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Muscle membrane integrity in Duchenne muscular dystrophy: recent advances in copolymer-based muscle membrane stabilizers

Muscle membrane integrity in Duchenne muscular dystrophy: recent advances in copolymer-based... The scientific premise, design, and structure-function analysis of chemical-based muscle membrane stabilizing block copolymers are reviewed here for applications in striated muscle membrane injury. Synthetic block copolymers have a rich history and wide array of applications from industry to biology. Potential for discovery is enabled by a large chemical space for block copolymers, including modifications in block copolymer mass, composition, and molecular architecture. Collectively, this presents an impressive chemical landscape to leverage distinct structure-function outcomes. Of particular relevance to biology and medicine, stabilization of damaged phospholipid membranes using amphiphilic block copolymers, classified as poloxamers or pluronics, has been the subject of increasing scientific inquiry. This review focuses on implementing block copolymers to protect fragile muscle membranes against mechanical stress. The review highlights interventions in Duchenne muscular dystrophy, a fatal disease of progressive muscle deterioration owing to marked instability of the striated muscle membrane. Biophysical and chemical engineering advances are presented that delineate and expand upon current understanding of copolymer- lipid membrane interactions and the mechanism of stabilization. The studies presented here serve to underscore the utility of copolymer discovery leading toward the therapeutic application of block copolymers in Duchenne muscular dystrophy and potentially other biomedical applications in which membrane integrity is compromised. Keywords: Duchenne muscular dystrophy, Block copolymers, Membrane stabilization Background will not be further elaborated on here. Rather, this review All eukaryotic cells are enveloped by a phospholipid bi- focuses on membrane protection from the perspective of layer membrane. An enormous literature exists that de- a chemical-based approach to preserve muscle membrane fines biological cell membrane form and function [1, 2]. integrity and how this unique cell extrinsic approach Regardless of biological cell type, the cell membrane could complement cell intrinsic membrane stabilization/ represents first and last line of defense for ensuring the repair pathways (Fig. 1). Numerous acquired and inherited normal function and ultimately the viability of the cell. diseases comprise, at some level, an etiology involving cell Accordingly, multiple cellular processes are present to membrane instability. Duchenne muscular dystrophy is help ensure the maintenance, repair and protection of the the archetype inherited disease of severe membrane fragil- cell membrane. There are numerous excellent expert re- ity and serves as the disease model focal point of this views detailing cell intrinsic mechanisms of membrane in- review. tegrity and repair [2–17] and mechanistic details on these Duchenne muscular dystrophy: a fatal disease of * Correspondence: metzgerj@umn.edu muscle membrane instability Department of Integrative Biology and Physiology, University of Minnesota Duchenne muscular dystrophy (DMD) is an X-linked re- Medical School, 6-125 Jackson Hall, 321 Church Street SE, Minneapolis, MN cessive disease of marked striated muscle deterioration, 55455, USA Full list of author information is available at the end of the article affecting 1 in 3500–5000 boys [18]. DMD results from © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Houang et al. Skeletal Muscle (2018) 8:31 Page 2 of 19 weakness in limb muscles and postural muscles [18], leading to spinal scoliosis and decrease in exercise capacity. Weak- ness of the knees and hip extensors are displayed through the Gower’s sign, a maneuver through which the affected child will right himself from a supine position by using his hands and arms to extend the hips and bring the torso to an upright position [20]. Other physical symptoms include re- duced muscle bulk, pseudo-hypertrophy, and contractures of the calf muscles and joints [21]. Bone fragility and osteopor- osis also contribute to the development of scoliosis [22]. Concurrent with the decline in orthopedic condition is loss of respiratory function brought on by significant diaphragm wasting [23] leading patients to be placed on positive pres- sure nocturnal ventilation. Loss of ambulation and wheel- chair dependency occur by the early teens [24], and DMD patients typically succumb in their 20s due to cardio- respiratory failure [25–28]. DMD patients develop a severe cardiomyopathy, pre- senting as dilated cardiomyopathy [29], with arrhythmias and eventually heart failure occurring in the second/ third decade of life [24]. With increases in patient life- span, as a result of palliative glucocorticoid treatment and improvements in respiratory care and orthopedic corrections [30, 31], cardiomyopathy is an increasingly important but underappreciated contributor to DMD mortality. It is now evident that cardiomyopathy is present in 90% of DMD patients by age 18 and is con- firmed by significant myocardial fibrosis in autopsies [32–35]. Interestingly, the cardiomyopathy usually re- mains subclinical at early age and cardiac disease pro- gression typically proceeds at a slower rate compared to the skeletal muscle degeneration [36]. The incidence and evolution of cardiomyopathy in Duchenne muscular dys- trophy is presumably due to lesser strain on the heart when physical activity is limited once the patient is wheelchair bound. Dystrophin Extensive genetic analysis of DMD patients determined that defects in the dystrophin gene are causal for the dis- ease [19]. The dystrophin gene spans 2.5 Mb of DNA on Fig. 1 Copolymer-based muscle membrane stabilization of dystrophic the X chromosome. Dystrophin’s 79 exons encode a muscle. a Representation of intact muscle membrane with dystrophin anchoring the DGC to the actin cytoskeleton. b Membrane instability 3685 amino acid cytoskeletal protein localized to the caused by the lack of dystrophin leads to pathological increases intracellular surface of the muscle membrane [19]. Dys- 2+ in intracellular Ca concentration. c Copolymer stabilization of trophin consists of four major functional domains: (1) the damaged membrane via insertion of its hydrophobic PPO 2+ an actin-binding domain at the N-terminus; (2) a central block (red) prevents entry of extracellular Ca into the cell rod domain consisting of 24 spectrin-like repeats sepa- rated by four hinge regions, that has been shown to un- the lack of the cytoskeletal protein dystrophin, a protein fold and give flexibility in response to mechanical indispensable for maintaining the structural integrity of stretch [19]; (3) a cysteine-rich domain that interacts the muscle cell membrane [19]. DMD disease onset with the transmembrane protein β-dystroglycan; and (4) typically occurs between the ages of 2 and 5 years and is a C-terminal domain, critical for dystrophin’s interaction characterized by a delay in achieving childhood motor with other sub-sarcolemmal proteins [37–39]. Detailed milestones. DMD presents as a prominent and progressive structure function-based transgenic animal studies have Houang et al. Skeletal Muscle (2018) 8:31 Page 3 of 19 determined that the domains most critical to DMD degree of packing of bilayer components, is collectively de- pathology are the cysteine-rich domain and the scribed as “membrane fluidity” [48, 56]. Membrane fluidity N-terminal domain, and those are directly associated is controlled by a number of factors, including lipid com- with mechanically linking the extracellular matrix and position, sterol enrichment, and temperature. Fluidity is the cytoskeleton [40]. generally assessed using fluorescence polarization methods, Dystrophin is part of a large membrane-spanning com- electron spin resonance, and other spectroscopic methods plex of glycoproteins (dystrophin-glycoprotein complex or [59–62]. Along with membrane fluidity, the structure and DGC) that also include sarcoglycans (α, β, γ, δ), dystrogly- composition of the bilayer can be described by parameters cans (α and β), dystrobrevins, syntrophins, and sarcospan such as rigidity, elasticity, and tensile strength, all of which [38, 39, 41](Fig. 1a). This dystrophin-associated protein make up the membrane physical property known as plasma complex is found and enriched at the muscle costamere, a membrane order [58, 63]. Various studies have suggested network of proteins that physically connect the extracellu- that an optimal level of membrane order is essential for lar matrix to the cytoskeleton, through the muscle mem- normal myocyte function [57, 64]. Of particular interest to brane or sarcolemma, and as such orchestrates the lateral muscle, nicotinic acetylcholine receptors which are present force transmission [42–44]. As such, one essential func- at neuromuscular junctions of muscle cells can be allosteri- tion of dystrophin in striated muscle is to stabilize the cally modulated by surrounding lipids and thus require an muscle membrane against the forces associated with optimal membrane microenvironment to retain normal contraction thereby acting as a “molecular shock ab- function [65, 66]. Therefore, alterations to the muscle sorber” or molecular force dampener of the muscle mem- membrane surrounding these receptors, either during brane [45, 46]. The importance of