Shear‐Responsive Drug Delivery Systems in Medical Devices: Focus on Thrombosis and Bleeding

Cardiovascular disease is the leading cause of death worldwide. Blood‐contacting medical devices provide critical support for patients with severe respiratory and/or cardiac failure. High shear stress zones and non‐biocompatible circuit can increase the risk of thrombus formation in patients. These thrombi restrict blood flow through the circuits and travel to the lungs and brain, creating significant consequences. Antithrombotic drugs are used to lower this risk, but they also raise the incidence of bleeding problems. This article explores the delicate balance between thrombosis and bleeding in medical devices by examining platelet activation and thrombosis formation under shear stress. The feasibility of a shear‐responsive nanomedicine‐based drug delivery system has been explored as a potential approach for targeted administration of antithrombotic medications to lower systemic drug levels and reduce the bleeding risk. Furthermore, the lack of in vitro platforms to investigate the biological behavior of antithrombotic nanoparticles is impeding their clinical translation. As a result, microfluidic technology offers a platform for investigating nanoparticle behavior in vitro and linking it to their performance in vivo. Finally, the challenges and factors that affect the functionality, stability, and circulation time of liposomal drugs are investigated to improve their efficacy for targeted drug administration in medical devices.


Introduction
Blood-contacting medical devices including stents, catheters, heart valves, and mechanical circulatory support (MCS) devices, such as left ventricular assist devices (LVADs), cardiopulmonary bypass (CPB), and extracorporeal membrane oxygenation (ECMO) have been widely used to treat various diseases (Figure 1). [1] Blood-contacting devices have varying durations of use, with different devices used for varying amounts of time. Typically, dialysis and CPB circuits are used for only a few hours, whereas ECMO and catheters are used for several weeks to months. [2] In contrast, stents and heart valves are intended to remain in place for many years, and typically for the duration of a person's life. The duration of LVAD implantation varies from a few weeks to several years, depending on whether the device is intended to serve as a bridge to heart transplantation or as a destination therapy. Regardless of the device's characteristics or length of use, it is inevitable that a foreign surface will meet the blood and high shear stress in those devices due to the high local blood velocity and narrowed blood flow area, potentially leading to the formation of thrombosis or immune responses. One of the major complications and a common cause of death for medical devices is thrombosis complications, such as ischemic stroke, right ventricular thrombus, [3] left ventricular thrombus, [4] pulmonary embolism [5] and bleeding. [6] In particular, it has been reported that the bleeding rate during ECMO is 20.8%-39.6% [6b,7] with the cannula site (13.2%), gastrointestinal tract (5.5%), lungs (6.1%), and central nervous system (3.9%) being the most prevalent sites, [8] while thrombosis formation occurs in 10%-46.1% of patients depending on the circuit type and age of the patient in various centers. [9] Antithrombotic agents are commonly used to prevent thrombosis in blood-contacting medical devices. However, the administration of these agents can lead to a systematic anticoagulant effect in the patient, thereby increasing bleeding complications. Therefore, additional techniques required to prevent thrombosis formation in medical devices without increasing the risk of bleeding complications.
Shear-responsive liposomes are a type of nanomedicine designed to respond to variations in shear stress. Liposomes are lenticular or spherical structures composed of phospholipids bilayer that can encapsulate drugs and other compounds. Conventional liposomes are relatively stable under shear stresses in normal physiological blood flow conditions. However, when exposed to high levels of shear stress, such as those found in medical devices, conventional liposomes can rupture and lose their contents. Importantly, this process occurs in an uncontrolled manner, rendering it challenging to manage. Shear-responsive liposomes, on the other hand, are made from a combination of lipids and polymers that are chosen to give the liposomes specific mechanical properties that allow them to respond to specific levels of shear stress in a more controlled and targeted manner. [10] This makes them more flexible and resistant to high shear stress, and also allows them to release their contents in response to changes in shear stress at a certain threshold. Shear-responsive liposomes have been proposed as a promising solution to prevent thrombosis in medical devices. These liposomes burst and release an antithrombotic agent at the site of high shear stress, decreasing the drug dosage required to prevent thrombosis and bleeding while increasing the drug's circulation time inside the body. [11] This article will focus on the potential application of shearresponsive nanomedicines to prevent thrombosis and reduce the bleeding complications in medical devices. To have a better understanding of the influence of the shear stress on thrombosis formation, the mechanism of platelet activation and thrombosis formation under shear stress is thoroughly discussed. Then, we explore the potential of shear-responsive liposomes in preventing thrombosis and bleeding in medical devices. The article will review the current literature on the use of shear-responsive liposomes and discuss their advantages and limitations. Additionally, we will discuss the current challenges and future directions for improving the stability of the shear-responsive liposomes in medical devices and their potential applications in clinical settings. In particular, Figure 2 depicts the use of shear sensitive nanoparticles in the context of ECMO to illustrate the practical application of shear-responsive nanoparticles.

