Self-Immolative Polymer Nanoparticles with Precise and Controllable pH-Dependent Degradation.

Polymer nanoparticles have generated significant interest as delivery systems for therapeutic cargo. Self-immolative polymers (SIPs) are an interesting category of materials for delivery applications, as the characteristic property of end-to-end depolymerization allows for the disintegration of the delivery system, facilitating a more effective release of the cargo and clearance from the body after use. In this work, nanoparticles based on a pH-responsive polymer poly(ethylene glycol)-b-(2-diisopropyl)amino ethyl methacrylate) and a self-immolative polymer poly[N,N-(diisopropylamino)ethyl glyoxylamide-r-N,N-(dibutylamino)ethyl glyoxylamide] (P(DPAEGAm-r-DBAEGAm)) were developed. Four particles were synthesized based on P(DPAEGAm-r-DBAEGAm) polymers with varied diisopropylamino to dibutylamino ratios of 4:1, 2:1, 2:3, and 0:1, termed 4:1, 2:1, 2:3, and 0:1 PGAm particles. The pH of particle disassembly was tuned from pH 7.0 to pH 5.0 by adjusting the ratio of diisopropylamino to dibutylamino substituents on the pendant tertiary amine. The P(DPAEGAm-r-DBAEGAm) polymers were observed to depolymerize (60-80%) below the particle disassembly pH after ∼2 h, compared to <10% at pH 7.4 and maintained reasonable stability at pH 7.4 (20-50% depolymerization) after 1 week. While all particles exhibited the ability to load a peptide cargo, only the 4:1 PGAm particles had higher endosomal escape efficiency (∼4%) compared to the 2:3 or 0:1 PGAm particles (<1%). The 4:1 PGAm particle is a promising candidate for further optimization as an intracellular drug delivery system with rapid and precisely controlled degradation.

Tramadol is a performance-enhancing drug in highly trained cyclists: a randomized controlled trial.

Tramadol is a potent narcotic analgesic reportedly used in multiple sports to reduce exertional pain and confer a performance advantage. This study sought to identify whether tramadol enhances performance in time trial cycling. Twenty-seven highly trained cyclists were screened for tramadol sensitivity and then attended the laboratory across three visits. Visit 1 identified maximal oxygen uptake, peak power output, and gas exchange threshold through a ramp incremental test. Participants returned to the laboratory on two further occasions to undertake cycling performance tests following the ingestion of either 100 mg of soluble tramadol or a taste-matched placebo control in a double-blind, randomized, and crossover design. In the performance tests, participants completed a 30 min non-exhaustive fixed intensity cycling task at a heavy exercise intensity (272 ± 42 W), immediately followed by a competitive self-paced 25-mile time trial (TT). Following removal of two outlier data sets, analysis was completed on n = 25. Participants completed the TT significantly faster (d = 0.54, P = 0.012) in the tramadol condition (3758 s ± 232 s) compared with the placebo condition (3808 s ± 248 s) and maintained a significantly higher mean power output (+9 W) throughout the TT (ηp2 = 0.262, P = 0.009). Tramadol reduced perception of effort during the fixed intensity trial (P = 0.026). The 1.3% faster time in the tramadol condition would be sufficient to change the outcomes of a race and is highly meaningful and pervasive in this cohort of highly trained cyclists. The data from this study suggests that tramadol is a performance-enhancing drug.NEW & NOTEWORTHY In the current study, when cycling with tramadol participants completed a time trial on average 50 s faster and at a 9 W higher power output than the placebo control. The study used both a fixed intensity and self-paced time trial exercise tasks to reflect the demands of a stage race. The outcomes from this study were used by the World Anti-Doping Agency to inform their addition of tramadol to the Prohibited List in 2024.

Intra- and interindividual reliability of muscle pain induced by an intramuscular injection of hypertonic saline injection into the quadriceps.

