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Background: Clinical collaboration between spine professionals in high-income countries (HICs) and low-and-middle-income countries (LMICs) may provide improvements in the accessibility, efficacy, and safety of global spine care. Currently, the scope and effectiveness of these collaborations remain unclear. In this review, we describe the literature on the current state of these partnerships to provide a framework for exploring future best practices., Methods: PubMed, Embase, and Cochrane Library were queried for articles on spine-based clinical partnerships between HICs and LMICs published between 2000 and March 10, 2023. This search yielded 1528 total publications. After systematic screening, nineteen articles were included in the final review., Results: All published partnerships involved direct clinical care and 13/19 included clinical training of local providers. Most of the published collaborations reviewed involved one of four major global outreach organizations with the majority of sites in Africa. Participants were primarily physicians and physicians-in-training. Only 5/19 studies reported needs assessments prior to starting their partnerships. Articles were split on evaluative focus, with some only evaluating clinical outcomes and some evaluating the nature of the partnership itself., Conclusions: Published studies on spine-focused clinical partnerships between HICs and LMICs remain scarce. Those that are published often do not report needs assessments and formal metrics to evaluate the efficacy of such partnerships. Toward improving the quality of spine care globally, we recommend an increase in the quality and quantity of published studies involving clinical collaborations between HICs and LICs, with careful attention to reporting early needs assessments and evaluation strategies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Although interspinous and supraspinous ligaments of the lumbar spine are thought to contribute to spinal stability, little is known about their dynamic biomechanics. We demonstrate that shear wave elastography (SWE) offers a novel technique to noninvasively and quantitatively evaluate posterior spinous ligament complex functional loading and stiffness in different physiologic positions., Methods: We performed SWE and measured the length of the interspinous/supraspinous ligament complex in cadaveric torsos ( N = 5), isolated ligaments ( N = 10), and healthy volunteers ( N = 9) to obtain length and shear wave velocity measurements. For cadavers and volunteers, SWE was utilized in 2 lumbar positions: lumbar spine flexion and extension. In addition, SWE was performed on isolated ligaments undergoing uniaxial tension to correlate shear wave velocities with experienced load., Results: Average shear wave velocity in cadaveric supraspinous/interspinous ligament complexes increased for lumbar levels (23%-43%) and most thoracic levels (0%-50%). This corresponded to an average increase in interspinous distance from extension to flexion for the lumbar spine (19%-63%) and thoracic spine (3%-8%). Volunteer spines also demonstrated an average increase in shear wave velocity from extension to flexion for both the lumbar spine (195% at L2-L3 to 200% at L4-L5) and thoracic spine (31% at T10-T11). There was an average increase in interspinous distance from extension to flexion for the lumbar spine (93% at L2-L3 to 127% at L4-L5) and thoracic spine (11% at T10-T11). In isolated ligaments, there was a positive correlation between applied tensile load and average shear wave velocity., Conclusion: This study creates a foundation to apply SWE as a noninvasive tool for assessing the mechanical stiffness of posterior ligamentous structures and has potential applications in augmenting or evaluating these ligaments in patients with spine pathology., Clinical Relevance: The interspinous and supraspinous ligaments are critical soft tissue supports of the posterior lumbar spine. Disruption of these structures is thought to have a negative impact on spinal stability in trauma and spine deformities., Competing Interests: Declaration of Conflicting Interests : The authors report no conflicts of interest in this work., (This manuscript is generously published free of charge by ISASS, the International Society for the Advancement of Spine Surgery. Copyright © 2023 ISASS. To see more or order reprints or permissions, see http://ijssurgery.com.)

Background: Pediatric spinal fusion may be associated with significant intraoperative blood loss, leading to complications from transfusion, hypoperfusion and coagulopathy. One emerging strategy to mediate these risks is by utilization of the anti-fibrinolytic agent tranexamic acid (TXA). However, concerns regarding potential adverse reactions, specifically postoperative seizures and thrombotic events, still exist. To assess these risks, we examined the perioperative morbidity of TXA use in a large national database., Methods: Retrospective data from pediatric patients (age 18 years or younger), discharged between January 2013 to December 2015, who underwent primary or revision posterior spinal fusions, was collected from the Premier Perspective database (Premier, Charlotte, NC). Patients were stratified by TXA use and records were assessed for complications of new onset seizures, strokes, pulmonary embolisms (PE) or deep vein thromboses (DVT) occurring during the perioperative period., Results: In this cohort of 2,633 pediatric patients undergoing posterior spinal fusions, most often to treat adolescent idiopathic scoliosis, 15% received TXA. Overall, adverse events were rare in this patient population. The incidence of seizure, stoke, PE, or DVT in the control group was 0.54% (95% CI, 0.31% to 0.94%) and not significantly different from the TXA group. There was no significant difference in the incidence of DVTs, and no incidences of stroke in either group. There were no new-onset seizures or PEs in patients who received TXA., Conclusions: The use of TXA was not associated with an increased risk of adverse events including seizure, stroke, PE, and DVT. Our findings support the safety of TXA use in pediatric patients undergoing spinal fusion surgery., (© 2022. The Author(s).)

