Branched-Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH.

The potential of mRNA therapeutics will be realized only once safe and effective delivery systems are established. Unfortunately, delivery vehicle development is stymied by an inadequate understanding of how the molecular properties of a vehicle confer efficacy. Here, a small library of lipidoid materials is used to elucidate structure-function relationships and identify a previously unappreciated parameter-lipid nanoparticle surface ionization-that correlates with mRNA delivery efficacy. The two most potent materials of the library, 306O10 and 306Oi10 , induce substantial luciferase expression in mice following a single 0.75 mg kg-1 mRNA dose. These lipidoids, which have ten-carbon tails and identical molecular weights, vary only in that the 306O10 tail is straight and the 306Oi10 tail has a one-carbon branch. Remarkably, this small difference in structure conferred a tenfold improvement in 306Oi10 efficacy. The enhanced potency of this branched-tail lipidoid is attributed to its strong surface ionization at the late endosomal pH of 5.0. A secondary lipidoid library confirms that Oi10 materials ionize more strongly and deliver mRNA more potently than lipidoids containing linear tails. Together, these data highlight the exquisite control that lipid chemistry exerts on the mRNA delivery process and show that branched-tail lipids facilitate protein expression in animals.

Lipid nanoparticle siRNA cocktails for the treatment of mantle cell lymphoma.

Mantle cell lymphoma is an aggressive and incurable subtype of non-Hodgkin B cell lymphoma. Patients typically present with advanced disease, and most patients succumb within a decade of diagnosis. There is a clear and urgent need for novel therapeutic approaches that will affect mantle cell lymphoma through a unique mechanism compared to current therapies. This study examined the use of RNA interference (RNAi) therapy to attack mantle cell lymphoma at the mRNA level, silencing genes associated with cancer cell proliferation. We identified a lipid nanoparticle formulated with the lipidoid 306O13 that delivered siRNA to JeKo-1 and MAVER-1 mantle cell lymphoma cell lines. Three therapeutic gene targets were examined for their effect on lymphoma growth. These included Cyclin D1, which is a cell cycle regulator, as well as Bcl-2 and Mcl-1, which prevent apoptosis. Gene knockdown with siRNA doses as low at 10 nM increased lymphoma cell apoptosis without carrier-mediated toxicity. Silencing of Cyclin D1 induced apoptosis despite a twofold "compensation" upregulation of Cyclin D2. Upon simultaneous silencing of all three genes, nearly 75% of JeKo-1 cells were apoptosing 3 days post-transfection. Furthermore, cells proliferated at only 15% of their pretreatment rate. These data suggest that lipid nanoparticles-formulated, multiplexed siRNA "cocktails" may serve as a beneficial addition to the treatment regimens for mantle cell lymphoma and other aggressive cancers.

Lipidoid nanoparticle mediated silencing of Mcl-1 induces apoptosis in mantle cell lymphoma.

Conventional chemo-immunotherapy fails to cure the majority of mantle cell lymphoma patients and causes substantial toxicity. Resistant mantle cell lymphoma cells commonly overexpress and are dependent on the anti-apoptotic protein, Mcl-1, for survival. In this study, we use potent lipidoid nanoparticles to deliver siRNA to silence Mcl-1 expression. Studies were conducted using two different mantle cell lymphoma cell lines, a normal (JeKo-1) and an aggressive (MAVER-1) line, to assess the ability of lipidoid nanoparticles to be used broadly in the treatment of mantle cell lymphoma. Mcl-1 mRNA silencing and protein knockdown was observed as early as one day after treatment and the lipidoid nanoparticles achieved sustained silencing of Mcl-1 mRNA for at least four days in both JeKo-1 and MAVER-1 cells. Eighty percent silencing was achieved at three days post-transfection in JeKo-1 cells while 50% silencing was achieved in MAVER-1 cells, which are more resistant to transfection. Interestingly, silencing of Mcl-1 induced apoptosis in nearly 30% of both JeKo-1 and MAVER-1 cells three days post-transfection. Additionally, Mcl-1 silencing and the resultant apoptosis in mantle cell lymphoma cells were dose dependent. These data suggest that lipidoid nanoparticles siRNA therapy targeting Mcl-1 has potential as a new treatment modality for mantle cell lymphoma and many other cancers that overexpress Mcl-1. The combination of anti-Mcl-1 lipidoid nanoparticles with other forms of targeted therapy offers hope for reducing or replacing cytotoxic chemotherapy as standard treatment for mantle cell lymphoma.

Lipidoid Nanoparticles for siRNA Delivery to the Intestinal Epithelium: In Vitro Investigations in a Caco-2 Model.