BACKGROUND: Intramuscular injections of hypertonic saline are commonly used to induce experimental muscle pain, but reliability data on this technique are lacking. This study investigated the intra- and interindividual reliability of pain measures from a hypertonic saline injection into the vastus lateralis. METHODS: Fourteen healthy participants (6 female) attended three laboratory visits where they received an intramuscular injection of 1 mL hypertonic saline into the vastus lateralis. Changes in pain intensity were recorded on an electronic visual analogue scale, and pain quality was assessed after pain had resolved. Reliability was assessed with the coefficient of variation (CV), minimum detectable change (MDC) and intraclass correlation coefficient (ICC) with 95% CIs. RESULTS: Mean pain intensity displayed high levels of intraindividual variability (CV = 16.3 [10.5-22.0]%) and 'poor' to 'very good' relative reliability (ICC = 0.71 [0.45-0.88]) but had a MDC of 11 [8-16] au (out of 100). Peak pain intensity exhibited high levels of intraindividual variability (CV = 14.8 [8.8-20.8]%) with 'moderate' to 'excellent' levels of relative reliability (ICC = 0.81 [0.62-0.92]), whereas the MDC was 18 [14-26] au. Measures of pain quality exhibited good reliability. Interindividual variability in pain measures was high (CV > 37%). CONCLUSIONS: Intramuscular injections of 1 mL of hypertonic saline into the vastus lateralis display substantial levels of interindividual variability, but MDC is below the clinically important changes in pain. This model of experimental pain is suitable for studies involving repeated exposures. SIGNIFICANCE: Many pain research studies have performed intramuscular injections of hypertonic saline to investigate responses to muscle pain. However, the reliability of this technique is not well established. We examined the pain response over three repeated sessions of a hypertonic saline injection. The pain induced by hypertonic saline has considerable interindividual variability but has largely acceptable intraindividual reliability. Therefore, the injections of hypertonic saline to induce muscle pain are a reliable model of experimental muscle pain.

The effect of hypertonic saline evoked muscle pain on neurophysiological changes and exercise performance in the contralateral limb.

Non-local muscle pain may impair endurance performance through neurophysiological mechanisms, but these are relatively unknown. This study examined the effects of muscle pain on neuromuscular and neurophysiological responses in the contralateral limb. On separate visits, nine participants completed an isometric time to task failure (TTF) using the right knee extensors after intramuscular injection of isotonic saline (CTRL) or hypertonic saline (HYP) into the left vastus lateralis. Measures of neuromuscular fatigue were taken before, during and after the TTF using transcranial magnetic stimulation (TMS) and peripheral nerve stimulation. Mean pain intensity was greater in the left leg in HYP (3.3 ± 1.9) compared to CTRL (0.4 ± 0.7; P < 0.001) which was combined with a reduced TTF by 9.8% in HYP (4.54 ± 0.56 min) compared to CTRL (5.07 ± 0.77 min; P = 0.005). Maximum voluntary force was not different between conditions (all P > 0.05). Voluntary activation was lower in HYP compared to CTRL (P = 0.022). No difference was identified between conditions for doublet amplitude (P > 0.05). Furthermore, no difference in MEP·Mmax-1 or the TMS silent period between conditions was observed (all P > 0.05). Non-local pain impairs endurance performance of the contralateral limb. This impairment in performance is likely due to the faster attainment of the sensory tolerance limit from a greater amount of sensory feedback originating from the non-exercising, but painful, left leg.

Understanding the Biological Interactions of pH-Swellable Nanoparticles.

pH-responsive nanoparticles have generated significant interest for use as drug delivery systems due to their potential for inducible release at low pH. The pH variation from the bloodstream (pH 7.4) to intracellular compartments of cells called endosomes/lysosomes (pH < 5.0) has been of particular interest. However, one of the limitations with nanoparticle delivery systems is the inability to migrate out of these compartments to the cytosol or other organelles, via a process termed endosomal escape. Previous studies have postulated that pH-responsive nanoparticles can facilitate endosomal escape through a range of mechanisms including membrane interaction, pH-induced swelling, and the proton-sponge effect. In this study, a series of pH-swellable nanoparticles (85-100 nm) are designed and their impact on biological interactions, particularly endosomal escape, are investigated. The particles exhibit tunable pH-induced swelling (from 120% to 200%) and have good buffering capacity. The cellular association is studied using flow cytometry and endosomal escape is determined using a calcein leakage assay. Interestingly, no endosomal escape with all nanoparticle formulations is found, which suggests there are limitations with both the proton-sponge effect and pH-induced swelling mechanism as the primary methods for inducing endosomal escape.

