Novel nanobased therapeutic approaches for thrombolysis

PhD Course

Translational Medicine

Curriculum

Drug Discovery

Academic Year

2018/2019

NOVEL NANOBASED

THERAPEUTIC APPROACHES

FOR THROMBOLYSIS

Author

M

C

Supervisor

P

.

M

E

Tutor

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S

Chapter 1: Introduction ... 5

Thrombosis ... 5

Coagulation Pathway ... 5

Pathogenesis of Thrombotic Diseases ... 8

Anti-thrombotic Drugs ... 14

Nanomedicine ... 16

Innovative Anti-Thrombotic Strategies ... 20

Abstract ... 24

Chapter 2: Discoidal Polymeric Nanoconstructs ... 27

Experimental Procedures ... 27

Synthesis of Discoidal Polymeric Nanoconstructs (DPNs) Coated with tissue Plasminogen Activator (tPA) ... 27

Physico-Chemical Characterization of DPNs and tPA-DPNs ... 28

Drug Encapsulation Efficiency ... 28

tPA Stability ... 29

Enzymatic Activity Test ... 29

Interaction of tPA-DPNs with Endothelial Cells ... 29

Confocal Analysis ... 29

FACS Analysis ... 30

Cytotoxicity Studies ... 30

Experimental Results ... 31

Synthesis and Physico-Chemical Characterization of tPA-DPNs ... 31

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Chapter 3: Spherical Polymeric Nanoconstructs ... 43

Experimental Procedures ... 43

Synthesis and Characterization of Spherical Polymeric Nanoconstructs (SPNs) Coated with tissue Plasminogen Activator (tPA) ... 43

Experimental Results ... 45

Chapter 4: In vitro Thrombolytic Efficacy ... 47

Experimental Procedures ... 47

In Vitro Efficacy of tPA-DPNs and tPA-SPNs in Static Conditions .... 47

Blood Clot Characterization ... 48

Fabrication of Microfluidic Chip ... 48

In Vitro Efficacy of tPA-DPNs and tPA-SPNs in Dynamic Conditions 49 Fabrication of the Leaf Microfluidic Chip ... 50

Blood Clot Dissolution with tPA and tPA-DPNs in the Leaf Microfluidic Chip ... 51

Statistical Analysis ... 51

Experimental Results ... 62

In Vitro Efficacy under Static Conditions ... 62

In Vitro Efficacy under Dynamic Conditions ... 69

Leaf-Microfluidic Chip for Assessing Thrombolytic Efficacy ... 74

Chapter 5: In vivo Thrombolytic Efficacy ... 79

Experimental Procedures ... 79

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Biodistribution of DPNs and SPNs in Immunocompetent Mice ... 80

Statistical Analysis ... 80

Experimental Results ... 83

In Vivo Efficacy of tPA-DPNs and tPA-SPNs ... 83

Biodistribution of Nanoparticles ... 87

Chapter 6: Discussion and Conclusions ... 89

Discussion ... 89

Conclusions ... 92

Chapter 7: Future directions ... 93

Introduction ... 93

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CHAPTER 1

I

1.1

T

1.1.1 Coagulation Pathway

Coagulation, also known as clotting, is the process involved in the formation of a blood clot, first stage of wound healing. The repair of an injured tissue comprises four events:[1]

1. the vascular constriction that decreases the blood flow in the area of the injury;

2. the activation of the platelets and their aggregation to form a platelet plug;

3. the formation of a fibrin network (clot) to entrap the plug; 4. the dissolution of the clot to resume a normal blood flow.

To start the hemostatic process, platelets must first adhere to exposed collagen, release their granules, and aggregate.[2] The bond with the collagen, and the transduction cascade induced by thrombin which leads to the release of intracellular Ca2+, activate the phospholipase A2.[3] The latter

then hydrolyzes membrane phospholipids, causing arachidonic acid liberation. The release of arachidonic acid determines the increase of production and activation of thromboxane A2 which is a potent

vasoconstrictor and platelet aggregation inducer.

The formation of the fibrin clot can be achieved through an “extrinsic” and an “intrinsic” mechanism,[4]

as depicted in Figure.1. These two biochemical cascades differentiate in the initial steps, but, at the end, they converge into a common pathway. The extrinsic pathway is activated to solve tissue injury.

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Conversely, the intrinsic pathway is activated in response to an abnormal vessel in the absence of tissue injury.

Figure.1 Blood Coagulation Pathway.

The intrinsic pathway initiates when prekallikrein, factor XI and factor XII are exposed to a negatively charged surface (contact phase).[5] This contact results in the conversion of prekallikrein to kallikrein, which converts factor XII into factor XIIa. Factor XIIa then activates factor XI to factor XIa. Besides, factor XIIa hydrolyzes more prekallikrein into kallikrein, establishing a reciprocal activation cascade. In the presence of Ca2+, factor XIa activates factor IX to factor IXa. Finally, factor IXa activates factor X to factor Xa. This last activation requires factor VIIIa that acts as a receptor and it is activated by small quantities of thrombin. The thrombin has a dual action that limits the extent of the coagulation cascade: as the concentration of thrombin increases, factor VIIIa is cleaved and inactivated. The intrinsic and extrinsic coagulation cascades converge due to the activated factor Xa. The extrinsic

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pathway is initiated in response to the release of tissue factor (TF), that is a factor VII cofactor in the activation of factor X.[6]

Prothrombin is activated on the surface of activated platelets by a prothrombinase complex composed by platelet phospholipids (phosphatidylinositol and phosphatidylserine), Ca2+, factor Va and Xa, and prothrombin. Factor V is converted into factor Va by small amounts of thrombin and is inactivated by high thrombin levels. Within the prothrombinase complex, prothrombin is cleaved at 2 sites by factor Xa. This cleavage generates a 2-chain active thrombin molecule containing an A and a B chain, which are held together by a single disulfide bond. Active thrombin is a serine protease that hydrolyses fibrinogen. Thrombin-mediated release of the fibrino-peptides generates fibrin monomers.[7] These monomers spontaneously aggregate in a regular array, forming a fibrin clot.

The dissolution of the fibrin clots (Figure.2) is mainly performed by plasmin, a serine protease that circulates as the inactive proenzyme, plasminogen.[8] The presence of plasmin in circulation is rapidly inhibited by α2-antiplasmin.

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin. Inactive tPA is released from vascular endothelial cells following injury; it binds to fibrin and is consequently activated. Active tPA cleaves plasminogen to plasmin which then digests the fibrin. Once the blood clot is finally dissolved, tPA activity is inhibited by 4 specific inhibitors: Plasminogen Activator-Inhibitor type 1 (PAI-1), and type 2 (PAI-2) are the most physiologically relevant.[9, 10]

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Figure.2 Role of Tissue Plasminogen Activator.

1.1.2 Pathogenesis of Thrombotic Diseases

Hemostasis is the perfect balanced process which leads to the maintenance of the integrity of a closed circulatory system after vascular damage.[11] Within seconds from the injury, this process is activated to form a thrombus, localized to the site of injury and with proportionated size.[4] Under normal conditions, regulatory mechanisms contain thrombus formation temporally and spatially. When a pathologic process is involved, it overwhelms the regulatory system initiating thrombosis. Thrombosis is the formation of a blood clot inside a blood vessel with the consequent obstruction of the blood flow.[12] The Virchow’s triad describes the main causes of thrombosis which can be defined in three categories of factors: endothelial injury or dysfunction, hypercoagulability, and hemodynamic changes (stasis).[13] In particular, endothelial injury includes vessel piercings and damages arising from shear stress or hypertension; hypercoagulability is an abnormality of blood coagulation caused by an excessively easy clotting of blood; and, stasis is a condition of slow blood flow.

