A Systematic Review and Meta-analysis of Biceps Tenodesis Fixation Strengths: Fixation Type and Location Are Biomechanically Equivalent.

PURPOSE: The purpose of this meta-analysis and systematic review was to critically evaluate the biomechanical outcomes of different fixation constructs for a variety of biceps tenodesis techniques in cadaveric models based on both type of fixation and location. METHODS: A PROSPERO-registered systematic review (CCRD42018109243) of the current literature was conducted with the terms "long head of biceps" AND "tenodesis" AND "biomechanics" and numerous variations thereof in the PubMed, Embase, and Cochrane databases, yielding 1,460 abstracts. After screening by eligibility criteria, 18 full-text articles were included. The individual biomechanical factors evaluated included ultimate load to failure (in newtons), stiffness (in newtons per millimeter), and cyclic displacement (in millimeters). After reviewing the included literature, we performed a quality analysis of the studies (Quality Appraisal for Cadaveric Studies scale score) and a meta-analysis comparing raw mean differences in data between the suprapectoral and subpectoral fixation location groups, as well as between the fixation construct groups. RESULTS: Among the 18 included studies, 347 cadaveric specimens were evaluated for ultimate load to failure, stiffness, and cyclic displacement when comparing both location (suprapectoral vs subpectoral) and tenodesis fixation type (interference screw vs cortical button, suture anchor, or all-soft-tissue techniques). Interference screw fixation showed significantly greater mean stiffness by 8.0 N/mm (P = .013) compared with the other grouped techniques but did not show significant differences when evaluated for ultimate load to failure and cyclic displacement (P = .28 and P = .18, respectively). Additionally, no difference in construct strength was seen when comparing the fixation strength of suprapectoral versus subpectoral techniques for stiffness, ultimate load to failure, and cyclic loading (P = .47, P = .053, and P = .13, respectively). CONCLUSIONS: In this meta-analysis, no significant biomechanical differences were found when the results were stratified by specific surgical technique (interference screw vs other tenodesis techniques) and location (suprapectoral vs subpectoral biceps tenodesis). CLINICAL RELEVANCE: As a result of this study, when biomechanically evaluating specific tenodesis constructs, the individual clinician has the liberty of choosing the fixation technique based on his or her preference and knowledge of shortcomings of each type of fixation construct.

Did school characteristics affect the uptake of meningococcal quadrivalent vaccine in Greater Manchester, United Kingdom?

OBJECTIVES: The objective of this study was to assess if school characteristics were associated with the uptake of the meningococcal ACWY (MenACWY) vaccine in Greater Manchester in 2017/18. STUDY DESIGN: This is an ecological cross-sectional study. METHODS: We analysed data on all 129 schools in seven local authorities in Greater Manchester from the Department for Education and from local child health information systems to determine whether school characteristics, including school type and Ofsted effectiveness score, were associated with vaccine uptake. Schools with no eligible pupils were excluded. We undertook single-variable and multivariable analysis and considered key interactions. RESULTS: The overall uptake rate was 80.7%, with a median uptake per school of 80.6% (interquartile range, 69.0%-87.4%). Lower vaccination rates were associated with lower overall effectiveness scores (odds ratio [OR]: 3.54, 95% confidence interval [CI]: 3.00-4.19) and lower numbers of pupils eligible for vaccination (OR: 1.39, 95% CI: 1.28-1.51). Schools with a lower percentage of pupils for whom English is a second language and high deprivation were associated with lower uptake (OR: 1.58, 95% CI: 1.41-1.78). In addition, community schools (the schools with the most local authority oversight) had lower vaccination rates than other categories of schools. CONCLUSIONS: In this study, uptake rates of the MenACWY vaccine were associated with all five school characteristics considered. Effectiveness scores for schools had the largest association with vaccine uptake, with poorer schools having lower uptake. These characteristics should be used by vaccination providers to prioritise their interventions to increase immunisation rates.

Lower-crustal intrusion on the North Atlantic continental margin.