Biomaterial and Shear Stress-Induced Thrombosis
According to Virchow's triad, thrombosis is triggered by a variety of factors, including hypercoagulability, medical device materials, and hemodynamic parameters (high shear stress/high residence time. [12] Non-biocompatible circuit surfaces, such as those made from synthetic materials, can trigger an immune response and promote platelet adhesion, aggregation, and ultimately thrombosis formation. [13] In addition, high shear stress and intensive blood flow regions, and prolonged blood contact time with medical devices, especially those in MCS, further contribute to these complications. These thrombi can obstruct blood flow across the circuit and harm blood vessels and organs. Thrombosis can form in the medical devices and embolize to other parts of the body, such as the lungs or brain, resulting in serious complications including stroke and pulmonary embolism, as well as organ damage. [14] Biomaterial thrombosis is the process by which a thrombus forms on the surface of a biomaterial, such as a medical device component surface (Figure 3). [15] This phenomenon can occur due to the foreign body effect of the biomaterial and the activation of the haemostatic pathways, including platelet activation, coagulation activation, and the immune response. [16] The interactions between these pathways and the biomaterial surface are influenced by various factors, including the presence of plasma proteins, such as fibrinogen, high molecular weight kininogen (HMWK), Pre-kallikrein (PK), Factor XII (FXII), complement proteins, von Willebrand factor (vWF), immunoglobulins, and tissue factor (TF). [17] The surface of the biomaterial activates platelets, leading to the initial adhesion of platelets to the biomaterial surface. This process is mediated by the exposure of vWF and other adhesive molecules, such as collagen and glycoprotein VI (GPVI), on biomaterial surface. [18] Platelet activation triggers the release of platelet granules, including adenosine diphosphate (ADP), which further stimulates platelet activation Figure 2. Illustration of nanoparticle rupturing in ECMO setup. Extracorporeal membrane oxygenation is a temporary support system for critically ill patients. The ECMO circuit comprises a blood pump, an oxygenator, tubing set, and two cannulas. The blood is propelled through the tubing set and the oxygenator, where it undergoes gas exchange to remove carbon dioxide and add oxygen. The ECMO pump generates the necessary blood flow to maintain perfusion and support vital organ function, while various monitoring devices track patient hemodynamic and oxygenation. Figure created with BioRender.com. and aggregation. The activation of platelets and the exposure of vWF on the biomaterial surface also initiates the coagulation cascade, leading to the formation of a fibrin mesh that provides structural support for the blood clot. This process is mediated by the conversion of prothrombin to thrombin, which in turn cleaves fibrinogen to form fibrin. [19] The activation of platelets and the presence of a foreign body also triggers an inflammatory response, which contributes to thrombosis by promoting the adhesion and activation of leukocytes, which release cytokines and other proinflammatory molecules. [20] Platelets continue to aggregate and accumulate on the biomaterial surface, strengthening the blood clot. This process is mediated by the binding of fibrin to the platelets, which functions as a bridge between the platelets and the biomaterial surface. The accumulation of platelets and fibrin on the biomaterial surface forms a thrombus. [21] Platelet function can be greatly affected by fluid dynamics in medical devices. Shear stress is defined as the force per unit area between layers of materials in contact, and has been identified as the main mechanical force that determines platelet-mediated haemostasis and thrombosis. [22] Shear stress is typically measured in units of pascals (Pa) or dynes per square centimeter (dyn/cm 2 ). Shear rate, on the other hand, refers to the rate at which adjacent layers of fluid are moving relative to each other. In other words, Shear rate refers to the velocity gradient of a fluid as it moves along a surface and describes how fast the fluid is flowing. Shear rate is typically measured in units of reciprocal seconds (s −1 ). Activation of platelets by high shears and the artificial surface of the medical devices have been proposed as the main causes of coagulopathy in patients. [23] Hence, it is vital to understand the function of platelets as the main cause of coagulopathy in patients with medical devices. This is because the interactions between platelet receptors and ligands, such as fibrinogen and vWF, are highly shear-dependent. [24] VWF is a protein that is involved in blood coagulation and plays an important role in the formation of blood clots. Under high shear rates, the mechanical force of the flowing blood causes vWF to undergo conformational changes, which alter its shape and structure. These changes result in the elongation of vWF, exposing the A1 domain. The A1 domain is the site where vWF binds to the platelet glycoprotein receptor Ib (GPIb). This binding allows the platelets to adhere to the blood vessel wall and initiate the formation of a clot. The exposure of the A1 domain and the subsequent binding of vWF to the platelets under high shear rates can increase the risk of thrombosis. This can lead to the occlusion of the blood vessels and medical devices and the subsequent formation of a thrombus. Moreover, under high shear stress conditions, the mechanical force of the flowing blood can cause erythrocytes to deform and become more elongated in shape. This deformation can cause the erythrocytes to become more adhesive, which can increase the risk of thrombosis. Low shear stress can cause the erythrocytes to aggregate and form rouleaux, [25] which increases the blood viscosity and consequently the shear stress that blood experiencing while under high shear stress these aggregates disintegrate and red blood cells (RBCs) may change shape to align with the flow direction, resulting in a reduction in viscosity via a process known as shear thinning. [26] In addition, the deformation of erythrocytes under high shear stress can cause them to release haemoglobin, which can interact with other blood components and contribute to the clot formation. [27]

Conventional Methods for Preventing Thrombosis and Bleeding in Medical Devices
Healthcare providers use different strategies to manage the risk of thrombosis and bleeding complications in medical devices, depending on the patient's condition. While heparin is the most used anticoagulant for patients using medical devices, it is associated with serious adverse problems including Heparin-induced thrombocytopenia (HIT) and heparin resistance. Heparin-induced thrombocytopenia can lead to the formation of blood clots, which can be dangerous and even life-threatening if they occur in the lungs, heart, or brain. [28] Heparin resistance is a term used to describe the condition in which a patient does not respond to therapeutic levels of heparin. [29] HIT and heparin resistance can be managed by using alternative anticoagulants, such as argatroban [28] or bivalirudin [30] but they can also show anticoagulant resistance requiring dose escalation. Factor XII inhibitors are promising anticoagulant agents that are currently under investigation in animal models, yet further approvals are required for their applications in human. [31] Moreover, utilizing low-dosage anticoagulation is another strategy that has been used in clinical settings to reduce bleeding problems while thrombosis formation is still a concern. [32] Additionally, the concept of an anticoagulant-free method, which involves coating the medical devices with biocompatible substances to prevent blood clotting, is being researched as an alternative, however it's still under investigation and not yet widely available or proven to be as safe and effective as traditional anticoagulation methods in medical devices and may not be appropriate for all patient populations. [33]

Novel Shear-Responsive Drug Delivery Systems
Despite the existence of various methods aimed at reducing thrombosis and bleeding complications in medical devices, there is still a need for more effective ways to lower the incidence of these issues. The use of shear-responsive drug delivery systems has the potential to improve the efficiency and specificity of drug  54] delivery, as the release of the drug can be precisely controlled in response to changes in shear stress. [34] Shear-responsive drug delivery systems refer to a class of drug delivery systems that are designed to respond to shear stress gradient, which is the force exerted on a fluid as it flows through a surface, such as blood flowing through a vessel or the blood flow within the medical devices. These systems are typically made up of a carrier, such as a nanoparticle or micelle, that contains the drug, and a shear-responsive coating or shell that surrounds the carrier. When these systems are introduced into a flowing liquid, such as blood, the shear stress on the coating or shell causes it to change its properties, such as its shape or surface charge, which in turn affects the release of the drug from the carrier. [35] Shear-responsive drug delivery systems rely on the reversible material deformation or disaggregation to elicit drug release. [36] In cardiovascular systems, shear stress is generated by the blood flow as it moves through the blood vessels. The range of shear stress in cardiovascular systems can vary depending on the location and size of the blood vessel, as well as the velocity and viscosity of the blood flow. Moreover, occlusion of the vessels due to cardiovascular diseases such as atherosclerosis, thrombosis, and embolism will induce higher shear stress to the flowing blood [37] providing the opportunity of localized vasodilators [38] and antithrombotic [39] drug delivery. [40] In particular, atherosclerosis is a common vascular disease that increases the risk of thrombosis by inducing shear stress at plaques. [41] Shear-responsive nanoparticles are a promising therapeutic approach for the treatment of atherosclerosis; however, the heterogeneity of lesions poses a significant challenge for the design of effective therapies, as various types of lesions can exist in the same patient under varying levels of shear stress. [42] To overcome this challenge, shear-responsive liposomes can be designed to target various types of lesions with varying shear stress levels. Alternately, nanoparticles may be functionalized with antibodies or other ligands that selectively bind to specific cell types or molecular markers associated with various types of lesions, thereby facilitating the targeted delivery of therapeutic agents to regions of high shear stress or specific lesion types.
