RGD-based ([68Ga]Ga-NOTA-G3-RGD2) radiopeptides was carried out in HT1080 tumour xenografts ) was investigated by Máté and co-workers [68Ga]Ga-NOTA-c(NGR) (NOTA-c(NGR)/c[Lys(NOTA)-Asn-Gly-Arg-Glu]-NH2) in experimental rat model systems. The synthesis procedure of the newly constructed [68Ga]Ga-labelled c[KNGRE]-NH2 cyclic peptide linked to the chelator p-SCN-Bn-NOTA ([68Ga]Ga-NOTA-c(NGR)) was also detailed. Cyclic peptides are reported to be more resistant to proteolytic activity (exo- and endoproteases) as well as chemical degradation compared to linear ones Ga-NOTA-c(NGR) Ga-labelled APN/CD13 targeting vectors: [68Ga]Ga-NODAGA-c(NGR), [68Ga]Ga-NODAGA-c(NGR) (MG1), [68Ga]Ga-NODAGA-c(NGR) (MG2), [68Ga]Ga-NODAGA-YEVGHRC, [68Ga]Ga-NOTA-G3-NGR, and [68Ga]Ga-NOTA-G3-NGR2 -Asn-Gly-Arg-Cys]-NH2 and c[CH2-CO-Lys(NODAGA)-Asn-N(Me)Gly-Arg-Cys]-NH2; respectively . The LogP values of the listed radiopharmaceuticals are displayed in The in vitro experiments revealed the hydrophilic nature of [A-c(NGR) . Confirm68Ga]Ga-NOTA-c(NGR). Further, the imaging properties of the NGR derivative were compared with those of the RGD (arginine-glycine-aspartate) ligand containing radiotracer ([68Ga]Ga-NODAGA-c(RGD)]2) targeting ανβ3 integrin, which is another pivotal biomarker of angiogenesis. The in vivo imaging behaviour and the ex vivo organ distribution were also studied in healthy control Fischer-344 rats. For the heterotopic experimental model, the tumour cells were subcutaneously inoculated into the left thighs of the rats, while for the orthotopic tumour generation, SRCA-based  tumour cell transplantation was performed [68Ga]Ga-NOTA-c(NGR) or [68Ga]Ga-NODAGA-[c(RGD)]2 into the lateral tail vein of both normal control and tumour-bearing rats. Ex vivo data were displayed from the radioactivity measurements of cardiac blood samples and tissue extracts from the liver, spleen, kidneys, intestine, heart, stomach, muscle, lung, and tumour mass. To further verify the APN/CD13 affinity of [68Ga]Ga-NOTA-c(NGR), a non-radioactive blocking peptide compound (200 µg of NOTA-c(NGR)) was employed before the application of the radiolabelled match. Thereafter, in vivo and ex vivo uptake inhibition studies were conducted.Adult male Fischer-344 rats bearing APN/CD13 positive orthotopic and heterotopic transplanted mesoblastic nephroma (Ne/De) tumours were used for the accomplishment of in vivo miniPET and ex vivo biodistribution studies with [erformed . The APN68Ga]Ga-NOTA-c(NGR) accumulation was recorded in the investigated abdominal organs, including the liver, the spleen, and the intestines, whereas the kidneys and the urine were depicted with prominent radiotracer accretion. In accordance with the results of Máté et al., Zhang et al.—68Ga]Ga-DOTA-NGRinvestigating the biodistribution pattern of Ga-NOTA-G3-NGR and [68Ga]Ga-NOTA-G3-NGR2 conducted by Shao et al. [68Ga]Ga-NOTA-G3-NGR2 uptake,—unlike the results of Máté et al.—no statistically significant difference could be noted between the in vivo organ distribution characteristics of the NGR and the RGD derivatives Ga-labelled c(NGR) peptides such as [68Ga]Ga-NODAGA-c(NGR), [68Ga]Ga-NODAGA-c(NGR) (MG1), [68Ga]Ga-NODAGA-c(NGR) (MG2), and [68Ga]Ga-NODAGA-YEVGHRC also found renal ways of clearance Ga-NOTA-c(NGR) was further verified by in vivo blocking experiments that showed that the quantitative uptake figures of the tumours significantly diminished after the application of the blocking molecule (T/M SUVmean blocked: 2.50 ± 0.39 vs. unblocked: 12.25 ± 3.08 and T/M SUVmax blocked: 2.48 ± 1.14 vs. unblocked 16.28 ± 2.48).The tumour targeting potential of [68Ga]Ga-NOTA-c(NGR) were also authenticated in the SRCA tumour model by the in vivo PET distribution examinations and the in vivo blocking studies, respectively. Consistent with the findings of Máté et al., the diagnostic feasibility of [68Ga]Ga-NOTA-c(NGR) was also confirmed by Szabó and colleagues in SRCA-induced Ne/De tumours [max and the T/M SUVmax values of [68Ga]Ga-NOTA-c(NGR)  and [68Ga]Ga-NODAGA-[c(RGD)]2 . On the contrary, no remarkable difference was shown between the T/M ratios of the NOTA-linked [68Ga]Ga-labelled dimeric Gly3-CNGRC cyclic peptide ([68Ga]Ga-NOTA-G3-NGR2) and [68Ga]Ga-NOTA-G3-RGD2 in the study of Shao et al. applying female BALB/c mice bearing HT1080 tumours with simultaneous expression of APN/CD13 and ανβ3/ανβ5 integrin [The diagnostic ability and the APN/CD13 binding selectivity of [ tumours . Compariintegrin .p ≤ 0.01) after the injection of the unlabelled compound (T/M SUVmean blocked: 1.71 ± 0.24 vs. unblocked: approx. 