Effect of Micromixer Design on Lipid Nanocarriers Manufacturing for the Delivery of Proteins and Nucleic Acids.

Lipid-based nanocarriers have emerged as helpful tools to deliver sensible biomolecules such as proteins and oligonucleotides. To have a fast and robust microfluidic-based nanoparticle synthesis method, the setup of versatile equipment should allow for the rapid transfer to scale cost-effectively while ensuring tunable, precise and reproducible nanoparticle attributes. The present work aims to assess the effect of different micromixer geometries on the manufacturing of lipid nanocarriers taking into account the influence on the mixing efficiency by changing the fluid-fluid interface and indeed the mass transfer. Since the geometry of the adopted micromixer varies from those already published, a Design of Experiment (DoE) was necessary to identify the operating (total flow, flow rate ratio) and formulation (lipid concentration, lipid molar ratios) parameters affecting the nanocarrier quality. The suitable application of the platform was investigated by producing neutral, stealth and cationic liposomes, using DaunoXome®, Myocet®, Onivyde® and Onpattro® as the benchmark. The effect of condensing lipid (DOTAP, 3-10-20 mol%), coating lipids (DSPE-PEG550 and DSPE-PEG2000), as well as structural lipids (DSPC, eggPC) was pointed out. A very satisfactory encapsulation efficiency, always higher than 70%, was successfully obtained for model biomolecules (myoglobin, short and long nucleic acids).

Mini- and macro-scale direct perfusion bioreactors with optimized flow for engineering 3D tissues.

Bioreactors enabling direct perfusion of cell suspensions or culture media through the pores of 3D scaffolds have long been used in tissue engineering to improve cell seeding efficiency as well as uniformity of cell distribution and tissue development. A macro-scale U-shaped bioreactor for cell culture under perfusion (U-CUP) has been previously developed. In that system, the geometry of the perfusion chamber results in rather uniform flow through most of the scaffold volume, but not in the peripheral regions. Here, the design of the perfusion chamber has been optimized to provide a more homogenous perfusion flow through the scaffold. Then, the design of this macro-scale flow-optimized perfusion bioreactor (macro-Flopper) has been miniaturized to create a mini-scale device (mini-Flopper) compatible with medium-throughput assays. Computational fluid dynamic (CFD) modeling of the new chamber design, including a porous scaffold structure, revealed that Flopper bioreactors provide highly homogenous flow speed, pressure, and shear stress. Finally, a proof-of-principle of the functionality of the Flopper systems by engineering endothelialized stromal tissues using human adipose tissue-derived stromal vascular fraction (SVF) cells has been offered. Preliminary evidence showing that flow optimization improves cell maintenance in the engineered tissues will have to be confirmed in future studies. In summary, two bioreactor models with optimized perfusion flow and complementary sizes have been proposed that might be exploited to engineer homogenous tissues and, in the case of the mini-Flopper, for drug testing assays with a limited amount of biological material.

Fast Approximate Quantification of Endovascular Stent Graft Displacement Forces in the Bovine Aortic Arch Variant.

PURPOSE: Displacement forces (DFs) identify hostile landing zones for stent graft deployment in thoracic endovascular aortic repair (TEVAR). However, their use in TEVAR planning is hampered by the need for time-expensive computational fluid dynamics (CFD). We propose a novel fast-approximate computation of DFs merely exploiting aortic arch anatomy, as derived from the computed tomography (CT) and a measure of central aortic pressure. MATERIALS AND METHODS: We tested the fast-approximate approach against CFD gold-standard in 34 subjects with the "bovine" aortic arch variant. For each dataset, a 3-dimensional (3D) model of the aortic arch lumen was reconstructed from computed tomography angiography and CFD then employed to compute DFs within the aortic proximal landing zones. To quantify fast-approximate DFs, the wall shear stress contribution to the DF was neglected and blood pressure space-distribution was averaged on the entire aortic wall to reliably approximate the patient-specific central blood pressure. Also, DF values were normalized on the corresponding proximal landing zone area to obtain the equivalent surface traction (EST). RESULTS: Fast-approximate approach consistently reflected (r2=0.99, p<0.0001) the DF pattern obtained by CFD, with a -1.1% and 0.7° bias in DFs magnitude and orientation, respectively. The normalized EST progressively increased (p<0.0001) from zone 0 to zone 3 regardless of the type of arch, with proximal landing zone 3 showing significantly greater forces than zone 2 (p<0.0001). Upon DF normalization to the corresponding aortic surface, fast-approximate EST was decoupled in blood pressure and a dimensionless shape vector (S) reflecting aortic arch morphology. S showed a zone-specific pattern of orientation and proved a valid biomechanical blueprint of DF impact on the thoracic aortic wall. CONCLUSION: Requiring only a few seconds and quantifying clinically relevant biomechanical parameters of proximal landing zones for arch TEVAR, our method suits the real preoperative decision-making process. It paves the way toward analyzing large population of patients and hence to define threshold values for a future patient-specific preoperative TEVAR planning.

