Intracellular delivery and deep tissue penetration of nucleoside triphosphates using photocleavable covalently bound dendritic polycations.

Nucleoside triphosphates (NTPs) are essential in various biological processes. Cellular or even organismal controlled delivery of NTPs would be highly desirable, yet in cellulo and in vivo applications are hampered owing to their negative charge leading to cell impermeability. NTP transporters or NTP prodrugs have been developed, but a spatial and temporal control of the release of the investigated molecules remains challenging with these strategies. Herein, we describe a general approach to enable intracellular delivery of NTPs using covalently bound dendritic polycations, which are derived from PAMAM dendrons and their guanidinium derivatives. By design, these modifications are fully removable through attachment on a photocage, ready to deliver the native NTP upon irradiation enabling spatiotemporal control over nucleotide release. We study the intracellular distribution of the compounds depending on the linker and dendron generation as well as side chain modifications. Importantly, as the polycation is bound covalently, these molecules can also penetrate deeply into the tissue of living organisms, such as zebrafish.

Spatiotemporal Regulation of FMNL2 by N-Terminal Myristoylation and C-Terminal Phosphorylation Drives Rapid Filopodia Formation.

The actin nucleating and polymerizing formin-like 2 (FMNL2) is upregulated in several cancers and has been shown to play important roles in cell migration, invasion, cell-cell adhesion and filopodia formation. Here, using structured illumination microscopy we show that FMNL2 promotes rapid and highly dynamic filopodia formation in epithelial cells while remaining on the tip of the growing filopodia. This filopodia tip localization depends fully on its N-terminal myristoylation. We further show that FMNL2-dependent filopodia formation requires its serine 1072 phosphorylation within the diaphanous-autoregulatory domain (DAD) by protein kinase C (PKC) α. Consistent with this, filopodia formation depends on PKC activity and PKCα localizes to the base of growing filopodia. Thus, a PKCα-FMNL2 signaling module spatiotemporally controls dynamic filopodia formation.

Vesicle-Associated Actin Assembly by Formins Promotes TGFß-Induced ANGPTL4 Trafficking, Secretion and Cell Invasion.

Vesicle trafficking has emerged as an important process driving tumor progression through various mechanisms. Transforming growth factor beta (TGFß)-mediated secretion of Angiopoietin-like 4 (ANGPTL4) is important for cancer development. Here, Formin-like 2 (FMNL2) is identified to be necessary for ANGPTL4 trafficking and secretion in response to TGFß. Protein kinase C (PKC)-dependent phosphorylation of FMNL2 downstream of TGFß stimulation is required for cancer cell invasion as well as ANGPTL4 vesicle trafficking and secretion. Moreover, using super resolution microscopy, ANGPTL4 trafficking is actin-dependent with FMNL2 directly polymerizing actin at ANGPTL4-containing vesicles, which are associated with Rab8a and myosin Vb. This work uncovers a formin-controlled mechanism that transiently polymerizes actin directly at intracellular vesicles to facilitate their mobility. This mechanism may be important for the regulation of cancer cell metastasis and tumor progression.

A systematic review of patient-reported nasal obstruction scores: defining normative and symptomatic ranges in surgical patients.