dystrophin’sscaffolding mechanical stress or in diseased states, such as in DMD, support at the membrane is evident in studies showing have important ramifications for ion conductance and thus that dystrophin-deficient muscle fibers where the mem- ultimately affecting action potential generation and propa- brane was experimentally removed show no difference in gation during muscle contraction. contractile function compared to normal skeletal muscle From a structural perspective, the lipid bilayer alone is fibers, indicating a defect in the membrane-cytoskeleton not sufficient to counteract the significant forces placed linkage rather than in the contractile apparatus [47]. on the membrane during muscle contraction [67]. Mech- anical integrity of the sarcolemma is further supported by Striated muscle membrane fragility in DMD key cytoskeletal proteins, including dystrophin, spectrin, Biological membranes are asymmetrical bilayers approxi- and F-actin [68, 69]. Electron microscopy analysis of dys- mately 5–6 nm thick and comprised of various lipids, in- trophic muscle directly shows disruptions in the muscle cluding phospholipids, sphingolipids, glycolipids and membrane, termed delta lesions [70, 71]. This discovery sterols [48–51]. Phospholipid composition can vary sig- led to the theory that the loss of dystrophin and associated nificantly between different cell types and also in disease proteins at the sarcolemma renders the membrane leaky states [48, 49, 52, 53]. The eukaryotic cell membrane is and the muscle susceptible to contraction-induced injury. also typically composed of 20–30% proteins responsible Indeed, serum detection of the soluble enzyme creatine for ion conduction, various signaling pathways, and kinase as it is released from the injured muscle is a clinical structural integrity [53]. Irrespective of cell type and hallmark of the disease [72]. Membrane permeability is function, the primary role of the cellular membrane is to further exacerbated by mechanical stress, particularly with segregate the intracellular milieu from the outside envir- lengthening contractions of skeletal muscles such as dur- onment to actively preserve intracellular homeostasis. ing downhill walking/running [73]. Lengthening contrac- Transmembrane proteins are essential for normal con- tions occur when the force applied to the muscle exceeds duction of ions, allowing maintenance of physiological the force generated by the muscle, resulting in lengthening ionic gradients at affordable metabolic cost. Failure to of the muscle during active contraction. Repetitive length- maintain barrier function leads to exhaustion of the ening contractions cause significant damage to dystrophic metabolic energy of the cell, biochemical arrest, and muscle by injuring the membrane and downstream ele- eventual cellular demise. ments, including the EC coupling machinery [74, 75]. The membrane bilayer is held together via hydrophobic In DMD patients, muscle biopsies show active degen- effect among phospholipids and their interaction with the eration and regeneration of skeletal muscle fibers and surrounding polar solvent environment, involving van der creatine kinase is persistently elevated [18, 27, 76, 77]. Waals forces, hydrogen bonding, and electrostatic interac- Presently, it is unclear the precise nature of membrane tions [50, 51, 53, 54, 55, 56, 57]. Membrane constituents disruptions caused by lengthening contractions. How- are allowed various intra-bilayer motions, including lateral ever, the release of intracellular enzymes such as creatine diffusion, rotation of lipids around their major axes, and kinase and the uptake of large proteins such as albumin oscillations [56–58]. Intra-bilayer motion, as well as the and vital dyes like Procion orange [73] and Evans blue Houang et al. Skeletal Muscle (2018) 8:31 Page 4 of 19 [78] into non-necrotic muscle fibers indicate that the function and reduction of ATP production leading to cellu- membrane disruptions are sufficiently large to permit lar energy deprivation and cell death. Oxidative stress and 2+ the transmembrane passage of sizable macromolecules elevated intracellular Ca signaling are evident in hearts of which can be monitored as biomarkers of muscle injury mdx mice before pathological manifestations of cardiomy- [72]. Lengthening injury is also particularly apparent in opathy, and there is increasing evidence of mitochondrial the diaphragm which contracts to expand the lungs dur- dysfunction in dystrophic striated muscle [89]. Conse- 2+ ing breathing. Ventilatory muscles of DMD patients and quently, maintaining intracellular Ca homeostasis by pre- 2+ in animal models have impaired contractility and in- venting the deleterious influx of extracellular Ca is crucial creased fibrosis [79]. Dystrophin also plays a crucial role to the survival of dystrophic striated muscle. Moreover, 2+ in buffering against cardiac myocyte extension [80]. This another recent study indicates that Ca influx can progres- occurs when the ventricle fills with blood during diastole sively increase in dystrophic muscle and lead to mitochon- to cause passive lengthening of myocytes. In dystrophin drial dysfunction. This, in turn, further compromises the deficiency, this passive lengthening leads to membrane endogenous membrane repair ability of dystrophin- 2+ dysfunction as evidenced by Ca entry and uptake of deficient myofibers. This negative feedback loop limits the extracellular molecules [80]. Moreover, the conse- cell intrinsic membrane repair machinery resulting in quences of membrane disruptions and increased perme- exacerbation of muscle deterioration in DMD [95]. ability are intrinsically different between cardiac and 2+ 2+ skeletal muscle as the process of Ca − induced Ca re- Current DMD therapeutic strategies: cell intrinsic/cell lease is predominant in the heart [81]. As such, with in- extrinsic strategies creases in contractility and larger passive extensions, There is no cure for DMD nor an effective treatment clin- 2+ subsequently more unregulated Ca entry into the cell ically demonstrated to halt, prevent, or reverse DMD stri- eventually results in terminal contracture of the dys- ated muscle deterioration. Glucocorticoids have been the trophic myocyte [80]. standard of care for DMD but are accompanied by several adverse effects such as excessive weight gain, behavioral Muscle membrane barrier function is severely disrupted issues, growth retardation, osteoporosis, and impairment in DMD of glucose metabolism, all associated with chronic 2+ Owing to membrane dysfunction, Ca homeostasis is per- long-term use [30, 96]. Prednisolone and deflazacort are 2+ turbed in dystrophic muscle (Fig. 1b). This Ca dysregula- regularly administered soon after diagnosis and have been tion is an important component of the pathological shown to slow the progression of the disease by improving processes leading to muscle cell death. Intracellular calcium muscle strength and exercise capacity thereby delaying levels are elevated in both mdx skeletal muscle fibers and loss of ambulation and improving both pulmonary and cardiac myocytes [80, 82–84]. It is still unclear what causes cardiac functions. Several ongoing experimental DMD 2+ this rise in intracellular Ca , with some studies suggesting therapeutics feature gene and cell-based strategies [97, 2+ Ca entering the cell due to increased membrane perme- 98], including exon-skipping strategies to restore dys- ability or “tears” [80], and other studies showing evidence trophin production [99–102]. Exon skipping strategies 2+ for the activation of Ca leak channels or stretch-activated using small molecules have been shown to ameliorate the channels [85]. Regardless of the initial mechanism of entry, severe dystrophic phenotype in both canine and murine 2+ this abnormal elevation in Ca has consequences to DMD models [99, 100, 102–104] while being well tolerated muscle structure and function due to activation of patho- and non-immunogenic. One significant caveat is that this 2+ logical Ca sensitive cellular pathways, including activation strategy is only applicable to the subset of DMD patients of the calpain proteases [86] and perturbation of calcium- with the corresponding targeted mutation. Additionally to activated signaling pathways including calmodulin [87], cal- date, most of these approaches have not yet been translated cineurin [88], and the mitochondrial permeability transition successfully in human patients [105, 106]. One exon pore [89]. Of importance, activation of calpains by extracel- skipping treatment, eteplirsen (Sarepta Therapeutics Inc.), 2+ lular Ca influx leads to cleavage of the transmembrane has recently been approved by the FDA through its acceler- protein dysferlin, a crucial mediator in the cell intrinsic ated approval pathway. A clinical trial in a small cohort of membrane repair machinery [90, 91]. A pathological rise in DMD patients resulted in a dose-dependent partial restor- 2+ cytosolic Ca also contributes to membrane damage via ation of dystrophin production with upregulation of other activation of phospholipase A2 and promotion of reactive dystrophin-associated proteins at the membrane, along oxygen species (ROS) production by the mitochondria [92]. with some improvement in patient walking ability com- ROS in turn leads to peroxidation of membrane lipids paredtoplacebocontrols[107, 108]. However, this 2+ [93, 94]. Additionally, mitochondrial Ca overload improvement was only observed in a small subset of the promotes irreversible opening of the mitochondrial patient group, with dystrophin levels observed to be highly permeability transition pore, aberration of mitochondrial variable among all patients, and a larger clinical trial is Houang et al. Skeletal Muscle (2018) 8:31 Page 5 of 19 currently underway to confirm these results across a larger topologically distinct hydrophilic and lipophilic compo- patient group. Unfortunately, eteplirsen is only targeted to nents. A wide range of block copolymers with distinct approximately 13% of DMD patients with a mutation physicochemical properties can be designed by varying the amenable to exon 51 skipping [108]leaving alarge popula- lengths of the PEO and PPO blocks. Poloxamers were the tion of DMD patients currently without treatment options. first commercially produced block copolymers, synthesized Many experimental therapeutic efforts preferentially tar- by Wyandotte Chemical Corporation in the late 1940s for get dystrophic skeletal muscles, leaving the diseased heart industrial purposes, and now widely found in both indus- untreated [29]. Skeletal muscle-centric strategies to im- trial and consumer products. Poloxamers span ~ 10–80% prove ambulation for DMD patients could lead to in- wt.