9 and T/M SUVmax blocked: 1.96 ± 0.56 vs. unblocked: 12.47 ± 2.19) that authenticates the APN/CD13 adhering capacity of the tracer. These blocking results—similarly to prior literature findings on other [68Ga]Ga-labelled NGR-based molecular probes—suggest that these diagnostic agents specifically target the APN/CD13 receptors Ga-NOTA-c(NGR) in the recent experiment of Szabó et al. Ga-labelled tracer.Similarly to the previous findings, the successful prohibition of tracer uptake proved the APN/CD13 targeting specificity of the [68Ga]Ga-NOTA-c(NGR) accumulation was noted in the Ne/De tumours in comparison with the uptake of [68Ga]Ga-NODAGA-c(RGD)]2. Given the difference between the T/M values of the RGD and NGR-based probes, we may state that the angiogenic targeting ability of [68Ga]Ga-NOTA-c(NGR) surpasses that of the RGD compound. The above-detailed results of Máté and colleagues further strengthen the idea that the [68Ga]Ga-labelled NGR-motif containing diagnostic vectors could be potential weapons not only in the timely identification of primary tumours with high APN/CD13 density but also in related metastatic diseases. Moreover, the negligible background activity along with the prominent uptake of the tumours ensure the acquisition of high-contrast images, which is of critical importance regarding PET scan reporting. An overview of the study by Máté et al. is given in Both the primary and metastatic tumours exhibited considerable NGR tracer uptake, and more enhanced [68Ga]Ga-NOTA-c(NGR), Kis and co-workers dealt with the in vivo assessment of the temporal changes of the APN/CD13 pattern in rat hepatocellular carcinoma (He/De) [68Ga]Ga-NOTA-c(NGR) with ανβ3 integrin-affine 68Ga-NODAGA-[c(RGD)]2 was also carried out. For the accomplishment of their study, rat hepatocellular (He/De) carcinoma models Ga-NOTA-c(NGR) uptake of the primary experimental hepatic (He/De) tumours was more elevated relative to the accumulation of [68Ga]Ga-NODAGA-[c(RGD)]2 [68Ga]Ga-NOTA-c(NGR) in relation to tumour growth, Kis et al. confirmed the relationship between the size extension of angiogenic tumour regions and the enlargement of the whole tumour mass. Furthermore, based on the meaningful connection between the accumulation of hypoxia-specific [68Ga]Ga-DOTA-nitroimidazol and [68Ga]Ga-NOTA-c(NGR), Kis and colleagues stated that the extent of hypoxia is proportional to tumour progression and angiogenic processes. Contrarily—evaluating the expression profile of αvβ3 integrin, endoglin, and vascular endothelial growth factor receptor 2 (VEGFR2) in subcutaneous breast, ovarian, and pancreatic cancer xenografts—Deshpande et al. published a reverse correlation between tumour size and the presence of angiogenic biomarkers [68Ga]Ga-NOTA-c(NGR) as an imaging probe in the diagnostic armamentarium of angiogenesis imaging. Moreover, [68Ga]Ga-NOTA-c(NGR)-based tracking of the change in the appearance of APN/CD13 over time may be the basis for efficient anti-tumour therapeutic planning. A summary of this study is presented in Also using [ (He/De) . In addia models  were estc(RGD)]2 . Strengtc(RGD)]2 . Unlike c(RGD)]2 . Thus, tomarkers . The difomarkers . These r68Ga]Ga-labelled NGR peptide compounds was compared using chemically induced syngeneic He/De and Ne/De preclinical rat tumour models: [68Ga]Ga-NOTA-c(NGR), [68Ga]Ga-NODAGA-c(NGR), [68Ga]Ga-NODAGA-c(NGR) (MG1) or [68Ga]Ga-NODAGA-c(NGR) (MG2) -Asn-Gly-Arg-Cys]-NH2 and c[CH2-CO-Lys(NODAGA)-Asn-N(Me)Gly-Arg-Cys]-NH2; respectively. For this purpose, in vivo PET/MRI imaging and ex vivo organ distribution assessment took place in 16 weeks old male Fischer rats bearing He/De Ga-DOTA-NGR, [68Ga]Ga-NOTA-NGR, and [68Ga]Ga-NOTA-c(NGR), the [68Ga]Ga-labelled c(NGR) derivatives showed increased accumulation in the kidneys, whereas discrete radiotracer concentration was found in the abdominal and thoracic organs [68Ga]Ga-NODAGA-NGR [68Ga]Ga-NOTA-c(NGR) in He/De tumour-bearing rats [68Ga]Ga-NODAGA-c(NGR) in mice bearing HT1080 and B16F10 tumours Ga-labelled