IMPORTANCE A gold standard objective measure of nasal airway obstruction (NAO) does not currently exist, so patient-reported measures are commonly used, particularly the Nasal Obstruction Symptom Evaluation (NOSE) scale and the visual analog scale (VAS). However, questions remain regarding how best to use these instruments. OBJECTIVES To systematically review studies on NOSE and VAS scores in patients with NAO and to compile and standardize the data to (1) define symptomatic and normative values for presurgical and postsurgical patients with NAO, asymptomatic individuals, and the general population; (2) determine if postsurgical scores are comparable with asymptomatic scores; and (3) determine if there is a clinically useful preoperative and postoperative score change. EVIDENCE REVIEW A systematic review of the literature was performed through PubMed for studies assessing NOSE and VAS scores in patients with chronic NAO. Strict inclusion criteria were applied to focus on anatomic obstruction only. For statistical analysis, the patients were classified as asymptomatic, presurgical and postsurgical with NAO, and the general population. FINDINGS The mean (SD) NOSE and VAS scores for a patient with NAO were 65 (22) and 6.9 (2.3), respectively. The mean postsurgical NOSE and VAS scores were 23 (20) and 2.1 (2.2), respectively. The mean asymptomatic individual NOSE and VAS scores were 15 (17) and 2.1 (1.6). The mean NOSE and VAS scores for the general population were 42 (27) and 4.6 (2.6), respectively. The mean presurgical to postsurgical change was more than 40 for NOSE scores and more than 4.0 for VAS scores. CONCLUSIONS AND RELEVANCE We have shown that normative and abnormal value ranges for NOSE and VAS can be established for clinical use. Given the consistency of both scales, we conclude that these measures can be used as a clinically meaningful measure of successful surgical outcomes.

Quantification of airflow into the maxillary sinuses before and after functional endoscopic sinus surgery.

BACKGROUND: The effects of increases in maxillary sinus (MS) airflow following functional endoscopic sinus surgery (FESS) are unknown. The goal of this study was to quantify the effects of FESS on airflow into the MS in a cohort of patients with chronic rhinosinusitis, and compare MS flow rate with patient-reported outcome measures. METHODS: A pilot study was conducted in which preoperative and postoperative computed tomography scans of 4 patients undergoing bilateral or unilateral FESS were used to create 3-dimensional (3D) reconstructions of the nasal airway and paranasal sinuses using Mimics™ (Materialise, Inc.). The size of the maxillary antrostomies post-FESS ranged from 107 to 160 mm(2). Computational meshes were generated from the 3D reconstructions, and steady-state, laminar, inspiratory airflow was simulated in each mesh using the computational fluid dynamics (CFD) software Fluent™ (ANSYS, Inc.) under physiologic, pressure-driven conditions. Airflow into the MS was estimated from the simulations and was compared preoperatively and postoperatively. In addition, patients completed preoperative and postoperative Rhinosinusitis Outcome Measure-31 (RSOM-31) questionnaires and scores were compared with MS airflow rates. RESULTS: CFD simulations predicted that average airflow rate into post-FESS MS increased by 18.5 mL/second, and that average flow velocity into the MS more than quadrupled. Simulation results also showed that MS flow rate trended with total RSOM-31 and all domain scores. CONCLUSION: CFD simulations showed that the healed maxillary antrostomy after FESS can greatly enhance airflow into the MS. Our pilot study suggests that to some extent, increasing airflow into the MS may potentially improve chronic rhinosinusitis patients' quality of life pre-FESS and post-FESS.

Modeling nasal physiology changes due to septal perforations.

OBJECTIVE: To use computational fluid dynamics (CFD) technology to help providers understand (1) how septal perforations may alter nasal physiology and (2) how these alterations are influenced by perforation size and location. STUDY DESIGN: Computer simulation study. SETTING: Facial plastic and reconstructive surgery clinic. SUBJECTS AND METHODS: With the aid of medical imaging and modeling software, septal perforations of 1 and 2 cm in anterior, posterior, and superior locations were virtually created in a nasal cavity digital model. The CFD techniques were used to analyze airflow, nasal resistance, air conditioning, and wall shear stress. RESULTS: Bilateral nasal resistance was not significantly altered by a septal perforation. Airflow allocation changed, with more air flowing through the lower-resistance nasal cavity. This effect was greater for anterior and posterior perforations than for the superior location. At the perforation sites, there was less localized heat and moisture flux and wall shear stress in superior perforations compared with those in anterior or posterior locations. For anterior perforations, a larger size produced higher wall shear and velocity, whereas in posterior perforations, a smaller size produced higher wall shear and velocity. CONCLUSION: Septal perforations may alter nasal physiology. In the subject studied, airflow allocation to each side was changed as air was shunted through the perforation to the lower-resistance nasal cavity. Anterior and posterior perforations caused larger effects than those in a superior location. Increasing the size of anterior perforations and decreasing the size of posterior perforations enhanced alterations in wall shear and velocity at the perforation.