% poly(ethylene oxide) and 1000 – 15000 g/mol molecu- creased stress on the untreated dystrophic myocardium as lar weight with complex interfacial behavior. Poloxamers a result of increased cardiac demands [29, 109, 110]. This have numerous biological applications, including as drug interplay between the progression of DMD cardiomyop- delivery adjuvants, enhancers of drug penetration in the athy and the skeletal myopathy as a pathophysiological treatment of multiple drug resistant tumors [113, 114], load on the heart underscores the importance of a thera- and membrane interacting agents, either as lysis deter- peutic strategy to effectively treat all striated muscles. In gents [115–117]or cellmembrane stabilizers [80, 118, this context, it is worth considering additional approaches 119] depending on structure. This latter feature is directly that target the primary defect of DMD: severe muscle attributed to poloxamers varying affinity for both the sur- membrane fragility. As the primary pathophysiological de- rounding solvent and with the similarly amphiphilic fect in DMD is the marked susceptibility to contraction- phospholipid membranes [120–123]. An excellent com- induced membrane stress, and the subsequent muscle prehensive review detailing copolymer physical and chem- damage and degeneration that occurs due to loss of ical properties, as well as safety, has been published [124]. muscle membrane barrier function, a unique therapeutic In the context of biomedical investigation, poloxamer approach is the use of synthetic membrane stabilizers to 188 (P188), with a PPO/PEO ratio of 0.20 and a molecu- prevent muscle damage by directly stabilizing the lar weight of 8400 Da, is the most widely studied tri- dystrophin-deficient muscle membrane (Fig. 1c). block copolymer (Table 1). P188’s earliest reported use was in 1952 as an additive to enhance blood oxygenation Copolymers as cell extrinsic muscle membrane [125]. It was found to reduce fat emboli and hemolysis stabilizers in patients under extended cardiopulmonary bypass The triblock copolymer class of membrane-interacting syn- [126–128] and as a priming agent in heart-lung bypass thetic molecules, known as poloxamers or pluronics, are [129]. P188 was also incorporated as a wetting agent linear structures comprised of a hydrophobic polypropylene [130, 131] and an emulsifier for clinically tested drug oxide (PPO) core block flanked on both sides by hydro- formulations [132, 133] as well as used as a solubilizing philic polyethylene oxide (PEO) chains (Fig. 2)(Table 1) agent of perfluorochemicals which have significant O [111, 112]. This constitutes the triblock copolymer A-B-A carrying capacity to create an emulsion used as an architecture. Poloxamers are non-ionic amphiphiles having artificial blood substitute [134]. P188 functions as a Fig. 2 Schematic representation of a triblock and diblock copolymers chemical structures. Chemical structures and representations of the triblock copolymer P188 (PEO –PPO –PEO ) and diblocks of P188 (PEO –PPO ) with differing end groups (–H and –C(CH ) ) where a and b represent 75 30 75 75 15 3 3 the number of repeating PEO and PPO group respectively Houang et al. Skeletal Muscle (2018) 8:31 Page 6 of 19 Table 1 Chemical properties of representative synthetic block copolymers a a b c d Architecture Polymer PEO PPO End group Mass PEO% Triblock copolymer/P188 PEO PPO PEO 150 30 – 8400 80 75 30 75 Triblock copolymer/P338 PEO PPO PEO 280 44 – 8400 84 140 44 140 Triblock copolymer/P331 PEO PPO PEO 14 54 – 3700 26 7 54 7 Diblock copolymer PEO PPO −H75 15 −H 4200 80 75 15 PEO PPO − C4 75 15 −C(CH ) 4430 77 75 15 3 3 Homopolymer PEO 198 0 – 8700 100 Total number of EO or PO monomer units Chemical end group at terminal PO c 1 Average molecular weight in g/mol by H NMR end-group analysis PEO weight percent to total molecular weight Manufacturer BASF Number average molecular weight rheological agent to reduce blood viscosity and platelet Following these reports, a seminal study by Yasuda aggregation [135–138]. It was also reported that P188 et al. [80] demonstrated that the acute application of reduces membrane fluidity and improves cell survivabil- P188 to isolated dystrophic mdx cardiac myocytes re- ity during shear stress in HB-32 hybridoma cell lines, stored myocyte cellular compliance to wild-type levels presumably through direct membrane interaction [61]. by blocking passive stretch-mediated calcium overload. P188 was subsequently widely deployed as a shear pro- Dystrophic mdx cardiac myocytes demonstrated in- tective agent used in cell bioreactors [139]. Additionally, creased passive tension during extension, resulting, in 2+ P188 was determined to reduce endothelial adherence part, by the influx of extracellular Ca during physio- and improves the rheology of sickled red blood cells logical passive myocyte lengthening. P188 fully normal- [140], leading to P188 in clinical trial as a therapeutic ized myocyte passive compliance to normal levels [80]. agent for sickle cell anemia [141–143]. A main outcome At the level of the whole organ, P188 decreased passive of a ~ 350 patient sickle cell anemia trial was its safety tension and thereby improved myocardial relaxation, profile in long-term use. P188’s first FDA approved use allowing for complete filling of the ventricles and return in humans was as a skin wound cleanser that has dem- to normal working end diastolic and end systolic onstrated lack of toxicity to the cellular components of volumes [29]. blood and lack of interference to the wound’s ability to heal and resist infection after being tested in more than Copolymer-based membrane stabilizers in vivo 1000 patients [144, 145]. Yasuda et al. further showed that in vivo systemic ad- ministration of P188 to mdx mice improved ventricular Copolymer-based muscle membrane stabilization: cellular geometry and prevented acute cardiac failure during a studies dobutamine cardiac stress test protocol [80]. In the The first applications of P188 in muscle demonstrated sig- golden retriever dystrophic canine model, chronic P188 nificant reduction in electroporation-induced leakage of administration prevented left-ventricular remodeling, re- carboxyfluorescein dye from isolated skeletal muscle cells duced myocardial fibrosis, and blocked cardiac troponin [118]. In parallel experiments, the hydrophilic control mol- I release [148]. In addition, long-term intermittent ad- ecule Dextran showed no membrane protective effect ministration of P188 was shown to confer protection [118], suggesting that P188 interacts with the damaged during isoproterenol-induced cardiomyopathy in mdx membrane in a way that alters membrane properties and mice [149]. promotes stability. Other reports produced similar results The ability of synthetic membrane stabilizers to protect in in vitro models of acute radiation injury which involves fragile DMD skeletal muscles had, up until recently, been the generation of reactive oxygen species which can rapidly less clear. Early investigations with P188 showed little to alter the structure and organization of the cell membrane no efficacy in protecting dystrophic limb skeletal muscle leading to cell necrosis. In a study by Hannig et al. [146], function in vivo [150, 151], even though P188 had been P188 was shown to retard cytoplasmic calcein leakage from shown effective in protecting hindlimb skeletal muscle in isolated rat skeletal muscle cells undergoing radiopermeabi- a range of other conditions, including electrocution injury lization. Greenebaum et al. [147] further showed that skel- [118, 152], hindlimb ischemia-reperfusion injury [153, etal muscle cells treated with P188 manifested enhanced 154], and in a model of dysferlin-deficiency [155]. Interest- viability and survival following high-dose irradiation. ingly, a recent study evaluating the pharmacodynamics of Houang et al. Skeletal Muscle (2018) 8:31 Page 7 of 19 P188 demonstrated P188 can fully protect dystrophic skel- measurable changes in membrane dynamics or structure, etal muscle against mechanical stress in vivo [156]. This as detected by electron paramagnetic resonance and iso- study showed how in vivo membrane protection is critic- thermal calorimetry techniques. Collectively, this is evi- ally dependent on delivery route [156] wherein subcutane- dence that P188 does not fully insert in the intact bilayer ous