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Pediatric intensive care unit (PICU) survivors experience a collection of physical, cognitive, emotional, and social symptoms named post-intensive care syndrome (PICS). We aimed to quantify PICS morbidities from multiple stakeholder perspectives across domains. Using physician, neuropsychologist, and parent/caregiver-completed outcome measures across domains in a cross-sectional analysis of 186 children between the ages of 8 and 19 years with data from a PICU follow-up clinic 1 to 3 months after PICU discharge, we quantified PICS morbidities and used Spearman correlation to evaluate interdependence of PICS domains. Compared with pre-hospitalization functioning, new PICS-related morbidities were observed and significant correlations were identified between the PICS domains. Results highlight the need to identify and support patients and families for multiple morbidities simultaneously across all relevant PICS domains; moreover, an important divergence between caregiver report, physician, and neuropsychologist assessment was also found, showcasing the importance of multiple assessments and perspectives. New PICS morbidities across domains are common after discharge from the PICU, and timely follow-up care is needed that involves collaboration/integration of physicians, neuropsychologists, youth, and families to effectively identify and treat PICS-related issues.

The androgen receptor plays a critical role in the progression of prostate cancer. Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor. Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration. Through these features, and by using a structurally refined cationic lipid and an optimized small interfering RNA payload, a lipid nanoparticle system with improved potency and significant therapeutic potential against prostate cancer and potentially other solid tumors was developed. Decreases in serum prostate-specific antigen, tumor cellular proliferation, and androgen receptor levels were observed in a mouse xenograft model following intravenous injection. These results support the potential clinical utility of a prostate-specific membrane antigen–targeted lipid nanoparticle system to silence the androgen receptor in advanced prostate cancer.

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using short-hairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP AR-siRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo.

Adsorption of i-butane, p-xylene, and benzene shrinks the size of MFI crystals in polycrystalline MFI membranes at room temperature. The crystal shrinkage, which is over different loading ranges for the three molecules, increases the sizes of the defects (gaps between the crystals). Two membranes, one with 90% of its helium flux through defects, and one with 9% were studied in detail. For the membrane with 90% of its helium flow through defects, the flux through the defects increased by as much as 44%, depending on the permeating molecule. The defect sizes increased for p-xylene loadings below 4 molec./u.c. and for i-butane loading between 3 and 8 molec./u.c. At higher loadings (7 molec./u.c. for p-xylene and 9 molec./u.c. for i-butane) p-xylene and i-butane expanded MFI crystals and shrank the defect sizes so that the helium flow through defects decreased by as much as two orders of magnitude. Benzene did not significantly change the flux through defects at loadings below 8 molec./u.c. Near a loading of 8 benzene molec./u.c. the flux through the defects increased by approximately 18% for one membrane.

Two polycrystalline MFI membranes with significant flow through defects (non-zeolitic pores that are gaps between the crystals) are shown to have high ideal and mixture selectivities. The membranes were characterized at room temperature by permporosimetry, pervaporation, separations, and single-gas permeation. These measurements indicate that one membrane (B-ZSM-5) had a relatively large number of smaller defects, whereas the second membrane (silicalite-1) had a smaller number of somewhat larger defects. The relative contributions of these defects to the overall flux changed dramatically in the presence of n-alkanes and SF6. These molecules caused adsorption-induced expansion of the crystals, and this expansion shrank the defect sizes and thus changed the membrane permeation characteristics. The B-ZSM-5 membrane had 90% of its helium flux through defects at room temperature, but it had a H2/SF6 ideal selectivity as high as 260 because of SF6-induced swelling that stopped 99% of the flux through the defects. In contrast, the silicalite-1 membrane had only 9% of its helium flux through defects, but the defects were large enough that crystal swelling only decreased the flux through them by 30%. Thus its selectivities were lower. These studies show that n-hexane, n-pentane, n-butane, n-propane, and SF6 swell MFI crystals when they adsorb, but benzene and CO2 do not. The changes in membrane microstructure due to crystal expansion not only significantly affect membrane separation ability, but also have implications on how to select appropriate characterization techniques for evaluating MFI membrane quality.

Adsorption isotherms and diffusion coefficients for n-C4 and i-C4 in the transport pathways through tubular ZSM-5 zeolite membranes were determined over a range of temperatures by a transient permeation method. The permeate response to step changes in the feed were measured, and the transport was modeled as Maxwell−Stefan diffusion with single site Langmuir adsorption in the zeolite. The heats of adsorption and the Langmuir parameters were comparable to values reported for MFI powders. The similarity of the membrane and powder isotherms indicates that butanes diffuse mainly through zeolite pores. Maxwell−Stefan diffusion coefficients for n-C4 and i-C4 in the ZSM-5 membranes were similar to those measured by other macroscopic techniques for zeolite crystals and membranes, and diffusion coefficients increased with increasing feed partial pressure for a high-quality membrane. The effective membrane thickness was also estimated from these transient measurements.