The shear stress threshold differs among pathological environments and medical devices. In the arterial system, the shear stress is relatively high due to the high blood flow and the relatively small diameter of the vessels (10-20 dyn cm −2 ). The peak shear stress occurs near the vessel wall, where the blood is moving at its fastest, and decreases as the distance from the wall increases. In contrast, the venous system has lower blood flow and larger diameter vessels, resulting in lower shear stress (1-6 dyn cm −2 ). [43] Shear stress inside healthy vasculature ranges from 0.1 to 7 pa (1-− 70 dyn cm −2 [44] whereas it is in the range of 150-1000 dyn cm 2 in constricted coronary arteries. [45] In MCS, the shear stress range is typically 1000 to 6000 dyn cm −2 depending on the blood flow rate that varies in neonates and adult patients. [46] The typical range of shear stress in pathological conditions and medical devices presented in Table 1. There are several types of shear-responsive nanocarriers including nanogels, [55] polymeric nanoparticles, [56] inorganic nanoparticles, [57] and liposomes. [58] Among those, liposomes are widely used in targeted drug delivery, gene therapy, and vaccine development due to their ease of preparation, biocompatibility and biodegradability. [59] Shear-responsive liposomes are small, spherical structures composed of a phospholipid bilayer that mimic the structure of cells and encapsulate a hydrophilic core. Liposomes are one of the most used nanocarriers for the encapsulation of thrombolytic drugs and other medicines. By encapsulating drugs, liposomes can protect them from enzymatic degradation and increase their half-life, resulting in improved selectivity and targeted delivery to the site of thrombus. Consequently, the use of nanocarriers can boost the efficacy of antithrombotic drugs such as antiplatelets, anticoagulants, and thrombolytics. [60] Typically, there are two different methods introduced for delivering antithrombotic agents including nanoparticles deformation [58] and nanoparticle disaggregation (Figure 4). [39] The first technique employs lipid nanoparticles, which can release their content drugs in response to various shear stresses, while in second method, shear stress can shatter the micron-sized nanoparticle aggregates, allowing the released nanoparticles to stick to the surface of injury. To minimize the contact surface between water and the hydrophobic area of the liposomes, they normally create spherical shapes with a specific range of shear sensitivity, so that their membrane disrupts in shear ranges greater than 400 dyn cm −2 .
[36b] This is ideal for usage in medical devices since the shear stress in medical devices exceeds this range. However, since shear stress inside the body is less than this level, this could limit their use in in vivo experiments without medical devices. In this situation, non-spherical (lentil) particles can be used to Micron-sized nanoparticle aggregates dissociate and release antithrombotic drug-encapsulated or coated nanoparticles in areas of high shear stress, such as narrowed blood vessels or components in medical devices (e.g., stent, pump, membrane oxygenator, and cannula) (right top). Targeted drugloaded nanoparticles can deliver the drug to high shear stress zones where thrombosis forms and release the drug based on the drug release efficiency of nanoparticles (right bottom). Figure created with BioRender.com. modify the shear sensitivity of liposomes so that their membrane can be disrupted and the drug within them can be released under a wide range of shear stress, therefore resolving the problem. [38,61] Table 2 summarizes the shear stress range and test exposure time, payload, carrier composition and size, and the type of experimental methods of different shear-responsive liposomal drug delivery systems.

Shear-Responsive Nanoparticle Deformation
Shear-responsive nanoparticle deformation is the alteration of the shape and structure of nanoparticles in response to shear forces, and it is being investigated as a potential method of controlling drug release in response to variations in blood flow. Holme et al. [36b] classified nanoparticles based on their drug release properties, with natural phospholipids in liquid state, such as EggPC (EPC) being leaky under static condition and shear stress, while natural vesicles in the gel state including Dipalmitoylphosphatidylcholine (DPPC) and N-palmitoyl-D-erythro-sphingosyl-phosphorylcholine (16:0 SM) do not release their cargo in either static or shear-induced circumstances. They synthesized Pad-PC-Pad, a lenticular artificial phospholipid that is stable under static conditions but releases cargo (5(6)carboxyfluorescein) at elevated shear stress. It is also stated that lenticular shape of the liposomes is responsible for their sensitivity to shear stress that results in favored breaking points along the equator inside the body or external high shear stress areas. They also utilized an extracorporeal circulation pump to investigate the effect of the number of passes on the drug release properties of nanoparticles exposed to shear stress. They found that Pad-PC-Pad nanoparticles were shear sensitive and released their content under high shear stress conditions due to shear-induced transient pore formation. There was a clear distinction in drug release between healthy and constricted models using Pad-PC-Pad, while no significant difference was observed in dye-loaded EPC nanoparticles.