iNGR and NGR  peptide—Possessing a C-terminal arginine or lysine with a free carboxyl group, the CendR motif (R/KXXR/K) enhances the circulation of the peptide and regulates its movement across biological membranes through its engagement with neuropilin-1 (NRP-1) ,65,66. T68Ga]Ga-DOTA-iNGR and [68Ga]Ga-DOTA-NGR [Zhao et al. applied DOTA for the conjugation of the investigated NGR derivatives. CD13 expressing HT1080 and receptor-negative HT29 mouse xenograft models were established for the in vitro and in vivo imaging characterisation of [DOTA-NGR .The competitive cell binding affinity study revealed no effects of the CendR motif either on the structure or the APN/CD13 selectivity of the NGR peptide. Higher iNGR uptake of the HT1080 cells—experienced at every investigated time point of the cell binding assay where the mechanism of NRP-1 could be expressed—indicates the better tumour-homing ability of the internalising peptide compared to the NGR counterpart.68Ga]Ga-iNGR: 3.41 ± 0.28, 2.97 ± 0.30, and 2.64 ± 0.31%ID/g at 0.5, 1, and 1.5 h post-injection; respectively, [68Ga]Ga-NGR: 2.68 ± 0.35, 1.91 ± 0.32, and 1.45 ± 0.30 %ID/g at 0.5, 1, and 1.5 h post-injection; respectively). The enhanced in vivo (1.6 times higher 1 h after injection) and ex vivo (1.9 times higher) tumour uptake of the iNGR derivative is consistent with previous research, where the two-fold higher cellular uptake ratio of the dual-modified liposomes was due to attachment to NRP-1 Ga-labelled DOTA-NGR was largely comparable to that of [68Ga]Ga-NOTA-c(NGR) [68Ga]Ga-DOTA-NGR occurs mostly through the kidneys [68Ga]Ga-NOTA-c(NGR)/[68Ga]Ga-NODAGA-c(NGR))([ to DOTA 68Ga]Ga-DOTA-NGR) or Technetium-99m (99mTc)—[68Ga]Ga-DOTA-NGR presented improved biodistribution and pharmacokinetical profiles [68Ga]Ga-labelled NGR derivatives serve as promising agents in the detection of APN/CD13 overexpressing tumours [68Ga]Ga-DOTA-NGR compared to [64Cu]Cu-DOTA-NGR into consideration, the radiolabelling of the NGR motif containing peptides with 68Ga might be superior to labelling with 64Cu. This research by Zhang and co-workers is detailed in 68Ga and 64Cu.Similarly to Zhao et al., Zhang and co-workers also used DOTA for the chelation of the NGR peptide . AlthougA-c(NGR) . Similar kidneys ,38,39,40 kidneys ,36,37. HAPN/CD13 . In addiprofiles . Overall tumours . Taking 68Ga]Ga-labelled c(NGR) peptide and linear LN peptide YEVGHRC -based radiotracers was studied at the preclinical level , 12 weeks old male C57BL/6 mice bearing B16F10 melanoma tumours in their left shoulder area were applied. The APN/CD13 positivity of the B16F10 tumour cell lines is well-established [68Ga]Ga-NOTA-c(NGR) or [68Ga]Ga-NODAGA-YEVGHRC and were subjected to a 20 min long static PET examination. Representative decay-corrected coronal PET images of healthy control mice are displayed in 68Ga]Ga-NODAGA-YEVGHRC and [68Ga]Ga-NOTA-c(NGR), the radioactivity of the blood and urine samples as well as different organs and tissues of interest was measured. According to the urinary route of excretion, PET data of the control small animals showed increased renal tracer uptake 90 min after the injection of both assessed probes (as presented in 68Ga]Ga-labelled c(NGR) derivatives: [68Ga]Ga-NOTA-c(NGR), [68Ga]Ga-NODAGA-c(NGR), [68Ga]Ga-NODAGA-c(NGR) (MG1), and [68Ga]Ga-NODAGA-c(NGR) (MG2) [68Ga]Ga-NOTA-c(NGR) and [68Ga]Ga-NODAGA-YEVGHRC in any of the selected organs and tissues (p ≤ 0.05). Although the subcutaneously growing tumours could be clearly delineated from the surrounding tissues with both angiogenesis-directed probes, the c(NGR) containing tracer showed higher tumour uptake ([68Ga]Ga-NOTA-c(NGR) SUVmean: 0.12 ± 0.02) than the YEVGHRC compound ([68Ga]Ga-NODAGA-YEVGHRC SUVmean: 0.03 ± 0.01). Therefore, based on these uptake values, the tumour specificity of the labelled YEVGHRC derivative was not appropriate. Structural modifications or the addition of targeting sequences to the molecule may improve its imaging properties. Conversely, Jia et al. strengthened the APN/CD13 tumour-homing ability of YEVGHRC peptide-functionalised liposomes [68Ga]Ga-NOTA-c(NGR) accumulation (%ID/g: 1.60 ± 0.30) in the tumourous alterations relative to [68Ga]Ga-NODAGA-YEVGHRC (%ID/g: 0.13 ± 0.06). Studying the target specificity of various [68Ga]Ga-labelled NGR-based imaging molecules in hepatocellular carcinoma (He/De), mesoblastic nephroma (Ne/De), and melanoma tumours, Kis et al., Máté et al., and Satpati et al. reported comparable findings to those of Dénes et al. [68Ga]Ga-labelled cyclic NGR (cyclic(lysyl-asparaginyl-glycyl-argininyl-glutamic acid amide)/c(KNGRE)-NH2) was found to present the foremost tumour-targeting affinity. Although the optimisation of the pharmacokinetics of YEVGHRC tracers is part of future work, the pioneering observations of Dénes et al. may initiate further investigations on more resistant forms of targeting vectors.With the aim of exploring the in vivo angiogenesis-targeting capability of the newly constructed APN/CD13 affine Ga-labelled NGR-based radiopharmaceuticals, Israel et al. intended to evaluate the effect of NGR dimerisation on the imaging characteristics of the radiolabelled APN/CD13 targeting compounds [68Ga]Ga-NODAGA-NGR (NGR monomer) vs. [68Ga]Ga-NOTA-(NGR)2 (NGR dimer)) under in vitro and in vivo circumstances Ga-NODAGA-NGR and [68Ga]Ga-NOTA-(NGR)2 for 15, 30, or 60 min, a gamma-counter was used for the determination of the cellular radiotracer accumulation. Although increased radiopharmaceutical accretion was recorded in all three cancer cell lines investigated with the NGR dimer in comparison with its monomeric counterpart, both probes demonstrated low in vitro uptake (less than 1%).The cellular uptake of the two investigated imaging probes was evaluated in vitro. Following the incubation of each cell line with [CD1-Foxn1nu-mice bearing A549 or SKHep-1 tumours in their front flank to study the in vivo diagnostic efficacy of [68Ga]Ga-NODAGA-NGR and [68Ga]Ga-NOTA-(NGR)2. Since the best tumour-to-background ratios could be attained 50 min after tracer injection, the microPET examinations took place 50 min post-administration of 7.8 ± 3.5 or 7.4 ± 3.4 MBq [68Ga]Ga]NODAGA-NGR or [68Ga]Ga-NOTA-(NGR)2, respectively, at an average tumour diameter of ≥0.5 cm. In the case of both assessed imaging probes, 10 A549 and 16 SKHep-1 tumourous mice were injected. Tumour-to-liver, tumour-to-lung, and T/M ratios were registered to ensure quantitative parameters for appropriate lesion detection. Although minor differences could be observed between the in vivo characteristics of the monomer and the dimer NGR, these were statistically not significant. Both APN/CD13 expressing tumour types could be definitely identified with both NGR-based tracers. Upon visual assessment, Israel et al. observed that the monomer NGR derivative seemed to accumulate more in the selected tissues and organs compared to its dimer sister. Moderately higher T/M ratios of the A549  and SKHep-1  tumours were observed in the case of the NGR dimer relative to the monomeric match; however, this was statistically not meaningful. Regardless of the fact that the monomer probe tended to present more elevated uptake in both tumours compared to the dimer, based on its higher concentration in the muscle tissue, Israel et al. concluded that improved T/M ratios could be attained using the dimer NGR instead of the monomer one. Tumour-to-liver ratios were depicted to be lower for the NGR dimer than for the NGR monomer in both the A549 and the SKHep-1 tumours. Identically to the T/M ratios, an increased tumour-to-lung ratio was registered for [68Ga]Ga-NOTA-(NGR)2 (5.1 ± 1.7) than for [68Ga]Ga-NODAGA-NGR (4.1 ± 1.0). Besides microPET imaging, the in vivo binding affinities of the radiopharmaceuticals were further evaluated with the administration of the APN/CD13 inhibitor bestatin in the A549 lung tumour-bearing mice. The co-injection of bestatin managed to induce tumour tracer uptake inhibition; however, it was not significant.Ten-minute-long microPET imaging studies were performed in eight-week-old 68Ga]Ga-NODAGA-NGR or [68Ga]Ga-NOTA-(NGR)2; respectively. After the tracer injection, blood samples were gathered every 10 min. Sixty minutes post-injection, various organs were harvested, and these organs and the blood samples were measured for radioactivity using a gamma counter. Based on the ex vivo data, both tracers demonstrated prompt, largely identical clearance from the blood stream. The elimination of the imaging probes occurred mostly through the urinary tract. In line with the in vivo