Wright State University Recent Advances in the Drug Carriers for Thrombolytic Article Review

Review www.small-journal.com Thrombolytic Agents: Nanocarriers in Controlled Release Soodabeh Hassanpour, Han-Jun Kim, Arezoo Saadati, Peyton Tebon, Chengbin Xue, Floor W. van den Dolder, Jai Thakor, Behzad Baradaran, Jafar Mosafer, Amir Baghbanzadeh, Natan Roberto de Barros, Mahmoud Hashemzaei, Kang Ju Lee, Junmin Lee, Shiming Zhang, Wujin Sun, Hyun-Jong Cho, Samad Ahadian, Nureddin Ashammakhi, Mehmet R. Dokmeci, Ahad Mokhtarzadeh,* and Ali Khademhosseini* Thrombosis is a life-threatening pathological condition in which blood clots form in blood vessels, obstructing or interfering with blood flow. Thrombolytic agents (TAs) are enzymes that can catalyze the conversion of plasminogen to plasmin to dissolve blood clots. The plasmin formed by TAs breaks down fibrin clots into soluble fibrin that finally dissolves thrombi. Several TAs have been developed to treat various thromboembolic diseases, such as pulmonary embolisms, acute myocardial infarction, deep vein thrombosis, and extensive coronary emboli. However, systemic TA administration can trigger non-specific activation that can increase the incidence of bleeding. Moreover, protein-based TAs are rapidly inactivated upon injection resulting in the need for large doses. To overcome these limitations, various types of nanocarriers have been introduced that enhance the pharmacokinetic effects by protecting the TA from the biological environment and targeting the release into coagulation. The nanocarriers show increasing half-life, reducing side effects, and improving overall TA efficacy. In this work, the recent advances in various types of TAs and nanocarriers are thoroughly reviewed. Various types of nanocarriers, including lipid-based, polymer-based, and metal-based nanoparticles are described, for the targeted delivery of TAs. This work also provides insights into issues related to the future of TA development and successful clinical translation. S. Hassanpour Department of Analytical Chemistry Faculty of Science Palacky University Olomouc 17. Listopadu 12, Olomouc 77146, Czech Republic Prof. H.-J. Kim, P. Tebon, Dr. C. Xue, F. W. van den Dolder, J. Thakor, Dr. N. R. de Barros, Dr. K. Lee, Dr. J. Lee, Dr. S. Zhang, Dr. W. Sun, Prof. H-J. Cho, Prof. S. Ahadian, Prof. N. Ashammakhi, Prof. M. R. Dokmeci, Prof. A. Khademhosseini Department of Bioengineering Center for Minimally Invasive Therapeutics (C-MIT) and California NanoSystems Institute University of California-Los Angeles Los Angeles, CA 90095, USA E-mail: [email protected] Prof. H.-J. Kim, Prof. S. Ahadian, Prof. M. R. Dokmeci, Prof. A. Khademhosseini Terasaki Institute for Biomedical Innovation Los Angeles, CA 90024, USA A. Saadati Pharmaceutical Analysis Research Center Tabriz University of Medical Sciences Tabriz 516614731, Iran F.W. van den Dolder Division Heart and Lungs Department of Cardiothoracic Surgery University Medical Center Utrecht Utrecht, GA 3508, The Netherlands The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.202001647. DOI: 10.1002/smll.202001647 Small 2020, 16, 2001647 F.W. van den Dolder Regenerative Medicine Center Utrecht University Medical Center Utrecht Utrecht, CT 3584, The Netherlands Prof. B. Baradaran, A. Baghbanzadeh, Dr. A. Mokhtarzadeh Immunology Research Center Tabriz University of Medical Sciences Tabriz 516614731, Iran E-mail: [email protected] Dr. J. Mosafer Research Center of Advanced Technologies in Medicine Torbat Heydariyeh University of Medical Sciences Torbat Heydariyeh 9519633787, Iran Dr. M. Hashemzaei Department of Pharmacodynamics and Toxicology School of Pharmacy Zabol University of Medical Sciences Zabol 9861618335, Iran Prof. H.-J. Cho College of Pharmacy Kangwon National University Chuncheon, Gangwon 24341, Republic of Korea Prof. N. Ashammakhi, Prof. M. R. Dokmeci, Prof. A. Khademhosseini Jonsson Comprehensive Cancer Center Department of Radiology and Department of Chemical and Biomolecular Engineering University of California-Los Angeles Los Angeles, CA 90095, USA Prof. A. Khademhosseini Department of Chemical and Biomolecular Engineering Henry Samueli School of Engineering and Applied Sciences University of California – Los Angeles Los Angeles, CA 90095, USA 2001647 (1 of 19) © 2020 Wiley-VCH GmbH www.small-journal.com www.advancedsciencenews.com 1. Introduction Thrombosis is a life-threatening pathological condition in which unwanted blood clots occlude blood vessels. It is an acute disorder caused by an accumulation of platelets within a blood vessel and is a hallmark of many cardiovascular diseases.[1] Among other symptoms, thrombosis frequently results in obstructive cardiovascular disorders, which cause ischemic injury to blood vessels and surrounding tissue.[2] Thoracic outlet syndrome, deep vein thrombosis (DVT), pulmonary embolism (PE), acute myocardial infarction (AMI), and stroke are lifethreatening thrombus-related diseases especially in developed countries.