Thrombosis may occur both in veins and in arteries. Arterial thrombosis (AT) requires a vessel wall damage for thrombus formation, while in venous thrombosis (VT) the blood clot forms without any injured epithelium.[14] Thrombi can be classified depending on their composition. In VT, the main

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components of the thrombus are red blood cells and fibrin, while in arterial thrombosis, platelets constitute the core of the thrombus. Thrombi characterized by predominance of platelets, such as AT thrombi, are called white thrombi; thrombi characterized by predominance of red blood cells, such as VT thrombi, are called red thrombi.

Blood clots in the veins develop due to two main reasons: immobility, and genetic errors in the clotting mechanism. A person bedridden due to illness or surgery or a person who is doing a long trip, forced to pass hours without the possibility to walk or stretch, has a high probability to have muscles not anymore able to strongly contract and push blood back to the heart. This is the typical scenario in which the stagnant blood can form small clots along the walls of the vein that can grow and partially or completely occlude the vein. Moreover, there may be a genetic or inborn error in the clotting mechanism, making a person hypercoagulable and at greater risk for forming clots.[15] Venous thrombosis leads to congestion of the affected part of the body.

The valves of the veins are recognized as a site of VT initiation.[16] Due to the blood flow pattern, the base of the valve sinus is particularly deprived of oxygen. Stasis exacerbates hypoxia, and this state activates white blood cells (leukocytes) and the endothelium. In particular, hypoxia activates hypoxia-inducible factor-1 (HIF-1), and early growth response 1 (EGR-1).[17] HIF-1 and EGR-1 pathways lead to monocyte association with endothelial proteins (P-selectin) and to the release from monocytes of macrovesicles filled with tissue factor, which initiate fibrin deposition (via thrombin) after binding the endothelial surface.

A common type of venous thrombosis is a deep vein thrombosis (DVT) depicted in Figure.3, specifically a blood clot in the deep veins usually of the leg.[18] The most frequent complication of DVT is post-thrombotic syndrome, which is caused by a reduction in the return of venous blood to the heart.[19] If a thrombus breaks and flows towards the lungs, it can become a pulmonary embolism (PE). PE is the most serious complication of DVT, and its risk is higher when clots are present in the thigh and pelvis.

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Figure.3 Deep Vein Thrombosis.(modified from [20])

Pulmonary embolism (Figure.4) is a medical emergency and can cause serious illness or death because of the blockage of an artery in the lungs.[21,

22]

Because of the blockage, the affected area of lung tissue may infarct or die, and hypoxia throughout the body may occur. In particular, lung infarction is caused by smaller pulmonary emboli which tend to lodge in more peripheral areas without collateral circulation, while largest pulmonary emboli typically cause dyspnea, hypoxia, low blood pressure, fast heart rate and fainting. Prognosis depends on the amount of lung affected.

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Arterial thrombosis occurs through a different mechanism. In the presence of atherosclerotic disease, plaques accumulate along arteries vessel walls and grow. The possible rupture of the plaque determines the formation of a blood clot, and the consequent partial or complete occlusion of the vessel.[24, 25] This disease process may cause heart attack (when it occurs in the coronary arteries that supply blood to the heart), stroke (when it occurs in arteries within the brain), or peripheral artery disease (occurring in the arteries of the legs).

The severity of a myocardial infarction (MI), commonly known as a heart attack (Figure.5), often depends on the amount of heart muscle that is damaged.[26] In many cases, only a small part of the heart muscle is damaged and then heals as a small patch of scar tissue. In this scenario, the heart can usually function normally. A larger heart attack is more likely to be life-threatening or cause complications. Coronary artery disease, the main cause of MIs, has as risk factors: high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet, and excessive alcohol intake. In fewer occasions, MIs are caused by coronary artery spasms, which may be due to cocaine, significant emotional stress, and extreme cold.[27]

The prognosis varies depending on the extent and location of the affected heart muscle, and the development and management of complications. Prognosis is worse with older age, and social isolation. Anterior infarcts, persistent ventricular tachycardia or fibrillation, development of heart blocks, and left ventricular impairment are all associated with poorer prognosis. Without treatment, about a quarter of those affected by MI die within minutes, and about 40% within the first month.[28]

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Figure.5 Myocardial Infarction.(modified from [29])

Stroke is the condition in which insufficient blood flow to the brain causes cell death. As depicted in Figure.6, there are two main kinds of stroke: the ischemic one, caused by lack of blood flow, and the hemorrhagic one, caused by bleeding. Both of them result in damage to the brain.[30]

Ischemic stroke occurs because of a loss of blood supply to part of the brain, initiating the ischemic cascade. Brain ceases to function if deprived of oxygen for more than 60 to 90 seconds, and after 3 h it can suffer irreversible injuries. There are four reasons why this might happen:

1. Thrombosis 2. Embolism

3. Systemic hypoperfusion

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In thrombotic stroke, a thrombus usually forms around atherosclerotic plaques. Two types of thrombosis can cause stroke:

- Large vessel disease involves the common and internal carotid arteries, the vertebral artery, and the Circle of Willis;

- Small vessel disease involves the smaller arteries inside the brain. An embolic stroke refers to an arterial embolism by an embolus originating from elsewhere. An embolus is most frequently a thrombus, but it can be composed of fat, cancer cells or clumps of bacteria.

Cerebral hypoperfusion is the reduction of blood flow to all parts of the brain. It is most commonly due to heart failure from cardiac arrest or arrhythmias, or from reduced cardiac output as a result of myocardial infarction, pulmonary embolism, pericardial effusion, or bleeding.

Cerebral venous sinus thrombosis is the presence of a blood clot in the dural venous sinuses, which drain blood from the brain. It leads to stroke due to locally increased venous pressure.

Hemorrhagic strokes can be caused by hypertensive hemorrhage, ruptured aneurysm, and drug-induced bleeding. Brain tissue is injured due to high compression derived from an expanding hematoma. The pressure may lead to a loss of blood supply with a consequent infarct. Moreover, the blood released by brain hemorrhage appears to have direct toxic effects on brain tissue and vasculature. There are two types of hemorrhagic stroke:

1. Intracerebral hemorrhage, which is bleeding within the brain itself;[31] 2. Subarachnoid hemorrhage, which is bleeding that occurs outside the

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Figure.6 Stroke. Hemorrhagic (left) and Ischemic (right) Stroke.(modified from [32])

Peripheral artery disease (PAD) is an abnormal narrowing of arteries which usually affects the legs.[33] Atherosclerosis is the most common cause of PAD; other causes include arterial spasm, thrombosis, and fibromuscular dysplasia. The most common symptom of PAD is intermittent claudication, which causes pain and severe cramping when walking or exercising. This occurs because muscles need more oxygen and the arteries are unable to meet the increased demand for oxygen by the muscles.