results, the dimer [68Ga]Ga-NOTA-(NGR)2 showed lower tracer uptake in all investigated organs—apart from the hepatic and the lienal activity—in comparison with the monomer [68Ga]Ga-NODAGA-NGR. In accordance with the diminished tumour-to-liver ratio of the dimer [68Ga]Ga-NOTA-(NGR)2, the ex vivo data showed higher hepatic tracer accretion in the case of the dimeric compound, which could be explained by its more prominent hepatobiliary excretion or more lengthened retention in the liver tissue compared to the monomeric derivative.In a bid to investigate the radiotracer uptake of different organs and tissues, an ex vivo organ distribution pattern was defined. Healthy control mice were intravenously administered with 6.7 ± 1.1 MBq or 7.6 ± 0.7 MBq of Ga-labelled dimeric cNGR ([68Ga]Ga-DOTA-c(NGR)2) using ES2 and SKOV3 ovarian tumour cell lines and corresponding mouse xenografts (seen in 68Ga]Ga-DOTA-c(NGR)2 accumulation was observed in the ES2 cells than in the SKOV3 cells, which confirmed the receptor selectivity and affinity of the probe. Analysing the microPET scans of nude mice carrying ovarian cancer, [68Ga]Ga-DOTA-c(NGR)2 exhibited significant accumulation in APN/CD13 overexpressing ES2 tumours . In line with this result, the same research group had similar findings when the receptor targeting ability of [68Ga]Ga-DOTA-NGR was evaluated in intensively receptor positive pulmonary malignancies [99mTc]Tc-labelled c(NGRyk) peptide ([99mTc]Tc-MAG3-PEG8-c(NGRyk)) accumulation was found in vivo in human ovarian adenocarcinoma (OVCAR-3)-bearing nude mice by Faintuch et al. . Ac. Ac68Ga]gnancies . Furtherh et al. . Moreoveimaging) . Upon MRications ,80.64Cu) can be a promising alternative to [68Ga]Ga-labelling. Chen et al. reported the feasibility of [64Cu]Cu-labelled monomeric and dimeric NGR compounds in the non-invasive identification of APN/CD13 receptors at the preclinical level  [64Cu]Cu-NGR1 and [64Cu]Cu-NGR2 in the major organs with high hepatic and renal tracer uptake 1-, 2-, and 4-h post-injection and—apart from the intestines—slight tracer accumulation in the rest of the organs at all time points. Contrarily, in the study of Israel et al., the investigated tissues and organs exhibited higher uptake of the [68Ga]Ga-NGR monomer than the [68Ga]Ga-NGR dimer Cu-labelled dimeric NGR peptide using sarcophagine cage (Sar) as a macrocyclic chelating agent [64Cu are stable enough to avoid the dissociation of the radiometal [64Cu2+ within an extensive range of pH values [64Cu]Cu-Sar-NGR2 was tested both in vitro and in vivo. The comparable receptor binding affinity of [64Cu]Cu-Sar-NGR2 (1.04 ± 0.26 nM) and the earlier synthesized dimeric NGR peptide indicates the augmentation of APN/CD13 receptor adhering capability induced by peptide multimerization Cu-Sar-NGR2 uptake (0.4 ± 0.03% 2 h after incubation). These in vitro findings further strengthen the specificity of the NGR homing sequence. Similarly to the in vivo results of Chen et al., the receptor positive HT1080 tumours were visualised with high contrast in comparison with the background; however, the APN/CD13 negative MCF-7 tumours showed only slight tracer accumulation in the bilateral tumour-bearing female athymic nude mice (HT1080 and MCF-7) [64Cu]Cu-DOTA-NGR1 and [64Cu]Cu-DOTA-NGR2 [64Cu]Cu-Sar-NGR2 uptake was registered at the early time points in the liver and in the kidneys, and—except for the intestines—minimal radioactivity was experienced in the other evaluated organs. High hepatic radioactivity may be underpinned by the expression of the APN/CD13 biomarker in the mouse liver as well as the prolonged hepatic retention of the radioisotope.As expected, significant tracer accretion was observed in HT1080 human fibrosarcoma cells with APN/CD13 overexpression (1.72 ± 0.24% 2 h post-incubation), while CD13-negative MCF-7 human breast adenocarcinoma cells did not exhibit meaningful Cu-Sar-NGR2 activity was noted ex vivo in the liver and in the kidneys; however, in the rest of the organs, the accumulation remained at a negligible level, and this is consistent with the organ distribution analysis of Chen et al. Ga and [64Cu]Cu-labelled NGR probes may stem from the disparities between the two radiometals and the biochemical structure of the assessed NGR sequences Cu-DOTA-NGR2 over [64Cu]Cu-DOTA-NGR1 