Further investigations were conducted to explore the impact of shear rate, surfactant-to-lipid molar ratio, lipid composition, and PEGylation of liposomes on the shear-induced leakage and fu-  The experiment was carried out at a temperature of 25°C and a shear rate of 7800 s −1 for a duration of 1000 s. C) Shows the fractional conversion of glucose over time for the liposomal GO-catalase system at various average shear rates, compared to a micellar enzyme system. Reproduced with permission. [65] Copyright 2014, ACS Publications.
sion of lipid nanoparticles. The researchers developed a method to control membrane permeability and morphology of unilamellar lipid vesicles with varying sizes under shear stress using nonionic surfactant (Brij 76). [61] They investigated the effect of vesicle size (50-400 nm), shear rate, and surfactant-to-lipid molar ratio on shear-induced leakage and fusion of lipid nanoparticles. Their findings demonstrate that introducing a small amount of detergent in EPC suspension induces leakage of hydrophilic species through the membrane in a dose-dependent manner, while no significant leakage is observed without detergent. Increasing the shear rate up to a threshold level enhances the leakage rate, due to the higher collision frequency between vesicles and increased Brij 76 concentration at vesicle poles. Furthermore, they utilized a Couette viscometer to expose the lipid vesicles to shearing for 20 min under various shear rates (0-10000 s −1 ). They observed that smaller unilamellar vesicles are less sensitive to shear stress than larger ones, and that leakage gradually increases with shearing time as demonstrated by UV absorbance results. In another study, Fujie and Yoshimoto examined the release rate of 5(6)-carboxyfluorescein dye from PEGylated liposomes using two different liposome compositions. [55] The impact of shear stress on PEGylated liposomes was studied using a cone-and-plate viscometer. The results indicate that dye release rises with time, peaks after 3 min, and then remains constant at a shear rate of 750 s −1 . PEGylation increases dye release from liposomes, as compared to non-PEGylated DOPC nanoparticles. Natsume and Yoshimoto highly synthesized shear-responsive liposome nanoparticles with lipid membrane composed of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and cholesterol (in a molar ratio of 50:20:30). Glucose oxidase (GO) and catalase were loaded into the liposomes to initiate the catalytic oxidation of glucose and evaluate the shear sensitivity of the nanoparticles. To determine the enzyme activity, GO and catalase encapsulated liposomes were introduced into a glucose solution. Laminar shear flow was generated at relatively high shear rates (≈7800 s −1 ) using microtubes with inner diameters of 190 and 380 μm equipped with a peristaltic pump (Figure 5A). The results revealed that the encapsulated liposomes were monodispersed (polydispersity index (PI) = 0.062) with a mean diameter of 150 nm ( Figure 5B). In static conditions, liposomal GO had low reactivity due to the significant penetration resistance of lipid membranes to substrate molecules. However, the glucose conversion rate increased with time (0-180 min) and shear rate (0-7800 s −1 ) ( Figure 5C), which demonstrated the shear sensitivity of the liposomal GO. [65] Chen et al. have developed a drug delivery system based on nanobowls, which are nanoparticles characterized by a bowl-like shape, with a hollow center and an outer liposomal shell. These nanobowls can be loaded with drugs and then released in a controlled manner to target cells or tissues in the body. The incorporation of nanobowls in the water cavity of the liposome structure improves the stability of liposomes in the blood circulation, preventing drug leakage induced by plasma proteins and blood flow shear stress. This will result in enhanced drug delivery to tumor sites ( Figure 6A). In comparison to conventional liposomes, it has been demonstrated that adopting Doxorubicin (DOX)-loaded nanobowl liposomes improves encapsulation efficiency and reduces drug leakage ( Figure 6B). Moreover, in vivo experiments in mice models have shown that the half-life of DOX-loaded nanobowl liposomes is 19.4 h, which is significantly longer than that of free DOX (1.3 h) and DOX-loaded liposomes (14.1 h). The shorter half-life of DOX-loaded liposomes is attributed to the considerable drug leakage in the circulation ( Figure 6C). Ex vivo re-sults further illustrate that DOX leakage from liposomes gradually increases after exposure to shear rate of 1500 s −1 for 4 h in serum, whereas there is no significant difference in drug leakage in the nanobowl-liposome formulation after 4 h of exposure to shear stress. This indicates the superior stability of nanobowlliposomes in the blood circulation. [62]

Shear-Responsive Aggregates
The second strategy for targeted drug delivery involves nanoparticle aggregates that can disintegrate in regions with elevated shear stress (Figure 4). Through modulation of drug release kinetics, shear-responsive aggregates can be used to achieve localized drug delivery and precise control of drug concentration. The Hagen-Poiseuille equation indicates that the magnitude of shear stress is inversely proportional to the size of the particles (d −3 ), hence stenotic regions in vessels and high shear stress zones in medical devices generate elevated shear stress. Micron-sized aggregates release nanoparticles carrying drugs upon experiencing high shear stress. Smaller particles experience less hydrodynamic force, allowing nanoparticles to adhere to the target zone. C) The schematic of shear-targeted thrombolytic drug delivery in a mouse artery thrombosis model. Ferric chloride injury leads to a partially obstructed thrombus. Injected SA-NTs accumulate near the thrombus due to local shear stress, coated with tPA for clot interaction and progressive blockage clearance. D) Intravenous fluorescence microscopy images reveal that tPA platelet mimetics, given as a bolus injection, eliminate a thrombus in a partially blocked mesenteric artery within 5 min. E) The pressure recovery efficiency of nanoparticle aggregates at lower doses and bolus delivery. F) Mice survival rate for tPA-coated SA-NTs and control mice group. Reproduced with permission. [39] Copyright 2012, Science Publications.