[3] There are two types of thrombosis: arterial thrombosis, a common characteristic of advanced atherosclerosis, and venous thrombosis, encompassing DVT (with or without PE), renal vein thrombosis, portal vein thrombosis, cerebral venous sinus thrombosis, and hepatic vein thrombosis (Budd-Chiari syndrome). Arterial thrombosis occurs when the atherosclerotic plaque ruptures from a vessel wall and provokes the formation of a thrombus or embolus. Venous thrombosis, on the other hand, is a blood clot originating in a vein and is known to form in places where blood flow slows or changes. Blood clots from both arterial and venous thrombosis can partially or completely occlude blood vessels, causing damage and dysfunction of the organs supplied by the affected blood vessels.[4] When the vessel wall is damaged, the thrombogenic cascade is immediately activated to reduce bleeding. Vasoconstriction occurs as one of the first steps followed by an injury to prevent blood loss from the affected area. Following that, the formation of a fibrin-platelet composite is the primary component of the typical blood clot.[5] In these coagulation cascades, thrombin is the serine protease enzyme generated to convert fibrinogen into fibrin, which forms the thrombus.[6] There are two exosites in thrombin that are essential for proper function; the first one binds thrombomodulin and fibrinogen while the second one interacts with heparin, factor VII, glycoprotein (GP) Ib-IX, and factor V, a platelet GP.[7] Thrombin is also a multifunctional enzyme that contributes to the anticoagulant, procoagulant, mitogenic, and inflammatory responses. Each of these assists in the maintenance of hemostatic balance.[6] Activation of fibrinolytic systems (Figure 1) through the administration of thrombolytic agents Figure 1. Illustration of the principles of thrombolysis in a fibrin surface and circulating blood environment. This figure describes the catalytic principle of the conversion of plasminogen to plasmin according to the binding method of the plasminogen activator (e.g., tissue-type plasminogen activator [t-PA], urokinase [UK], and streptokinase [SK]). Plasminogen specifically binds to the surface of the fibrin blood clot. In direct activation, t-PA preferentially attaches to plasminogen, resulting in the formation of a ternary complex. On the other hand, in indirect activation, SK cannot directly bind to the plasminogen but induce conformational changes of the plasminogen to form a streptokinase-plasminogen complex. Subsequently, these complexes form plasmin through cleavage of the fibrin-associated plasminogen. Plasmin formed by direct/indirect activation breaks down fibrin into fibrin degradation products (FDP), which eventually dissolves blood clots. The thrombolytic process in circulating blood is triggered by non-fibrin-specific or less fibrin-specific plasminogen activators. Plasminogen activators such as the UK and SK induce plasmin production by cleavage of circulating plasminogen. Subsequently, plasmin degrades fibrinogen factor VIII instead of fibrinogen. Plasmin activator inhibitor-1 acts on plasminogen, blocking cleavage into plasmin, causing blood clot formation. α2-antiplasmin acts only on circulating blood, can inhibit thrombolysis by interfering with plasmin binding sites with fibrinogen factor VIII. Small 2020, 16, 2001647 2001647 (2 of 19) © 2020 Wiley-VCH GmbH www.small-journal.com www.advancedsciencenews.com (TA) causes the pharmacological dissolution of the blood clot, termed thrombolysis. In the thrombolysis reaction, the enzyme plasmin is activated and cleaves the fibrin mesh, which constitutes coagulation. To complement the primary thrombolytic mechanism, TA development research has primarily focused on the inhibition of thrombus formation, as well as thrombolysis.[8] However, the current clinical application of TAs still has several limitations such as low targeting capability, short half-life, and increased risk of uncontrolled bleeding. Systemic delivery and non-specific activation of TA increase the likelihood of hemorrhage and limit the use of certain therapeutic drugs. Moreover, protein-based TAs are rapidly inactivated upon injection resulting in the need for large doses.[9] Accordingly, research has focused on surmounting these challenges through selective delivery systems capable of targeting the site of vascular occlusion.[10] Targeting drug delivery to the clotting site is an appealing approach as it could reduce side effects and minimize the impact of short molecular half-life. An efficient methodology for delivering TAs is a drug delivery system (DDS) which utilizes carrier materials to maintain drug stability in transit to the site of action.[11] These nanocarrier-based DDS technologies can deliver optimized concentrations of medication to the target tissue, minimizing systemic exposure and overdose, facilitating a reduction in administered dosage and accompanying side effects.