1.1.3 Anti-thrombotic Drugs

An anti-thrombotic drug can reduce the formation of blood clots by affecting different blood clotting processes. In particular, we can distinguish three different categories of drugs:

1. Antiplatelet drugs: decrease platelet aggregation by reversibly or irreversibly inhibit the process involved in platelet activation, resulting in a decreased tendency of platelets to adhere to one another. Antiplatelet drugs effect may be increased or decreased depending on

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patient's medications, current medical conditions, food and supplements taken. The increase of their effects would increase the risk of bleeding, while the decrease of their effects would potentially lead to thromboembolic risk;[34]

2. Anticoagulants: chemical substances that prolong the clotting time, preventing or reducing the coagulation of blood. Since the biggest complication is the increased risk of bleeding, the use of anticoagulants is a decision based upon the risks and benefits of anticoagulation;[35]

3. Thrombolytic drugs: dissolve blood clots, typically by targeting fibrin. Patients must be selected to receive this drug. Only the ones with the least risk of having a fatal complication, usually as a consequence of bleeding, will be treated.

The currently approved thrombolytic drugs are derived from

Streptococcus species, or, more recently, from recombinant biotechnology.[36] In the latter case, tissue plasminogen activator (tPA) is manufactured using cell culture, resulting in a recombinant tPA (rtPA). tPA is a serine protease which catalyzes the conversion of plasminogen into plasmin, the enzyme assigned to clot breakdown.[37] His recombinant version, rtPA, is sold under multiple brand names: - Activase (Alteplase): FDA-approved for the treatment of myocardial

infarction, acute ischemic stroke, and acute massive pulmonary embolism;[38]

- Reteplase: FDA-approved for acute myocardial infarction, it has a more convenient administration and a faster thrombolysis than Alteplase. This is because its half-life is up to 20 minutes and this allows it to be administered as a bolus injection rather that an infusion like Alteplase;[39, 40]

- Tenecteplase: indicated in acute myocardial infarction, showing fewer bleeding complications but otherwise similar mortality rates after one year compared to Alteplase;[41]

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1.2

N

Nanomedicine is the application of nanotechnology to medicine.[43-45] Nanotechnology is possible due to the merging of different knowledges coming from chemistry, biology, physics, mathematics, and engineering.[46] The idea of nanotechnology rises from the fact that biological, physical, chemical, mechanical, and optical properties at the nanoscale level differ from those of the larger, “bulk”, counterparts.

The most important concept in this field is the possibility to design multifunctional nanoparticles (NPs) that can provide numerous benefits. In particular, compared to conventional medicines, NPs can improve efficacy, bioavailability, targeting ability, and safety.[43, 47-49]

The physical characteristics of NPs can differ in many ways.[50] In particular: - Size: NPs have at least one dimension in the range of 1 to 100 nm,

although they can also be micrometer-sized particles. In this way they have novel structural, optical, and electronic properties. They also have improved solubility and increased bioavailability and circulation time;

- Shape: NPs can be spheres, discs, hemispheres, cylinders, cones, tubes, and wires. In this way, different interactivity, loading capacity, and transport capabilities can be reached;

- Surface area: as particles size decreases, the total surface area increases exponentially. An increase in surface area means that a greater portion of atoms are located on the particles surface relative to the core. This makes NPs more reactive and more prone to be conjugated with electrostatic charges or biomolecules selected for targeting or other purposes;

- Permeability: NPs small size can facilitate the crossing of biological barriers that are normally not accessible. For example, NPs can transport anti-cancer drugs into tumors according to the Enhanced Permeation and Retention (EPR) effect, through which drug delivery systems smaller than 200 nm should target passively tumors;[51] or

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they can cross the blood-brain barrier through the use of different uptake mechanisms.

One of the most interesting capability in nanomedicine is the functionalization of NPs by altering NPs properties through chemical or physical modifications applied to achieve a specific effect. Various approaches to functionalize NPs include:

- Targeting: NPs can be actively targeted using cell-specific ligands, magnetic localization, and/or size based selectivity.[52, 53] The targeting property can enhance the efficacy of NP drug-delivery systems while significantly reducing toxicity;[51]

- Surface Conjugation: the NP surface can be conjugated to a lot of biomolecules for different purposes. Some candidates are fluorescent dyes for imaging,[54] agents for genetic therapy such as small inhibitory RNA (siRNA),[55] targeting molecules that bind to highly expressed tumor cell receptors to facilitate the transport of imaging contrast agents, which aid in tumor detection;

- Improved bioavailability: opsonins, immune system proteins, are the proteins in charge of activating the immune complement to mark the NPs for destruction by macrophages and other phagocytes.[56] Opsonins recognize less neutral NPs than charged particles, and hydrophilic particles less than hydrophobic particles. It is possible to design NPs to be neutral or conjugated with hydrophilic polymers (such as polyethylene glycol - PEG) to prevent opsonization, prolong circulation time, reduce RES uptake, and/or enhance biocompatibility.[57, 58]

- Controlled release: NPs can also be designed to be activated to release therapeutic or diagnostic molecules in response to a site-specific trigger such as pH, temperature, magnetic field, enzymatic activity, and light or radiofrequency signals.[43] For example, the acidic environment inside inflammatory and tumor tissues (pH 6.8) and cellular vesicles, such as endosomes (pH 5.5–6.0) and lysosomes (pH 4.5–5.0), can be exploited to trigger drug release.[59]

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Nanoplatforms are commonly classified in some principal categories: the most widely used are dendrimers, micelles, nanotubes, nanoshells, liposomes, and polymeric nanoparticles (Figure.7).[50]

Figure.7 Schematic Representation of Nanoparticles. A. Dendrimers. B. Micelles. C. Nanotubes. D. Nanoshells. E. Liposomes. F. Polymeric

nanoparticles.

Figure.7A depicts the schematic of dendrimers. They are branched polymeric macro-molecules forming a star-like structure made of a repetition of starting small units.[60-62] Dendrimers present a size ranging from 1 to 20 nm and due to their dimensions, they are rapidly uptaken by cells and provoke a reduced activation of the immune system. Their propensity to form complex with biomolecules present in the circulation limits their use in clinic. Micelles (Figure.7B) are composed by an inner hydrophobic core, suitable for the entrapment of non-polar molecules, and an external hydrophilic layer, crucial for their solubility in aqueous solutions. They are made of a single layer of phospholipids and present a size of about 7-35 nm. The main advantage in their use is the great easiness of the synthesis process.

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However, the main limitation regards their impossibility to transport hydrophilic molecules.[63]

Carbon nanotubes, depicted in Figure.7C, are composed of a distinct molecular form of carbon atoms that give them unusual thermal, mechanical, and electrical properties.[64] If modified with PEG, they are surprisingly stable

in vivo, with long circulation times and low uptake by the reticuloendothelial system (RES).[48] Carbon nanotubes have been used for the delivery of imaging and therapeutic agents and in the transport of DNA molecules into cells.

Carbon nanoshells (Figure.7D) are composed of a silica core that is covered by a thin metallic shell, usually composed of gold. Their primary use is in thermal ablation therapy but they also scatter light, a property which is useful for cancer imaging.[65]

Nevertheless, the most common drug delivery systems are liposomes and polymeric nanoparticles.