Background: Prior studies have demonstrated an increase in the performance of outpatient anterior cervical surgery. The degree to which this increase is due to volume increase per individual surgeon versus increase in individual surgeons performing outpatient cervical surgery is unknown., Methods: Patients undergoing anterior cervical discectomy and fusion (ACDF) or cervical disk arthroplasty (CDA) between 2010 and 2018 in NY state were identified. As a comparison we also evaluated trends for inpatient ACDF and CDA. Annual outpatient case volumes were calculated and defined as being high (> 20/year), intermediate (>5 and ≤ 20/year) or low (>1 and ≤ 5/year). Descriptive statistics were used to report temporal trends and Poisson regression was used to test for statistical significance. We also analyzed trends in various operative metrics by surgeon volume., Results: In 2010, there were 96 surgeons who performed outpatient ACDF or CDA on a total of 1,855 patients. In 2018, this increased to 253 surgeons performing outpatient ACDF or CDA on a total of 3,372 patients. In comparison, there were 350 surgeons performing 6,783 inpatient cases in 2010 and 376 surgeons performing 6,796 inpatient cases in 2018. The average annual outpatient case volume decreased from 18.8 (95% CI, 13.5 - 24.1) to 12.2 (95% CI, 10.0 - 14.3) surgeries per surgeon. The percentage of surgeons with a high case volume also decreased from 30.2% in 2010 to 10.7% in 2018, whereas the percentage with a low case volume increased (32.3% to 49.8%). Differences between high and low volume surgeons in operative time, length of stay and total charges widened over time., Conclusion: The increase in outpatient anterior cervical surgery appears to be primarily driven by a greater number of surgeons performing ACDF and CDA on an outpatient basis, as opposed to increased case volumes for each surgeon. In contrast, trends for inpatient anterior cervical surgery were stable., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Published by Elsevier Ltd on behalf of North American Spine Society.)

Abstract: Globally, the burden of musculoskeletal conditions continues to rise, disproportionately affecting low and middle-income countries (LMICs). The ability to meet these orthopaedic surgical care demands remains a challenge. To help address these issues, many orthopaedic surgeons seek opportunities to provide humanitarian assistance to the populations in need. While many global orthopaedic initiatives are well-intentioned and can offer short-term benefits to the local communities, it is essential to emphasize training and the integration of local surgeon-leaders. The commitment to developing educational and investigative capacity, as well as fostering sustainable, mutually beneficial partnerships in low-resource settings, is critical. To this end, global health organizations, such as the Consortium of Orthopaedic Academic Traumatologists (COACT), work to promote and ensure the lasting sustainability of musculoskeletal trauma care worldwide. This article describes global orthopaedic efforts that can effectively address musculoskeletal care through an examination of 5 domains: clinical care, clinical research, surgical education, disaster response, and advocacy., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJS/G840)., (Copyright © 2021 The Authors. Published by The Journal of Bone and Joint Surgery, Incorporated. All rights reserved.)