[12] Here, we thoroughly review the recent advances in various types of TAs and nanocarriers for targeted delivery. We also provide insights on barriers and discussions on expanding aid for developing on TAs and minimally invasive therapeutics. 2. Types of TAs Plasminogen activators (PAs) are proenzymes, precursors to a class of proteolytic enzymes, that are broadly applied as TAs to remedy blockages caused by thrombi. PAs have great specificity for plasminogen and convert plasminogen into plasmin.[13] Then, the newly formed plasmin degrades insoluble fibrin clots into soluble fibrin molecules.[14] Plasmin also enzymatically degrades any peptide or protein with accessible arginine (Arg)-lysine (Lys) sequences, which include fibrin, fibrinogen, and albumin, blood coagulation factors V, VIII, IX, and X.[15] Due to these thrombolytic potentials, PAs are used to treat cerebrovascular and cardiovascular obstructions that lead to stroke and heart attack.[16] PAs can be classified as either direct or indirect. Indirect PAs include streptokinase (SK), staphylokinase (SAK), and vampire bat PA (bat-PA).[17] SK and SAK are produced by bacteria and do not show proteolytic activity. These proteins indirectly facilitate plasmin formation by generating a 1:1 stoichiometric compound with plasminogen.[18] Direct PAs include tissue PA (t-PA), urokinase (UK), prourokinase (ProUK), acylated plasminogen–streptokinase activator complex (APSAC), alteplase (rt-PA), reteplase (r-PA), tenecteplase (TNK-t-PA), monteplase, and lanoteplase (n-PA).[17] The most prevalent direct PA is t-PA, a single-chain GP synthesized in endothelial cells, which is activated by fibrin to boost the conversion of plasminogen to plasmin.[18,19] Small 2020, 16, 2001647 2.1. Streptokinase (SK) The first TA to reach the commercial market was SK. As the first drug of its kind, it was a significant advance that contributed to a 50% decrease in mortality related to lethal disorders.[20] SK was first isolated in 1933 and used as a therapeutic agent in the mid-1940s. In 1947, Dr. Charles Dotter prescribed SK to treat peripheral arterial occlusive disease.[18] It currently remains as one of the most inexpensive and broadly prescribed fibrinolytic agents to treat thromboembolic diseases by preventing thrombus formation.[21] SK is a 47kDa single-chain polypeptide composed of 414 amino acids. Various strains of β-hemolytic streptococci produce SK as an extracellular protein. It forms a complex after binding to plasminogen, and induces the substrate’s conversion to plasmin.[18] After intravenous delivery into the physiological environment, SK has a short half-life of only 20–30 min as it is quickly bound by antibodies and removed from circulation by the reticuloendothelial system.[22] 2.2. Urokinase (UK) The fibrinolytic activity of urine was discovered by Dr. J. Pilling and Dr. R. G. Macfarlane in 1947.[23] In 1951, Dr. J. R. B. Williams identified that the activity was due to the presence of a PA now known as UK.[24] UK is a serine protease that is found in human urine and can also be isolated from human parenchyma in kidney tissue culture.[18,25] The PA consists of two polypeptide chains and has a molecular weight between 32 and 54kDa. The low-molecular-weight protein was isolated from cultured kidney cells; whereas, the high-molecular-weight molecule was isolated from urine. UK is a direct PA that has a half-life of approximately 15 min in the blood. Additionally, a glycosylated, recombinant form of UK has also been harvested from murine hybridoma cells; this recombinant protein features a higher molecular weight and a shorter half-life.[18,25] 2.3. Staphylokinase (SAK) Although SK is the most notable microbe-based PA, it is not the only one. SAK is an alternative derived from Staphylococcus aureus bacteria with a half-life of approximately 6.3 min.[25,26] SAK binds to plasminogen, forming an inactive complex, and requires a PA to activate it.[26c] S. aureus produces a recombinant protein consisting of a single 136-amino acid polypeptide chain weighing approximately 16.5kDa.[18,27] Early studies of SAK in the 1960’s demonstrated the drug’s pros and cons as they proved the protein’s efficacy as a TA while also demonstrating its ability to cause severe side effects due to impurities resulting from poor purification. Major advances in bacterial cloning and recombinant gene expression in the 1980s facilitated the production of pure SAK to be evaluated as a TA in humans.[28] These studies demonstrated one of the unique, attractive properties of SAK-specificity to fibrin. α2-antiplasmin rapidly neutralizes SAK when fibrin is not included in the reaction; however, in the presence of fibrin, SAK does not easily neutralize at the surface of the clot when fibrin is present.[28] 2001647 (3 of 19) © 2020 Wiley-VCH GmbH www.small-journal.com www.advancedsciencenews.com 2.4. Prourokinase (proUK) A novel fibrinolytic agent currently undergoing clinical trials is proUK – a 414 amino acid, 49kDa, single polypeptide chain.