Exploring Mechanisms of Lipid Nanoparticle-Mucus Interactions in Healthy and Cystic Fibrosis Conditions.

Mucus forms the first defense line of human lungs, and as such hampers the efficient delivery of therapeutics to the underlying epithelium. This holds particularly true for genetic cargo such as CRISPR-based gene editing tools which cannot readily surmount the mucosal barrier. While lipid nanoparticles (LNPs) emerge as versatile non-viral gene delivery systems that can help overcome the delivery challenge, many knowledge gaps remain, especially for diseased states such as cystic fibrosis (CF). This study provides fundamental insights into Cas9 mRNA or ribonucleoprotein-loaded LNP-mucus interactions in healthy and diseased states by assessing the impact of the genetic cargo, mucin sialylation, mucin concentration, ionic strength, pH, and polyethylene glycol (PEG) concentration and nature on LNP diffusivity leveraging experimental approaches and Brownian dynamics (BD) simulations. Taken together, this study identifies key mucus and LNP characteristics that are critical to enabling a rational LNP design for transmucosal delivery.

RNA therapeutics to control fibrinolysis: review on applications in biology and medicine.

Regulation of fibrinolysis, the process that degrades blood clots, is pivotal in maintaining hemostasis. Dysregulation leads to thrombosis or excessive bleeding. Proteins in the fibrinolysis system include fibrinogen, coagulation factor XIII, plasminogen, tissue plasminogen activator, urokinase plasminogen activator, α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, plasminogen activator inhibitor-1, α2-macroglobulin, and others. While each of these is a potential therapeutic target for diseases, they lack effective or long-acting inhibitors. Rapid advances in RNA-based technologies are creating powerful tools to control the expression of proteins. RNA agents can be long-acting and tailored to either decrease or increase production of a specific protein. Advances in nucleic acid delivery, such as by lipid nanoparticles, have enabled the delivery of RNA to the liver, where most proteins of coagulation and fibrinolysis are produced. This review will summarize the classes of RNA that induce 1) inhibition of protein synthesis, including small interfering RNA and antisense oligonucleotides; 2) protein expression, including messenger RNA and self-amplifying RNA; and 3) gene editing for gene knockdown and precise editing. It will review specific examples of RNA therapies targeting proteins in the coagulation and fibrinolysis systems and comment on the wide range of opportunities for controlling fibrinolysis for biological applications and future therapeutics using state-of-the-art RNA therapies.

Lipid nanoparticles and siRNA targeting plasminogen provide lasting inhibition of fibrinolysis in mouse and dog models of hemophilia A.

Antifibrinolytic drugs are used extensively for on-demand treatment of severe acute bleeding. Controlling fibrinolysis may also be an effective strategy to prevent or lessen chronic recurring bleeding in bleeding disorders such as hemophilia A (HA), but current antifibrinolytics have unfavorable pharmacokinetic profiles. Here, we developed a long-lasting antifibrinolytic using small interfering RNA (siRNA) targeting plasminogen packaged in clinically used lipid nanoparticles (LNPs) and tested it to determine whether reducing plasmin activity in animal models of HA could decrease bleeding frequency and severity. Treatment with the siRNA-carrying LNPs reduced circulating plasminogen and suppressed fibrinolysis in wild-type and HA mice and dogs. In HA mice, hemostatic efficacy depended on the injury model; plasminogen knockdown improved hemostasis after a saphenous vein injury but not tail vein transection injury, suggesting that saphenous vein injury is a murine bleeding model sensitive to the contribution of fibrinolysis. In dogs with HA, LNPs carrying siRNA targeting plasminogen were as effective at stabilizing clots as tranexamic acid, a clinical antifibrinolytic, and in a pilot study of two dogs with HA, the incidence of spontaneous or excess bleeding was reduced during 4 months of prolonged knockdown. Collectively, these data demonstrate that long-acting antifibrinolytic therapy can be achieved and that it provides hemostatic benefit in animal models of HA.