Liposomes (Figure.7E) are constituted by one or multiple layer of lipid bilayer and thus, in the latter case, present two aqueous phases, one in the core and one between the bi-layers in which it is possible to entrap polar molecules.[66] Conversely, in the lipid bilayer non-polar molecules can be loaded. To increase their circulation half-life, liposomes can present also PEG in the external layer and this addition has been shown to increase by ten-fold the circulation half-life of liposomes.[67] Moreover, by using lipids of different fatty-acid-chain lengths, scientists can construct liposomes to be temperature-sensitive or pH-temperature-sensitive, thereby permitting the controlled release of their contents only when they are exposed to specific environmental conditions. In 1995, “Doxil”, the first liposomal platform has been approved by FDA as therapeutic agent for the treatment of AIDS-associated Kaposi’s sarcoma. It represents a liposome formulation loaded with Doxorubicin.[68, 69]

Finally, polymeric nanoparticles (Figure.7F) are colloidal system realized by amphiphilic molecules. This platform usually presents a spherical like structure with a size range of approximately 50-250 nm and it is made by

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using block of co-polymers to have both the hydrophobic and the hydrophilic part. Based on the synthesis procedure, it is possible to obtain two different systems: nanoparticles with a polymeric hydrophobic core and nanoparticles with an aqueous core surrounded by the polymer.[51] Several clinical studies have already used polymeric nanoparticles.[70] The choice of the material used is fundamental for the physico-chemical characterization, the pharmaco-kinetic properties, and the biodegradability of NPs. Biodegradable polymers are of particular interest, since they can be fully metabolized and removed from the body, creating non-toxic NPs.[71]Poly-lactic-co-glycolic acid (PLGA) is an especially intriguing example of a biodegradable polymer, since relative proportions of his components, polylactic acid (PLA) and polyglycolic acid (PGA), can be used to tune the biodegradability of PLGA.[72]

More recently, another nano-platform has been developed. It consists of an inner hydrophobic core made of the polymeric matrix (PLGA) and an external layer made of lipids monolayer. Some of these lipids are also conjugated with PEG to stabilize more the nanoparticle in circulation.[57, 58] This platform combine higher rate of loading efficiency of non-polar molecules, but presents also a lipid layer to resemble more to biologic vesicles.[73]

1.2.1 Innovative Anti-Thrombotic Strategies

In thrombolytic therapies, the rapid recanalization of occluded blood vessels is a key requirement to prevent extensive cell death and permanent tissue damage, and subsequent organ dysfunction and impair patient prognosis. Currently, major thrombotic events, such as ischemic stroke, myocardial infarction, pulmonary embolism, and venous thrombosis, are mostly treated with the systemic or local infusion of clot busting drugs.[74-76] Recombinant tissue plasminogen activator (rtPA) is the most effective and sole clinically approved of these drugs. This protein catalyzes the conversion of blood plasminogen into plasmin, which eventually breaks down the fibrin network holding together the cellular component of the clot.[37] However, this same protein is known to induce bleeding and modulate the permeability of the

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blood brain barrier leading to intracranial hemorrhages. For this reason, clinical protocols suggest the slow intravenous infusion of tPA for up to 2 hours, and only within the first 3 to 4.5 hours after symptom’s onset.[77]

Delay administrations are associated with a higher risk of developing intracranial hemorrhages, neuronal damage and permanent disabilities, and fatal outcome, as a consequence of the progressive non-specific deposition of tPA within the brain parenchyma.[78] All these safety-related restrictions significantly limit the number of patients that could benefit of tPA-based therapies. In the case of acute ischemic strokes, it is estimated that less than 5% of the patients would receive a tPA treatment and, out of them, 60% would either suffer permanent disabilities or die.[79, 80]

A more efficient clinical exploitation of thrombolytic therapies would require the administration of agents with a high affinity with blood clots and negligible deposition within the brain parenchyma in order to realize selective thrombolysis with no hemorrhagic complications. Three different strategies have been proposed to improve the specific delivery of clot busting agents to vascular occlusions: conjugation with ligand molecules; loading into nanoparticles; and association with erythrocytes.

Within the first strategy, Haber and colleagues conjugated tPA molecules to an anti-fibrin monoclonal antibody and achieved successful fibrinolysis in a rabbit thrombosis model with an overall 3-fold increase in potency, as compared to free tPA.[81] Collen et al. developed a series of antibodies against activated platelets which were used in vitro and in vivo for treating platelet rich clots.[82] The group of Yang devised a modular approach for originating thrombolytic compounds resulting from the electrostatic interaction between a negatively charged heparin–antifibrin complex and a modified tPA molecule.[83, 84] Although these specifically targeted molecular agents have been demonstrated over 15 years ago, their clinical application has been limited, due to their complexity, stability, immunogenicity, and reduced thrombolytic activity as compared to the original free active molecule.[85]

More recently, nanoparticles have been proposed as an alternative strategy. After the pioneering work of Heeremans and colleagues, who loaded tPA

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molecules in the aqueous core of conventional liposomes showing a 5-fold increase in thrombolytic potency,[86] a variety of nanoparticles with clot-specific targeting ability and triggered release of thrombolytic agents have been demonstrated.[87-89] For instance, the group of Lanza and Wickline prepared fibrin-targeted liquid perfluorocarbon nanoparticles for the delivery of streptokinase demonstrating in vitro enhanced thrombolysis upon ultrasound stimulation.[87] Vaidya et al. developed target-sensitive liposomes that would rapidly release tPA in the presence of activated platelets, thus realizing a selective drug release.[88] Pegylated liposomes were used to increase up to 20-times the half-life of tPA in the circulation.[89] Recently, the group of Letourneur demonstrated that fucoidan-labeled polymeric nanoparticles exhibit a strong tropism for the clot fibrin network and facilitate vessel recanalization.[90] Non-organic nanoparticles have also been used for thrombolysis. Clusters of iron oxide nanocubes, coated and stabilized by a tPA multi-layer, were proposed by Voros et al. for the combined chemical and thermal treatment of blood clots upon stimulation with exogenous alternating magnetic fields.[91] Ultrasmall superparamagnetic iron oxide nanoparticles, coated with fucoidan, were proposed for the magnetic resonance imaging (MRI) of blood clots.[92] The team led by Kim developed fibrin-targeted glycol chitosan–coated gold nanoparticles for assessing the clot stability via

computed tomography (CT) imaging.[93] The advantage of nanoparticles over the free or targeted tPA stays in the larger amounts of lytic agents that could be deployed at the clot site; the multivalent adhesive interactions with the clot surface; the higher stability and longer circulation half-life in vivo; the ability to trigger the release via exogenous and endogenous stimuli, and to image and treat clots simultaneously.

Micron-sized particles and blood cells have been also proposed for the specific delivery of thrombolytic molecules to blood clots. A notable example is given by the work of Korin and colleagues who developed  4 µm spherical polymeric microparticles resulting from the aggregation of multiple, smaller  200 nm tPA-carrying nanoparticles. The microscale aggregates were shown to be intact under physiological flow conditions and break apart under high shear stresses (> 100 dyne/cm2), which are typical of partially occluded

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vessels.[94] Although these shear-activated particles induced a 3-fold faster vessel recanalization in abdominal clots and rescued almost all tested mice with pulmonary emboli, they exhibited a short circulation half-life (< 5 min), which is comparable to free tPA, and a rapid accumulation in the liver (> 70% ID/g). At even a larger scale, erythrocytes (RBCs) were elegantly used by the group of Muzykantov to deliver thrombolytic agents to nascent clots.[95-97] These blood cells have a characteristic size of  7 µm but their deformability allows them to squeeze through the smallest capillaries in the lungs and splenic parenchyma, supporting their circulation for several days. Indeed, tPA-RBCs cannot infiltrate or firmly attach onto mature clots, because of their large size. However, they can certainly impair the formation of new clots and, as such, were proposed for thromboprophylaxis rather than for acute treatments.[95] Moreover, the large size of tPA-RBCs prevented the accumulation of free tPA within the brain tissue, dramatically reducing the risk of cerebral hemorrhages.[97] Indeed, a major technical challenge for the clinical implementation of this approach is the coupling of tPA with autologous RBCs and their reinjection into the patient.