When continents break apart, the rifting is sometimes accompanied by the production of large volumes of molten rock. The total melt volume, however, is uncertain, because only part of it has erupted at the surface. Furthermore, the cause of the magmatism is still disputed-specifically, whether or not it is due to increased mantle temperatures. We recorded deep-penetration normal-incidence and wide-angle seismic profiles across the Faroe and Hatton Bank volcanic margins in the northeast Atlantic. Here we show that near the Faroe Islands, for every 1 km along strike, 360-400 km(3) of basalt is extruded, while 540-600 km(3) is intruded into the continent-ocean transition. We find that lower-crustal intrusions are focused mainly into a narrow zone approximately 50 km wide on the transition, although extruded basalts flow more than 100 km from the rift. Seismic profiles show that the melt is intruded into the lower crust as sills, which cross-cut the continental fabric, rather than as an 'underplate' of 100 per cent melt, as has often been assumed. Evidence from the measured seismic velocities and from igneous thicknesses are consistent with the dominant control on melt production being increased mantle temperatures, with no requirement for either significant active small-scale mantle convection under the rift or the presence of fertile mantle at the time of continental break-up, as has previously been suggested for the North Atlantic Ocean.

Mapping low intake of micronutrients across Europe.

Achieving an understanding of the extent of micronutrient adequacy across Europe is a major challenge. The main objective of the present study was to collect and evaluate the prevalence of low micronutrient intakes of different European countries by comparing recent nationally representative dietary survey data from Belgium, Denmark, France, Germany, The Netherlands, Poland, Spain and the United Kingdom. Dietary intake information was evaluated for intakes of Ca, Cu, I, Fe, Mg, K, Se, Zn and the vitamins A, B1, B2, B6, B12, C, D, E and folate. The mean and 5th percentile of the intake distributions were estimated for these countries, for a number of defined sex and age groups. The percentages of those with intakes below the lower reference nutrient intake and the estimated average requirement were calculated. Reference intakes were derived from the UK and Nordic Nutrition Recommendations. The impact of dietary supplement intake as well as inclusion of apparently low energy reporters on the estimates was evaluated. Except for vitamin D, the present study suggests that the current intakes of vitamins from foods lead to low risk of low intakes in all age and sex groups. For current minerals, the study suggests that the risk of low intakes is likely to appear more often in specific age groups. In spite of the limitations of the data, the present study provides valuable new information about micronutrient intakes across Europe and the likelihood of inadequacy country by country.

Revision Reverse Total Shoulder Arthroplasty for Failed Anatomic Total Shoulder Arthroplasty With Massive Irreparable Rotator Cuff Tear.

Anatomic total shoulder arthroplasty (TSA) has become more common as surgical indications have expanded. However, the burden of revision shoulder arthroplasty has inevitably increased as well. Multiple studies have examined the use of reverse total shoulder arthroplasty (rTSA) as a revision option for failed anatomic TSA with a massive irreparable rotator cuff tear. Successful reconstruction of failed TSA with rTSA requires sufficient glenoid bone to place the glenoid segment, enough proximal humeral bone to allow for implantation of the humeral component, and sufficient tension in the soft-tissue envelope to ensure implant stability. In this article, we describe our preferred rTSA revision technique for the treatment of a failed TSA.

Core polymer optimization of ternary siRNA nanoparticles enhances in vivo safety, pharmacokinetics, and tumor gene silencing.

Gene silencing with siRNA nanoparticles (si-NPs) is promising but still clinically unrealized for inhibition of tumor driver genes. Ternary si-NPs containing siRNA, a single block NP core-forming polymer poly[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] (DMAEMA-co-BMA, 50B), and an NP surface-forming diblock polymer 20 kDa poly(ethylene glycol)-block-50B (20kPEG-50B) have the potential to improve silencing activity in tumors due to the participation of both 50B and 20kPEG-50B in siRNA electrostatic loading and endosome disruptive activity. Functionally, single block 50B provides more potent endosomolytic activity, while 20kPEG-50B colloidally stabilizes the si-NPs. Here, we systematically explored the role of the molecular weight (MW) of the core polymer and of the core:surface polymer ratio on ternary si-NP performance. A library of ternary si-NPs was formulated with variation in the MW of the 50B polymer and in the ratio of the core and surface forming polymeric components. Increasing 50B core polymer MW and ratio improved si-NP in vitro gene silencing potency, endosome disruptive activity, and stability, but these features also correlated with cytotoxicity. Concomitant optimization of 50B size and ratio resulted in the identification of lead ternary si-NPs 50B4-DP100, 50B8-DP100, and 50B12-DP25, with potent activity and minimal toxicity. Following intravenous treatment in vivo, all lead si-NPs displayed negligible toxicological effects and enhanced pharmacokinetics and tumor gene silencing relative to more canonical binary si-NPs. Critically, a single 1 mg/kg intravenous injection of 50B8-DP100 si-NPs silenced the tumor driver gene Rictor at the protein level by 80% in an orthotopic breast tumor model. 50B8-DP100 si-NPs delivering siRictor were assessed for therapeutic efficacy in an orthotopic HCC70 mammary tumor model. This formulation significantly inhibited tumor growth compared to siControl-NP treatment. 50B8-DP100 si-NPs were also evaluated for safety and were well-tolerated following a multi-dose treatment scheme. This work provides new insight on ternary si-NP structure-function relationships and identifies core polymer optimization strategies that can yield safe si-NP formulations with potent oncogene silencing.

Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles.

The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.

Preventing and treating childhood obesity: time to target fathers.

OBJECTIVE: To examine the long-term effects of having one overweight or obese parent on child weight status and determine whether these effects vary according to parent sex. DESIGN: Prospective study: Longitudinal Study of Australian Children (LSAC). SUBJECTS: Two-parent families (N=3285) from the LSAC were included if height and weight data were available for both parents and their child at the 2004 and 2008 time points. MEASUREMENTS: Child weight status category (healthy, overweight, obese) in 2008 when the child was aged 8-9 years. Regression modelling was used to investigate how self-reported parent weight status in 2004 influenced measured child weight status 4 years later. RESULTS: Parent body mass index (BMI) was significantly correlated with child BMI, but there was no evidence of sex-specific associations between parent and child BMI correlations. The results from the regression analysis showed that having an overweight or obese father, but a healthy weight mother, significantly increased the odds of child obesity (odds ratio: 4.18, 95% confidence interval (CI): 1.01-17.33 and odds ratio: 14.88, 95% CI: 2.61-84.77, respectively), but the reverse scenario (overweight or obese mother with a healthy weight father) was not a significant predictor of child overweight or obesity (odds ratio: 2.52, 95% CI: 0.38-16.71 and odds ratio: 2.56, 95% CI: 0.31-21.26, respectively). CONCLUSIONS: Children with overweight or obese fathers are at a higher risk of becoming obese. This suggests that interventions are urgently required to test the efficacy of treating overweight fathers as a key strategy for childhood obesity prevention and/or treatment.

Oral rosmarinic acid-enhanced Mentha spicata modulates synovial fluid biomarkers of inflammation in horses challenged with intra-articular LPS.

A biological extract of high-rosmarinic acid mint (HRAM) has previously demonstrated inhibitory effects on lipopolysaccharide (LPS)-induced prostaglandin E(2) (PGE(2)), nitric oxide (NO) and glycosaminoglycan (GAG) release in vitro. This study was undertaken to determine whether HRAM added to feed produces similar effects in horses challenged with intra-articular LPS. Eight horses received HRAM (0 or 28.1 ± 1.3 g/day; n = 4 per group) in their feed for 24 days in a blinded manner. On day 21, all horses received an intra-articular injection of LPS (0.3 ng) into their left or right intercarpal joint. Synovial fluid (SF) samples were taken on postinjection day (PID)-21 (i.e. prior to commencement of supplementation), PID0, PID0.25, PID0.5, PID1 and PID3 and analysed for PGE(2), GAG, NO, protein and total nucleated cells counts. Blood biochemistry and haematology screens were conducted at PID-21, PID0, PID1 and PID3. There was a significant reduction in LPS-induced PGE(2) and GAG in SF in horses supplemented with HRAM compared with controls and a tendency to increase complement recognition protein accumulation in synovial fluid of HRAM horses. Plasma from HRAM horses had reduced total white blood cells, segmented neutrophils (compared with baseline concentrations) and lymphocytes (compared with controls), and increased SF nucleated cell count (compared with baseline concentrations and controls). It is concluded that HRAM offered as part of the feed alter biomarkers of inflammation in SF of LPS-challenged horses. Larger studies that seek to clarify effects of HRAM on synovial fluid cell counts and possible role of HRAM-induced interference with complement signalling are warranted.