Genetically engineered transfusable platelets using mRNA lipid nanoparticles.

Platelet transfusions are essential for managing bleeding and hemostatic dysfunction and could be expanded as a cell therapy due to the multifunctional role of platelets in various diseases. Creating these cell therapies will require modifying transfusable donor platelets to express therapeutic proteins. However, there are currently no appropriate methods for genetically modifying platelets collected from blood donors. Here, we describe an approach using platelet-optimized lipid nanoparticles containing mRNA (mRNA-LNP) to enable exogenous protein expression in human and rat platelets. Within the library of mRNA-LNP tested, exogenous protein expression did not require nor correlate with platelet activation. Transfected platelets retained hemostatic function and accumulated in regions of vascular damage after transfusion into rats with hemorrhagic shock. We expect this technology will expand the therapeutic potential of platelets.

Lipid Nanoparticle-Mediated Hit-and-Run Approaches Yield Efficient and Safe In Situ Gene Editing in Human Skin.

Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for in situ gene editing. We observed distinct LNP composition and cell-specific effects such as an extended presence of RNP in slow-cycling epithelial cells for up to 72 h. While obtaining similar gene editing rates using Cas9 RNP and mRNA with MC3-based LNPs (10-16%), mRNA-loaded LNPs proved to be more cytotoxic. Interestingly, ionizable lipids with a pKa ∼ 7.1 yielded superior gene editing rates (55%-72%) in two-dimensional (2D) epithelial cells while no single guide RNA-dependent off-target effects were detectable. Unexpectedly, these high 2D editing efficacies did not translate to actual skin tissue where overall gene editing rates between 5%-12% were achieved after a single application and irrespective of the LNP composition. Finally, we successfully base-corrected a disease-causing mutation with an efficacy of ∼5% in autosomal recessive congenital ichthyosis patient cells, showcasing the potential of this strategy for the treatment of monogenic skin diseases. Taken together, this study demonstrates the feasibility of an in situ correction of disease-causing mutations in the skin that could provide effective treatment and potentially even a cure for rare, monogenic, and common skin diseases.

Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency.

The transfection potency of lipid nanoparticle (LNP) mRNA systems is critically dependent on the ionizable cationic lipid component. LNP mRNA systems composed of optimized ionizable lipids often display distinctive mRNA-rich "bleb" structures. Here, it is shown that such structures can also be induced for LNPs containing nominally less active ionizable lipids by formulating them in the presence of high concentrations of pH 4 buffers such as sodium citrate, leading to improved transfection potencies both in vitro and in vivo. Induction of bleb structure and improved potency is dependent on the type of pH 4 buffer employed, with LNP mRNA systems prepared using 300 mm sodium citrate buffer displaying maximum transfection. The improved transfection potencies of LNP mRNA systems displaying bleb structure can be attributed, at least in part, to enhanced integrity of the encapsulated mRNA. It is concluded that enhanced transfection can be achieved by optimizing formulation parameters to improve mRNA stability and that optimization of ionizable lipids to achieve enhanced potency may well lead to improvements in mRNA integrity through formation of the bleb structure rather than enhanced intracellular delivery.

Elimination of fibrin polymer formation or crosslinking, but not fibrinogen deficiency, is protective against diet-induced obesity and associated pathologies.