delivery of P188 led to dramatic improvement in mdx interior nor does it affect overall lipid packing [123]. hindlimb muscle function during lengthening contractions As DMD pathophysiology is exacerbated by lengthening and decreased uptake of Evans blue dye in vivo. In con- contractions, it is important to compare results from trast, in this model, neither intraperitoneal nor intraven- non-stressed membranes to mechanically stressed mem- ous delivery, which were routes used in previous studies, branes. To mimic bilayer mechanical stress using artificial led to improvement in muscle function [156]. Thus, the membranes in vitro, studies have used Langmuir troughs. lack of skeletal muscle efficacy reported in previous stud- This approach permits fine control of the surface area and ies using P188 [150, 151] could be attributed to subopti- therefore lipid packing density of supported phospholipid mal mode of delivery of P188, rather than a fundamental monolayers at the air/water interface [121, 158]. Maskari- limitation in the mechanism by which the block copoly- nec et al. [159] focused on P188 insertion as a function of mer stabilizes fragile dystrophic skeletal muscle mem- surface pressure, which directly correlates to lipid packing branes. This was further supported by another recent density. Here, using either anionic dipalmitoylphosphati- study showing that chronic dosing of P188 using subcuta- dylglycerol (DPPG) or zwitterionic dipalmitoylphosphati- neous delivery improves diaphragm function in mdx and dylcholine (DPPC) monolayers, results showed P188 −/− mdx:utr mouse models in vivo [157]. In that study, inserts into both lipid types at a surface pressure (π) ≤ P188 improved dystrophic mouse respiratory parameters 22 mN/m, which is lower than that of a healthy cell mem- in vivo, including tidal volume/body weight and minute brane (~ 30–35 mN/m) [160, 161]. P188 was found to re- volume/body weight, as well as decreased central nucle- main inserted until the surface pressure increased back to ation and decreased collagen deposition in treated dia- threshold surface pressure equivalent to that of an intact phragm muscle fibers [157]. These results are promising membrane [158, 159]. X-ray reflectivity results further in indicating that chronic P188 treatment may be benefi- showed that at high surface pressure lipid films, in the cial in preserving respiratory and limb muscle functions. presence and absence of P188 in the subphase, exhibit Taken together, these findings are evidence that synthetic similar electron density profiles [121, 162]. membrane stabilizers provide a unique first-in-class treat- Morphologically, P188 insertion appears to tighten lipid ment strategy for simultaneously treating all affected stri- packing via physical occupation of surface area in localized ated muscles in DMD. A summary of in vivo studies patches rather than uniformly across the whole membrane testing block copolymers as a therapeutic strategy in [121, 159]. The hypothesis follows that only when lipid DMDmodelsispresented in Table 2. packing density is low, and the hydrophobic core of the monolayer is exposed, that P188 partitions to the mem- Elucidating the copolymer-muscle membrane interface brane via hydrophobic interactions between the acyl The mechanism underlying copolymer-lipid bilayer inter- chains of the bilayer and the copolymer hydrophobic PPO action has not been delineated. Elucidating copolymer block. Inability to remain inserted above a threshold sur- chemical and structural characteristics are essential to de- face pressure suggests that P188 does not insert into nor- termine membrane stabilizer function, under both normal mal intact cell membranes and only inserts once lipid and disease conditions. Because biological membranes are density is decreased. This leads to a dynamic interaction, structurally complex, artificial phospholipid-based mem- wherein P188 is “squeezed out” from the cell bilayer when branes are an invaluable model to study the biophysical normal membrane structure is restored (Fig. 3). Copoly- basis of copolymer-membrane interactions. To investigate mer “squeeze out” upon normalization of membrane lipid the physical nature of P188-membrane interactions, packaging density is an important concept driving thera- Cheng et al. employed H Overhauser dynamic nuclear peutic applications. In this context, copolymers only insert polarization/Nuclear Magnetic Resonance spectroscopy to into areas of the membrane that are damaged. This work- determine local hydration dynamics at the P188-lipid ing model hypothesizes that when copolymer insertion membrane interface [123]. The high spatial resolution re-establishes membrane barrier function and prevents 2+ afforded by this technique allows for probing the local Ca overload during muscle contraction, the endogenous water diffusivity in lipid bilayer systems. Here, P188 cell membrane repair response would be able to patch the weakly adsorbed to the intact vesicle membrane surface. membrane [1]. Upon repair, the copolymer would then This was shown by membrane hydration dynamics and disengage from the membrane (Fig. 3). This copolymer intra-bilayer water diffusivity, both at the membrane sur- squeeze out at normal surface pressure would be benefi- face and bilayer interior. Furthermore, P188 weakly cial in the context of biomedical applications of damaged adsorbed at the membrane surface and produced no cellular membranes where copolymers selectively insert Houang et al. Skeletal Muscle (2018) 8:31 Page 8 of 19 Table 2 Summary of studies using block copolymers as a treatment in DMD models in vivo Copolymer Pathophysiology DMD Treatment Dosage Delivery Results References model time route P188 Cardiomyopathy mdx Pre-treatment 460 mg/kg i.v. P188 significantly improved cardiac hemodynamic response and Yasuda et al. (30 min) animal survival during cardiac stress testing (2005) [80] P188 Skeletal muscle mdx Pre-treatment 600–1800 mg/kg i.p. No significant difference in % EBD penetration in rectus femoris Quinlan et al. (30 min) muscle fibers in P188 treated mdx mice exercised by downhill (2006) [150] treadmill running P188 Cardiomyopathy GRMD 8 weeks 60 mg/kg/hr i.v. Chronic P188 treatment normalized serum cTnI levels, blocked Townsend et al. increases in heart failure marker BNP, significantly decreased (2010) [148] cardiac fibrosis, and prevented dilated cardiomyopathy. Cardiac hemodynamic function in response to dobutamine stress was significantly improved compared to saline treatment. Serum CK levels were not affected. P188 Cardiomyopathy mdx 2–4 weeks 460 mg/kg i.p. P188 treatment prevented a decrease in cardiac function in Spurney et al. response to isoproterenol stress testing. Treated mice did (2010) [149] not show significant differences in cardiac fibrosis but had increase in EBD positive fibers, these hearts showed increased systolic function compared to untreated hearts. P188 Skeletal muscle mdx Pre-treatment 30 mg/kg, 460 mg/ i.p. Single dose P188 treatment induced an increase in specific force Terry et al. 2-week daily kg and decreased the number of IgG positive fibers in both non-stressed (2014) [151] and stressed muscles. P188 treatment improved the histological appearance in TA muscles under some conditions. 2-week P188 did not affect TA force. During lengthening contraction injury, it was reported that in a subset of contractions the P188 treatment group had slightly but statistically significant lower force than saline control. P188, P338 Skeletal muscle mdx Pre-treatment 60–460 mg/kg i.p., i.v., s.c., Subcutaneous but not intravenous nor intraperitoneal injection of P188 Houang et al. (0.5–3h) i.m. significantly decreased the force loss during and after lengthening (2015) [156] contractions of hindlimb mdx muscle and significantly decreased EBD uptake into TA myofibers post-injury. Subcutaneous delivery of PEO8000 had no protective effect. Lower dosage of intraperitoneal and intramuscular but not subcutaneous or intravenous injections of P338 shows significant protective effect. P188 Respiratory mdx Q.D., 22 weeks 3 mg/kg s.c. Chronic delivery of P188 had significant positive effects on respiratory Markham et al. function parameters and improved diaphragm histological parameters (2015) [217] and caused improvement in cardiac hemodynamics of treated mdx mice −/− Cardiomyopathy mdx/utr Q.D., 8 weeks 1 mg/kg s.c. P188 treatment slowed the loss of respiratory function and improved diaphragm histological parameters in double knockout mice diP188 diP188-CH Skeletal muscle mdx Pre-treatment 1000 mg/kg i.p. A diblock copolymer architecture confers membrane stabilization. Houang et al. diP188-(CH ) (0.5–3h) The addition of a single hydrophobic tert-butoxy end-group to the (2017) [183] 3 3 PPO core significantly enhanced membrane protection against lengthening contractions. The less hydrophobic methoxy and hydrophilic hydroxyl end groups did not confer membrane protection in vivo. i.v., intravenous; i.p., intraperitoneal; s.c., subcutaneous; i.m., intramuscular; EBD, Evans blue dye; GRMD, golden retriever muscular dystrophy; cTnI, cardiac troponin I; BNP, brain natriuretic peptide; CK, creatine kinase; TA, tibialis anterior; Q.D., daily; diP188, diblock P188 Houang et al. Skeletal Muscle (2018) 8:31 Page 9 of 19 Fig. 3 Model of copolymer-based membrane stabilization. a In DMD, susceptibility to sarcolemmal damage from lengthening muscle contraction 2+ 2+ renders the muscle cell membrane leaky to extracellular Ca (pink circles). Subsequent intracellular Ca overload leads to