Background: Considered the gold standard of study designs, randomized controlled trials' (RCTs) results shape clinical practice, effect policy, and influence reimbursement. The fragility index (FI) can be used to quantitate the relative robustness of RCT results, with higher scores indicating more stout results. Unfortunately, most RCTs in surgery have fragile results. The aim of this study was to report on the FI in addition to a qualitative assessment of recent RCTs within the field of shoulder and elbow surgery., Methods: A systematic review was performed identifying recently published shoulder/elbow RCTs that included 1:1 allocated parallel study arms, dichotomous primary outcome variables, and statistical significance. The FI was calculated by sequentially modifying outcome groups by exchanging a nonevent in one group to an event until the P value for the outcome comparison, as calculated by the Fisher exact test, was increased above the .05 threshold., Results: Thirty RCTs were included. The median FI was 4. Sixty percent trials had a FI of 2 or less. Fifty-three percent studies reported that participants were lost to follow-up. In 87.5% of these studies, the losses to follow-up exceeded their respective FIs. Only 53% of studies defined a primary outcome variable and 60% studies performed a prestudy power analysis., Conclusions: The median FI reported in the recent shoulder/elbow literature is 4; however, a high proportion of included RCTs display significant methodological concerns. The FI is a useful adjunct to analyze RCT results, but careful analysis of trial methods should be employed in each circumstance before drawing conclusions., (Copyright © 2019 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Purpose: Randomized controlled trials (RCTs) are the gold standard for comparing clinical interventions. Statistical significance as reported via a P value has been used to determine if a difference between clinical interventions exists in an RCT. However, P values do not clearly convey information about the robustness of a study's conclusions. An emerging metric, called the fragility index (the number of subjects who would need to change outcome category to raise the P value above the .05 threshold), is an indirect measure of how likely a repeat of the trial would reach the same conclusions. This study addressed the fragility of RCTs using dichotomous outcomes in hand surgery., Methods: Using systematic searching of the MEDLINE database, we identified hand surgery RCTs published in 11 high-impact journals published in the last decade (2007-2017). Studies were identified that involved 2 parallel arms, allocated patients to treatment and control in a 1:1 ratio, and reported statistical significance for a dichotomous variable. The fragility index was calculated using Fisher's exact test, using previously published methods., Results: Five hand surgery RCTs were identified for inclusion reporting a range of fragility indices from 0 to 26. Two of the trials (40%) had a fragility index of 2 or less. Two of the trials (40%) reported that the number of patients lost to follow-up exceeded the fragility index, meaning that results of the patients lost to follow-up could theoretically completely reverse the study conclusions., Conclusions: The range of fragility indices reported in the recent hand surgery literature is consistent with previous reporting within orthopedic surgery., Clinical Relevance: The fragility index is a useful metric to analyze the robustness of the study conclusions that should complement other methods of critical evaluation including the P value or effect sizes. Our results emphasize the need for future efforts to strengthen the robustness of RCT conclusions., (Copyright © 2019 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Study Design: Retrospective database analysis., Objective: To identify preoperative risk factors for emergency department (ED) visit and unplanned hospital readmission after primary anterior cervical discectomy and fusion (ACDF) at 30 and 90 days., Summary of Background Data: Limited data exist to identify factors associated with ED visit or readmission after primary ACDF within the first 3 months following surgery., Methods: Patients undergoing ACDF from 2005 to 2012 were identified in the Statewide Planning and Research Cooperative System database. Multivariable regression models were created based on patient-level and surgical characteristics to identify independent risk factors for hospital revisit., Results: Of 41,813 patients identified, 2514 (6.0%) returned to the ED within 30 days of discharge. Risk factors included age < 35, black race (OR 1.19), Charlson Comorbidity index score > 1, length of stay (LOS) greater than 1 day (OR 1.23), and fusion of > 2 levels (OR 1.17). Four thousand six hundred nine (11.0%) patients returned to the ED within 90 days. Risk factors mirrored those at 30 days. Patients having private insurance or those discharged to rehab were less likely to present to the ED. One thousand three hundred ninety-four (3.3%) patients were readmitted by 30 days. Risk factors included male sex, Medicare, or Medicaid insurance (OR 1.71 and 1.79 respectively), Charlson comorbidity index > 1, discharge to a skilled nursing facility (OR 2.90), infectious/pathologic (OR 3.296), or traumatic (OR 1.409) surgical indication, LOS > 1 day (OR 1.66), or in-hospital complication. 2223 (5.3%) patients were readmitted by 90 days. Risk factors mirrored those at 30 days. No differences in readmission were seen based on race or number of levels fused. Patients aged 18 to 34 were less likely to be readmitted versus patients older than 35., Conclusion: Insurance status, comorbidities, and LOS consistently predicted an unplanned hospital visit at 30 and 90 days. Although nondegenerative surgical indications and in-hospital complications did not predict ED visits, these factors increased the risk for readmission., Level of Evidence: 3.