into account, the construction of dimer peptide-based radiopharmaceuticals other than NGR is warranted.Although erations . Alternaerations ,86,87,88equences . Table 399mTc-pertecnetate (99mTc) is also widely applicable for the radiolabelling of NGR peptides [99mTc]Tc-labelled monomeric and dimeric NGR derivatives was investigated in vivo in receptor positive HepG2 hepatoma-bearing female BALB/c nude mice using microSPECT Tc-NGR2) compared to the monomer ([99mTc]Tc-NGR1), which was similar to the previous results of Chen et al. and Israel et al. Ga-NOTA-(NGR)2, [64Cu]Cu-DOTA-NGR1, [64Cu]Cu-DOTA-NGR2, and [64Cu]Cu-Sar-NGR2 [99mTc]Tc-labelled probes, both renal and hepatic excretion were present.Tc-labelled NGR peptides was conducted by Faintuch et al., who explored the in vitro and in vivo behaviour of pegylated cyclic NGRyk compounds appended with 99mTc ([99mTc]Tc-MAG3-PEG8-c(NGRyk)) in human lung carcinoma (A549), androgen-independent human prostate carcinoma (PC-3), and ovarian carcinoma (OVCAR-3) cell lines and corresponding mouse xenograft models (as presented in 99mTc]Tc-labelled cyclic pegylated pentapeptide NGRyk ([99mTc]Tc-MAG3-PEG8-c(NGRyk)), the most prominent accumulation was also found in the kidneys, which was the major route of elimination (99mTc]Tc-MAG3-PEG8-c(NGRyk) accumulation in both studies [99mTc]Tc-NGR1 and [99mTc]Tc-NGR2—the hepatobiliary system also plays a role in the elimination of [99mTc]Tc-MAG3-PEG8-c(NGRyk) [99mTc]Tc-MAG3-PEG8-c(NGRyk) activity in the A549 tumours than in the OVCAR-3 tumours, images with higher contrast and better T/M ratios were obtained in the case of the lung carcinoma bearing mice . Likewise, high T/M ratios were registered for [99mTc]Tc-MAG3-PEG8-c(NGRyk) in the B16F10 (5.0) and SKMEL28 (approximately 3) melanoma tumourous mice Tc-labelled derivatives of a wild-type endogenous angiogenic inhibitor—named human plasminogen kringle 5 (hPK5)—and its CNGRC-attached match (NGR-hPK5) in Mouse Lewis lung carcinoma (LLC) carrying female C57BL/6J mice ; HYNIC: hydrazinonicotinamide), and biodistribution studies were performed in B16F10 melanoma tumour-bearing C57BL6 mice. The rapid tumour uptake (maximum at 30 min post-injection), suitable T/M/tumour-to-blood ratios, and prompt clearance from the non-target normal tissues support the diagnostic value of [99mTc]Tc-HYNIC-CLB-c(NGR) as well. Their study is detailed in Based on the fact that more significant tumour growth inhibition was achieved with the NGR-modified compound both in the LLC and Colo 205 tumour-bearing mice, NGR peptides could be pioneering in the targeted delivery of anti-cancer drugs. Moreover, cisplatin administration in combination with NGR-attached hPK5 led to increased suppression of tumour neovascularization compared to the application of hPK5 alone plus cisplatin. This strengthened the role of the NGR motif in increasing the anti-tumour effect of the peptide. Accordingly, immunofluorescence staining using CD31 antibody also proved that the microvessel density of the LLC tumours was reduced to a greater extent upon NGR-hPK5 administration in comparison with the wild type hPK5. Examining the cytotoxicity of the NGR peptide connected to DNA alkylating nitrogen mustard chlorambucil (CLB), Vats et al. also stated that the attachment of target-specific peptides to chemotherapeutic agents could enhance the anti-tumour effect . They re99mTc was used for the in vivo detection of APN/CD13 expression in nude mice transplanted with HT1080 human fibrosarcoma tumour cells -gluconate Tc-labelled APN/CD13 targeting probes as well as the easy accessibility and applicability of the radiometal, 99mTc serves as a valuable alternative for the radiolabelling of NGR ligands. Further, these findings collectively demonstrate that the therapeutic efficacy of anti-cancer drugs could be augmented by attaching them to angiogenesis-specific peptide ligands; moreover, this could be a novel pharmacological approach to battling cancer. 