activation of pathological cellular pathways. Further membrane damage overloads the repair capacity of endogenous cell membrane repair mechanisms and ultimately leads to cell death. b Copolymer insertion driven by hydrophobic interactions (red PPO block of the copolymer with the hydrophobic part of the membrane 2+ that is now exposed due to instability). Membrane stabilization prevents pathological Ca entry into the cell and prevents activation of cellular death pathways. c While the copolymer stabilizes the membrane and prevents further damage, intrinsic cell membrane repair mechanisms can repair lesions at damaged sites [215]. d Once the membrane integrity is restored, the copolymer membrane stabilizer is “squeezed out” of the membrane. Here, the membrane is resealed, its lipid packing density is restored, and its hydrophobic portion is now enclosed [159, 216] only onto localized areas of the membrane where the local or PEG), the hydrophilic constituent of poloxamers, has been lipid density is reduced, and thus only where the mem- well investigated in the fusion of model membranes and for brane is structurally impaired, and not interact with intact its ability to lower water molecule activity at the membrane- with healthy areas of the membrane. solvent interface [165]. While PEO-mediated membrane stabilization has been shown to be effective, the very high Copolymer structure-function analysis concentrations (mM-M) required for effectiveness indicate Mechanistic investigation via the structure−function relation- that the hydrophobic block plays an essential role in ship of block copolymer chemistry is required to define the copolymer-membrane interactions [166]. basis of copolymer-based membrane interaction. This is The relationship between copolymer chemical struc- crucial in the long-term to guide the design of an optimal ture and the kinetics of adsorption, insertion, and subse- membrane stabilizer. There is considerable interest in block quent squeeze out from lipid monolayers has been copolymers as membrane stabilizers due to their overall sur- investigated by Frey et al. via Langmuir trough experi- face active and solvent-selective characteristics and intrinsic ments and Monte Carlo simulations [120]. Here, upon thermodynamic properties and architectures [163, 164]. compression of the monolayer, copolymers with higher P188 is part of a large family of poloxamers, each with dis- PPO/PEO ratio favored a higher squeeze out pressure. tinct physicochemical properties. Polyethylene glycol (PEO Moreover, higher molecular weight copolymers were Houang et al. Skeletal Muscle (2018) 8:31 Page 10 of 19 observed to squeeze out at higher surface pressures, pressures before being squeezed out [120, 158]. More- while at constant PPO/PEO ratios smaller copolymers over, hydrophobic copolymers with bulkier PPO blocks squeezed out at lower pressures. Results showed that the were found to increase flippase activity compared to co- ratio dictates the equilibrium spreading pressure of polymers with shorter PPO blocks [117]. Copolymer- copolymers at the phospholipid interface. Hydrophobic bilayer interactions have been investigated using pulse copolymers were less soluble resulting in a higher field gradient nuclear magnetic resonance to quantify proportion of adsorption at the monolayer interface and copolymer diffusion in the presence and absence of uni- thus higher equilibrium spreading pressure [120]. These lamellar liposomes [171]. Here, the binding percentage findings demonstrate the relationship between the PPO/ of copolymers to liposomes was quantified, and results PEO ratio and molecular weight in determining further confirmed that increased copolymer molecular copolymer-membrane interactions. weight and increased relative hydrophobicity cause in- Overall, copolymer hydrophobicity has a principal role creased binding and liposome coverage relative to in affecting membrane bilayer physical structure. Thus, smaller, more hydrophilic copolymers. Another recent more hydrophobic copolymers decrease membrane study using surface plasmon resonance to probe and microviscosity [117, 167] and increase the rate of lipid compare binding of P188 and a PEO homopolymer of motion across the outer and inner leaflets of vesicular similar size provides direct evidence of binding onto membranes [117], causing membrane leakiness [115, 168]. supported intact lipid bilayers with comparable binding Chang et al. [169] showed that surface pressure-area kinetics. Moreover, this study provides biophysical isotherms exhibited by P188 (PEO –PPO –PEO )com- evidence that copolymer adsorption alone does not fully 75 30 75 pared to the highly hydrophobic P181 (PEO –PPO – account for membrane protection efficacy. [172]A 2 30 PEO ) are significantly different. P181 exhibits condensed- schematic summary of structure-function of copolymer- film-like surface behavior whereas P188 exhibits an based membrane stabilization is presented in (Fig. 4). expanded-like behavior. This was confirmed by Cheng et al. [123] using dynamic light scattering, isothermal cal- Molecular dynamics analysis of copolymer- orimetry, and small molecule-directed lipid peroxidation membrane interactions of liposomes. The PPO/PEO ratio was shown to be a key Mechanistic insights into copolymer-membrane interaction feature in effectively protecting intact liposomes from are aided by studies pursued at the atomistic level. Molecular peroxidation. Copolymers that adsorb at the membrane dynamics (MD) simulations have been recently developed to surface, without penetration into the bilayer core, such as investigate copolymer-phospholipid bilayer interactions P188 and PEG8000, presumably affect the hydration shell [173, 174]. MD simulations are physics-based computa- of the bilayer. This would suppress the diffusion of the tional methods to simulate and observe the interactions free radical lipid peroxidation initiator into the lipid bi- of atoms and molecules at resolutions that are currently layer, thereby preventing the initiation of lipid peroxida- hard or impossible to probe experimentally. In general, tion. The more hydrophobic poloxamers, for example, MD simulations of large macromolecules, such as P335 (PEO –PPO –PEO ), P333 (PEO –PPO – copolymers, are computationally challenging to per- 38 54 38 20 54 PEO ), and P181 (PEO –PPO –PEO ), have significant form. Past MD efforts have focused on coarse-grained 20 2 30 2 heat of partitioning indicative of insertion into the liposo- [120, 175, 176] and united atom [168, 177, 178]models, mal membrane [123]. These hydrophobic copolymers do which are models that reduce the total number of de- not prevent initiation of lipid peroxidation [170] indicating grees of freedom in the system by representing mole- that copolymer hydrophobicity affects kinetics of inser- cules and their interactions at lower resolution. This tion. More hydrophobic copolymers insert at faster rates allows for significantly increased simulation timescale by initially embedding below the lipid head group region, at lower computational cost but in exchange for the opening up the packing of acyl chains and accelerating the loss of atomistic level details. passage of water across the membrane, thus increasing An in silico model of copolymer adsorption using permeability [123, 166]. coarse-grained force field showed copolymer-membrane The size of the hydrophobic PPO block influences in- insertion, followed by percolation across the unstressed sertion of the copolymer into lipid films. Poloxamers at lipid bilayers [179]. Here, copolymers containing a PPO fixed 80% PEO composition and different molecular block with a length comparable to that of the bilayer weights (P108, P238, P188, and P338) have been tested thickness tended to span across, or percolate across, the for their relative ability to insert into lipid monolayers lipid bilayer. In comparison, copolymers with shorter [158]. Copolymers with high PPO content required PPO blocks inserted partially, with the PEO blocks lower surface pressure for insertion. Additionally, once remaining in water on one side of the bilayer. Moreover, inserted, high mass copolymers are able to retain pos- total percolation of copolymers across the bilayer led to ition within the monolayer at much higher surface reduction in membrane thickness and an increase in the Houang et al. Skeletal Muscle (2018) 8:31 Page 11 of 19 Fig. 4 Schematic representation of structure-function of copolymer-membrane interaction. Triblock copolymer membrane stabilization occurs via insertion of the hydrophobic PPO core block (red) and balanced by flanking of the two hydrophilic PEO blocks (blue) that are required to prevent complete translocation across the membrane. Without a second flanking PEO chain, diblock copolymers can also insert into the membrane, but insertion is at least in part dictated by the PPO end group. Here, the more hydrophobic end group, such as –C(CH ) (†), driving insertion and 3 3 anchoring and the more hydrophilic end groups, such as –OH, retained at the solvent-polar head group interface. Variation in PEO (blue) and PPO (red) block lengths alters the hydrophobic/hydrophilic balance that is required for optimal membrane insertion and stabilization. Too high a PPO/PEO ratio and large size PPO group drives the copolymer deeper into the membrane and further exacerbates damage to the membrane area per lipid. Goliaei et al. [177] used an united-atom molecule (A ) to model bilayer mechanical stress [181]. force field-based MD model to show that P188 can pas- P188 interaction