Background: Statistical significance, as reported by the P value, has traditionally been the most commonly reported way to determine whether a difference exists between clinical interventions. Unfortunately, P values alone confer little about the robustness of a study's conclusions. An emerging metric, the fragility index (FI), helps to address this challenge by quantifying the number of events per outcome group that would need to be reversed to the alternative outcome in order to raise the P value above the 0.05 threshold., Methods: Using systematic search strategy, we identified randomized controlled trials (RCTs) pertaining to clavicular fractures published in the last decade (2007-2017). Studies included for analysis involved 2 parallel arms, were published in English, allocated patients to treatment and control arms in a 1:1 ratio, and reported statistical significance (P < .05) for dichotomous variables. The FI was determined based on the Fisher exact test, using previously published methods., Results: Fifteen RCTs were included. The median FI was 2 (range, 0-17). Eleven studies (73.3%) had an FI of 2 or less. Seven of the trials (46.7%) reported that the number of patients lost to follow-up exceeded the FI., Conclusions: The median FI reported in the recent literature on clavicular fractures is only 2. The FI is a useful metric to analyze the robustness of study conclusions that should complement other methods of critical data evaluation, including the P value or effect sizes. Future efforts are needed to increase institutional collaboration and patient recruitment to strengthen the robustness of RCT conclusions, especially in the realm of clavicular fracture management., (Copyright © 2018 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Background Context: Postdischarge care is a significant source of cost variability after posterior lumbar fusion surgery. However, there remains limited evidence associating postdischarge inpatient services and improved postoperative outcomes, despite the high cost of these services., Purpose: To determine the association between posthospital discharge to inpatient care facilities and postoperative complications., Study Design: A retrospective review of all 1- to 3-level primary posterior lumbar fusion cases in the 2010-2014 National Surgical Quality Improvement Program registry was conducted. Propensity scores for discharge destination were determined based on observable baseline patient characteristics. Multivariable propensity-adjusted logistic regressions were performed to determine associations between discharge destination and postdischarge complications, with adjusted odds ratios (OR) and 95% confidence intervals (CI)., Results: A total of 18,652 posterior lumbar fusion cases were identified, 15,234 (82%) were discharged home, and 3,418 (18%) were discharged to continued inpatient care. Multivariable propensity-adjusted analysis demonstrated that being discharged to inpatient facilities was independently associated with higher risk of thromboembolic complications (OR [95% CI]: 1.79 [1.13-2.85]), urinary complications, (1.79 [1.27-2.51]), and unplanned readmissions (1.43 [1.22-1.68])., Conclusions: Discharge to continued inpatient care versus home after primary posterior lumbar fusion is independently associated with higher odds of certain major complications. To optimize clinical outcomes as well as cost savings in an era of value-based reimbursements, clinicians and hospitals should consider further investigation into carefully investigating which patients might be better served by home discharge after surgery., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The randomized controlled trial (RCT) is the gold standard study design allowing critical comparison of clinical outcomes while minimizing bias. Traditionally clinical trials are evaluated through statistical significance, expressed by P-values and confidence intervals. However, until recently, the robustness of a study's conclusions has been given little attention. A new metric, the fragility index, quantifies the number of patients theoretically required to switch outcomes in order to reverse the study conclusions. The primary aim of our work was to determine the fragility index of RCTs in the pediatric orthopaedic literature. The secondary aim was to determine study factors associated with lower fragility index., Methods: Pubmed and Embase were systematically searched for pediatric orthopaedic RCTs published September 1, 2006 to September 1, 2016. Two independent reviewers screened titles, abstracts, and manuscripts to identify studies published in English involving 2 treatment arms. Trials without dichotomous primary or secondary outcomes or with patients >18 years were excluded. Data were extracted from each eligible article in duplicate and the fragility index was determined using Fisher exact test, with previously published methods. Univariate analysis was used to determine factors associated with lower fragility index., Results: Seventeen trials were eligible for inclusion. The median treatment arm size was 58 and overall sample size was 116 patients. The median fragility index was 3 (range, 0 to 18). A fragility index of 3 means that just 3 patients would need to switch treatment outcomes in order for the trial results to become statistically nonsignificant. In 1 study, the number of patients lost to follow-up exceeded the fragility index, such that the study conclusions could be completely reversed purely depending on the outcomes of the patients lost to follow-up. Lower fragility index was associated with smaller patient sample sizes and greater P-values., Conclusions: The fragility index is a useful adjunct metric to the P-value and confidence intervals, allowing analysis of the robustness of study conclusions. RCTs in pediatric orthopaedics often have small sample sizes, many with low fragility indices. Future efforts could focus on encouraging institutional collaboration and patient recruitment with the ultimate goal of improving RCT sample sizes, and potentially improving the robustness of RCT results., Level of Evidence: Level I.