As the nanoparticles adhere to the high shear stress zone, drugs are subsequently released following drug release kinetics. The disintegration of more aggregates results in a greater release of nanoparticles and drugs. [66] Marosfoi et al. present a novel technique for treating emergent large vessel occlusion (ELVO) in stroke patients using shearactivated nanoparticles. The approach involves delivering recombinant tissue plasminogen activator (rtPA) to the clot site, activated by the shear stress induced by blood flow. To provide an effective therapy option for ELVO, they combined temporary endovascular bypass (TEB) with shear-activated nanotherapeutic (SA-NT). To make high shear stress inside the rabbit body and temporary clot, the authors utilized stent retriever. Shearresponsive aggregates containing nanoparticles have the size of 4 μm and 200 nm, respectively coated with rtPA, can adhere to surfaces under pathological shear stress (>100 dyn cm −2 ) while remaining intact under physiological shear stress. [34] In another study, Korin et al. used this approach by synthesizing micro-sized poly (lactic-co-glycolic acid) (PLGA) shear-activated nanotherapeutics (SA-NTs) aggregates coated with tissue plasminogen activator (tPA) using a spray-drying technique and biotin-streptavidin coating method ( Figure 7A). The stability of these aggregates in aqueous solutions is attributed to their hydrophobic nature, which limits the release of drugs embedded on their surface to high shear stress zones such as occlusion zones. A microfluidic device was used to assess the efficacy of their SA-NT approach in selectively targeting agents to stenotic regions under hemodynamic flow conditions and it was found that perfusion of SA-NTs through these microfluidic devices resulted in a 16-fold increase in the release of free NPs downstream of the obstruction ( Figure 7B). Also, the in vitro rheometer investigation demonstrates that aggregates can only be broken down to nanoparticles under shear stress greater than 100 dyn cm −2 , which corresponds to the shear stress caused by a 60% lumen blockage of coronary artery, and the thrombolytic agent tPA can be released. The aggregates were administered intravenously into a mesenteric injury mouse model, demonstrating their efficient thrombolysis effect by delaying the time to full vessel occlusion in a FeCl 3 mouse arterial thrombus model, reversing the debilitating hemodynamic changes of acute pulmonary embolism ex vivo at a concentration 100-times lower than native rtPA, and having a higher survival rate compared to the mouse pulmonary embolism model ( Figure 7C,D). Fluorescently-labelled tPA exhibited preferential accumulation of shear-dispersed nanoparticles near the stenosed vessel, resulting in rapid reopening of the vessel at a 100-fold lower dose than similar active tPA ( Figure 7E). In mouse models, 86% of mice treated with tPA-coated SA-NTs survived, whereas all control mice died within 45 min of injection due to acute emboli formation caused by fibrin clots, and no adverse bleeding effects were detected, as well as rapid clearance of micro-aggregates from the systemic circulation ( Figure 7F). [39]

Toward Drug-Loaded Liposomes for Thrombosis Prevention in Medical Devices
To prevent thrombosis formation and bleeding complications in medical devices, several classes of medications have been proposed and investigated: anticoagulants, antiplatelets, and thrombolytic agents. Liposomal formulations of these drugs can enhance their stability, targeting, and reduce their systemic side effects. [67] Anticoagulant, such as unfractionated heparin (UFH), [68] low molecular weight heparin (LMWH), [69] and bivalirudin [70] are the most commonly used drugs to lower the risk of thrombosis. Antiplatelet medications, such as aspirin, clopidogrel, ticagrelor and prasugrel can be used in combination with other anticoagulant medications to achieve optimal thromboprophylaxis and reducing the risk of thrombosis formation. [71] Thrombolytic agents such as tPA and urokinase can dissolve clots that have formed in the medical devices by enhancing their targeting to the clot site. [72]

Anticoagulants
Anticoagulants are commonly used in medical devices to prevent blood clots. The commonly used anticoagulants commonly includes heparin, direct thrombin inhibitors (DTIs) (bivalirudin, argatroban, lepirudin), factor Xa inhibitors (danaparoid and fondaparinux), direct oral anticoagulants (DOAC) (DTI (dabigatran), Xabans (rivaroxaban, apixaban, darexaban, edoxaban, and betrixaban)), factor-XIIa inhibitors, nafamostat mesylate (NM), and warfarin so that heparin and DTIs are the most prevalent anticoagulants used in critically ill patients. [33b] Even though anticoagulants are commonly used in medical devices to reduce the risk of thrombosis, their systemic administration increases the risk of bleeding complications. [73] Red blood cells (RBCs) have gained attention as a promising drug delivery vehicle due to their innate properties such as biocompatibility, biodegradability, nonimmunogenicity, and extended circulation time. Their large surface area also enables effective drug loading. RBCs can enclose drugs either within their lipid bilayer or within the haemoglobin, allowing for sustained drug release. Furthermore, engineering RBCs to express specific receptors on their surface facilitates targeted drug delivery to specific cells or tissues. The use of RBCs as nanocarriers in medical devices can potentially prevent thrombosis. These RBCs could be customized to target specific thrombosis sites within the medical devices or release drugs in response to changes in shear stress, like shear-responsive liposomes.
Chen et al. utilized electrostatic forces to bind heparinencapsulated nanoparticles onto RBCs to prolong heparin circulation in the body. This extended blood circulation time is advantageous because it increases the chances of the nanoparticles reaching their target site and delivering their payload. The nanoparticles, made up of heparin and thiolated poly-L-lysine (PLL), exhibit increased stability through the formation of disulfide bonds (Figure 8A). The positively charged heparin nanoparticles are attracted to the negatively charged RBCs. Since blood vessels at thrombus sites are narrowed, the magnitude of shear stress exerted on them increases significantly, resulting in the targeted release of heparin-containing nanoparticles (cNPs) from RBCs. Under low shear stress (1 Pa), scanning electron microscope imaging revealed that ≈80% of the particles were attached to the RBCs while under high shear-stress (10 Pa), ≈50% of the particles remained attached to the RBCs after 24 h. After 48 h, nearly all the particles were detached, demonstrating the effect of shear stress on nanoparticle detachment from RBCs ( Figure 8B). The size and charge of cNPs at the optimal ratio of poly-L-lysine to heparin, as well as microscopic images of cNPs and RBCs adsorbed with cNPs in both aqueous and dry states, were also characterized ( Figure 8C). [64]

Antiplatelet Agents
Due to high shear stress sites, the ECMO circuit can activate platelets and increase the risk of thrombosis. By inhibiting platelet aggregation, antiplatelet drugs can help prevent thrombosis and reduce the risk of thrombosis formation. Antiplatelet medications can prevent clot formation or stop existing clots from growing further, and they particularly target platelets by blocking their adherence or aggregation via different mechanisms. Since platelets play a crucial role in haemostasis, the use of antiplatelet medications for a variety of clinical disorders has grown in popularity in recent years. Aspirin, integrin GPIIb/IIIa ( IIb 3) antagonists, thienopyridines, dipyridamole, and ticagrelor are the most often utilized antiplatelet medicines. [74] GPIIb/IIIa antagonists work by preventing fibrinogen from binding to integrin IIb3. However, bleeding and thrombocytopenia are the most common adverse effects. [75] Abciximab, Eptifibatide, and Tirofiban are three parenteral GPIIb/IIIa inhibitors that have been developed and licensed for use in North America, particularly to prevent thrombotic reocclusion following percutaneous coronary intervention (PCI). [76] They impede fibrinogen and vWF binding, and thereby platelet aggregation, by blocking GPIIb/IIIa receptors on activated platelets. Physiologic platelet agonists such as ADP, epinephrine, collagen, or thrombin generate intracellular signals that convert the inactive GPIIb/IIIa on resting platelets to an active conformation in response to disturbed vascular endothelium. This reveals a highaffinity binding location for ligands like fibrinogen on the extracellular domain of GPIIb/IIIa. When fibrinogen and/or vWF bind to active GPIIb/IIIa in the presence of calcium ions, adjacent platelets aggregate. [77] Molloy et al. addressed the high risk of bleeding complications associated with antiplatelet therapy by utilizing shear-responsive nanocapsules (NCs) for targeted drug delivery to thrombosis sites. In vivo and in vitro experiments were conducted to compare this approach to systematic antiplatelet therapy. Phosphatidylcholine (PC)-based nanocapsules containing eptifibatide were tested for shear sensitivity using microfluidic devices with 80% stenosis (Figure 9A). Fluorescence microscopy was used to observe the inhibitory effect of drug-loaded nanocapsules on thrombosis ( Figure 9D). The results indicate that nanocapsules are only ruptured at the stenosis area with a shear rate of 8000 s −1 , but not in the upstream area with a shear rate of 1000 s −1 (Figure 9B,C). The study also demonstrated that eptifibatide-loaded nanoparticles have antithrombotic properties without prolonging bleeding time in a mouse model of vessel wall damage. [63]