with lipid bilayers was demonstrated to sively insert into the 1,2-dilauroyl-sn-glycero-3-phospho- be dependent on A with insertion of the PPO block oc- 0, choline (DLPC) lipid bilayer under non-stressed curring at a ~ 15–20% increase in A Additionally, P188 conditions after extensive simulation time (> 500 ns). insertion into the membrane significantly increased the Here, the PPO block inserted into the hydrophobic part lateral pressure required for membrane rupture under of the bilayer and the PEO chains remained solvated mechanical stress [181]. Further, membrane insertion and outside the membrane [177]. Moreover, using a 3 nm stabilization efficacy appeared dependent on the PPO/ water pore model to simulate a damaged lipid bilayer, PEO ratio. MD simulations of hydrophobic copolymers, the PPO block of P188 inserted adjacent to the water such as P331 (PEO –PPO –PEO ), inserted at signifi- 7 54 7 pore and “pushed” water molecules out of the pore to cantly lower A , as well as decreased the lateral pressure reduce pore size. required to rupture the membrane. This is consistent with Simplified force field models allow for larger timescale the results of Nawaz et al. [168] who demonstrated perco- simulation; however, they yield only a partial view of lation across the bilayer of highly hydrophobic copolymers membrane structural properties and limit atomic reso- causing membrane bending and an increase in local per- lution insights [180]. Importantly, previous MD studies meability allowing water molecule penetration into the have focused on copolymer-bilayer interactions under hydrophobic region of the membrane. The timescale for constant pressure and temperature (NPT) and constant percolation was inversely proportional to the PEO block area and temperature conditions (NPAT), and thus are length [168]. Moreover, another all-atom MD study by computational models of membranes under normal non- Zaki and Carbone showed that incorporation of multiple stressed conditions. Recently, an all-atom MD simulation copolymer units within the bilayer hinders lipid diffusion model was developed to investigate copolymer-lipid mem- and forced nearby lipids to remain closely packed, even brane interaction under conditions of varied lateral mech- during lateral mechanical stress [182]. anical stress. This in silico approach correlates to the Overall, the results from MD studies are consistent physiological state to lengthening contraction muscle in- with experimental observations from Langmuir trough jury in DMD. Here, an increase in surface tension (γ) was studies in that P188 inserts into areas of low lipid dens- applied to induce expansion in the bilayer area per lipid ity and at low surface pressures [158, 159]. MD studies Houang et al. Skeletal Muscle (2018) 8:31 Page 12 of 19 feature a simplified phospholipid bilayer as a basic for sensitive modulation of the diblock PPO block hydropho- model of the biological membrane, which is comprised bicity. This strategy has precedence in the surfactancy litera- of proteins, complex mixtures of lipid types, and other ture where novel terminal functional groups have been macromolecules, all organized in a tightly regulated shown to influence solution and bulk phase behavior [187, manner. Nonetheless, all atom MD results are qualita- 188]. Diblock copolymers have never been investigated for tively comparable to results derived in cells and animals biological membrane stabilization until a recent report dem- [156, 183, 184]. Complementation of findings from in onstrating that diblock PEO–PPO architectures can confer silico to in vivo methods underscores MD simulations as membrane stabilization in both in vitro and in vivo DMD a powerful tool to further mechanistic understanding of models [156, 171, 183, 184]. This establishes that specific copolymer-bilayer interactions and to ultimately guide PPO end group chemistries play a critical role in defining design and optimization of copolymers for physiological muscle membrane stabilization [183, 184]. membrane stabilization. Recent diblock studies have advanced an “anchor and chain” model of membrane stabilization (Fig. 4)[156, Copolymer architecture: diblock copolymers as 171, 183, 184]. Here, the addition of a small hydrophobic membrane stabilizers end group “anchor,” as demonstrated by tert-butoxy to Block copolymers can be designed with two or more dis- the PPO block, discretely increases the hydrophobic tinct polymer blocks covalently bonded together. These character of the end of the PPO block, without signifi- can exist in a variety of molecular sizes, relative degree of cantly increasing the overall mass of the copolymer. polymerization of each block (composition), hydrophobi- From these results, it is hypothesized that discrete alter- city, chemical moieties, and architectures, from diblock ations in the structure of the PPO terminal functional and triblock to multi-blocks. This broad landscape leads group, such as replacing tert-butoxy with n-butoxy or to a nearly infinite number of possible distinct chemical other non-polar end groups, will further influence the configurations [112]. Previously, from a practical perspec- packing and interaction strength with the lipid core. The tive, the use of poloxamers has been generally constrained PEO chain appears to be required to preserve the to those made available commercially. This limitation pro- amphiphilic behavior of the copolymer and to maintain vides an impetus for advancing discovery of the copolymer the copolymer at the solvent-membrane interface. De- chemical landscape beyond that of the triblock architec- tailed structure-function analysis of the PEO block, in- ture. As above, P188 is reported to be weakly adsorbed to cluding length, structure, and chemical characteristics, the lipid bilayer [123, 170] and it is hypothesized that this has not yet been initiated and this will be important to weak association is due to steric constraints imposed by determine in further experimentation. Taken together, the flanking PEO chains [162]. The removal of one of the these proof-of-principle results establish physiological flanking PEO chains to form the diblock PEO–PPO archi- relevance to diblock copolymers and support further in- tecture (Fig. 2) allows for facile assessment of the associ- vestigation of this expansive copolymer chemical space. ation of the hydrophobic PPO core with the lipid bilayer. Firestone et al. employed small- and wide-angle X-ray scattering techniques to examine the structure of a lipid Clinical applications, challenges, and ongoing bilayer and the phase produced by either the triblock developments P188 or a PEO–PPO diblock with an equivalent PPO P188 was first approved by the FDA as an anti-viscosity block length [185]. P188-synthetic lipid bilayer interaction agent added to blood before transfusions [135, 189]. P188 produced an aggregate phase structure suggesting limited (labeled as RheothRx, Glaxo Wellcome Inc.) has been previ- insertion of the copolymer into the lipid bilayer. On the ously tested in clinical trials for both sickle cell anemia [141, other hand, the PEO–PPO diblock produced a well- 142] and myocardial infarction [190, 191]. Due to its nature ordered lamellar phase suggesting enhanced interfacing as a nonionic surfactant and demonstrated hemorrheologic within the bilayer [185]. This suggests that removing one properties, a randomized, double-blind, placebo-controlled of the flanking PEO chains facilitates PPO block inter- pilot study in 50 patients was initiated in the early 1990s to action with the hydrophobic acyl chain region of the lipid determine the safety and efficacy of P188 in treating acute bilayer to strengthen copolymer-bilayer interaction. vaso-occlusive crises in sickle cell anemia disease. Treated The PEO–PPO diblock architecture offers several advan- patients showed a significant decrease in painful episodes, re- tages for advancing copolymer-membrane structure-function duced hospital stay, requirement of analgesics, and reported studies. These include an easier and more controlled chem- pain [142]. Moreover, continuous RheothRx intravenous in- ical synthetic process [186], the more precise control of PPO fusion over 48 h (60-min loading dose of 300 mg/kg and PEO block sizes and the ability to design specific func- followed by a 47-h maintenanceinfusion of 30 mg/kg) was tional end groups to the hydrophobic PPO core to finely well tolerated with the exception of a mild increase in serum tune membrane interactions. This latter modification allows creatinine in one patient with underlying renal dysfunction. Houang et al. Skeletal Muscle (2018) 8:31 Page 13 of 19 Pharmacokinetic study of P188 injection in healthy Conclusions males has been conducted in a cohort of volunteers and From a conceptual perspective for clinical application, determined that elimination occurs primarily through synthetic muscle membrane stabilizers for treating DMD renal clearance [192]. RheothRx (P188) clinical trial in patients have several attractive features. These include (1) patients tested adjunctive therapy during thrombolytic treatment strategy targeting the primary defect in DMD— therapy for acute myocardial infarction at time of severe muscle membrane instability causing muscle de- hospitalization. Initial reports showed P188 resulted in terioration and cell death, (2) copolymers as muscle mem- significantly smaller-sized infarcts, greater myocardial brane interfacing molecules could in principle treat all salvage, and improved median ejection fraction [191]. DMD