Background: To determine if new-onset perioperative atrial fibrillation during arthroplasty represents a benign response to intraoperative cardiac stress or is a risk factor for stroke, we evaluated the subsequent risk of ischemic stroke in patients with new-onset atrial fibrillation occurring during primary total knee arthroplasty (TKA) and total hip arthroplasty (THA)., Methods: Discharge data of all adult patients undergoing primary TKA or THA from 1997 to 2013 were queried via the New York Statewide Planning and Research Cooperative System database to find patients with new-onset perioperative atrial fibrillation. These patients were then followed up over time to determine their risk of ischemic stroke., Results: Of the 312,636 TKA and 215,610 THA unique patient admissions, 3646 (0.7%) had a diagnosis of new-onset perioperative atrial fibrillation. The cohort of patients with this finding was 58.9% female with an average age of 73.6 years and higher prevalence of vascular risk factors. Adjusting for validated stroke risk factors, the risk of ischemic stroke within 1 year after THA or TKA in patients with new-onset atrial fibrillation was 2.7 times higher than in those without a history of atrial fibrillation (odds ratio: 2.7, 95% confidence interval: 1.5-4.8). Hospital length of stay and charges for patients with new-onset atrial fibrillation were also greater than patients with either a prior diagnosis or no diagnosis of atrial fibrillation., Conclusion: New-onset atrial fibrillation during TKA and THA may indicate risk of ischemic stroke following surgery that should warrant medical follow-up and may increase hospital length of stay and charges., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Modularity in total hip arthroplasty facilitates intraoperative restoration of patient anatomy. Although dual-taper modular total hip arthroplasty offers potential advantages for optimizing the hip center of rotation, it has been associated with modular taper corrosion. This corrosion has led to adverse local tissue reactions (pseudotumors) at the neck-stem junction and elevated metal-ion levels. However, the occurrence of taper-corrosion-related pseudotumors in patients who remain asymptomatic following total hip arthroplasty with a dual-taper modular femoral stem remains largely unknown. The aims of this study were (1) to determine the prevalence of asymptomatic pseudotumors by utilizing metal artifact reduction sequence magnetic resonance imaging (MARS-MRI) and (2) compare serum metal-ion levels between symptomatic and asymptomatic patients with a dual-taper modular stem total hip replacement., Methods: We performed a retrospective cross-sectional study of 97 consecutive patients who had been treated with a dual-taper modular femoral stem total hip arthroplasty. Eighty-three patients were stratified into symptomatic and asymptomatic groups and evaluated with MARS-MRI, measurement of serum metal-ion levels, and the University of California at Los Angeles (UCLA) functional hip score., Results: The prevalence of pseudotumors as determined with MARS-MRI was 15% in our asymptomatic patients and 36% in the overall cohort. The median serum cobalt level and cobalt/chromium ratio were significantly higher in patients with a pseudotumor than in those without a pseudotumor (8.0 versus 2.0 μg/L [p = 0.004] and 10.3 versus 2.4 μg/L [p = 0.012], respectively). However, there was no significant difference in the serum cobalt level or cobalt/chromium ratio between symptomatic patients with a pseudotumor and asymptomatic patients with a pseudotumor (7.6 versus 6.2 μg/L [p = 0.37] and 8.3 versus 10.6 μg/L [p = 0.46], respectively). The UCLA scores of asymptomatic patients with a pseudotumor were similar to those of patients without a pseudotumor (6.7 versus 6.6)., Conclusions: The prevalence of asymptomatic taper-corrosion-related pseudotumors on MARS-MRI in this study demonstrated that the absence of symptoms does not exclude the presence of adverse local tissue reactions. Elevated cobalt levels and cobalt/chromium ratios were associated with the presence of pseudotumors in asymptomatic and symptomatic patients. Cross-sectional imaging such as MARS-MRI is indicated for patients with elevated metal-ion levels. A longitudinal study is required to determine whether asymptomatic patients with taper-corrosion-related pseudotumors will develop symptoms with time., Level of Evidence: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence., (Copyright © 2016 by The Journal of Bone and Joint Surgery, Incorporated.)