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1.3

A

A thrombus, or blood clot, is the final product of the blood coagulation step in hemostasis. It is a healthy response to injury intended to prevent bleeding, but can be harmful in thrombosis, when clots obstruct blood flow through healthy blood vessels. A plethora of risk factors and pathologic conditions can lead to blood clot formation. The obstruction of the blood flow due to the presence of a thrombus in the arterial vessels can induce myocardial infarctions, ischemic strokes or trigger peripheral arterial diseases; whereas obstructions on the venous side can lead to deep vein thrombosis and pulmonary embolism.

Tissue plasminogen activator (tPA) is the sole approved therapeutic molecule for the treatment of acute ischemic stroke. Yet, only a small percentage of patients could benefit from this life-saving treatment because of medical contraindications and severe side effects, including brain hemorrhage, associated with delayed administration.

To overcome these side effects and enhance drug therapeutic efficacy, the reformulation of thrombolytic molecules into nanotechnological platforms is proposed here (Figure.8). A nano therapeutic agent is realized by directly associating the clinical formulation of tPA to the porous structure of soft discoidal polymeric nanoconstructs (tPA-DPNs). The porous matrix of DPNs protects tPA from rapid degradation, allowing tPA-DPNs to preserve over 70% of the tPA original activity after 3 h of exposure to serum proteins. Under dynamic conditions, tPA-DPNs dissolve clots more efficiently than free tPA, as demonstrated in a microfluidic chip where clots are formed mimicking in vivo conditions. At 60 min post treatment initiation, the clot area reduces by half (57 + 8%) with tPA-DPNs, whereas a similar result (56 + 21%) is obtained only after 90 min for free tPA. In murine mesentery venules, the intravenous administration of 2.5 mg/kg of tPA-DPNs resolves almost 90% of the blood clots, whereas a similar dose of free tPA successfully recanalize only about 40% of the treated vessels. At about 1/10 of the clinical dose (1.0 mg/kg), tPA-DPNs still effectively dissolve 70% of the clots, whereas free tPA works efficiently only on 16% of the vessels. In vivo, discoidal tPA-DPNs

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outperform the lytic activity of 200 nm spherical tPA-coated nanoconstructs in terms of both percentage of successful recanalization events and clot area reduction. The conjugation of tPA with preserved lytic activity, the deformability and blood circulating time of DPNs, together with the faster blood clot dissolution, make tPA-DPNs a promising nanotool for enhancing both potency and safety of thrombolytic therapies.

Figure.8 Graphical Abstract. Tissue plasminogen activator-conjugated discoidal polymeric nanoconstructs (tPA-DPNs) enhance the thrombolytic activity, extend the circulation half-life and prevent the rapid degradation of

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CHAPTER 2

D

P

N

2.1

E

P

2.1.1 Synthesis of Discoidal Polymeric Nanoconstructs (DPNs) Coated with tissue Plasminogen Activator (tPA)

Particles were synthesized using a top-down approach.[99, 100] A silicon master template was fabricated using a Laser Writer Lithography technique which allows to transfer on the silicon a specific pattern made out of discoidal wells with a diameter and a height characteristic for the nanoparticles. Then, a polydimethylsiloxane (PDMS – Sylgard 184) solution was transferred onto the master to obtain a template with the opposite shape of the Si master. After 4 h of polymerization at 60 °C, PDMS was peeled away and the cylindrical pillars on the template were covered with a poly(vinyl alcohol) (PVA) solution and left in the oven at 60 °C for 3 h. Once polymerized, the sacrificial PVA template presented the same cylindrical wells of the original Si master.

DPNs are composed by a mixture of (poly(lactic acid-co-glycolic acid) (PLGA) and poly(ethylene glycol) diacrylate (PEG diacrylate) polymers. 30 mg of PLGA were dissolved in 600 μl of dichloromethane and chloroform, and mixed with 6 mg of PEG. Then, 0.6 mg of a photo-initiator (2-Hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone) were added into the polymeric solution to allow a further polymerization of PEG diacrylate. The polymeric mixture was spread into the PVA wells and the loaded templates were then exposed to UV-light for 10 min. Hydrophilic PVA templates were dissolved in deionized water for 3 h under stirring conditions at room temperature. Nanoparticles were released from the wells of PVA, and then collected

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through centrifugation (3,900 rpm for 20 min) and purified from PVA debris with 2 μm filters.

Purified DPNs were incubated with EDC/NHS - in a molar ratio of 3:1 (EDC/NHS:PLGA) - for 5 h under rotation at room temperature. Unlinked activators were removed with washing steps (20 min of centrifuge at maximum speed). Activated DPNs were incubated O/N with 60 µg of tPA. Unlinked drug was removed with washing steps (20 min of centrifuge at maximum speed).

2.1.2 Physico-Chemical Characterization of DPNs and tPA-DPNs DPNs and tPA-DPNs size and concentration were obtained using a Multisizer 4E Coulter Particle Counter (Beckman Coulter, USA). Particles were synthesized and suspended in isotone solution. Superficial charge (ζ) was measured using Zetasizer Nano (Malvern, UK). Particles were synthesized and suspended in 1 ml of deionized water in a cuvette after shortly sonication. DPNs size and shape were observed using a Jem-1011 Transmission Electron Microscope (Jeol), a JSM 6490LA Scanning Electron Microscope (Jeol), and a MCF-MC-23 Atomic Force Microscope (Park Systems). In particular, for TEM analysis, DPNs were synthesized, drop casted on a carbon grid and analyzed; for SEM analysis, DPNs were synthesized, sputtered with 10 nm of gold, and analyzed operating at an acceleration voltage of 100 kV. Fluorescent DPNs were synthesized adding 30 µg of Rhodamin-B (lipid-RhodB) to the polymeric mix made of PLGA and PEGDA. RhB-DPNs were activated, incubated with FITC-tPA O/N, and suspended in PBS. Confocal images were collected with a Nikon A1 Confocal Microscope.

2.1.3 Drug Encapsulation Efficiency

To analyze the encapsulation efficiency, DPNs were incubated with different amount of tPA: 10, 20, 30, 40, 50, 60, and 70 µg. The amount of tPA loaded

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on DPNs was measured using the Bicinchoninic protein assay kit (BCA). 200 µl of Reagent were added to 25 µl of each sample of tPA-DPNs. The optical density (OD 562) was read at the micro-plate reader (Tecan, CH) after 45 min of incubation at 37 °C. The concentration of the drug was extrapolated by a calibration curve prepared before with different concentration of tPA dissolved in PBS.

2.1.4 tPA Stability

The release profile was measured analyzing the drug still loaded on DPNs with BCA kit at different time points: 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h. In particular, particles were synthesized and incubated in PBS at 37 °C. At each time point, tPA-DPNs were centrifuged to eliminate the released drug. The pellets were suspended in PBS and the optical density (OD 562) was read.