Reverse Total Shoulder Arthroplasty for Treatment of Massive, Irreparable Rotator Cuff Tear.

Massive tears of the rotator cuff can result in severe functional deficits due to loss of the axial force couple and effective fulcrum that the intact cuff normally provides. For massive, irreparable rotator cuff tears, especially in the setting of early to moderate degenerative changes, reverse total shoulder arthroplasty functions to modify the center of joint rotation, allowing the deltoid and intact components of the cuff to carry out shoulder function more effectively. Our preferred technique uses a standard open deltopectoral shoulder approach with a 3-dimensional glenoid baseplate model and a 135° prosthesis in an onlay configuration to reduce the risk of scapular notching and increase lateralization of the humerus.

Shoulder strength outcomes after reverse total shoulder arthroplasty: a systematic review and descriptive synthesis.

Hypothesis and Background: There is no differences in abduction, internal rotation, or external rotation strength after reverse total shoulder arthroplasty (rTSA) with or without subscapularis repair. Repair of the subscapularis can be effective in the setting of rTSA. However, consensus has yet to be reached on whether postoperative strength after rTSA differs based on subscapularis management. The purpose of this review is to evaluate shoulder strength outcomes after rTSA with and without subscapularis tendon repair. Methods: A comprehensive literature review was conducted using the key terms "subscapularis" AND "reverse total shoulder arthroplasty" AND "muscle strength" in PubMed, Embase, Web of Science, Cochrane Reviews and Trials, and Scopus. Original, English-language studies evaluating shoulder strength outcomes after rTSA published from January 1, 2000, to present were evaluated. Strength outcomes reported included abduction strength (kg) and internal rotation strength (kg) using an electric spring balance and external rotation strength (lb) using a handheld dynamometer. Heterogeneity of data in the included studies did not allow for meta-analysis. Resuts: The search yielded 4253 unique results, which were screened for inclusion according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Two articles met eligibility criteria and were included in the final full-text review. A total of 267 shoulders were represented, 111 with subscapularis repair and 156 without subscapularis repair. No significant differences in abduction (P = .39), internal rotation (P = .09), and external rotation (P = .463) strength were observed between subscapularis repair and nonrepair groups. Conclusion: There were no differences in abduction, internal rotation, or external rotation strength after rTSA with or without subscapularis repair. The literature on postoperative strength outcomes after rTSA is limited, and further study in this area is warranted.

Subscapularis repair in reverse total shoulder arthroplasty: a systematic review and descriptive synthesis of cadaveric biomechanical strength outcomes.

Hypothesis/Background: There is no consensus on whether to repair the subscapularis in the setting of reverse total shoulder arthroplasty (rTSA). There have been an assortment of studies showing mixed results regarding shoulder stability and postoperative strength outcomes when looking at subscapularis repair in rTSA. The purpose of this systematic review was to investigate differences in biomechanical strength outcomes of cadaveric subscapularis repair vs. no repair in rTSA.Increased force will be required to move the shoulder through normal range of motion (ROM) in cadaveric rTSA shoulders with the subscapularis repaired when compared with no subscapularis repair. Methods: A comprehensive literature review was conducted in accordance with the 2009 Preferred Reporting Items for Systematic Review and Meta-Analysis statement. The databases used to search the keywords used for the concepts of subscapularis, reverse total shoulder arthroplasty, and muscle strength were PubMed (includes MEDLINE), Embase, Web of Science, Cochrane Reviews and Trials, and Scopus. Original, English-language cadaveric studies evaluating rTSA and subscapularis management were included, with subscapularis repair surgical techniques and strength outcomes being evaluated for each article meeting inclusion criteria. Results: The search yielded 4113 articles that were screened for inclusion criteria by 4 authors. Two articles met inclusion criteria and were subsequently included in the final full-text review. A total of 11 shoulders were represented between these 2 studies. Heterogeneity of the data across the 2 studies did not allow for meta-analysis. Hansen et al found that repair of the subscapularis with rTSA significantly increased the mean joint reaction force and the force required by the posterior deltoid, total deltoid, infraspinatus, teres minor, total posterior rotator cuff, and pectoralis major muscles. Giles et al found that rotator cuff repair and glenosphere lateralization both increased total joint load. Conclusion: The present review of biomechanical literature shows that repair of the subscapularis in the setting of rTSA can effectively restore shoulder strength by increasing joint reactive forces and ROM force requirements of other rotator cuff muscles and of the deltoid muscle. Available biomechanical evidence is limited, and further biomechanical studies evaluating the strength of various subscapularis repair techniques are needed to evaluate the effects of these techniques on joint reactive forces and muscle forces required for ROM.