Thrombolytic Drugs
Thrombolytic (clot-busting) drugs dissolve blood clots within blood vessels and are used in medical devices to prevent damage to components from clot formation. In particular, tPA, [78] rtPA, [79] urokinase plasminogen activator (uPA), [80] and streptokinase are commonly used thrombolytic drugs in ECMO. These drugs activate plasminogen, which degrades fibrin, the main component of blood clots. [81] However, the use of thrombolytic drugs in medical devices must be considered on an individual basis due to the risk of bleeding and other comorbidities. Fac-tors such as the patient's clinical condition, duration of device support, underlying medical condition, and other comorbidities should be considered. Liposome-loaded thrombolytic drugs are designed to target and deliver the thrombolytic drug specifically to the site of the blood clot, while minimizing exposure to healthy tissue. [82] This approach can enhance the efficacy of thrombolytic drugs and decrease the risk of bleeding complications by increasing drug stability through protecting it from enzymatic degradation and inactivation in the body. [83] It also extends the drug's circulation time, and improves drug specificity via the incorporation of receptor-binding molecules. [84] The activated-platelet-sensitive nanocarrier is designed to release its contents when encountering activated platelets, enabling targeted delivery of antithrombotic drugs to the site of the clot. This targeted delivery of antithrombotic drugs can increase the effectiveness of thrombolytic therapy and reduce the risk of bleeding complications. [85] Huang et al. developed a low-toxicity targeted delivery system using PEGylated liposomes coated with cyclic RGD (cRGD) peptide for tPA. The interaction between cRGD peptides on liposomes and GPIIb/IIIa integrins on activated platelets results in the fusion of platelet membrane and liposomes and the release of tPA (Figure 10A). This approach resulted in significantly higher and faster release of tPA compared to untargeted liposomes. [86] Figure 10B displays the results Figure 9. Antiplatelet nanocapsules target thrombi in stenotic vessel segments. A) In vitro microfluidic channels have a semicircular protrusion on the sidewall with an occlusion of 80%. B) Citrate anticoagulated whole blood loaded with either phosphate-buffered saline (PBS) NCs (PBS-NCs) or eptifibatide NCs (E-NCs) was perfused at 1000 s −1 input shear rates and imaged upstream and within the stenosis. C) PBS, PBS-NCs, E-NCs, or free eptifibatide were used to measure platelet aggregate surface area in the upstream section with a shear rate of 1000 s −1 and the stenotic region with 8000 s −1 . D) Fluorescent images of DiOC6-labeled platelet thrombi (red) on collagen type I in the presence of NBD-PE-labelled NCs following blood perfusion (green). Reproduced with permission. [63] Copyright 2012, Elsevier Publications. of a study on the binding affinity of different liposomal formulations to resting and activated platelets. The mean fluorescence intensity (MFI) of resting and activated platelets incubated with various formulations was analyzed. The results suggest that tPA-PEG-cRGD-lip displayed significantly enhanced staining of activated platelets compared to the other liposomal formulations, indicating its ability to efficiently facilitate targeted drug delivery to activated platelets at a thrombus site. The results also suggest that PEG-cRGD-lip showed significantly enhanced dissolution of blood clots compared to Lip and PEG-lip ( Figure 10C). Zhang et al. combined active thrombus targeting with cRGD and gradual drug release from liposomes to improve in vivo thrombolytic efficacy 4-fold over free uPA while also shortening the bleeding time of the tail bleeding assay of hemostasis, potentially reducing side effects associated with uPA by reducing the amount of uPA required. [87]

Novel Microfluidic Devices to Assess Thrombosis and Drug Release
Nanoparticles hold promise as therapeutic agents for treating cardiovascular disease. However, their development faces obstacles due to a lack of in vitro platforms that precisely emulate in vivo environment for analyzing their behavior. [88] Designing and optimizing effective antithrombotic nanoparticles requires understanding their reactions in the dynamic bloodstream environment and how they interact with blood components and medical devices. Their efficiency in vivo, however, is influenced by a variety of parameters, including their size, shape, surface chemistry, and ability to interact with blood components. [89] To optimize the design and formulation of antithrombotic nanoparticles for maximum therapeutic efficacy, it is necessary to understand the circulation time, uptake by target cells, and toxicity affect their biological behavior.
Microfluidic technology offers a promising in vitro platform for studying the behavior of nanoparticles, particularly for the development of effective nanoparticle-based therapies for cardiovascular disease. [90] It can replicate the complex flow and shear conditions of the bloodstream and provide controlled and welldefined conditions for nanoparticle testing and linking in vitro measurements of nanoparticle behavior with their performance in vivo. With the potential to model a range of biological systems, microfluidic devices can save valuable and costly samples, minimize experimentation costs, and can be highly automated, expediting drug testing and development. [91] Zilberman-Rudenko et al. designed a multi-bypass microfluidics ladder network to investigate the patterns of blood clot formation under complex shear. They simulated complex blood fluid dynamics by assuming that red blood cells and platelets are non-interacting spherical particles transported by bulk fluid flow. They were able to predict the patterns of blood clot formation at specific locations in the device, and their experimental data was used to adjust the model to account for the dynamic presence of thrombus formation. A distinct flow and thrombus formation patterns were observed in the microfluidic device, implying a specific correlation between microvascular geometry and thrombus formation dynamics under shear. This model has the potential to identify regions susceptible to intravascular thrombus formation within the microvasculature as well as in therapeutic devices. [92] In another study, Marcial et al. [93] developed a microfluidic platform with multiple stenoses to study thrombosis formation under the hemodynamic conditions of medical devices, such as ECMO, in which blood passes through the circuit multiple times (Figure 11). They confirmed thrombus formation at severe stenosis with 85% narrowing and high shear rates. Clots were found in all stenoses, but the first stenosis had the highest percentage of aggregation. The findings suggest that the first stenosis should be treated earlier to prevent total occlusion. These findings may aid in the assessment and treatment of patients with multiple lesions in the same blood vessel, as well as the long-term effect of multiple blood passes inside the medical devices. Figure 11. A) The setup used to monitor the formation of thrombosis. B) Fabricated microfluidic chip. The chip contained channels with one (a), two (b), and three (c) lesions. Reproduced with permission. [93] Copyright 2022, Springer Nature.