patients regardless of their genetic lesion, (3) However, in follow up large-scale clinical studies, pre-clinical studies provide evidence of copolymer protec- Rheothrx administration did not significantly decrease tion in other applications, (4) first-in-class membrane infarct size or favorably alter outcome [193]. Moreover, stabilizer P188 NF has a favorable safety profile in cardiac, in a subset of elderly patients with pre-existing renal dis- respiratory, and limb striated muscles, as derived from hu- ease increased renal dysfunction was reported. This ad- man clinical trial data in humans. The inherent limitation verse effect was later determined to be due to small with membrane stabilizers as a potential therapy for DMD molecular weight impurities in the P188 formulation, is that this approach is not a cure and would necessitate which was manufactured as an excipient-grade product chronic treatment for DMD patients. following National Formulary specifications [194]. Sub- The ultimate goal for membrane stabilizing therapy is to sequent clinical studies using the purified formulation of significantly improve and prolong patient quality of life P188 significantly improved the renal safety profile and while awaiting a potential effective cure for DMD. As tolerability [194]. DMD is a chronic progressive disease, membrane Purified formulation of P188 was repackaged as MST-188 stabilization treatment would require life-long administra- or vepoloxamer (Mast Therapeutics, Inc.) which was then tion. In the best case scenario, this clinical treatment further evaluated in another interventional clinical trial would effectively manage the disease, analogous, for ex- (EPIC trial) in children with sickle cell disease. In a recent ample, to the highly effective life-long daily insulin treat- large-scale phase 3 clinical trial, vepoloxamer did not meet ment used by type I diabetic patients. One could envision primary efficacy endpoints of demonstrating a statistically chronic copolymer treatment starting soon after diagnosis significant reduction in the mean duration of vaso-occlusive with the aim to preserve striated muscle function before crisis events. However, this clinical trial did show that vepo- muscle degeneration and wasting occurs. Membrane sta- loxamer was generally well tolerated with no statistically sig- bilizers may also be envisioned in acute settings for DMD nificant differences in treatment-related adverse events in the patients, for example, during orthopedic surgery or other vepoloxamer group compared to the placebo group (https:// stress-inducing events [148]. Another setting where co- clinicaltrials.gov/ct2/show/NCT01737814). polymer administration could make a significant positive For membrane stabilizers in DMD, Phrixus Pharmaceuti- impact is during exercise training protocols for DMD pa- cals, Inc. has initiated a Phase 2 single site, open-label trial tients implemented to oppose the loss of functional abil- for respiratory, cardiac and skeletal limb muscle end points ities as a result of muscle disuse [195]. It is still unclear in non-ambulatory DMD patients (ClinicalTrials.gov Iden- whether exercise training and which exercise protocols tifier: NCT03558958). Drug P-188 NF (Carmeseal-MD) is could be beneficial to DMD patients or other patients with directed toward DMD patients with early heart failure and myopathic disorders, at least in part due to the potential respiratory dysfunction who are currently on stable regi- detrimental effects of strenuous exercise and muscle con- men of background therapies. Phrixus Pharmaceuticals traction on the muscle membrane [196]. Treating DMD and Ethicor Pharma Ltd. have made Carmeseal-MD avail- patients with membrane stabilizers prior to an exercise able in 2015 as a “special” or unlicensed medicinal product training bout may support striated muscle membranes in the European Union prior to regulatory approval. This during strength exercise and abrogate deleterious effects allows access to Carmeseal-MD to DMD patients with re- that would occur while supporting muscle repair and spiratory and cardiac deficits through physician request. As strength building. of the end of 2017, one patient under the Expanded Access It is also likely that effective DMD treatment will ultim- Program has been reported to have met the 15-month ately require a combination of approaches to achieve opti- treatment mark with treatment reported to have been well mal outcomes. One example where bundled therapies tolerated and reductions in creatine kinase and cardiac containing P188 has already shown promise is cardiac ar- troponin I observed (Phrixus Pharmaceuticals). Moving rest and resuscitation [197]. Block copolymers have been in forward, larger scale human clinical data will be required use as vehicles for enhanced gene delivery in other applica- to fully evaluate membrane stabilizer treatment efficacy in tions [198, 199], and the prospect of bundled therapies of DMD patients. block copolymers and gene-directed strategies would be of Houang et al. Skeletal Muscle (2018) 8:31 Page 14 of 19 significant interest to pursue in future works. Another strat- Abbreviations DGC: Dystrophin-glycoprotein complex; DLPC: 1,2-dilauroyl-sn-glycero-3- egy where copolymer-based membrane stabilizers could be phosphocholine; DMD: Duchenne muscular dystrophy; combined would be stem cell therapy to regenerate muscle. DPPC: Dipalmitoylphosphatidylcholine; DPPG: Dipalmitoylphosphatidylglycerol; Induced pluripotent stem cell (iPSC) technology allows MD: Molecular dynamics; NPAT: Constant pressure, area, and temperature; NPT: Constant pressure and temperature; NPγT: Constant pressure, surface derivation of patient-derived stem cells which obviates tension, and temperature; PEG: Polyethylene glycol; PEO: Polyethylene oxide; immunological concerns. One recent study showed PPO: Polypropylene oxide; ROS: Reactive oxygen species proof-of-principle application of ex vivo genetic correction Acknowledgements of dystrophic iPS cells with a micro-utrophin transgene be- We thank our colleagues at the University of Minnesota, in particular the fore transplantation back into dystrophin/utrophin double UMN Biomedical Block Copolymer Research Consortium for dynamic knockout mice [200]. They observed that engrafted muscle interactions and discussions. had large numbers of corrected myofibers, restoration of Funding the dystrophin and associated proteins complex and im- This work was supported by grants from the National Institutes of Health proved contractile strength. While these results are positive (J.M.M.), the Lillehei Heart Institute, the Muscular Dystrophy Association (J.M.M.), and the American Heart Association Predoctoral Fellowship (E.M.H.). and exciting, this strategy still has to overcome multiple im- portant hurdles, such as improved survival of the cells Authors’ contributions post-injection, effective migration to the compromised EMH, FSB, YYS, and JMM all contributed to the writing of this manuscript. All muscles, and successful engraftment. Copolymer-based authors read and approved the final manuscript. membrane stabilizers injected alongside iPS-derived myo- Ethics approval and consent to participate cytes may help improve survival of these cells post- Not applicable injection. Synthetic membrane stabilizers may ultimately extend to Consent for publication Not applicable numerous other inherited or acquired diseases in which cell membrane integrity is compromised. In the last few years, Competing interests many preclinical studies using P188 as cell membrane stabi- The authors declare the following potential conflict of interest: J.M.M. is on the scientific advisory board of and holds zero value equity shares in Phrixus lizers have been published in a variety of pathological set- Pharmaceuticals Inc., a company developing novel therapeutics for heart tings, including amyotrophic lateral sclerosis [201], traumatic failure and DMD, and this is actively managed by the UMN Office of brain injury [202], aggregation of unfolded protein [203– Institutional Compliance. 207], hypoxia and ischemia-reperfusion injury [154, 208, 209], irradiation and burn injury [152, 210, 211], cartilage Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in damage, and joint degeneration following blunt impact published maps and institutional affiliations. [212–214]. Based on the potential novel uses of copolymer- based membrane stabilizers in various other diseases where Author details Department of Integrative Biology and Physiology, University of Minnesota the cell membrane is damaged, one could anticipate that in- Medical School, 6-125 Jackson Hall, 321 Church Street SE, Minneapolis, MN creased academic and clinical interest in this therapeutic 55455, USA. University of Minnesota Informatics Institute, MN, USA. strategy will help promote faster translation to human clin- Bioinformatics and Computational Biology Program, University of Minnesota, MN, USA. Department of Chemical Engineering and Materials Science, ical applications. University of Minnesota, MN, USA. Finally, as detailed in this review, first-in-class copolymer-based membrane stabilizer P188 has a long Received: 30 May 2018 Accepted: 13 September 2018 history. 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Muscle membrane integrity in Duchenne muscular dystrophy: recent advances in copolymer-based muscle membrane stabilizers

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Springer Journals
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Copyright © 2018 by The Author(s).