2.1.5 Enzymatic Activity Test

tPA activity was tested after the conjugation to DPNs using a chromogenic activity assay test (abcam108905). Particles were synthesized and incubated up to 2 h with the assay mix composed by plasminogen and plasmin substrate. The assay measures the ability of Tissue type Plasminogen Activator to activate the plasminogen to plasmin. The amount of plasmin produced is measured using a specific plasmin substrate releasing a yellow para-nitroaniline (pNA) chromophore. The change in absorbance of the pNA in the reaction solution at 405 nm is directly proportional to the Tissue type Plasminogen Activator enzymatic activity.

2.1.6 Interaction of tPA-DPNs with Endothelial Cells 2.1.6.1 Confocal Analysis

tPA-DPNs internalization into HUVECs was analyzed via a confocal fluorescent microscopy. DPNs were again synthesized with lipid-RhB and

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incubated with FITC-tPA overnight. HUVECs were seeded in 8-well dishes and incubated with FITC-tPA-RhB-DPNs for 24 h. In particular, 103 HUVECs were incubated with 10 DPNs per cell. After 24 h, cells were fixed with 3.7% of PFA for 10 min, washed with PBS, and stained with 4',6-diamidino-2-phenylindole (DAPI) staining and Wheat Germ Agglutinin (WGA) staining for nuclei and glycosylated membrane proteins, respectively.

2.1.6.2 FACS Analysis

DPNs and tPA-DPNs internalization into Human Umbilical Vein Endothelial Cells (HUVECs) was also analyzed via Flow Cytometry analysis. DPNs were synthesized with lipid-RhB. HUVEC cells were seeded in 6-well dishes and incubated with DPNs and tPA-DPNs for 24 h. In particular, 120  103 HUVECs were incubated with 10 DPNs per cell. After 24 h, cells were washed with PBS, detached from the dishes with a scraper, suspended in a final volume of 1 ml of medium, and then analyzed on Becton Dickinson FACSAria (BD-Biosciences).

2.1.7 Cytotoxicity Studies

To assess DPN toxicity, HUVECs were incubated with DPNs and tPA-DPNs. Briefly, 103 cells were seeded in 96-well plates. After 36 h, fresh endothelial cells medium containing different concentrations of DPNs was added to cells, subsequently incubated for 24 and 72 h. In particular, HUVECs were treated with 1, 5, 10, and 20 DPNs per cell. After 24 and 72 h, MTT-dye was mixed with medium (50 mg/ml) and added to the samples. After 4 h of incubation, the MTT solution was replaced with Ethanol (90%) to create formazan dye, and the optical density (OD 490) was read using a micro-plate reader. MTT test was also performed to analyze the toxicity of tPA on HUVEC cells. In particular, cells were treated for 3, 6, and 9 h with 5, 10, 20, 30, and 40 µg/ml of tPA.

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2.2

E

2.2.1 Synthesis and Physico-Chemical Characterization of tPA-DPNs

The synthesis of DPNs is a multistep, top-down process.[99-101] Briefly, laser writing lithography is used to realize a pattern with billions of wells, reproducing the characteristic geometry of DPNs, in a silicon master template (Figure.9A). Then, via replica molding, this template is used to generate an intermediate PDMS template (Figure.9B) and eventually a sacrificial PVA template (Figure.9C).

Figure.9 Synthesis of Discoidal Polymeric Nanoconstructs. A-C. Electron microscopy images of the original master Si template (A), intermediate PDMS template (B) and final sacrificial PVA template (C)

employed for realizing 1,000 x 400 nm DPNs.

In the current configuration, DPNs are circular disks with a diameter of 1,000 nm, a height of 400 nm, and are composed by an homogeneous mixture of Poly(D,L-lactide-co-glycolide)-acid carboxylic terminated (PLGA-COOH) and poly(ethylene glycol) diacrylate (PEG-DA). Note that these polymers are among the most studied and best characterized polymers for biomedical applications, and have been approved by various regulatory agencies for diverse clinical applications.[102, 103]

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This polymeric mixture, together with a photoinitiator, is deposited into the wells of the PVA template and exposed to a UV-light source for crosslinking. After polymerization, the sacrificial PVA template is dissolved into an aqueous solution at room temperature, under gentle stirring, and, upon centrifugation and filtration, DPNs are collected. Note that DPNs are hydrogel-based particles with a porous structure. Finally tPA is directly associated to the resuspended DPNs. Specifically, the carboxylic groups on the PLGA chains are activated, via an EDC/NHS reaction, and then covalently coupled to one of the amine groups on the tPA molecules (Figure.10A). A schematic representation of tPA-DPNs is depicted in Figure.10B.

Figure.10 Synthesis of Discoidal Polymeric Nanoconstructs. A. Reaction steps for the covalent conjugation of tissue Plasminogen Activator molecules to the DPN surface. B. Schematic representation of tPA-DPNs, highlighting

the porous structure of DPNs and their direct conjugation with tPA.

The geometrical features of DPNs and tPA-DPNs were assessed via a Multisizer Particle Counter, a Zetasizer Nano, electron microscopy, and atomic force microscopy (Figure.11-12). The Multisizer Particle Counter spectra for the DPNs and tPA-DPNs are almost perfectly overlapped, as shown in Figure.11 (orange: tPA-DPNs; green: empty DPNs). After the reaction with tPA, DPNs presented a negligible increase in average size (<

- 33 -

3%), from 784 + 10 nm (DPNs) to 803 + 15 nm (tPA-DPNs). On the other hand, the change in surface ζ potential was more relevant, increasing from a negative value of -21 + 0.6 mV for DPNs to a positive value of 16 + 0.4 mV for tPA-DPNs. This large variation in surface ζ potential has to be ascribed to the tPA conjugation with the carboxylic groups presented on the PLGA chains and the progressive neutralization of the originally negative surface electrostatic charge of DPNs. As such, the change in surface ζ potential can be also used as an indirect method to quantify the efficiency of tPA conjugation.

Figure.11 Physico-Chemical Properties of Discoidal Polymeric Nanoconstructs. Multisizer analysis of DPNs (green) and tPA-DPNs

(orange).

A representative Transmission electron Microscopy (TEM) image of DPNs is given in Figure.12A, which confirms the shape with a circular base of about 1 µm in diameter. In the same figure, the upper-left inset shows an image deriving from the superimposition of an electron microscopy and a fluorescent microscopy picture for the same DPN, which was loaded with lipid-Rhodamine B (RhB). A Scanning Electron Microscopy (SEM) image and

- 34 -

an Atomic Force Microscopy (AFM) image of DPNs are given in Figure.12B-C, respectively, which document a DPN diameter of  1.2 µm, for both SEM and AFM, and height of  347 nm for SEM and  300 nm for AFM.

Figure.12 Microscope Analysis of tPA-DPNs. A. TEM image of DPNs demonstrating the circular shape with a base diameter of  1,000 nm. The

upper-left inset shows a fluorescent microscopy image of a RhB-DPN superimposed on its TEM image. B. SEM image of DPNs demonstrating the

diameter of  1,200 nm and the height of  347 nm. C. AFM image of DPNs demonstrating the diameter of  1,100 nm and the height of  300 nm.

Finally, as depicted in Figure.13, tPA-DPNs were demonstrated to be geometrically stable, upon storage in saline at 37 °C, with an overall variation in diameter of 6% over six days.