Challenges and Factors Impacting the Efficacy of Liposomal Drug Delivery Systems
Liposomes are widely used for drug delivery due to their ability to encapsulate both hydrophilic and hydrophobic compounds. Liposomal drugs have the potential to improve the efficiency and specificity of drug delivery by protecting drugs from enzymatic degradation and providing targeted delivery to the site of action. [94] Despite their potential to improve drug efficiency and specificity, liposomal drugs face challenges such as hemolysis, toxicity, stability, and targeting issues. Additionally, the composition of the liposomes, environmental conditions, and the presence of other medical conditions can significantly affect their functionality. Moreover, patients using medical devices will typically receive support for several days or even weeks. During this time, the liposomes must remain stable to effectively deliver drugs and prevent thrombosis formation. Liposomes that are not stable will tend to leak their contents or be rapidly removed from circulation, reducing their efficacy in preventing thrombosis. This section examines the challenges and factors influencing the effectiveness of liposomal drugs for targeted drug delivery in medical devices.
Liposomes are generally non-hemolytic and non-toxic, [95] but under specific circumstances they can cause hemolysis, the rupture of red blood cells. Hemolysis is influenced by liposome size, surface charge, concentration, and composition. Higher concentrations, larger size, and positive surface charge induce greater hemolysis. [96] However, uncharged liposomes can lower hemolysis. [97] Liposomal drugs are considered as foreign nanoparticles that can be attacked by immune system. They can trigger complement activation-related pseudo-allergy (CARPA), [98] an adverse immune phenomenon that can cause acute hypersensitivity reactions (HSRs) in up to 45% of individuals. [99] European Medicines Agency recommends in vivo and in vitro assessment of CARPA as a preclinical immune toxicity analysis prior to developing liposomal drugs. It has been shown that Pad-PC-Pad liposomes with a concentration of 20 mg mL −1 were shown to induce complement cascade via the alternative pathway, while with a lower lipid concentration of 2 mg mL −1 , no complement activation was observed in both in vitro human and porcine sera and animal porcine model. The study found that Pad-PC-Pad demonstrates less risk of CARPA compared to food and drug administration (FDA)-approved liposomal drugs. However, the low www.advancedsciencenews.com www.afm-journal.de transient phase temperature of Pad-PC-Pad in the human body limits its clinical application, and more stable liposomes should be explored. [100] The stability of the nanocarriers is a major concern in shearresponsive drug delivery systems. The prolonged use of MCS medical devices such as ECMO or LVAD for severely ill patients suffering from conditions such as acute respiratory distress syndrome (ARDS) or severe cardiac failure poses a challenge to liposome stability and circulation time that must be addressed. Liposomes are recognized and cleared by the mononuclear phagocyte system (MPS), reducing their therapeutic potential. [101] Strategies such as PEGylation, steric stabilization, stealth liposomes, targeting ligands, size, charge, and formulation can improve stability, circulation time and efficacy of liposomal drugs. PEGylation involves attaching PEG molecules to liposome surfaces, [102] which improves their stability and circulation time in vivo by reducing recognition and uptake by the MPS. [103] This phenomenon can be explained by PEG-induced steric hindrance that refers to the physical obstruction of the access of molecules to a surface by the presence of other molecules. When PEG molecules are attached to the surface of liposomes, they create a cloud of PEG molecules around the liposomes that physically obstructs the access of other molecules to the surface of the liposomes. This reduction in the rate of liposome-cell and liposome-protein interactions increases the circulation time of liposomes in the bloodstream, making them more stable and reducing the rate of their clearance by the MPS. [104] Liposome size and charge are important factors that can influence liposome stability. Smaller liposomes with a neutral or positive surface charge circulate longer than larger liposomes with a negative surface charge as they are less likely to be recognized by the MPS. [105] Depending on the lipid content, liposomes can be neutral, positive, or negative in charge. Neutral liposomes tend to aggregate and have less physical stability, and they do not interact strongly with cells, causing the drug to be released into the extracellular space. However, charged liposomes have several advantages over neutral liposomes, including the ability to prevent aggregation and enhance cell contact by generating zeta-potential, an electrostatic repulsion. [106] The incorporation of cholesterol into the membrane of liposomes can have a substantial effect on their stability and circulation time. [107] Cholesterol is a steroid molecule that naturally exist in cell membranes and is known to contribute to the fluidity and stability of membranes. Liposomes devoid of cholesterol in the bilayer are more susceptible to leakage and fusion with other vesicles, and they can reduce circulation time since they are rapidly cleared by the MPS. Cholesterol can affect the circulation time of liposomes by altering their interaction with the immune system of the body. Cholesterol can modify the surface properties of liposomes by decreasing their negative charge, thereby making them less susceptible to recognition and clearance by the MPS. This can increase the circulation time of liposomes, allowing them to remain at their target site for a longer duration. When cholesterol is incorporated into the liposomal membrane, it can increase the stability of the liposomes by reinforcing the integrity of the lipid bilayer. Cholesterol is a molecule that has both hydrophobic and hydrophilic regions, which allows it to span across the hydrophobic core of the bilayer and interact with the polar headgroups of the phospholipids. This interaction can help to reduce the fluidity of the bilayer and increase its packing density, making the liposome more resistant to leakage and fusion with other vesicles. [108] The effects of cholesterol are dose-dependent, and optimization of its concentration during formulation is necessary to achieve desired outcomes. Liposomes should also be characterized for size, shape, charge, and fluidity to ensure stability and suitability for their intended use. [109] Different natural and synthetic phospholipids have different phase transition temperature (T M ), which is a very important parameter influencing drug encapsulation efficiency, storage stability, and in vivo stability. Liposomes with higher T M are more thermally stable than those with lower T M . This is because when the temperature is below the T M , the lipids in the bilayer are in a solid-ordered phase that makes the bilayer more rigid and less permeable to small molecules. This increased rigidity of the bilayer makes the liposomes more resistant to environmental stimulus, such as temperature changes, pH changes, and shear stress. The T M of liposomes is dependent on the type of lipids that make up the bilayer. Liposomes composed of saturated lipids have a higher T M than those composed of unsaturated lipids. In particular, distearoyl phosphotidyl