Development and optimization of microfluidic assisted manufacturing process to produce PLGA nanoparticles.

Nanomedicine consists in the application of nanotechnology in medicine to revolutionize the healthcare sector through transformative new diagnostic and therapeutic tools. In this field, nanostructures or nanocarriers (i.e., nanoparticles) are extensively used as a drug delivery system. Despite the well-defined profits offered by nanomedicines based on poly (lactic-co-glycolic acid) (PLGA), the major barriers hampering the launch of a nanoparticles-based product on the market are batch-to-batch variations and its lack of reproducibility from the benchtop to an industrial scale production. Currently, microfluidics technology has emerged as potential tool to achieve a continuous manufacturing with a precise control over fluids mixing and particles quality attributes. This work aims at defining a tailored strategy to produce PLGA NPs, exploiting a new microfluidic device. Moreover, Design of Experiments (DoE) and computational fluid dynamics approaches were exploited to understand the main process parameters and material attributes affecting the quality of the final product as well as the NPs manufacturing process. Finally, the ability to incorporate a drug into the PLGA nanoparticles was investigated by using Curcumin as model payload reaching encapsulation efficiency in the rank 28-44%. This paper is proposed as useful guide for the preparation of PLGA NPs by microfluidic technique.

Non-invasive estimation of vascular compliance and distensibility in the arm vessels: a novel ultrasound-based protocol.

Background: Performance and durability of arterio-venous grafts depend on their ability to mimic the mechanical behavior of the anastomized blood vessels. To select the most suitable synthetic graft, in vivo evaluation of the radial deformability of peripheral arteries and veins could be crucial; however, a standardized non-invasive strategy is still missing. Herein, we sought to define a novel and user-friendly clinical protocol for in vivo assessment of the arm vessel deformability. Methods: A dedicated protocol, applied on 30 volunteers, was specifically designed to estimate both compliance and distensibility of the brachial and radial arteries, and of the basilic and cephalic veins. Bi-dimensional ultrasound imaging was used to acquire cross-sectional areas (CSAs) of arteries in clinostatic configuration, and CSAs of veins combining clinostatic and orthostatic configurations. Arterial pulse pressure was measured with a digital sphygmomanometer, while venous hydrostatic pressure was derived from the arm length in orthostatic configuration. Results: For each participant, all CSAs were successfully extracted from ultrasound images. The basilic vein and the radial artery exhibited the largest (21.5±8.9 mm2) and the smallest (3.4±1.0 mm2) CSAs, respectively; CSA measurements were highly repeatable (Bland-Altman bias <10% and Pearson correlation ≥0.90, for both arteries and veins). In veins, compliance and distensibility were higher than in arteries; compliance was significantly higher (P<0.0001) in the brachial than in the radial artery (3.52×10-4 vs. 1.3×10-4 cm2/mmHg); it was three times larger in basilic veins than in cephalic veins (17.4×10-4 vs. 5.6×10-4 cm2/mmHg, P<0.0001). Conclusions: The proposed non-invasive protocol proved feasible, effective and adequate for daily clinical practice, allowing for the estimation of patient-specific compliance and distensibility of peripheral arteries and veins. If further extended, it may contribute to the fabrication of biohybrid arterio-venous grafts, paving the way towards patient-tailored solutions for vascular access.

A Deep Learning-Based and Fully Automated Pipeline for Thoracic Aorta Geometric Analysis and Planning for Endovascular Repair from Computed Tomography.