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Figure.13 Size stability of tPA-DPNs. tPA-DPNs stability in PBS at 37 °C,

via DLS analysis. [n = 3]

In addition to the size, the surface -potential of tPA-DPNs has been characterized using a zeta-size system. As documented in Figure.14A below, the surface -potential reduces slowly with time, from + 11.5  1.03 mV at day 1 to + 6.97  1.29 mV at day 2, + 3.44  2.09 mV at day 3, and eventually to – 4.49  0.9 mV at day 5. This variation would be ascribed to the progressive degradation of tPA molecules. The Figure.14B documents the steady increase in the surface -potential of DPNs with the tPA amount. This should be ascribed to the progressive neutralization of the PLGA-COOH groups exposed on the DPN surface by tPA.

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Figure.14 Variation of the Surface -potential of tPA-DPNs. A. Progressive reduction of the tPA-DPN surface -potential to be mostly ascribed to tPA degradation. [n = 3] B. Progressive increase of the tPA-DPN

surface -potential to be mostly ascribed to the neutralization of the PLGA-COOH groups exposed on the DPN surface by tPA. [n = 3]

The pharmacological properties of DPNs were characterized for the tPA encapsulation efficiency and release profile. First, to confirm the firm conjugation of the thrombolytic agent to the particle structure, DPNs were grafted with FITC-tPA and loaded with lipid-RhB. This fluorescent lipid was dispersed uniformly within the PLGA/PEG matrix before particle synthesis. In Figure.15A, projections of tridimensional confocal microscopy images show the green fluorescence associated with FITC-tPA properly co-localized with the red fluorescence given by the lipid-RhB. The full reconstruction is

- 37 -

presented in the Figure.15B. Note that the green signal, associated with FITC-tPA, and red signal, associated with lipid-RhB, co-localize and are quite uniformly distributed within the DPN polymeric matrix. This implies that tPA interacts with the PLGA-COOH chains throughout the nanoconstruct matrix and is not solely restricted to the DPN surface. This also demonstrates the ability to load multiple agents within the tPA-DPN matrix.

Figure.15 Co-loading in Discoidal Polymeric Nanoconstructs. A. Confocal images of DPNs co-loaded with FITC-tPA (green) and lipid-RhB (red), demonstrating the uniform distribution of both compounds throughout the porous DPN matrix. B. Six-plane projection of confocal images for DPNs

- 38 -

Moreover, in order to assess the tPA encapsulation efficiency, 5 x 108 DPNs were incubated with different amounts of tPA, ranging from 10 to 70 µg. As depicted in Figure.16A, the amount of tPA conjugated to DPNs grows linearly with the input dose returning an almost 1:1 ratio (dashed line), thus implying an encapsulation efficiency close to 100%. For 5 x 108 DPNs, saturation is reached at about 60 µg of tPA. Indeed, the total tPA amount can be finely tuned by changing the number of DPNs. Given the hydrogel nature of these DPNs, tPA molecules are expected to react with the PLGA-COOH groups exposed on the surface as well as those available within the polymer matrix. Finally, the possible release of tPA from DPNs was analyzed upon particle incubation in PBS, at 37 °C, up to 72 h. Figure.16B shows that the largest majority of tPA (i.e. > 90%) is associated with the DPN structure throughout the observation period, which demonstrates the stable conjugation of the lytic agent. It is here important to highlight that this feature of tPA-DPNs together with their relatively large size would limit the non-specific accumulation of tPA molecules within the brain parenchyma and possibly reduce the risk of hemorrhagic events.

Figure.16 Loading and Stability of tPA-DPNs. A. Amount of tPA associated with DPNs (0.5109

) for different mass inputs of the thrombolytic agent. The dashed line represents a 1:1 ratio between associated tPA and input, demonstrating an association efficiency of almost 100%. [n = 3] B. tPA

release from tPA-DPNs over time, at 37 °C, demonstrating a stable association. [n = 3]

- 39 -

To assess if the conjugation of tPA to the DPNs surface affects the activity of the drug, an enzymatic assay of free and conjugated tPA was performed. This in vitro assay measures the ability of tPA to activate the plasminogen to plasmin, using a plasmin substrate releasing a yellow para-nitroaniline (pNA) chromophore. Three different concentrations were tested, namely 10, 20, and 50 µg/ml of drug, up to 2 h. As depicted in Figure.17, the activity of tPA after the conjugation with the carboxylic group of PLGA is retained. No statistically significant difference is observed between free or bound tPA.

Figure.17 Enzymatic Activity Test. [n = 3]

2.2.2 Characterization of tPA-DPNs in Biological Conditions

The propensity of DPNs to resist internalization by HUVEC cells was assessed using two different biological assays, including confocal and flow cytometry (FC) analysis.

Fluorescent tPA-DPNs were incubated with HUVEC cells seeded in 8-well dishes for 24 h. In particular, 103 HUVECs were incubated with 10 DPNs per cell. The Figure.18A documents no uptake of tPA-DPNs by HUVECs. It is here important to recall that DPNs, in different size and shape format, have been shown not be easily uptaken even by professional phagocytic cells.[100]

- 40 -

As a complementary and more quantitative assay, a flow cytometry analysis was performed. Fluorescent DPNs and tPA-DPNs were incubated with HUVEC cells seeded in 6-well dishes for 24 h. In particular, 120  103 HUVECs were incubated with 10 DPNs per cell. Results expressed in terms of percentage of cells associated with DPNs are summarized in Figure.18B. The FC results confirm the overall picture that has emerged through the confocal microscopy studies. No significant internalization was observed for both empty DPNs and tPA-DPNs (Figure.18B-D). This is remarkable, given the different surface potentials of the two nanoconstructs: slightly negative for the empty DPNs (-21  0.6 mV), and slightly positive for the tPA-DPNs (+16  0.4 mV).

Figure.18 Internalization Analysis of DPNs and tPA-DPNs. A Confocal microscopy analysis of tPA-DPNs incubated with HUVECs. B Percentage of DPN internalization into HUVECs quantified through flow cytometry analysis. [n = 3] C-D. Flow cytometry analysis of DPNs and tPA-DPNs interaction with

- 41 -

In the presence of HUVEC cells, the potential cytotoxicity of the DPNs and tPA-DPNs was tested using an MTT assay. In particular, HUVECs were treated with 1, 5, 10, and 20 DPNs per cell for 24 and 72 h. No significant toxicity was documented with both particles as shown in the Figure.19A. MTT test was also performed to analyze the toxicity of tPA on HUVEC cells. In particular, cells were treated for 3, 6, and 9 h with: 5, 10, 20, 30, and 40 µg/ml of tPA. The bar chart in Figure.19B confirms the lack of any toxicity with a cell survival of about 100%.