Choline (DSPC) and hydrogenated soybean phosphatidylcholine (HSPC) have a T M of 58 ○ C [110] and 52 ○ C, [111] respectively, while the T M of the dioleolyl phosphotidy I choline (DOPC) is − 20 ○ C. [112] Targeting liposomes with antibodies is a method of directing liposomes to specific cells or tissues in the body. [113] The effect of targeting liposomes with antibodies on their stability and circulation time can depend on various factors, including the type of antibody and the target. In general, targeting liposomes with antibodies can increase their stability and circulation time in the bloodstream by reducing the rate of liposome-cell and liposomeprotein interactions. This is achieved by reducing the rate of opsonization, which is the process of liposomes being marked by proteins, this marking allows the immune system to recognize and remove them. Additionally, targeting liposomes with antibodies can also increase their stability by enhancing the specificity of the liposomes to their target, resulting in a higher accumulation of liposomes at the target site and a reduced rate of clearance by the MPS. Thrombosis targeted liposomes could potentially be used to prevent or treat blood clots in medical devices. The liposomes can be coated with an antibody that binds to a protein found on the surface of the clot, such as tissue factor, which allows the liposomes to target the clot specifically. Once the liposomes reach the clot, the clot-dissolving agent, usually a plasminogen activator, is released, and it breaks down the clot. [114] However, it is worth noting that targeting liposomes with antibodies can also have some limitations. For example, antibodies can be expensive, and the conjugation process can alter the properties of liposomes, such as their size and charge, which can affect their stability and circulation time. [115] Therefore, the effect of targeting liposomes with antibodies on their stability and circulation time is complex and needs to be carefully evaluated.
Despite FDA approval of several liposomal drugs, clinical challenges and limitations remain. [116] Due to the sensitivity of liposomes to environmental factors such as temperature, pH, and humidity, and shear stress, determining the optimal dose is difficult. Therefore, proper storage and handling are essential to ensure the stability and shelf-life of liposomal drugs. Additionally, the formulation of liposomal drugs can also be a challenge, www.advancedsciencenews.com www.afm-journal.de as the composition and size of the liposomes can greatly affect their stability and effectiveness. Formulation optimization can help to identify the optimal liposome size, composition, and surface properties that are required to achieve the desired pharmacokinetic and pharmacodynamic properties. [117] Limited approval and insurance coverage, as well as high production costs, can reduce accessibility and affordability for patients. These challenges can be overcome by developing efficient production methods and advocating for favorable reimbursement policies. [118] Further research and careful clinical trial design can help to identify optimal dosing and administration methods, as well as to assess safety and efficacy in patients. Overall, while liposomes have great potential as drug delivery vehicles, more research is needed to address these challenges and improve their efficacy in vivo.

Conclusions and Outlook
Medical devices usually provide respiratory and/or cardiac support to critically ill patients. However, the use of medical devices is associated with a high risk of thrombosis formation and bleeding complications. Shear stress is an important mechanism in the medical devices, as it can cause injury to the blood cells and increase the risk of thrombosis. The use of shear-responsive liposomes, which are engineered to respond to the shear stress of blood flow, has the potential to further improve the specificity and efficacy of antithrombotic therapy in medical devices. These engineered liposomes can be designed to release their contents only when they reach the site of high shear stress, reducing the risk of bleeding complications. These liposomes can be coated with an antibody that binds to activated platelets or fibrin mesh. Once the liposomes reach the clot, the antithrombotic agent is released, and it either prevents platelet aggregation and thrombosis or breaks down the thrombosis.
The field of shear-responsive drug carriers is still in its early stages, and there are numerous challenges that must be addressed to advance this technology toward clinical applications. There is currently no standardized assay to characterize shear activation of such drug carriers, and such assays would need to be validated and approved. In this way, microfluidic devices can be utilized for mimicking the shear stress conditions in medical devices for liposomal drug testing and investigate their efficacy under controlled condition. The use of microfluidic technology in in vitro drug testing has a variety of advantages including the ability to precisely control fluid flow, reduced sample and reagent requirements, and automation that facilitates faster experimentation. Consequently, this platform holds great promise for the creation of effective drugs to treat a variety of medical conditions. Moreover, significant issues such as hemolysis, stability, and toxicity can compromise the efficacy of liposomal drugs in vivo. In addition, temperature, pH, and humidity, shear stress can cause liposomes to degrade and lose their efficacy, resulting in stability issues. Liposomes can be recognized and cleared by the immune system, which can result in low levels of the drug reaching the target site and toxicity. While developing liposome-based therapies for patients using medical devices, it is important to consider the stability of the liposomes. To effectively deliver drugs to the medical devises area and prevent thrombosis formation, the stability of liposomes over an extended period is crucial. In addition, optimization of size, charge, and formulation can help to determine the optimal liposome size, composition, and surface properties required to achieve the desired pharmacokinetic and pharmacodynamic properties. Additional research is required to optimize these particles for effective drug targeting, which is highly dependent on the used drugs. Therefore, choosing the appropriate drugs to use with this technology is crucial to its success.
Despite various achievements, further investigation is required to bridge the gap between current advances and their clinical application. Notably, the use of shear-responsive liposomes in medical devices is still under investigation and has not yet been approved for use in patients. Furthermore, it is important to note that the use of shear-responsive liposomes in conjunction with anticoagulation therapy, which is commonly used in medical devices, should be carefully evaluated to prevent adverse interactions. Finally, liposomal drugs have a variety of clinical challenges and limitations that must be addressed. These barriers include difficulties in achieving targeted drug delivery, determining the optimal dose, issues with stability and shelf-life, high production costs, administration routes, and regulatory approval challenges. Various strategies, such as targeted delivery systems, formulation optimization, the development of more efficient and costeffective manufacturing methods, and careful clinical trial design and monitoring, can help overcome these constraints.