Analyzing Modeled Torque Profiles to Understand Scale-Dependent Active Muscle Responses in the Hip Joint.

Animal locomotion is influenced by a combination of constituent joint torques (e.g., due to limb inertia and passive viscoelasticity), which determine the necessary muscular response to move the limb. Across animal size-scales, the relative contributions of these constituent joint torques affect the muscular response in different ways. We used a multi-muscle biomechanical model to analyze how passive torque components change due to an animal's size-scale during locomotion. By changing the size-scale of the model, we characterized emergent muscular responses at the hip as a result of the changing constituent torque profile. Specifically, we found that activation phases between extensor and flexor torques to be opposite between small and large sizes for the same kinematic motion. These results suggest general principles of how animal size affects neural control strategies. Our modeled torque profiles show a strong agreement with documented hindlimb torque during locomotion and can provide insights into the neural organization and muscle activation behavior of animals whose motion has not been extensively documented.

Hemorrhagic Resuscitation Guided by Viscoelastography in Far-Forward Combat and Austere Civilian Environments: Goal-Directed Whole-Blood and Blood-Component Therapy Far from the Trauma Center.

Modern approaches to resuscitation seek to bring patient interventions as close as possible to the initial trauma. In recent decades, fresh or cold-stored whole blood has gained widespread support in multiple settings as the best first agent in resuscitation after massive blood loss. However, whole blood is not a panacea, and while current guidelines promote continued resuscitation with fixed ratios of blood products, the debate about the optimal resuscitation strategy-especially in austere or challenging environments-is by no means settled. In this narrative review, we give a brief history of military resuscitation and how whole blood became the mainstay of initial resuscitation. We then outline the principles of viscoelastic hemostatic assays as well as their adoption for providing goal-directed blood-component therapy in trauma centers. After summarizing the nascent research on the strengths and limitations of viscoelastic platforms in challenging environmental conditions, we conclude with our vision of how these platforms can be deployed in far-forward combat and austere civilian environments to maximize survival.

The 'Healthy Dads, Healthy Kids' randomized controlled trial: efficacy of a healthy lifestyle program for overweight fathers and their children.

OBJECTIVE: To evaluate the feasibility and efficacy of the 'Healthy Dads, Healthy Kids' (HDHK) program, which was designed to help overweight fathers lose weight and be a role model of positive health behaviors for their children. DESIGN: Randomized controlled trial. PARTICIPANTS: A total of 53 overweight/obese men (mean (s.d.) age=40.6 (7.1) years; body mass index (BMI)=33.2 (3.9)) and their primary school-aged children (n=71, 54% boys; mean (s.d.) age=8.2 (2.0) years) were randomly assigned (family unit) to either (i) the HDHK program (n=27 fathers, n=39 children) or (ii) a wait-list control group (n=26 fathers, n=32 children). INTERVENTION: Fathers in the 3-month program attended eight face-to-face education sessions. Children attended three of these sessions. OUTCOMES: The primary outcome was fathers' weight. Fathers and their children were assessed at baseline, and at 3- and 6-month follow-up, for weight, waist circumference, BMI, blood pressure, resting heart rate (RHR), objectively measured physical activity and self-reported dietary intake. RESULTS: Intention-to-treat analysis revealed significant between-group differences at 6 months for weight loss (P<0.001), with HDHK fathers losing more weight (-7.6 kg; 95% confidence interval (CI) -9.2, -6.0; d=0.54) than control group fathers (0.0 kg; 95% CI -1.4, 1.6). Significant treatment effects (P<0.05) were also found for waist circumference (d=0.62), BMI (d=0.53), systolic blood pressure (d=0.92), RHR (d=0.66) and physical activity (d=0.91), but not for dietary intake. In children, significant treatment effects (P<0.05) were found for physical activity (d=0.74), RHR (d=0.51) and dietary intake (d=0.84). CONCLUSION: The HDHK program resulted in significant weight loss and improved health-related outcomes in fathers and improved eating and physical activity among children. Targeting fathers is a novel and efficacious approach to improving health behavior in their children.