BACKGROUND: Obesity predisposes individuals to metabolic syndrome, which increases the risk of cardiovascular diseases, non-alcoholic fatty liver disease (NAFLD), and type 2 diabetes. A pathological manifestation of obesity is the activation of the coagulation system. In turn, extravascular fibrin(ogen) deposits accumulate in adipose tissues and liver. These deposits promote adiposity and downstream sequelae by driving pro-inflammatory macrophage function through binding the leukocyte integrin receptor αM ß2 . OBJECTIVES: An unresolved question is whether conversion of soluble fibrinogen to a crosslinked fibrin matrix is required to exacerbate obesity-driven diseases. METHODS: Here, fibrinogen-deficient/depleted mice (Fib- or treated with siRNA against fibrinogen [siFga]), mice expressing fibrinogen that cannot polymerize to fibrin (FibAEK ), and mice deficient in the fibrin crosslinking transglutaminase factor XIII (FXIII-) were challenged with a high-fat diet (HFD) and compared to mice expressing a mutant form of fibrinogen lacking the αM ß2 -binding domain (Fib𝛾390-396A ). RESULTS AND CONCLUSIONS: Consistent with prior studies, Fib𝛾390-396A mice were significantly protected from increased adiposity, NAFLD, hypercholesterolemia, and diabetes while Fib- and siFga-treated mice gained as much weight and developed obesity-associated pathologies identical to wildtype mice. FibAEK and FXIII- mice displayed an intermediate phenotype with partial protection from some obesity-associated pathologies. Results here indicate that fibrin(ogen) lacking αM ß2 binding function offers substantial protection from obesity and associated disease that is partially recapitulated by preventing fibrin polymer formation or crosslinking of the wildtype molecule, but not by reduction or complete elimination of fibrinogen. Finally, these findings support the concept that fibrin polymerization and crosslinking are required for the full implementation of fibrin-driven inflammation in obesity.

Aortic intimal resident macrophages are essential for maintenance of the non-thrombogenic intravascular state.

Leukocytes and endothelial cells frequently cooperate to resolve inflammatory events. In most cases, these interactions are transient in nature and triggered by immunological insults. Here, we report that in areas of disturbed blood flow, aortic endothelial cells permanently and intimately associate with a population of specialized macrophages that are recruited at birth from the closing ductus arteriosus and share the luminal surface with the endothelium becoming interwoven in the tunica intima. Anatomical changes that affect hemodynamics, like in patent ductus arteriosus, alter macrophage seeding to coincide with regions of disturbed flow. Aortic resident macrophages expand in situ via direct cell renewal. Induced-depletion of intimal macrophages led to thrombin-mediated endothelial cell contraction, progressive fibrin accumulation and formation of microthrombi that, once dislodged, caused blockade of vessels in several organs. Together the findings revealed that intravascular resident macrophages are essential to regulate thrombin activity and clear fibrin deposits in regions of disturbed blood flow.

Suppression of fibrin(ogen)-driven pathologies in disease models through controlled knockdown by lipid nanoparticle delivery of siRNA.

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

Hypofibrinogenemia with preserved hemostasis and protection from thrombosis in mice with an Fga truncation mutation.

Genetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ∼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga-/- mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5'-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite ∼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ∼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga-/- mice. Decreasing the normal fibrinogen levels to ∼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier.

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils through the αMß2 integrin receptor and activates effector functions, including the production of reactive oxygen species and neutrophil extracellular trap formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis in mice and humans. Concordantly, genetic polymorphisms in PLG, encoding plasminogen, are associated with common forms of periodontal disease. Thus, fibrin is a critical regulator of neutrophil effector function, and fibrin-neutrophil engagement may be a pathogenic instigator for a prevalent mucosal disease.

Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles.

Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

Emerging gene therapies for enhancing the hemostatic potential of platelets.

Platelet transfusions are an integral component of balanced hemostatic resuscitation protocols used to manage severe hemorrhage following trauma. Enhancing the hemostatic potential of platelets could lead to further increases in the efficacy of transfusions, particularly for non-compressible torso hemorrhage or severe hemorrhage with coagulopathy, by decreasing blood loss and improving overall patient outcomes. Advances in gene therapies, including RNA therapies, are leading to new strategies to enhance platelets for better control of hemorrhage. This review will highlight three approaches for creating modified platelets using gene therapies: (i) direct transfection of transfusable platelets ex vivo, (ii) in vitro production of engineered platelets from platelet-precursor cells, and (iii) modifying the bone marrow for in vivo production of modified platelets. In summary, modifying platelets to enhance their hemostatic potential is an exciting new frontier in transfusion medicine, but more preclinical development as well as studies testing the safety and efficacy of these agents are needed.