A simple, efficient, and scalable manufacturing technique is required for developing siRNA-lipid nanoparticles (siRNA-LNP) for therapeutic applications. In this chapter we describe a novel microfluidic-based manufacturing process for the rapid manufacture of siRNA-LNP, together with protocols for characterizing the size, polydispersity, RNA encapsulation efficiency, RNA concentration, and total lipid concentration of the resultant nanoparticles.

A simple, efficient, and scalable manufacturing technique is required for developing siRNA-lipid nanoparticles (siRNA-LNP) for therapeutic applications. In this chapter we describe a novel microfluidic-based manufacturing process for the rapid manufacture of siRNA-LNP, together with protocols for characterizing the size, polydispersity, RNA encapsulation efficiency, RNA concentration, and total lipid concentration of the resultant nanoparticles.

Background Anxiety among patients in a pediatric emergency department (PED) can be significant, but often goes unaddressed. Objective Our aim was to determine whether exposure to Child Life (CL) or hospital clowning (HC) can reduce anxiety in children presenting to a PED. Methods Patients were randomized to CL, HC, or control and assessed upon entry to examination room (T1), before physician arrival (T2), and during physician examination (T3), using the modified Yale Preoperative Anxiety Scale (m-YPAS). CL and HC interventions occurred for 5 to 10 min before physician entry. Effects were analyzed using mixed analysis of variance. Results m-YPAS scores ranged from 23 to 59, with a higher score indicating increased anxiety. Mixed analysis of variance on the study sample (n = 113) showed a significant interaction between groups (CL, HC, control) and time (p = 0.02). Additional analyses indicated effect of group only at T2 (CL: mean = 23.8; 95% confidence interval [CI] 23.2–24.5; HC: mean 25.2; 95% CI 24.2–26.2; control: mean = 26.1; 95% CI 24.2–27.9; p = .02). Subanalysis of patients with T1 m-YPAS score ≥ 28 (n = 56) showed a significant interaction between group and time (p = 0.01). Additional analysis showed effect of group only at T2 (CL: mean 24.4; 95% CI 23.3–25.6; HC: mean 27.0; 95% CI 25.2–28.7; control: mean 29.2; 95% CI 25.6–32.7; p = 0.003). Conclusions CL services can reduce state anxiety for patients presenting to a PED with heightened anxiety at baseline. This reduction occurred immediately after CL intervention, but was not observed in patients exposed to HC or during physician examination.

The use of targeting ligands to enhance the delivery of liposomal nanoparticles (LNs) has moved slowly toward clinical application. This relative lack of clinical progression is further complicated by the existence of conflicting in vivo results in the literature. In this work, we describe new formulations of LNs that are targeted with an arginine-glycine-aspartic acid-containing peptide, cRGDfK, conjugated to the lipid distearoyl phosphatidylethanolamine (DSPE). These formulations may be able to circumvent some of the challenges encountered during the development of targeted-LNs. Of the constructs studied, a fluorescently labeled peptide-lipid conjugate was incorporated into LNs with high yield and accuracy. It is shown that the resulting targeted-LNs bind to human umbilical vein endothelial cells (HUVECs) with increasing avidity as the amount of peptide displayed on the LN surface increases. We specifically demonstrate the ability of targeted-LNs loaded with doxorubicin and incubated with HUVECs to deliver the drug to the cytosol. The cell does not internalize nontargeted LNs, supporting the notion that the RGD motif is associated with internalization of the targeted LN.

Prostate cancer is the most commonly diagnosed non-skin cancer in men and one of the leading causes of cancer-death. While frequently curable by surgical or radiation ablation, recurrent or metastatic disease is usually managed with androgen withdrawal therapies (medical castration), which block the growth promoting effects of androgens on the androgen receptor (AR). Unfortunately, the cancers eventually progress to the lethal castration-resistant state. Previously, we reported that knocking down the AR by RNA interference using small hairpin RNA (shRNA) inhibits this progression and, in about 50% of tumours, causes complete regression. In this study, we investigated the ability of small interfering RNA (siRNA) encapsulated in lipid nanoparticle (LNP) to silence AR in human prostate cancer cell lines and xenograft tumours following intravenous injections. In vitro screening studies with prostate cancer cell lines using a panel of cationic lipids demonstrated that siRNA formulated in LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to enter into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, we found that LNP AR-siRNA systems containing DLin-KC2-DMA could silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following intravenous injection. The 5′RACE experiments further confirmed that siRNA-induced specific cleavage of AR mRNA was the primary mechanism of action. In conclusion, these results indicate that in vivo delivery of siRNA to knockdown AR using our LNP formulations causes a decrease in serum PSA and provides proof in principle that this approach may offer a therapeutic strategy for treating advanced prostate cancer. (Funded by Canadian Institutes for Health Research and a PCF-STAR Project from Prostate Cancer Canada with the support of Safeway Canada) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1970. doi:1538-7445.AM2012-1970