Kupffer cell release of platelet activating factor drives dose limiting toxicities of nucleic acid nanocarriers.

This work establishes that Kupffer cell release of platelet activating factor (PAF), a lipidic molecule with pro-inflammatory and vasoactive signaling properties, dictates dose-limiting siRNA nanocarrier-associated toxicities. High-dose intravenous injection of siRNA-polymer nano-polyplexes (si-NPs) elicited acute, shock-like symptoms in mice, associated with increased plasma PAF and consequently reduced PAF acetylhydrolase (PAF-AH) activity. These symptoms were completely prevented by prophylactic PAF receptor inhibition or Kupffer cell depletion. Assessment of varied si-NP chemistries confirmed that toxicity level correlated to relative uptake of the carrier by liver Kupffer cells and that this toxicity mechanism is dependent on carrier endosome disruptive function. 4T1 tumor-bearing mice, which exhibit increased circulating leukocytes, displayed greater sensitivity to these toxicities. PAF-mediated toxicities were generalizable to commercial delivery reagent in vivo-jetPEI® and an MC3 lipid formulation matched to an FDA-approved nanomedicine. These collective results establish Kupffer cell release of PAF as a key mediator of siRNA nanocarrier toxicity and identify PAFR inhibition as an effective strategy to increase siRNA nanocarrier tolerated dose.

Diagnosing metal fume fever--an integrated approach.

BACKGROUND: Exposure to high concentrations of zinc oxide fume can cause metal fume fever (MFF). Two cases occurring following the oxy-acetylene cutting of galvanized steel frames are reported. AIMS: To show that integrated working is valuable as secondary health care staff may not be familiar with MFF. METHODS: Site visits by Her Majesty's Medical, Health and Safety (H&S) and Specialist Occupational Hygiene Inspectors to gather information and cooperation with occupational health, primary and secondary health care staff to diagnose MFF. RESULTS: Poor exposure control caused metal fume inhalation in these cases. Multidisciplinary working established diagnosis, causation and compliance with H&S legislation. CONCLUSIONS: Risk assessments are required to identify health risks and to determine required exposure control measures. Failure to do this resulted in an unusual case of MFF leading to hospital admission. Diagnosis can be delayed if a full occupational history is not taken. Successful investigation of such cases benefits from collaborative working.

Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides.

Rapid, facile, and noncovalent cell membrane modification with alkyl-grafted anionic polymers was sought as an approach to enhance intracellular delivery and bioactivity of cationic peptides. We synthesized a library of acrylic acid-based copolymers containing varying amounts of an amine-reactive pentafluorophenyl acrylate monomer followed by postpolymerization modification with a series of alkyl amines to afford precise control over the length and density of aliphatic alkyl side chains. This synthetic strategy enabled systematic investigation of the effect of the polymer structure on membrane binding, potentiation of peptide cell uptake, pH-dependent disruption of lipid bilayers for endosome escape, and intracellular bioavailability. A subset of these polymers exhibited pKa of ∼6.8, which facilitated stable membrane association at physiological pH and rapid, pH-dependent endosomal disruption upon endocytosis as quantified in Galectin-8-YFP reporter cells. Cationic cell penetrating peptide (CPP) uptake was enhanced up to 15-fold in vascular smooth muscle cells in vitro when peptide treatment was preceded by a 30-min pretreatment with lead candidate polymers. We also designed and implemented a new and highly sensitive assay for measuring the intracellular bioavailability of CPPs based on the NanoLuciferase (NanoLuc) technology previously developed for measuring intracellular protein-protein interactions. Using this split luciferase class of assay, polymer pretreatment enhanced intracellular delivery of the CPP-modified HiBiT peptide up to 30-fold relative to CPP-HiBiT without polymer pretreatment (p < 0.05). The overall structural analyses show that polymers containing 50:50 or 70:30 molar ratios of carboxyl groups to alkyl side chains of 6-8 carbons maximized peptide uptake, pH-dependent membrane disruption, and intracellular bioavailability and that this potentiation effect was maximized by pairing with CPPs with high cationic charge density. These results demonstrate a rapid, mild method for polymer modification of cell surfaces to potentiate intracellular delivery, endosome escape, and bioactivity of cationic peptides.