The effect of elevated muscle pain on neuromuscular fatigue during exercise.

PURPOSE: Muscle pain can impair exercise performance but the mechanisms for this are unknown. This study examined the effects of muscle pain on neuromuscular fatigue during an endurance task. METHODS: On separate visits, twelve participants completed an isometric time-to-task failure (TTF) exercise of the right knee extensors at ~ 20% of maximum force following an intramuscular injection of isotonic saline (CTRL) or hypertonic saline (HYP) into the vastus lateralis. Measures of neuromuscular fatigue were taken before, during and after the TTF using transcranial magnetic stimulation (TMS) and peripheral nerve stimulation. RESULTS: The mean pain intensity was 57 ± 10 in HYP compared to 38 ± 18 in CTRL (P < 0.001). TTF was reduced in HYP (4.36 ± 0.88 min) compared to CTRL (5.20 ± 0.39 min) (P = 0.003). Maximum voluntary force was 12% lower at minute 1 (P = 0.003) and 11% lower at minute 2 in HYP (P = 0.013) compared to CTRL. Voluntary activation was 4% lower at minute 1 in HYP compared to CTRL (P = 0.006) but not at any other time point (all P > 0.05). The TMS silent period was 9% longer at 100 s during the TTF in HYP compared to CTRL (P = 0.026). CONCLUSION: Muscle pain reduces exercise performance through the excacerbation of neuromuscular fatigue that is central in origin. This appears to be from inhibitory feedback from group III/IV nociceptors which acts to reduce central motor output.

Quantifying the Endosomal Escape of pH-Responsive Nanoparticles Using the Split Luciferase Endosomal Escape Quantification Assay.

All nanoparticles have the potential to revolutionize the delivery of therapeutic cargo such as peptides, proteins, and RNA. However, effective cytosolic delivery of cargo from nanoparticles represents a significant challenge in the design of more efficient drug delivery vehicles. Recently, research has centered on designing nanoparticles with the capacity to escape endosomes by responding to biological stimuli such as changes in pH, which occur when nanoparticles are internalized into the endo-/lysosomal pathway. Current endosomal escape assays rely on indirect measurements and yield little quantitative information, which hinders the design of more efficient drug delivery vehicles. Therefore, we adapted the highly sensitive split luciferase endosomal escape quantification (SLEEQ) assay to better understand nanoparticle-induced endosomal escape. We applied SLEEQ to evaluate the endosomal escape behavior of two pH-responsive nanoparticles: the first with a poly(2-diisopropylamino ethyl methacrylate) (PDPAEMA) core and the second with 1:1 ratio of poly(2-diethylamino ethyl methacrylate) (PDEAEMA) and PDPAEMA. SLEEQ directly measured the cytosolic delivery and showed that engineering the nanoparticle disassembly pH could improve the endosomal escape efficiency by fivefold. SLEEQ is a versatile assay that can be used for a wide range of nanomaterials and will improve the development of drug delivery vehicles in the future.

Acid-Responsive Poly(glyoxylate) Self-Immolative Star Polymers.

Self-immolative polymers have significant potential for applications such as drug or gene delivery. However, to realize this potential, such materials need to be customized to respond to specific variations in biological conditions. In this work, we investigated the design of new star-shaped self-immolative poly(ethyl glyoxylate)s (PEtGs) and their incorporation into responsive nanoparticles. PEtGs are a subclass of stimulus-responsive self-immolative polymers, which can be combined with different stimuli-responsive functionalities. Two different tetrathiol initiators were used for the polymerization in combination with a variety of potential pH-responsive end-caps, yielding a library of star PEtG polymers which were responsive to pH. Characterization of the depolymerization behavior of the polymers showed that the depolymerization rate was controlled by the end caps rather than the architecture of the polymer. A selection of the star polymers were modified with amines to allow introduction of charge-shifting properties. It was shown that pH-responsive nanoparticles could be prepared from these modified polymers and they demonstrated pH-dependent particle disruption. The pH responsiveness of these particles was studied by dynamic light scattering and 1H nuclear magnetic resonance spectroscopy.

Effects of experimentally induced muscle pain on endurance performance: A proof-of-concept study assessing neurophysiological and perceptual responses.