Spontaneous, solvent-free entrapment of siRNA within lipid nanoparticles.

Lipid nanoparticle (LNP) formulations of nucleic acid are leading vaccine candidates for COVID-19, and enabled the first approved RNAi therapeutic, Onpattro. LNPs are composed of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced using rapid-mixing techniques. These procedures involve dissolution of the lipid components in an organic phase and the nucleic acid in an acidic aqueous buffer (pH 4). These solutions are then combined using a continuous mixing device such as a T-mixer or microfluidic device. In this mixing step, particle formation and nucleic acid entrapment occur. Previous work from our group has shown that, in the absence of nucleic acid, the particles formed at pH 4 are vesicular in structure, a portion of these particles are converted to electron-dense structures in the presence of nucleic acid, and the proportion of electron-dense structures increases with nucleic acid content. What remained unclear from previous work was the mechanism by which vesicles form electron-dense structures. In this study, we use cryogenic transmission electron microscopy and dynamic light scattering to show that efficient siRNA entrapment occurs in the absence of ethanol (contrary to the established paradigm), and suggest that nucleic acid entrapment occurs through inversion of preformed vesicles. We also leverage this phenomenon to show that specialized mixers are not required for siRNA entrapment, and that preformed particles at pH 4 can be used for in vitro transfection.

Sustained depletion of FXIII-A by inducing acquired FXIII-B deficiency.

The activated form of coagulation factor XIII (FXIII-A2B2), FXIII-A*, is a hemostatic enzyme essential for inhibiting fibrinolysis by irreversibly crosslinking fibrin and antifibrinolytic proteins. Despite its importance, there are no modulatory therapeutics. Guided by the observation that humans deficient in FXIII-B have reduced FXIII-A without severe bleeding, we hypothesized that a suitable small interfering RNA (siRNA) targeting hepatic FXIII-B could safely decrease FXIII-A. Here we show that knockdown of FXIII-B with siRNA in mice and rabbits using lipid nanoparticles resulted in a sustained and controlled decrease in FXIII-A. The concentration of FXIII-A in plasma was reduced by 90% for weeks after a single injection and for more than 5 months with repeated injections, whereas the concentration of FXIII-A in platelets was unchanged. Ex vivo, crosslinking of α2-antiplasmin and fibrin was impaired and fibrinolysis was enhanced. In vivo, reperfusion of carotid artery thrombotic occlusion was also enhanced. Re-bleeding events were increased after challenge, but blood loss was not significantly increased. This approach, which mimics congenital FXIII-B deficiency, provides a potential pharmacologic and experimental tool to modulate FXIII-A2B2 activity.

Lipid nanoparticle technology for therapeutic gene regulation in the liver.

Hereditary genetic disorders, cancer, and infectious diseases of the liver affect millions of people around the globe and are a major public health burden. Most contemporary treatments offer limited relief as they generally aim to alleviate disease symptoms. Targeting the root cause of diseases originating in the liver by regulating malfunctioning genes with nucleic acid-based drugs holds great promise as a therapeutic approach. However, employing nucleic acid therapeutics in vivo is challenging due to their unfavorable characteristics. Lipid nanoparticle (LNP) delivery technology is a revolutionary development that has enabled clinical translation of gene therapies. LNPs can deliver siRNA, mRNA, DNA, or gene-editing complexes, providing opportunities to treat hepatic diseases by silencing pathogenic genes, expressing therapeutic proteins, or correcting genetic defects. Here we discuss the state-of-the-art LNP technology for hepatic gene therapy including formulation design parameters, production methods, preclinical development and clinical translation.

Potential of Xylanases to Reduce the Viscosity of Micro/Nanofibrillated Bleached Kraft Pulp.