Feasibility assessment and planning of thoracic endovascular aortic repair (TEVAR) require computed tomography (CT)-based analysis of geometric aortic features to identify adequate landing zones (LZs) for endograft deployment. However, no consensus exists on how to take the necessary measurements from CT image data. We trained and applied a fully automated pipeline embedding a convolutional neural network (CNN), which feeds on 3D CT images to automatically segment the thoracic aorta, detects proximal landing zones (PLZs), and quantifies geometric features that are relevant for TEVAR planning. For 465 CT scans, the thoracic aorta and pulmonary arteries were manually segmented; 395 randomly selected scans with the corresponding ground truth segmentations were used to train a CNN with a 3D U-Net architecture. The remaining 70 scans were used for testing. The trained CNN was embedded within computational geometry processing pipeline which provides aortic metrics of interest for TEVAR planning. The resulting metrics included aortic arch centerline radius of curvature, proximal landing zones (PLZs) maximum diameters, angulation, and tortuosity. These parameters were statistically analyzed to compare standard arches vs. arches with a common origin of the innominate and left carotid artery (CILCA). The trained CNN yielded a mean Dice score of 0.95 and was able to generalize to 9 pathological cases of thoracic aortic aneurysm, providing accurate segmentations. CILCA arches were characterized by significantly greater angulation (p = 0.015) and tortuosity (p = 0.048) in PLZ 3 vs. standard arches. For both arch configurations, comparisons among PLZs revealed statistically significant differences in maximum zone diameters (p < 0.0001), angulation (p < 0.0001), and tortuosity (p < 0.0001). Our tool allows clinicians to obtain objective and repeatable PLZs mapping, and a range of automatically derived complex aortic metrics.

Comparison of Four-Dimensional Magnetic Resonance Imaging Analysis of Left Ventricular Fluid Dynamics and Energetics in Ischemic and Restrictive Cardiomyopathies.

BACKGROUND: Time-resolved three-directional velocity-encoded (4D flow) magnetic resonance imaging (MRI) enables the quantification of left ventricular (LV) intracavitary fluid dynamics and energetics, providing mechanistic insight into LV dysfunctions. Before becoming a support to diagnosis and patient stratification, this analysis should prove capable of discriminating between clearly different LV derangements. PURPOSE: To investigate the potential of 4D flow in identifying fluid dynamic and energetics derangements in ischemic and restrictive LV cardiomyopathies. STUDY TYPE: Prospective observational study. POPULATION: Ten patients with post-ischemic cardiomyopathy (ICM), 10 patients with cardiac light-chain cardiac amyloidosis (AL-CA), and 10 healthy controls were included. FIELD STRENGTH/SEQUENCE: 1.5 T/balanced steady-state free precession cine and 4D flow sequences. ASSESSMENT: Flow was divided into four components: direct flow (DF), retained inflow, delayed ejection flow, and residual volume (RV). Demographics, LV morphology, flow components, global and regional energetics (volume-normalized kinetic energy [KEV ] and viscous energy loss [ELV ]), and pressure-derived hemodynamic force (HDF) were compared between the three groups. STATISTICAL TESTS: Intergroup differences in flow components were tested by one-way analysis of variance (ANOVA); differences in energetic variables and peak HDF were tested by two-way ANOVA. A P-value of <0.05 was considered significant. RESULTS: ICM patients exhibited the following statistically significant alterations vs. controls: reduced KEV , mostly in the basal region, in systole (-44%) and in diastole (-37%); altered flow components, with reduced DF (-33%) and increased RV (+26%); and reduced basal-apical HDF component on average by 63% at peak systole. AL-CA patients exhibited the following alterations vs. controls: significantly reduced KEV at the E-wave peak in the basal segment (-34%); albeit nonstatistically significant, increased peaks and altered time-course of the HDF basal-apical component in diastole and slightly reduced HDF components in systole. DATA CONCLUSION: The analysis of multiple 4D flow-derived parameters highlighted fluid dynamic alterations associated with systolic and diastolic dysfunctions in ICM and AL-CA patients, respectively. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 3.

Aortic hemodynamics assessment prior and after valve sparing reconstruction: A patient-specific 4D flow-based FSI model.