Computed intranasal spray penetration: comparisons before and after nasal surgery.

BACKGROUND: Quantitative methods for comparing intranasal drug delivery efficiencies pre- and postoperatively have not been fully utilized. The objective of this study is to use computational fluid dynamics techniques to evaluate aqueous nasal spray penetration efficiencies before and after surgical correction of intranasal anatomic deformities. METHODS: Ten three-dimensional models of the nasal cavities were created from pre- and postoperative computed tomography scans in 5 subjects. Spray simulations were conducted using a particle size distribution ranging from 10 µm to 110 µm, a spray speed of 3 m/second, plume angle of 68 degrees, and with steady state, resting inspiratory airflow present. Two different nozzle positions were compared. Statistical analysis was conducted using Student t test for matched pairs. RESULTS: On the obstructed side, posterior particle deposition after surgery increased by 118% and was statistically significant (p = 0.036), while anterior particle deposition decreased by 13% and was also statistically significant (p = 0.020). The fraction of particles that bypassed the airways either pre- or postoperatively was less than 5%. Posterior particle deposition differences between obstructed and contralateral sides of the airways were 113% and 30% for pre- and postsurgery, respectively. Results showed that nozzle positions can influence spray delivery. CONCLUSION: Simulations predicted that surgical correction of nasal anatomic deformities can improve spray penetration to areas where medications can have greater effect. Particle deposition patterns between both sides of the airways are more evenly distributed after surgery. These findings suggest that correcting anatomic deformities may improve intranasal medication delivery. For enhanced particle penetration, patients with nasal deformities may explore different nozzle positions.

Computed nasal resistance compared with patient-reported symptoms in surgically treated nasal airway passages: a preliminary report.

BACKGROUND: Nasal airway obstruction (NAO) is a common health condition impacting mood, energy, recreation, sleep, and overall quality of life. Nasal surgery often addresses NAO but the results are sometimes unsatisfactory. Evaluating surgical treatment efficacy could be improved if objective tests were available that correlated with patient-reported measures of symptoms. The goal of this study was to develop methods for comparing nasal resistance computed by computational fluid dynamics (CFD) models with patient-reported symptoms of NAO using early data from a 4-year prospective study. METHODS: Computed tomography (CT) scans and patient-reported scores from the Nasal Obstruction Symptom Evaluation (NOSE) scale and a visual analog scale (VAS) measuring unilateral airflow sensation were obtained pre- and postoperatively in two NAO patients showing no significant mucosal asymmetry who were successfully treated with functional nasal surgery, including septoplasty. Pre- and postsurgery CFD models were created from the CT scans. Numerical simulation of steady-state inspiratory airflow was used to calculate bilateral and unilateral CFD-derived nasal resistance (CFD-NR). RESULTS: In both subjects, NOSE and VAS scores improved after surgery, bilateral CFD-NR decreased, and unilateral CFD-NR decreased on the affected side. In addition, NOSE and VAS scores tracked with unilateral CFD-NR on the affected side. CONCLUSION: These preliminary results suggest a possible correlation between unilateral NR and patient-reported symptoms and imply that analysis of unilateral obstruction should focus on the affected side. A formal investigation of unilateral CFD-NR and patient-reported symptoms in a series of NAO patients is needed to determine if these variables are correlated.

Role of virtual surgery in preoperative planning: assessing the individual components of functional nasal airway surgery.