Pain arising from exercise potentiates fatigue and impairs the performance of endurance exercise. We assessed neurophysiological and perceptual responses to endurance exercise performed under experimentally induced muscle pain by a model that separates muscle pain from muscle fatigue. After a series of pilot studies investigating different hypertonic saline volumes, 17 healthy males performed a preliminary VO2PEAK test before performing a familiarization of the cycling time-to-exhaustion exercise (80% of the peak power output in the VO2PEAK test). Participants, performed a baseline exercise session before the sessions with hypertonic and isotonic saline injections in the vastus lateralis of both legs, in a crossover and counterbalanced design. Neurophysiological and perceptual responses such as electroencephalography (EEG) in frontal, prefrontal, parietal, and motor cortex, electromyography (EMG) of the vastus lateralis and biceps femoris muscles, ratings of perceived exertion (RPE), pain sensation, and affective valence were measured at rest and during exercise. The hypertonic injection reduced the resting EEG alpha-beta ratio in the frontal and prefrontal cortex. When compared to exercise performed after the isotonic injection (430.5 ± 152.6 s), hypertonic injection shortened the time-to-exhaustion (357.5 ± 173.0 s), reduced the EMG of the assessed muscles, and increased the muscle co-contraction during exercise. The hypertonic injection also reduced the EEG alpha-beta ratio in the prefrontal and parietal cortex, increased RPE and pain sensation, and reduced affective valence during exercise. This proof-of-concept study showed that hypertonic injection-induced muscle pain reduced endurance performance, promoting centrally mediated alterations in motor command and cortical activation, as well as an interplay of perceptual responses.

Muscle pain from an intramuscular injection of hypertonic saline increases variability in knee extensor torque reproduction.

The intensity of exercise-induced pain (EIP) reflects the metabolic environment in the exercising muscle, so during endurance exercise, this may inform the intelligent regulation of work rate. Conversely, the acute debilitating effects of EIP on motor unit recruitment could impair the estimation of force produced by the muscle and impair judgement of current exercise intensity. This study investigated whether muscle pain that feels like EIP, administered via intramuscular injection of hypertonic saline, interferes with the ability to accurately reproduce torque in a muscle group relevant to locomotive exercise. On separate days, 14 participants completed an isometric torque reproduction task of the knee extensors. Participants were required to produce torque at 15% and 20% maximal voluntary isometric torque (MVIT), without visual feedback before (baseline), during (pain/no pain), and after (recovery) an injection of 0.9% isotonic saline (Control) or 5.8% hypertonic saline (Experimental) into the vastus lateralis of the right leg. An elevated reported intensity of pain, and a significantly increased variance in mean contraction torque at both 15% (P = 0.049) and 20% (P = 0.002) MVIT was observed in the Experimental compared to the Control condition. Both 15 and 20% target torques were performed at a similar pain intensity in the Experimental condition (15% MVIT: 4.2 ± 1.9; 20% MVIT: 4.5 ± 2.2; P > 0.05). These findings demonstrate that the increased muscle pain from the injection of hypertonic saline impeded accurate reproduction of knee extensor torque. These findings have implications for the detrimental impact of EIP on exercise regulation and endurance performance.NEW & NOTEWORTHY We provide novel data demonstrating that the presence of muscle pain interferes with estimations of torque produced by the knee extensors, which could impair judgment of work rate during endurance exercise. The novelty of our study is in the application of the hypertonic saline experimental model into a quadriceps muscle during short, submaximal isometric contractions at an intensity that provides a more translatable assessment of the impact of exercise-induced pain on work-rate regulation during whole body exercise.

Muscle pain induced by hypertonic saline in the knee extensors decreases single-limb isometric time to task failure.