INTRODUCTION: Valve-sparing root replacement (VSRR) of the ascending aorta is a life-saving procedure for the treatment of aortic aneurysms, but patients remain at risk for post-operative events involving the downstream native aorta, the mechanism for which is uncertain. It is possible that proximal graft replacement of the ascending aorta induces hemodynamics alterations in the descending aorta, which could trigger adverse events. Herein, we present a fluid-structure interaction (FSI) protocol, based on patient-specific geometry and boundary conditions, to assess impact of proximal aortic grafts on downstream aortic hemodynamics and distensibility. METHODS: Cardiac magnetic resonance (CMR), including MRA, cine-CMR and 4D flow sequences, was performed prior and after VSRR on one subject. Central blood pressure was non-invasively acquired at the time of the CMR: data were used to reconstruct the pre- and post-VSRR model and derive patient-specific boundary conditions for the FSI and a computational fluid dynamic (CFD) analysis with the same settings. Results were validated comparing the predicted velocity field against 4D flow dataset, over four landmarks along the aorta, and the predicted distensibility against the cine-CMR derived value. RESULTS: Instantaneous velocity magnitudes extracted from 4D flow and FSI were similar (p > 0.05), while CFD-predicted velocity was significantly higher (p < 0.001), especially in the descending aorta of the pre-VSRR model (vmax was 73 cm/s, 76 cm/s and 99 cm/s, respectively). As measured in cine-CMR, FSI predicted an increase in descending aorta distensibility after grafting (i.e., 4.02 to 5.79 10-3 mmHg-1). In the descending aorta, the post-VSRR model showed increased velocity, aortic distensibility, stress and strain and wall shear stress. CONCLUSIONS: Our Results indicate that i) the distensibility of the wall cannot be neglected, and hence the FSI method is necessary to obtain reliable results; ii) graft implantation induces alterations in the hemodynamics and biomechanics along the thoracic aorta, that may trigger adverse vessel remodeling.

4D flow evaluation of blood non-Newtonian behavior in left ventricle flow analysis.

Blood is generally modeled as a Newtonian fluid, assuming a standard and constant viscosity; however, this assumption may not hold for the highly pulsatile and recirculating intracavitary flow in the left ventricle (LV), hampering the quantification of fluid dynamic indices of potential clinical relevance. Herein, we investigated the effect of three viscosity models on the patient-specific quantification of LV blood energetics, namely on viscous energy loss (EL), from 4D Flow magnetic resonance imaging: I) Newtonian with standard viscosity (3.7 cP), II) Newtonian with subject-specific hematocrit-dependent viscosity, III) non-Newtonian accounting for the effect of hematocrit and shear rate. Analyses were performed on 5 controls and 5 patients with cardiac light-chain amyloidosis. In Model II, viscosity ranged between 3.0 (-19%) and 4.3 cP (+16%), mildly deviating from the standard value. In the non-Newtonian model, this effect was emphasized: viscosity ranged from 3.2 to 6.0 cP, deviating maximally from the standard value in low shear rate (i.e., <100 s-1) regions. This effect reflected on EL quantifications: in particular, as compared to Model I, Model III yielded markedly higher EL values (up to +40%) or markedly lower (down to -21%) for subjects with hematocrit higher than 39.5% and lower than 30%, respectively. Accounting for non-Newtonian blood behavior on a patient-specific basis may enhance the accuracy of intracardiac energetics assessment by 4D Flow, which may be explored as non-invasive index to discriminate between healthy and pathologic LV.

Prediction of post-stenting biomechanics in coarcted aortas: A pilot finite element study.

Endovascular stenting has recently become a standard treatment for native coarctation of the aorta (CoA) in children and young adults, given the efficacy in relieving vessel obstruction with a low incidence of adverse events. Yet, despite the short-term success of the technique, late hypertension remains an endemic risk. To assess the impact of the percutaneous procedure on the aortic wall biomechanics, we designed a novel finite element (FE) protocol for the simulation of endovascular stenting in three patient-specific CoA anatomies, developing a remeshing procedure that allows for coping with different CoA severities. Our FE protocol was able to yield numerical results on stent distortions and stresses, as well as on changes in aortic wall stresses and distensibility. These results were consistent with intraprocedural in-vivo evidences and with previous findings from the literature, and they suggest that our numerical approach could be used to understand the role of patient specific anatomical features (CoA severity and arch type) on the post-stenting aortic biomechanics. If soundly validated on a vast cohort of patients, our approach could support patient selection for the procedure.

3-Dimensional personalized planning for transcatheter pulmonary valve implantation in a dysfunctional right ventricular outflow tract.