Context: The rising popularity and intensity of youth sports has increased the incidence of patellar dislocation. These sports-related injuries may be associated with significant morbidity in the pediatric population. Treatment requires understanding and attention to the unique challenges in the skeletally immature patient., Evidence Acquisition: PubMed searches spanning 1970-2013., Study Design: Clinical review., Level of Evidence: Level 5., Results: Although nonoperative approaches are most often suitable for first-time patellar dislocations, surgical treatment is recommended for acute fixation of displaced osteochondral fractures sustained during primary instability and for patellar realignment in the setting of recurrent instability. While a variety of procedures can prevent recurrence, the risk of complications is not minimal., Conclusion: Patellar stabilization and realignment procedures in skeletally immature patients with recurrent patellar dislocation can effectively treat patellar instability without untoward effects on growth if careful surgical planning incorporates protection of growth parameters in the skeletally immature athlete.

Monitoring for drug-induced liver injury (DILI) via serial transaminase measurements in patients on potentially hepatotoxic medications (e.g., for HIV and tuberculosis) is routine in resource-rich nations, but often unavailable in resource-limited settings. Towards enabling universal access to affordable point-of-care (POC) screening for DILI, we have performed the first field evaluation of a paper-based, microfluidic fingerstick test for rapid, semi-quantitative, visual measurement of blood alanine aminotransferase (ALT). Our objectives were to assess operational feasibility, inter-operator variability, lot variability, device failure rate, and accuracy, to inform device modification for further field testing. The paper-based ALT test was performed at POC on fingerstick samples from 600 outpatients receiving HIV treatment in Vietnam. Results, read independently by two clinic nurses, were compared with gold-standard automated (Roche Cobas) results from venipuncture samples obtained in parallel. Two device lots were used sequentially. We demonstrated high inter-operator agreement, with 96.3% (95% C.I., 94.3-97.7%) agreement in placing visual results into clinically-defined "bins" (<3x, 3-5x, and >5x upper limit of normal), >90% agreement in validity determination, and intraclass correlation coefficient of 0.89 (95% C.I., 0.87-0.91). Lot variability was observed in % invalids due to hemolysis (21.1% for Lot 1, 1.6% for Lot 2) and correlated with lots of incorporated plasma separation membranes. Invalid rates <1% were observed for all other device controls. Overall bin placement accuracy for the two readers was 84% (84.3%/83.6%). Our findings of extremely high inter-operator agreement for visual reading-obtained in a target clinical environment, as performed by local practitioners-indicate that the device operation and reading process is feasible and reproducible. Bin placement accuracy and lot-to-lot variability data identified specific targets for device optimization and material quality control. This is the first field study performed with a patterned paper-based microfluidic device and opens the door to development of similar assays for other important analytes.

The use of small interfering RNAs (siRNAs) to down-regulate the expression of disease-associated proteins carries significant promise for the treatment of a variety of clinical disorders. One of the main barriers to the widespread clinical use of siRNAs, however, is their entrapment and degradation within the endolysosomal pathway of target cells. Here we report the trafficking and function of PP75, a non-toxic, biodegradable, lipid membrane disruptive anionic polymer composed of phenylalanine derivatized poly(L-lysine iso -phthalamide). PP75 is readily endocytosed by cells, safely permeabilizes endolysosomes in a pH dependent manner and facilitates the transfer of co-endocytosed materials directly into the cytoplasm. The covalent attachment of siRNAs to PP75 using disulfide linkages generates conjugates that effectively traffic siRNAs to the cytoplasm of target cells both in vitro and in vivo . In a subcutaneous malignant glioma tumor model, a locally delivered PP75-stathmin siRNA conjugate decreases stathmin expression in tumor cells and, in combination with the nitrosourea chemotherapy carmustine, is highly effective at inhibiting tumor growth. PP75 may be clinically useful for the local delivery of siRNAs, in particular for the treatment of solid tumors.

The clinical potential of short interfering RNA (siRNA) based therapeutics remains hindered by the challenge of delivering enough siRNA into the cytoplasm to yield a clinically relevant effect. Although much research has focused on optimizing delivery vehicles for this class of molecules, considerably less is known about the microenvironmental influences on the response of target cells to siRNA. The substrate to which cells adhere is one component of the microenvironment that can modulate cellular behavior. Here, we tested the hypothesis that modulating the properties of cellular adhesion substrates can alter siRNA efficacy. Specifically, cationic lipid complexed siRNA particles were applied to U251 cells seeded on alginate hydrogel surfaces with systematic variation in elastic modulus and integrin ligand arginine-glycine-aspartate (RGD) peptide density. These experiments revealed no change in siRNA-mediated eGFP knockdown over the elastic modulus range tested (53-133 kPa). However, an eightfold increase in RGD content of the alginate growth substrate resulted in an increase in siRNA knockdown efficacy from 25 ± 12% to 52 ± 10%, a more than twofold increase in silencing. Our results identify control of the cell-adhesion substrate interaction as a modulator of siRNA protein silencing efficacy., (Copyright © 2012 Wiley Periodicals, Inc.)