Short interfering ribonucleic acid (siRNA) therapeutics show promise for the treatment of intestinal diseases by specifically suppressing the expression of disease relevant proteins. Recently, a class of lipid-like materials termed "lipidoids" have been shown to potently deliver siRNA to the liver and immune cells. Here, we seek to establish the utility of lipidoid nanoparticles (LNPs) in the context of siRNA delivery to the intestinal epithelium. Initial studies demonstrated that the siRNA-loaded LNPs mediated potent, dose dependent, and durable gene silencing in Caco-2 intestinal epithelial cells, with a single 10 nM dose depressing GAPDH mRNA expression for one week. Transfection with siRNA-loaded LNPs did not induce significant cytotoxicity in Caco-2 cells or alter intestinal barrier function. Protein silencing was confirmed by Western blotting, with the lowest levels of GAPDH protein expression observed five days post-transfection. Together, these data underscore the potential of LNPs for the treatment of intestinal disorders.

Introduction of a fresh cadaver laboratory during the surgery clerkship improves emergency technical skills.

BACKGROUND: Student acquisition of technical skills during the clinical years of medical school has been steadily declining. To address this issue, the authors instituted a fresh cadaver-based Emergency Surgical Skills Laboratory (ESSL). METHODS: Sixty-three medical students rotating through the third-year surgery clerkship participated in a 2-hour, fresh cadaver-based ESSL conducted during the first 2 days of the clerkship. The authors evaluated students utilizing both surgical skills and written examination before the ESSL and at 4 weeks post ESSL. RESULTS: Students demonstrated a mean improvement of 64% (±11) (P < .001) and 38% (±17) (P < .001) in technical skills and clinical knowledge, respectively. When technical skills were compared between cohorts, there were no differences observed in both pre- and post-testing (P = .08). CONCLUSIONS: A fresh cadaver laboratory is an effective method to provide proficiency in emergency technical skills not acquired during the clinical years of medical school.

In pursuit of a moving target: nanotherapeutics for the treatment of non-Hodgkin B-cell lymphoma.

INTRODUCTION: Non-Hodgkin lymphoma (NHL) is diagnosed in 70,000 Americans annually. Chemotherapy was the standard course of treatment until the addition of the monoclonal antibody (mAb) drug, rituximab, to therapy regimens in 1997. Although disease prognosis has improved dramatically since that time, nearly 20,000 patients succumb annually to the disease, with an average life expectancy beyond diagnosis of only 12 years. The advent of nanomedicine may fulfill the remaining need for novel therapy capable of eradicating solid tumor and disseminated B-cell lymphomas. AREAS COVERED: This review details the current landscape of B-cell NHL and nanoparticles now being developed for its treatment. Specifically, we discuss lipid, polymer and metal nanoparticles that deliver an array of drugs, including toxins, chemotherapeutic agents and nucleic acids. EXPERT OPINION: Because B-cell malignancies have responded quite well to new components in multi-drug regimens, nanomedicines that are mechanistically distinct from existing therapies hold significant promise. In our opinion, advancement of these technologies into the clinic will likely require significantly more effective targeting systems coupled with a better understanding of lymphoma biology. Furthermore, it is important for researchers to recognize the genetic and phenotypic heterogeneity of NHL and to develop therapeutic strategies for distinct subsets of NHL before attempting to generalize approaches.

OKN asymmetry in human subjects: a literature review.

Optokinetic nystagmus (OKN) is a reflex eye movement induced by motion of the whole or a large proportion of the visual field. It can be horizontal, vertical, and torsional in direction and consists of two basic components, a slow tracking movement and a rapid recovery saccade. Two forms of OKN exist: "look" and "stare" OKN. There is strong evidence that horizontal OKN is symmetrical in normal healthy adults and that the OKN gains can be influenced by a variety of different factors including target size, shape, contrast, and velocity. Vertical OKN on the other hand is less well understood, although there is a belief that vertical OKN is asymmetrical with an upward preference. Recent publications contradict this assertion. In this article a comprehensive literature review was carried out to determine whether a vertical OKN asymmetry exists in healthy subjects and to explain any anomalous findings.

Vertical optokinetic nystagmus in Parkinson's disease.

Parkinson's disease (PD) is associated with a number of oculomotor deficits; however, little is known about changes in vertical optokinetic nystagmus (OKN) associated with PD. We recorded eye movements in 14 PD patients and 14 age-matched controls in response to large field OKN stimulation using stimulus velocities of 20 degrees /second and 40 degrees /second. We compared asymmetry of horizontal and vertical responses in the two groups. We found vertical OKN to be strongly asymmetric in PD with reduced gains for downward-moving stimuli. This asymmetry was significantly greater than that recorded in control volunteers. We postulate that this could result from an abnormal pursuit/early OKN system in PD leading to greater influence of the delayed OKN system.

Effect of distance upon horizontal and vertical look and stare OKN.