99mTc]Tc-labelled radiotracers with selective APN/CD13 homing capability.Given the target specificity of the examined Bi-labelled radiotracers in therapeutic settings is the central focus of current oncological research [213Bi]Bi-DOTAGA-cKNGRE, Képes et al. assumed for the first time the usefulness of [213Bi]Bi-labelled molecular probes in angiogenesis-targeted cancer treatment -1,4,7,10-tetraazacyclododecane glutaric acid; demonstrated in 213Bi]Bi-DOTAGA-cKNGRE to test the effect of the agent on the body weight and the tumour volume of the animals. The tumour volumes of the treatment-naïve animals  and the treated tumourous animals  showed significant differences, which led to the conclusion that [213Bi]Bi-DOTAGA-cKNGRE exerted a meaningful anti-tumour effect.Given that the high linear energy transfer and short tissue penetration range of the alpha emitter Bismuth-213 (y (TRNT) ,100. Henresearch . Evaluat99mTc]Tc-labelled NGR derivatives [213Bi]Bi-DOTAGA-cKNGRE. Suitable image contrast/quality obtained at 90 min post-tracer administration suggest that the diagnostic value of the gamma rays of the radiometal could also be exploited.Similarly to the Ga-DOTAGA-cKNGRE—used for the identification of the HT1080 tumours—showed significant tumour uptake coupled with faint background activity . This was similar to the results of previous studies with different c(NGR) derivatives, such as [68Ga]Ga-DOTA-c(NGR)2 [68Ga]Ga-NOTA-c(NGR) Ga-DOTAGA-cKNGRE/[213Bi]Bi-DOTAGA-cKNGRE radiotracer pair as a theranostic agent in APN/CD13 positive tumour diagnostics, therapy, and therapeutic response assessment.These initial results with - particles (2.12 MeV) of Rhenium-188 (188Re), along with its gamma co-emission (155 keV), it is applicable for the labelling procedure of NGR molecules not only in imaging but also in radiotherapeutic settings Re-labelled NGR-VEGI fusion protein as a possible theranostic agent for the SPECT diagnostics and the targeted therapy of HT1080 xenografted female BALB/c nude mice. 188Re]Re-NGR-VEGI was confirmed by the satisfactory tumour-to-background tissue ratios experienced upon SPECT image interpretation and biodistribution data analysis.Based on literature data, radiolabelled NGR-drug conjugates in complexation with anti-angiogenic vascular endothelial growth inhibitor (VEGI) could be effectively used in theranostic applications . Ma et a188Re]Re-NGR-VEGI into account—it may emerge as a potentially new approach to cancer therapy. The anti-tumour activity of [188Re]Re-NGR-VEGI was more prominent than that of the NGR peptide, or NGR-VEGI. The successful cytotoxic effect of the probe was further confirmed by in vitro therapeutic studies presenting notably higher tumour cell necrosis and apoptosis in the case of [188Re]Re-NGR-VEGI than regarding the vector control, NGR peptide, VEGI protein, and the unlabelled NGR-VEGI protein. The lack of therapy associated systemic toxicity is another advantage of this agent.Taking the considerable in vivo tumour growth inhibition—induced by the administration of 18.5 MBq of Lu-DOTA-cNGR) as well as the peptide-free [177Lu]Lu-labelled CNS ([177Lu]Lu-DOTA-CNS) using B16F10 melanoma tumour-bearing C57BL6 mice. The more enhanced tumour uptake of [177Lu]Lu-DOTA-CNS-cNGR coupled with a more suitable tumour-to-background contrast compared with the other probes may allow us to draw the conclusion that the application of peptide-nanosphere complexes may herald a new era in target-specific nanomedicine.To assess the imaging properties of this nanosphere-peptide conjugate  for the imaging of APN/CD13 in infarcted myocardial tissue [111In]In-DOTA-cNGR combined with 99mTc-sestamibi was performed in Swiss mice 7 days after myocardial ischaemia induction by the ligation of the left anterior descending coronary artery (LAD) (MI mice). MicroSPECT imaging verified that [111In]In-DOTA-cNGR is eliminated by the kidneys and is rapidly washed out of the intact myocardium. Markedly higher [111In]In-DOTA-cNGR accumulation was experienced in the underperfused infarcted myocardial regions or at the border zone with diminished 99mTc-sestamibi uptake. Ex vivo gamma counting was carried out both in healthy control and MI mice for the qualitative measurement of myocardial [111In]In-DOTA-cNGR uptake and for the assessment of the normal distribution pattern. This study draws attention to the fact that radiolabelled NGR probes could be used not only for the imaging of cancer-related angiogenic processes but also for the imaging of APN/CD13 expression in perfusion-deficient myocardial regions. In addition, NGR-based molecules have also been