Figure.19 Cytotoxicity Analysis of DPNs and tPA-DPNs. A. Cytotoxicity analysis of DPNs and tPA-DPNs at 24 and 72 h post incubation with HUVECs. B. Cytotoxicity of free tPA on HUVECs at different time points. [n =

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CHAPTER 3

S

P

N

3.1

E

P

3.1.1 Synthesis and Characterization of Spherical Polymeric Nanoconstructs (SPNs) Coated with tissue Plasminogen Activator (tPA)

Spherical polymeric nanoconstructs (SPNs) were synthesized using an emulsion/solvent evaporation technique described previously.[104, 105] Briefly, the aqueous phase was obtained dissolving 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[Carboxy(Polyethylene Glycol)-2000] (DSPE-PEG) in a 4% ethanol solution, whereas the oil phase was obtained dissolving PLGA and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in chloroform. The oil phase was added in a dropwise manner to the aqueous solution under ultrasonication for 1 min and 30 sec at 100% amplitude (Q125 sonicator, Q-Sonica). To allow chloroform evaporation, the resulting emulsion was then gently stirred at room temperature for 3 h. Finally, nanoparticles were purified and collected through centrifugation steps. To conjugate tPA to SPNs, the same approach used for DPNs was adopted. In particular, SPNs were incubated with EDC/NHS in a molar ratio of 3:1 (EDC/NHS : DSPE-PEG) for 5 h under rotation, at room temperature. Unreacted activators were removed with washing steps (20 min of centrifuge at maximum speed). Activated SPNs were incubated O/N with 60 µg of tPA. Unconjugated tPA was removed with washing steps (20 min of centrifuge at maximum speed). SPNs and tPA-SPNs size and surface zeta potential were measured by dynamic light scattering (DLS). For scanning electron microscopy (SEM) analysis, the SPN solution was dropped directly onto a silicon wafer. After

- 44 -

drying, samples were sputter-coated with platinum prior imaging, to enhance polymer contrast.

- 45 -

3.2

E

For comparison with DPNs, Spherical polymeric nanoconstructs (SPNs) were also prepared. As depicted in the schematic representation of the particles in Figure.20, these nanoparticles comprise a PLGA core and a lipid surface layer, and are synthesized via an emulsion technique. Similarly to tPA-DPNs, carboxylic groups exposed on the SPN surface are activated, via an EDC/NHS reaction, and then covalently coupled to the amine groups on the tPA molecules returning the tPA-SPNs.

Figure.20 Schematic Representation of SPNs.[104]

Figure.21A shows a slight increase in SPN size upon tPA conjugation from 221 + 1.6 nm to 240 + 0.2 nm, and a decrease in surface zeta potential from -37 + 0.5 mV to -8 + 0.6 mV.

Moreover, tPA-SPNs were synthesized, sputter-coated with platinum prior imaging, to enhance polymer contrast, and analyzed at SEM (Figure.21B).

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Figure.21 Characterization of Spherical Polymeric Nanoconstructs. A. DLS analysis of SPNs (blue) and tPA-SPNs (red). B. SEM image of SPNs.

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CHAPTER 4

I

V

T

E

4.1

E

P

4.1.1 In Vitro Efficacy of tPA-DPNs and tPA-SPNs in Static Conditions

Blood was obtained from rats after standard procedures. Anesthesia was calculated on the base of an adult rat Sprague Dawley of about 300 gr. The anesthesia was inducted with a cotton swab full of isoflurane put inside the cage. Then, 80 μl of Xilazina i.m. (right gluteus), and, after 5 min, 300 μl of Ketamine were administered. After the complete sedation of the rat, confirmed with the podal reflection, the sternum was removed and a syringe of 5 ml with a hypodermic needle of 21 G was inserted into the left ventricle to gently aspirate the blood.

Blood clots were prepared adding into several tubes 50 U of thrombin solution with 100 μl of whole blood. After 30 min of maturation at 37 °C with continuous shaking at 50 rpm, clots were moved from each Eppendorf tube into a 24-multiwells dish with 1.5 ml of saline solution for each well.

Dissolution and dissolution rate of blood clots were measured estimating the amount of hemoglobin released from the clots over time. Clots were treated with PBS, free tPA, DPNs, SPNs, tPA-DPNs, and tPA-SPNs. In particular, they were treated with 10, 30, and 50 µg of tPA/clot. Clots were incubated at 37 °C with continuous shaking at 50 rpm for 300 min. At times 0, 30, 90, 180, and 300 min post treatments, 200 μl of supernatant were placed in a 96-multiwells dish and the optical density (OD 415) of the hemoglobin released in the dissolution was measured in a micro-plate reader. The dissolution rate (DR) was measured with the following formula:

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𝐷𝑅 = (𝑂𝐷_𝑖 − 𝑂𝐷₀) (𝑡⁄ _𝑖 − 𝑡₀) where i and 0 are two consecutive time points.

Moreover, to establish the possible interference of the protein corona, first, the same experiment was performed with FBS instead of PBS and, then, tPA-DPNs and tPA-SPNs were incubated for 0.5, 1, and 3 h with FBS and, then, tested to dissolve blood clots in PBS.

4.1.2 Blood Clot Characterization

To analyze the structural changing of blood clots before and after the treatment with tPA-DPNs, clots were fixed for 2 h in a fixative solution (2% Glutaraldehyde, 2% Paraformaldehyde in buffer Na-Cacodylate 0.1 M) and then post-fixed (2 h) in a solution 1% OsO4, 1.5% Hexacyanoferrate in Na-cacodylate buffer. Samples were then dehydrated with series of alcohols. After complete dehydration they were infiltrated with a scale of Ethanol, treated with hexametyldisilazane (HMDS) solution, left in HMDS for some hours and finally left under the hood over/night to let the evaporation of HMDS. Samples were then sputtered with 10 nm of gold.

SEM images were collected with a Jeol JSM 6490-LA (Jeol, Japan) electron microscope, operating at an acceleration voltage of 10 kV.

4.1.3 Fabrication of Microfluidic Chip

The chips were fabricated by using a replica molding approach as previously described.[106] It consists in two lithographic steps (etching of the pillars and etching of the whole chip) for the creation of a silicon master that was then replicated by polydimethylsiloxane (PDMS - Sylgard 182). The microfluidic chip consists in two micro-channels only connected via a 500 µm long array of pillars. The upper and lower channels have a height of 50 µm and a width of 200 µm.

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The Si template pattern was replicated by using a pre-polymer solution of Sylgard 182. PDMS was mixed with the curing agent in a ratio (w/w) 10:1. The solution was degassed in a vacuum chamber, and casted on the Si template. PDMS was cured in the oven at 60 °C for 4 h, cooled down to -20 °C for 1 h, and peeled-off from the Si template.

PDMS templates were punched to create four holes (2 inlets and 2 outlets) for the handling of the fluids. Templates were treated with O2 plasma and

bonded with a glass coversheet.

4.1.4 In Vitro Efficacy of tPA-DPNs and tPA-SPNs in Dynamic Conditions

Blood was collected in heparin containing tubes. To create the clot, two tubes, connected to the syringe pump, were inserted in the two inlets on the microfluidic chip. One tube was filled with whole blood, and the other with thrombin solution and CaCl2. Once the two solutions reached the pillars, they

entered in contact and the clot started to form.

Tubes with blood and thrombin were replaced with tubes containing the different treatments: PBS, DPNs, SPNs, tPA, tPA-DPNs, and tPA-SPNs. In particular, clots were treated with 30 µg/ml of tPA. The flow rate imposed on the syringe pump was 100 nl/min. A movie of the clot dissolution was acquired at the time-lapse microscope for 2 h. The area of the clots was measured over time with ImageJ. Since the initial size of the clots was different throughout the experiments, the average of the size percentage was weighted based on the initial size of each clot. The weighted area was calculated by using the following formula:

𝒙̅ = ∑ 𝑨𝒊𝑨𝟎

𝒏 𝒊=𝟏

∑𝒏 𝑨_𝟎

𝒊=𝟏