BACKGROUND: Identification of adequate landing zone for transcatheter pulmonary valve implantation (TPVI) is crucial to successfully treat an aneurysmatic native right ventricle outflow tract (RVOT); three-dimensional (3D) patient-tailored digital and physical printed models are available but their actual strengths and weaknesses still not well documented. The aim of the study was to tackle TPVI planning in the dysfunctional and borderline RVOT exploiting both digital and physical printed 3D patient-specific models. METHODS: Electrocardiographically gated computed tomography (CT) angiography was segmented and anatomical RVOT geometrical changes dynamically tracked throughout the cardiac cycle using in-house processing. A compliant 3D-printed model was manufactured from the diastolic rest phase to test in vitro the catheter-based procedure feasibility; results were compared against CT-derived in vivo measurements and the actual catheterization outcome. RESULTS: CT-gated analysis successfully quantified in vivo RVOT dynamic changes corroborating the feasibility of non-conventional pulmonary jailing percutaneous intervention. Clinicians used the 3D-printed model to test the steps of the jailing procedure; yet, the deformable 3D model printed at diastole underestimated the final implant dimensions obtained during cardiac catheterization by the same operators. CONCLUSIONS: Multidisciplinary synergy between CT-gated analysis and pre-procedural tests on 3D-printed phantoms can help the interventional team to tackle complex TPVI procedures. To fully exploit 3D-printed models, adequate selection of the still frame to print and tuning of printing material properties is crucial and can be aided by 3D dynamic virtual models.

Prediction of stenting related adverse events through patient-specific finite element modelling.

Right ventricular outflow tract (RVOT) calcific obstruction is frequent after homograft conduit implantation to treat congenital heart disease. Stenting and percutaneous pulmonary valve implantation (PPVI) can relieve the obstruction and prolong the conduit lifespan, but require accurate pre-procedural evaluation to minimize the risk of coronary artery (CA) compression, stent fracture, conduit injury or arterial distortion. Herein, we test patient-specific finite element (FE) modeling as a tool to assess stenting feasibility and investigate clinically relevant risks associated to the percutaneous intervention. Three patients undergoing attempted PPVI due to calcific RVOT conduit failure were enrolled; the calcific RVOT, the aortic root and the proximal CA were segmented on CT scans for each patient. We numerically reproduced RVOT balloon angioplasty to test procedure feasibility and the subsequent RVOT pre-stenting expanding the stent through a balloon-in-balloon delivery system. Our FE framework predicted the occurrence of CA compression in the patient excluded from the real procedure. In the two patients undergoing RVOT stenting, numerical results were consistent with intraprocedural in-vivo fluoroscopic evidences. Furthermore, it quantified the stresses on the stent and on the relevant native structures, highlighting their marked dependence on the extent, shape and location of the calcific deposits. Stent deployment induced displacement and mechanical loading of the calcific deposits, also impacting on the adjacent anatomical structures. This novel workflow has the potential to tackle the analysis of complex RVOT clinical scenarios, pinpointing the procedure impact on the dysfunctional anatomy and elucidating potential periprocedural complications.

Toward the Virtual Benchmarking of Pneumatic Ventricular Assist Devices: Application of a Novel Fluid-Structure Interaction-Based Strategy to the Penn State 12 cc Device.

The pediatric use of pneumatic ventricular assist devices (VADs) as a bridge to heart transplant still suffers for short-term major complications such as bleeding and thromboembolism. Although numerical techniques are increasingly exploited to support the process of device optimization, an effective virtual benchmark is still lacking. Focusing on the 12 cc Penn State pneumatic VAD, we developed a novel fluid-structure interaction (FSI) model able to capture the device functioning, reproducing the mechanical interplay between the diaphragm, the blood chamber, and the pneumatic actuation. The FSI model included the diaphragm mechanical response from uniaxial tensile tests, realistic VAD pressure operative conditions from a dedicated mock loop system, and the behavior of VAD valves. Our FSI-based benchmark effectively captured the complexity of the diaphragm dynamics. During diastole, the initial slow diaphragm retraction in the air chamber was followed by a more rapid phase; asymmetries were noticed in the diaphragm configuration during its systolic inflation in the blood chamber. The FSI model also captured the major features of the device fluid dynamics. In particular, during diastole, a rotational wall washing pattern is promoted by the penetrating inlet jet with a low-velocity region located in the center of the device. Our numerical analysis of the 12 cc Penn State VAD points out the potential of the proposed FSI approach well resembling previous experimental evidences; if further tested and validated, it could be exploited as a virtual benchmark to deepen VAD-related complications and to support the ongoing optimization of pediatric devices.