extensively investigated in the detection of myocardial angiogenesis. APN/CD13 affine nanoparticles, including cNGR peptides targeting quantum dots and liposomes, were proposed for the imaging of highly angiogenic myocardial regions with APN/CD13 overexpression [Hendrikx et al. applied for the first time a cNGR homing sequence furnished with Indium-111  [68Ga]Ga-NOTA-cNGR was subjected to in vivo PET studies in male Fischer-344 rats with DR induced by the ligation of the left bulbus oculi  and in control animals as well. Considerably higher [68Ga]Ga-NOTA-cNGR uptake of the ligated left eye was measured at all time points  compared to the radioactivity of the eyes of the control group, which proves the applicability of the tracer in the detection of IR-mediated APN/CD13 expression. Farkasinszky et al. concluded that the role of cellular, vascular, and physiological autoregulation mechanisms, including perfusion changes in the optic neurons or smooth muscle depolarisation, as well as alterations in the permeability of the blood-retinal barrier induced by APN/CD13-related extracellular matrix degradation, could be responsible for the significant [68Ga]Ga-NOTA-cNGR accretion experienced in the right bulbs (non-ligated) of the surgical cohort 10 days after IR induction Gnduction ,114,115.As these studies confirmed the diagnostic feasibility of NGR radiotracers in areas other than cancer-related angiogenesis, we expect to see more NGR-based radiopharmaceuticals being developed in the forseeable future in a wider range of non-cancerous applications.3H]H-CHE-labelled targeted NGR-SL[DXR] or non-targeted liposomal doxorubicin (SL[DXR]) indicated 10-fold higher tumour accumulation for NGR-SL[DXR] than for SL[DXR] 24 h after the injection. Based on this result, we may state that the addition of the NGR motif to SL[DXR] enhances its tumour-homing capability and therapeutic potential. Apart from the spleen, where significantly higher NGR-SL[DXR] uptake was found, the radioactivity of the two molecules did not differ in the investigated organs. We suppose that the increased lienal concentration of the targeted liposome could be caused by its relatively larger molecular size and related greater uptake by the reticuloendothelial cells. The following results of the in vivo therapeutic studies definitively strengthened the potential of NGR tripeptide in therapeutic settings: Rapid tumour volume reduction or even complete tumour remission, reduction of blood vessel density, and inhibition of metastatic spread were experienced in mice treated with NGR-SL[DXR] compared to the control cohorts receiving HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, untreated group), mismatched peptide ARA-targeted SL[DXR] (ARA-SL[DXR]) or NGR peptide-targeted liposome (NGR-SL) without DXR. In a similar manner, the histopathological findings—derived from the treated mice—such as disruption of tumour vasculature, suppression of blood vessel density, and apoptosis of the endothelial and tumour cells also support the feasibility of NGR in the development of targeted drug-delivery systems. Moreover, based on previous studies by Zhang et al. and Palmowski et al., ultrasmall superparamagnetic iron oxide USPIO nanoparticles and poly(butyl cyanoacrylate) PBCA-based microbubbles may emerge as tumour selective nanotheranostics that could be suitable for angiogenesis targeting, magnetic resonance (MR), ultrasound (US), and NIRF-based imaging, as well as for therapeutic purposes, due to their facile attachment to anti-cancer drugs and their favourable surface chemistry Ga-labelled heterodimeric tracer with NGR and RGD motifs ([68Ga]Ga-NGR-RGD) showed excellent in vivo imaging performance in mouse models of different breast cancers and pulmonary metastases [Considering that tumour cells use various pathways for their survival, the establishment of multi-targeted diagnostic probes has attracted the interest of many investigators in oncological research fields. For example, in the study of Gai et al., a [tastases . This enAlthough the investigated NGR probes display rapid tumour uptake together with slow washout, negligible off-target accumulation, and fast elimination from the blood, the fulfilment of chemical modifications to further optimize pharmacokinetics may be part of future work.