The endosomal membrane has proven to be a challenging barrier for the delivery of therapeutic biomacromolecules, including DNA, siRNA and proteins, which are taken up by endosomes but cannot freely diffuse across lipid bilayers. Anionic polymers that undergo conformational changes and become membrane disruptive in low-pH environments have the potential to assist in the delivery of these biomacromolecules across the endosomal membrane to the cytosol. Such endosomolytic polymers have been synthesized through the grafting of hydrophobic side-chains to a poly(L-lysine iso-phthalamide) backbone. The phenylalanine grafted form of poly(L-lysine iso-phthalamide) has a pH-sensitive membrane disruptive profile corresponding to the pH range of maturing endosomes and, thus, has a favourable endosomolytic profile. In order to understand the influence of hydrophobicity versus pi-pi interactions mediated by aromatic rings, a tyrosine grafted form of poly(L-lysine iso-phthalamide) was synthesized and its aqueous pH-sensitive properties, cytotoxicity and endosomal disruptive capacity were compared to phenylalanine-grafted poly(L-lysine iso-phthalamide). The similarity between these two polymers' properties, despite the large difference in hydrophobicity between their side-chains, supports the conclusion that the aromatic character of sidechains in poly(L-lysine iso-phthalamide) is an important property, as opposed to hydrophobicity alone, in determining the effectiveness of acidic pH triggered endosomolysis.

A series of pH-responsive polymers have been synthesized by grafting L-leucine onto the pendant carboxylic acid groups of the linear pseudopeptide, poly(L-lysine iso-phthalamide). The effect of the degree of grafting on aqueous solution properties, cell membrane-disruptive activity, and in vitro cytotoxicity was examined by UV-visible and fluorescence spectroscopy, hemolysis, alamar blue staining, and propidium iodide fluorescence assays. Modification of poly(L-lysine iso-phthalamide) with < or =23.6 mol % L-leucine caused a marginal effect on the pH-mediated hydrophobic association and hemolytic activity. Increasing the degree of grafting from 31.9 to 61.2 mol % resulted in polymers with progressively enhanced hydrophobic association and cell membrane disruption, thus confirming that the pH responsiveness and the extent of hydrophobic association and membrane disruption of the polymers can be modulated by varying the degree of grafting with hydrophobic amino acids. The pH responses were demonstrated to be concentration-dependent. At certain degrees of leucine grafting, the polymers were nonmembrane-lytic at physiological pH but mediated considerable membrane lysis at endosomal pH values (5.0-6.8), a feature critical for potential drug delivery applications.

pH-responsive polymers have been synthesised by grafting l-valine (PV-75), l-leucine (PL-75) and l-phenylalanine (PP-75) onto the pendant carboxylic acid moieties of a pseudo-peptide, poly(l-lysine iso-phthalamide), at a stoichiometric degree of substitution of 75 mol%. The effect of such modification on the pH-, concentration- and time-dependent cell membrane-disruptive activity of the grafted polymers has been investigated using a haemolysis model. At 0.025 mg mL(-1), the grafted polymers were almost non-haemolytic at pH 7.4, but mediated considerable membrane lysis after 60 min in the pH range characteristic of early endosomes, which ranked in the order: PP-75 > PL-75 > PV-75 > poly(l-lysine iso-phthalamide). PP-75 was 35-fold more lytic on a molar basis than the membrane-lytic peptide melittin. With increasing concentration, the grafted polymers showed an increased ability to lyse cell membranes and caused noticeable membrane disruption at physiological pH. The mechanism of the polymer-mediated membrane destabilisation has been investigated. The in-vitro cytotoxicity of the grafted polymers has been assessed using a propidium iodide fluorescence assay. It has been demonstrated by confocal microscopy that the grafted polymers can induce a significant release of endocytosed materials into the cytoplasm of HeLa cells, which is a feature critical for drug delivery applications.