Tuning Composition of Polymer and Porous Silicon Composite Nanoparticles for Early Endosome Escape of Anti-microRNA Peptide Nucleic Acids.

Porous silicon nanoparticles (PSNPs) offer tunable pore structure and easily modified surface chemistry, enabling high loading capacity for drugs with diverse chemicophysical properties. While PSNPs are also cytocompatible and degradable, PSNP integration into composite structures can be a useful approach to enhance carrier colloidal stability, drug-cargo loading stability, and endosome escape. Here, we explored PSNP polymer composites formed by coating of oxidized PSNPs with a series of poly[ethylene glycol-block-(dimethylaminoethyl methacrylate-co-butyl methacrylate)] (PEG-DB) diblock copolymers with varied molar ratios of dimethylaminoethyl methacrylate (D) and butyl methacrylate (B) in the random copolymer block. We screened and developed PSNP composites specifically toward intracellular delivery of microRNA inhibitory peptide nucleic acids (PNA). While a copolymer with 50 mol % B (50B) is optimal for early endosome escape in free polymer form, its pH switch was suppressed when it was formed into 50B polymer-coated PSNP composites (50BCs). We demonstrate that a lower mol % B (30BC) is the ideal PEG-DB composition for PSNP/PEG-DB nanocomposites based on having both the highest endosome disruption potential and miR-122 inhibitory activity. At a 1 mM PNA dose, 30BCs facilitated more potent inhibition of miR-122 in comparison to 40BC (p = 0.0095), 50BC (p < 0.0001), or an anti-miR-122 oligonucleotide delivered with the commercial transfection reagent Fugene 6. Using a live cell galectin 8-based endosome disruption reporter, 30BCs had greater endosomal escape than 40BCs and 50BCs within 2 h after treatment, suggesting that rapid endosome escape correlates with higher intracellular bioactivity. This study provides new insight on the polymer structure-dependent effects on stability, endosome escape, and cargo intracellular bioavailability for endosomolytic polymer-coated PSNPs.

Structural Optimization of Polymeric Carriers to Enhance the Immunostimulatory Activity of Molecularly Defined RIG-I Agonists.

RNA ligands of retinoic acid-inducible gene I (RIG-I) hold significant promise as antiviral agents, vaccine adjuvants, and cancer immunotherapeutics, but their efficacy is hindered by inefficient intracellular delivery to the cytosol where RIG-I is localized. Here, we address this challenge through the synthesis and evaluation of a library of polymeric carriers rationally designed to promote the endosomal escape of 5'-triphosphate RNA (3pRNA) RIG-I agonists. We synthesized a series of PEG-block-(DMAEMA-co-A n MA) polymers, where A n MA is an alkyl methacrylate monomer ranging from n = 2-12 carbons, of variable composition, and examined effects of polymer structure on the intracellular delivery of 3pRNA. Through in vitro screening of 30 polymers, we identified four lead carriers (4-50, 6-40, 8-40, and 10-40, where the first number refers to the alkyl chain length and the second number refers to the percentage of hydrophobic monomer) that packaged 3pRNA into ∼100-nm-diameter particles and significantly enhanced its immunostimulatory activity in multiple cell types. In doing so, these studies also revealed an interplay between alkyl chain length and monomer composition in balancing RNA loading, pH-responsive properties, and endosomal escape, studies that establish new structure-activity relationships for polymeric delivery of 3pRNA and other nucleic acid therapeutics. Importantly, lead carriers enabled intravenous administration of 3pRNA in mice, resulting in increased RIG-I activation as measured by increased levels of IFN-α in serum and elevated expression of Ifnb1 and Cxcl10 in major clearance organs, effects that were dependent on polymer composition. Collectively, these studies have yielded novel polymeric carriers designed and optimized specifically to enhance the delivery and activity of 3pRNA with potential to advance the clinical development of RIG-I agonists.