PURPOSE: Increased nociceptive activity and the experience of exercise-induced pain (EIP) may contribute to fatigue during endurance exercise. To investigate this, a pain model that produces pain similar to EIP and decouples its relationship to exercise intensity is required. This study (1) compared the quality of pain caused by a hypertonic saline injection into the vastus lateralis in resting and exercise conditions, and (2) investigated whether this pain contributes to changes in time to task failure. METHODS: On separate days, 18 participants completed a time to task failure at 20% maximal voluntary torque (MVT), a resting hypertonic saline intramuscular injection, and in a further three visits a time to task failure at 10% MVT following injection of isotonic saline, hypertonic saline or a control (no injection). RESULTS: In a subset of eligible participants (n = 12), the hypertonic saline combined with 10% MVT produced a qualitative experience of pain (assessed by the McGill Pain Questionnaire) that felt similar to EIP. 10% MVT with hypertonic saline significantly elevated pain intensity in the first 20% of the time to task failure and caused a significantly (P < 0.05) shorter time to task failure (448 ± 240 s) compared with the isotonic saline (605 ± 285 s) and control (514 ± 197 s) conditions. CONCLUSION: These findings demonstrate that hypertonic saline increases the intensity of pain during exercise, which results in a faster occurrence of exercise-induced fatigue. These results provide important evidence supporting pain as a limiting factor in endurance performance.

Engineered Polymeric Materials for Biological Applications: Overcoming Challenges of the Bio-Nano Interface.

Nanomedicine has generated significant interest as an alternative to conventional cancertherapy due to the ability for nanoparticles to tune cargo release. However, while nanoparticletechnology has promised significant benefit, there are still limited examples of nanoparticles inclinical practice. The low translational success of nanoparticle research is due to the series ofbiological roadblocks that nanoparticles must migrate to be effective, including blood and plasmainteractions, clearance, extravasation, and tumor penetration, through to cellular targeting,internalization, and endosomal escape. It is important to consider these roadblocks holistically inorder to design more effective delivery systems. This perspective will discuss how nanoparticlescan be designed to migrate each of these biological challenges and thus improve nanoparticledelivery systems in the future. In this review, we have limited the literature discussed to studiesinvestigating the impact of polymer nanoparticle structure or composition on therapeutic deliveryand associated advancements. The focus of this review is to highlight the impact of nanoparticlecharacteristics on the interaction with different biological barriers. More specific studies/reviewshave been referenced where possible.

The Endosomal Escape of Nanoparticles: Toward More Efficient Cellular Delivery.

Many emerging therapies rely on the delivery of biological cargo into the cytosol. Nanoparticle delivery systems hold great potential to deliver these therapeutics but are hindered by entrapment and subsequent degradation in acidic compartments of the endo/lysosomal pathway. Engineering polymeric delivery systems that are able to escape the endosome has significant potential to address this issue. However, the development of safe and effective delivery systems that can reliably deliver cargo to the cytosol is still a challenge. Greater understanding of the properties that govern endosomal escape and how it can be quantified is important for the development of more efficient nanoparticle delivery systems. This Topical Review highlights the current understanding of the mechanisms by which nanoparticles escape the endosome, and the emerging techniques to improve the quantification of endosomal escape.

Use of the real-time PCR assay in conjunction with MagNA Pure for the detection of mycobacterial DNA from fixed specimens.

Tuberculosis in immunocompromised patients is often caused by Mycobacterial species other than Mycobacterium tuberculosis. Thus, detection of and differentiation between M. tuberculosis and nontuberculosis species is necessary for diagnosis of disease in these patients. Furthermore, when tissue changes show granulomatous inflammation, quick confirmation testing for mycobacterial infection is needed for conclusive diagnosis. The aim of this study was to validate the utility of a real-time polymerase chain reaction (PCR) assay in conjunction with the MagNA Pure LC automated extraction system for the detection of mycobacterial DNA from formalin-fixed, paraffin-embedded specimens. A total of 46 archived, paraffin-embedded, fixed specimens showing granulomatous inflammation were studied for mycobacterial infection by real-time PCR. Bacterial DNA was extracted and isolated using the MagNA Pure extraction system. Real-time PCR was performed on the LightCycler using the Artus Real Art Mycob Diff ASR kit from Qiagen. Thirteen of the 46 patient specimens were positive for mycobacterial infection by acid-fast bacilli (AFB) stain. Of the13 reported positive by AFB stain, 12 where positive by real-time PCR. All 13 specimens reported positive by AFB were sent for culture confirmation. Eleven of 13 were returned positive by culture. Specimens reported as negative by culture and positive by real-time PCR were confirmed positive by a second PCR method from another reference laboratory. We believe that these studies are beneficial in the differential diagnosis of mycobacterial infection from fixed tissue specimens where tuberculosis might not have been clinically initially suspected and when specimens are not suitable for microbiologic examination.