OBJECTIVES: To demonstrate the effect of individual components of functional nasal airway surgery in a patient with multifactorial obstruction and to discuss the potential benefit of computational fluid dynamics (CFD)­aided virtual surgery. METHODS: A 53-year-old woman underwent septoplasty,turbinate reduction, and nasal valve repair. Presurgery and postsurgery digital nasal models were created from computed tomographic images, and nasal resistance was calculated using CFD techniques. The digital models were then manipulated to isolate the effects of the components of the surgery, creating a nasal valverepair alone model and a septoplasty/turbinate reduction alone model. RESULTS: Bilateral nasal resistance in the postsurgery model was approximately 25% less than presurgery values.Similarly, CFD analysis showed reductions in nasal resistance of the virtual models: 19% reduction with intranasal surgery alone and 6% reduction with nasal valve repair alone. CONCLUSIONS: Most of the reduction in nasal resistance was accomplished with performance of septoplasty and inferior turbinate reduction. The contribution of nasal valve repair was less in comparison but not insignificant.This pilot study implies that CFD-aided virtual surgery may be useful as part of preoperative planning inpatients with multifactorial anatomical nasal airwayobstruction

Computed nasal resistance compared with patient-reported symptoms in surgically treated nasal airway passages: A preliminary report.

BACKGROUND: Nasal airway obstruction (NAO) is a common health condition impacting mood, energy, recreation, sleep, and overall quality of life. Nasal surgery often addresses NAO but the results are sometimes unsatisfactory. Evaluating surgical treatment efficacy could be improved if objective tests were available that correlated with patient-reported measures of symptoms. The goal of this study was to develop methods for comparing nasal resistance computed by computational fluid dynamics (CFD) models with patient-reported symptoms of NAO using early data from a 4-year prospective study. METHODS: Computed tomography (CT) scans and patient-reported scores from the Nasal Obstruction Symptom Evaluation (NOSE) scale and a visual analog scale (VAS) measuring unilateral airflow sensation were obtained pre- and postoperatively in two NAO patients showing no significant mucosal asymmetry who were successfully treated with functional nasal surgery, including septoplasty. Pre- and postsurgery CFD models were created from the CT scans. Numerical simulation of steady-state inspiratory airflow was used to calculate bilateral and unilateral CFD-derived nasal resistance (CFD-NR). RESULTS: In both subjects, NOSE and VAS scores improved after surgery, bilateral CFD-NR decreased, and unilateral CFD-NR decreased on the affected side. In addition, NOSE and VAS scores tracked with unilateral CFD-NR on the affected side. CONCLUSION: These preliminary results suggest a possible correlation between unilateral NR and patient-reported symptoms and imply that analysis of unilateral obstruction should focus on the affected side. A formal investigation of unilateral CFD-NR and patient-reported symptoms in a series of NAO patients is needed to determine if these variables are correlated.

Effects of anatomy and particle size on nasal sprays and nebulizers.

OBJECTIVE: To study the effects of nasal deformity on aerosol penetration past the nasal valve (NV) for varying particle sizes using sprays or nebulizers. STUDY DESIGN: Computed mathematical nasal airway model. SETTING: Department computer lab. SUBJECTS AND METHODS: Particle deposition was analyzed using a computational fluid dynamics model of the human nose with leftward septal deviation and compensatory right inferior turbinate hypertrophy. Sprays were simulated for 10 µm, 20 µm, 50 µm, or particle sizes following a Rosin Rammler particle size distribution (10-110 µm), at speeds of 1, 3, or 10 meters per second. Nebulization was simulated for 1, 3.2, 6.42, or 10 µm particles. Steady state inspiratory airflow was simulated at 15.7 liters per minute. RESULTS: Sprays predicted higher NV penetration on the right side for particle sizes >10 µm, with comparable penetration on both sides at 10 µm. Nearly 100% deposited in the nasal passages for all spray characteristics. Nebulizer predictions showed nearly 100% of particles <6.42 µm and more than 50% of 6.42 µm bypassing both sides of the nose without depositing. Of the nebulized particles that deposited, penetration was higher on the right at 10 µm, with comparable penetration on both sides at 6.42 µm. Spray penetration was highest at 10 µm, with more than 96% penetrating on both sides at 1 and 3 meters per second. Nebulization penetration was also highest at 10 µm (40% on the left, >90% on the right). CONCLUSION: In the presence of a septal deviation, sprays or nebulizers containing 10-µm particles may have good penetration beyond the NV. Nebulized particles <10 µm are likely to be respirable. Additionally, spray speeds above 3 meters per second may limit penetration.