Previous reports suggest that distance influences horizontal stare OKN gains; however, the effect of distance on vertical OKN and look OKN is unknown. Horizontal and vertical look and stare OKN gains were recorded in 16 healthy volunteers (velocity 38.4 degrees /s) at three distances (0.3 m, 1 m, and 2.5 m) and two different stimulus sizes. Asymmetry of responses and correlation of gains in different directions were compared. Measurements at near were compared with and without glasses. Distance did not significantly affect horizontal look and stare OKN or vertical look OKN, however, downward stare OKN gains were reduced at greater distances (p = 0.002). Mean downward stare OKN gains recorded in each individual were strongly correlated to leftward and rightward gains but not upward gains. In contrast, upward OKN gains were not correlated to gains in leftward, rightward, or downward directions. Downward stare OKN responses are significantly sensitive to the effects of distance, whereas stare OKN in other directions and look OKN responses in all directions are not. Individual mean downward stare OKN gains are more closely related to horizontal responses rather than upward responses. This suggests that the downward OKN system is more functionally related to the horizontal system rather than the upward OKN system.

Horizontal and vertical look and stare optokinetic nystagmus symmetry in healthy adult volunteers.

PURPOSE: Look optokinetic nystagmus (OKN) consists of voluntary tracking of details in a moving visual field, whereas stare OKN is reflexive and consists of shorter slow phases of lower gain. Horizontal OKN is symmetrical in healthy adults, whereas symmetry of vertical OKN is controversial. Horizontal and vertical look and stare OKN symmetry was measured, and the consistency of individual asymmetries and the effect of varying stimulus conditions were investigated. METHODS: Horizontal and vertical look and stare OKN gains were recorded in 15 healthy volunteers (40 degrees /s) using new methods to delineate look and stare OKN. Responses with right and left eye viewing were compared to investigate consistency of individual OKN asymmetry. In a second experiment, the symmetry of stare OKN was measured in nine volunteers varying velocity (20 degrees /s and 40 degrees /s), contrast (50% and 100%), grating contrast profile (square or sine wave), and stimulus shape (full screen or circular vignetted). RESULTS: There was no horizontal or vertical asymmetry in look or stare OKN gain for all volunteers grouped together. However, individual vertical asymmetries were strongly correlated for left and right eye viewing (look: r = 0.77, P = 0.0008; stare: r = 0.75, P = 0.001) and for look and stare OKN (r = 0.66, P = 7.3 x 10(-5)) because of a strong correlation for downward moving stimuli (r = 0.73, P = 0.002). Horizontal and vertical asymmetries were not significantly affected by variations in stimulus parameter. CONCLUSIONS: Although no horizontal or vertical OKN asymmetries existed for volunteers grouped together, vertical OKN was characterized by idiosyncratic asymmetries that remained consistent for an individual. Look and stare OKN gain is strongly associated for downward moving stimuli.

The VF-14 and psychological impact of amblyopia and strabismus.

PURPOSE: To assess the impact of amblyopia, strabismus and glasses on subjective visual and psychological function among amblyopes. METHODS: Questionnaires were administered to 120 teenagers with amblyopia (cases), with residual amblyopia after treatment, or with or without strabismus and 120 control subjects (controls) Cases underwent ophthalmic examination including cycloplegic refraction. Two questionnaires (visual function 14 [VF-14] and a newly designed eight-item questionnaire) were administered to assess the psychological impact score of general daily life, having a weaker eye, glasses wear, and current noticeable strabismus. Questionnaires were validated in 60 subjects in each group by a second administration of the questionnaire. The VF-14 scores, psychological impact scores, and clinical data were compared. RESULTS: The VF-14 and psychological impact scores were highly reproducible. The mean VF-14 score for the control group was 95.5 and for the cases was 78.9 (P < 0.0001), but the scores did not correlate with the severity of amblyopia. The psychological impact score in general daily life was sensitive in discriminating between mild (median score 31) and moderate to severe (median score 56) amblyopes (P < 0.02). The cases segregated into two clear groups; those who scored high (large detrimental psychological impact) on psychological impact, with subjectively noticeable manifest strabismus, and those who scored low (low detrimental psychological impact), without noticeable strabismus. The subjective experience of patching treatment differentiated the two groups best of all. CONCLUSIONS: Subjective visual and psychological functions are altered compared with normal subjects due to amblyopia, strabismus, and a previous unpleasant patching experience. The mean VF-14 score was similar to that previously published for patients with glaucoma. The study underlines that amblyopia and/or strabismus have an impact on teenagers' subjective visual function and well-being.

Benign recurrent abducens (6th) nerve palsy in two children.

Benign recurrent abducens (6th) nerve palsy is rare. We found 23 cases in children reported in the literature; however, many of these cases followed immunization or were associated with viral illness. Here we report two cases of recurrent abducens nerve palsy with no obvious etiology. The diagnosis should be considered in any child who experiences abducens nerve palsy in the absence of any underlying pathology or precipitating factors.