The generally high viscosity of micro/nanofibrillated cellulose limits its applications in cream and fluid products. A bleached softwood Kraft (BSK) pulp was refined with increasing energy (500-2500 kWh t-1) to produce micro/nanofibrillated cellulose (MNBSK). Subsequent xylanase treatment was shown to influence the viscosity, gel point, aspect ratio, and fiber surface morphology of the MNBSK. It was apparent that the accessibility to xylanases was increased even at low refining energies (500 kWh t-1). Depending on the initial degree of cellulose fibrillation, xylanase treatment decreased the viscosity of the MNBSK from 4190-2030 to 681-243 Pa·s at a shear rate of 0.01 s-1, corresponding to the reduction in the aspect ratio from 183-296 to 163-194. It was likely that the xylanases were predominantly acting on the xylan present on the fiber surfaces, reducing the cross-linking points on the cellulose fibers and consequently resulting in the reduction in MNBSK viscosity.

On the role of helper lipids in lipid nanoparticle formulations of siRNA.

Onpattro, the first RNAi-based therapeutic to receive FDA approval, is enabled by a lipid nanoparticle (LNP) system that facilitates siRNA delivery into the cytoplasm of target cells (hepatocytes) following intravenous (i.v.) administration. These LNP-siRNA systems consist of four lipid components (ionizable cationic lipid, distearolyphosphatidycholine or DSPC, cholesterol, and PEG-lipid) and siRNA. The ionizable cationic lipid has been optimised for RNA encapsulation and intracellular delivery, and the PEG-lipids have been engineered to regulate LNP size and transfection potency. The roles of the other "helper" lipids, DSPC and cholesterol, remain less clear. Here we show that in empty LNP systems that do not contain siRNA, DSPC-cholesterol resides in outer layers, whereas in loaded systems a portion of the DSPC-cholesterol is internalised together with siRNA. It is concluded that the presence of internalised helper lipid is vital to the stable encapsulation of siRNA in the LNP and thus to LNP-siRNA function.

Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads.

The success of Onpattro™ (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-lipid solution with an acidic aqueous solution followed by dialysis into neutralizing buffer. A detailed understanding of the mechanism of LNP formation is crucial to improving LNP design. Here we use cryogenic transmission electron microscopy and fluorescence techniques to further demonstrate that LNP are formed through the fusion of precursor, pH-sensitive liposomes into large electron-dense core structures as the pH is neutralized. Next, we show that the fusion process is limited by the accumulation of PEG-lipid on the emerging particle. Finally, we show that the fusion-dependent mechanism of formation also applies to LNP containing macromolecular payloads including mRNA, DNA vectors, and gold nanoparticles.

Anterior segment optical coherence tomography changes with introduction and discontinuation of tamsulosin.

PURPOSE: The aim of this study was to quantify changes and reversibility in pupil dilation and iris dilator muscle region thickness associated with introduction and subsequent discontinuation of tamsulosin in patients naïve to this drug with the aid of an anterior OCT system. METHODS: The study was carried out on 7 patients (14 eyes) naïve to tamsulosin and with benign prostatic hypertrophy (BHP). Measurements taken by Vistante OCT were done pre- and post-dilation of the following: pupil size, iris dilator muscle region (DMR) thickness, sphincter muscle region (SMR) thickness, and anterior chamber depth. These measurement were taken at Day 0 (tamsulosin naive), Day 30 (after one month of tamsulosin, the treatment period) and day 60 (after one month of no tamsulosin, the discontinuation period). RESULTS: Post-dilation pupil diameter significantly increased during the discontinuation period (P = 0.047). Iris DMR thickness measurements post-dilation significantly decreased during treatment (P = 0.00044), discontinuation (0.00011), and combined periods (P = 0.000050). Anterior chamber depth measurements in post-dilation were significantly decreased during treatment (P = 0.0016), discontinuation (P = 0.017), and combined periods (P = 0.00022). CONCLUSION: Tamsulosin discontinuation effectively increases dilated pupil size, a measure that has been inversely linked to IFIS incidence pre-operatively. Decreased DMR thickness in this short term likely illustrates changes aside from atrophy, such as vascular changes. Decreased anterior chamber depths suggest aqueous humor production is decreased as well.