[25] Eight clinical trials have been conducted in China and the Netherlands while three of them are currently ongoing.[29] In the blood, proUK has a half-life of 7 to 8 min. In the physiological environment, plasmin partially converts proUK to an active 276 amino acid, low-molecular-weight, two-chain UK. Also, the unconverted proUK fraction directly activates plasminogen.[18] Currently, proUK is manufactured through recombinant processes in glycosylated mammalian cells or non-glycosylated E. coli.[25] 2.5. Acylated Plasminogen-Streptokinase Activator Complex (APSAC) two functional protein domains are deleted, leading to extended half-life in plasma, reduced fibrin specificity, and improved blood clot penetration.[39] The extended half-life is approximately 14 min.[18] The analogous function of the protein is due to its inclusion of kringle domain 2, the portion responsible for stimulating protease domains to degrade fibrin, instead of the kringle domain 1 and epidermal growth factor (EGF) found in t-PA.[19] While t-PA forms a complex with plasminogen to facilitate conversion to plasmin, the protein domain’s deletion in r-PA removes this requirement, facilitating an increase in thrombolytic activity. In some patients, the increased enzymatic activity can lead to complications as rapid thrombolysis impairs the formation of blood clots to stop bleeding.[19] 2.8. Tenecteplase (TNK-t-PA) The impact of acute myocardial infarction warranted the development of APSAC, which is suitable for intravenous bolus injection for treatment. It is composed of the acylated active site of human plasminogen in complex with bacteria-derived SK.[26a,30] APSAC has a molecular weight of 131 kDa and half-life observed as 70 to 120 min, which is greater than that of SK.[31] Acylation of the active site in human plasminogen inhibits the formation of plasmin, yet it does not impact the ability of APSAC’s Lys-binding residue to adhere to fibrin. Upon injection, the catalytic center of APSAC is immediately deacylated to activate the thrombolytic capacity of APSAC. A certain amount of plasminogen-SK complex is attached to fibrin during circulation. Circulating α2-antiplasmin protects the plasmin from neutralization, while the plasmin is now available to degrade fibrin in the thrombi. However, the circulating deacylated complex had previously formed free plasmin, which is neutralized until it exceeds the neutralizing capacity of the α2-antiplasmin.[29] 2.6. Alteplase (rt-PA) t-PA is a serine protease composed of 527 amino acids yielding a molecular weight of 68 kDa and a half-life of 4 to 6 min. t-PA is one of the two PAs found in the organs and blood of mammals that can convert plasminogen into active plasmin. The other endogenous enzyme is UK which differs in molecular structure.[32] rt-PA is manufactured using recombinant CHO cells transfected with cDNA that is transcribed and translated to express t-PA.[25,33] It was the first recombinant t-PA to cleave arginine-valine interactions to activate plasminogen into plasmin in the presence of fibrin.[19,31b] However, t-PA inhibits this transformation in the absence of fibrin.[19] rt-PA is widely used for controlling thrombosis, especially acute ischemic strokes which must be treated before 3 h from onset.[12a] TNK-t-PA is a modified t-PA designed to protect the fibrinolytic activity of wild type t-PA. T, N, and K refer to the three amino acids (threonine (Thr), asparagine (Asn), and Lys respectively) that vary from the native sequence of t-PA. The functional protein kringle domain 1 in TNK-t-PA contains a threonine as the 103rd residue, instead of an Asn, while the 117th residue, glutamine (Gln), is exchanged for Asn. Additional changes are made to the catalytic protease domain; the Lys-histidine (His)-Arg-Arg (296299) sequence in the active site is substituted for four alanines (Ala).[17,25] These modifications to TNK-t-PA expand the molecule, leading to greater half-life (t-PA:TNK-t-PA, 3.5:22 min).[34] Despite the modifications, the altered form of human t-PA still has functional protease activity which attaches to fibrin and catalyzes the formation of plasmin.[18] 2.9. Monteplase Monteplase is the bioengineered modified form of rt-PA, in which a single amino acid is exchanged in the EGF protein domain (84th residue cysteine (Cys) to serine (Ser)), and has a molecular weight of 68 kDa.[35] The single substitution results in a drastic increase in half-life to more than 20 min compared to the 4-min half-life of natural rt-PA.[36] Monteplase has been used in clinical trials in combination with coronary angioplasty and has shown higher patency rates than angioplasty alone.[37] 2.10. Lanoteplase (n-PA) n-PA is in the third generation of engineered PAs. It is modified by mutations in which the glycosylated position in the kringle domain 1 has been altered (117th residue Asn to Gln), and the EGF protein domain and finger domain …

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