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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2044-5040
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10.1186/s13395-018-0177-7
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Abstract

The scientific premise, design, and structure-function analysis of chemical-based muscle membrane stabilizing block copolymers are reviewed here for applications in striated muscle membrane injury. Synthetic block copolymers have a rich history and wide array of applications from industry to biology. Potential for discovery is enabled by a large chemical space for block copolymers, including modifications in block copolymer mass, composition, and molecular architecture. Collectively, this presents an impressive chemical landscape to leverage distinct structure-function outcomes. Of particular relevance to biology and medicine, stabilization of damaged phospholipid membranes using amphiphilic block copolymers, classified as poloxamers or pluronics, has been the subject of increasing scientific inquiry. This review focuses on implementing block copolymers to protect fragile muscle membranes against mechanical stress. The review highlights interventions in Duchenne muscular dystrophy, a fatal disease of progressive muscle deterioration owing to marked instability of the striated muscle membrane. Biophysical and chemical engineering advances are presented that delineate and expand upon current understanding of copolymer- lipid membrane interactions and the mechanism of stabilization. The studies presented here serve to underscore the utility of copolymer discovery leading toward the therapeutic application of block copolymers in Duchenne muscular dystrophy and potentially other biomedical applications in which membrane integrity is compromised. Keywords: Duchenne muscular dystrophy, Block copolymers, Membrane stabilization Background will not be further elaborated on here. Rather, this review All eukaryotic cells are enveloped by a phospholipid bi- focuses on membrane protection from the perspective of layer membrane. An enormous literature exists that de- a chemical-based approach to preserve muscle membrane fines biological cell membrane form and function [1, 2]. integrity and how this unique cell extrinsic approach Regardless of biological cell type, the cell membrane could complement cell intrinsic membrane stabilization/ represents first and last line of defense for ensuring the repair pathways (Fig. 1). Numerous acquired and inherited normal function and ultimately the viability of the cell. diseases comprise, at some level, an etiology involving cell Accordingly, multiple cellular processes are present to membrane instability. Duchenne muscular dystrophy is help ensure the maintenance, repair and protection of the the archetype inherited disease of severe membrane fragil- cell membrane. There are numerous excellent expert re- ity and serves as the disease model focal point of this views detailing cell intrinsic mechanisms of membrane in- review. tegrity and repair [2–17] and mechanistic details on these Duchenne muscular dystrophy: a fatal disease of * Correspondence: metzgerj@umn.edu muscle membrane instability Department of Integrative Biology and Physiology, University of Minnesota Duchenne muscular dystrophy (DMD) is an X-linked re- Medical School, 6-125 Jackson Hall, 321 Church Street SE, Minneapolis, MN cessive disease of marked striated muscle deterioration, 55455, USA Full list of author information is available at the end of the article affecting 1 in 3500–5000 boys [18]. DMD results from © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Houang et al. Skeletal Muscle (2018) 8:31 Page 2 of 19 weakness in limb muscles and postural muscles [18], leading to spinal scoliosis and decrease in exercise capacity. Weak- ness of the knees and hip extensors are displayed through the Gower’s sign, a maneuver through which the affected child will right himself from a supine position by using his hands and arms to extend the hips and bring the torso to an upright position [20]. Other physical symptoms include re- duced muscle bulk, pseudo-hypertrophy, and contractures of the calf muscles and joints [21]. Bone fragility and osteopor- osis also contribute to the development of scoliosis [22]. Concurrent with the decline in orthopedic condition is loss of respiratory function brought on by significant diaphragm wasting [23] leading patients to be placed on positive pres- sure nocturnal ventilation. Loss of ambulation and wheel- chair dependency occur by the early teens [24], and DMD patients typically succumb in their 20s due to cardio- respiratory failure [25–28]. DMD patients develop a severe cardiomyopathy, pre- senting as dilated cardiomyopathy [29], with arrhythmias and eventually heart failure occurring in the second/ third decade of life [24]. With increases in patient life- span, as a result of palliative glucocorticoid treatment and improvements in respiratory care and orthopedic corrections [30, 31], cardiomyopathy is an increasingly important but underappreciated contributor to DMD mortality. It is now evident that cardiomyopathy is present in 90% of DMD patients by age 18 and is con- firmed by significant myocardial fibrosis in autopsies [32–35]. Interestingly, the cardiomyopathy usually re- mains subclinical at early age and cardiac disease pro- gression typically proceeds at a slower rate compared to the skeletal muscle degeneration [36]. The incidence and evolution of cardiomyopathy in Duchenne muscular dys- trophy is presumably due to lesser strain on the heart when physical activity is limited once the patient is wheelchair bound. Dystrophin Extensive genetic analysis of DMD patients determined that defects in the dystrophin gene are causal for the dis- ease [19]. The dystrophin gene spans 2.5 Mb of DNA on Fig. 1 Copolymer-based muscle membrane stabilization of dystrophic the X chromosome. Dystrophin’s 79 exons encode a muscle. a Representation of intact muscle membrane with dystrophin anchoring the DGC to the actin cytoskeleton. b Membrane instability 3685 amino acid cytoskeletal protein localized to the caused by the lack of dystrophin leads to pathological increases intracellular surface of the muscle membrane [19]. Dys- 2+ in intracellular Ca concentration. c Copolymer stabilization of trophin consists of four major functional domains: (1) the damaged membrane via insertion of its hydrophobic PPO 2+ an actin-binding domain at the N-terminus; (2) a central block (red) prevents entry of extracellular Ca into the cell rod domain consisting of 24 spectrin-like repeats sepa- rated by four hinge regions, that has been shown to un- the lack of the cytoskeletal protein dystrophin, a protein fold and give flexibility in response to mechanical indispensable for maintaining the structural integrity of stretch [19]; (3) a cysteine-rich domain that interacts the muscle cell membrane [19]. DMD disease onset with the transmembrane protein β-dystroglycan; and (4) typically occurs between the ages of 2 and 5 years and is a C-terminal domain, critical for dystrophin’s interaction characterized by a delay in achieving childhood motor with other sub-sarcolemmal proteins [37–39]. Detailed milestones. DMD presents as a prominent and progressive structure function-based transgenic animal studies have Houang et al. Skeletal Muscle (2018) 8:31 Page 3 of 19 determined that the domains most critical to DMD degree of packing of bilayer components, is collectively de- pathology are the cysteine-rich domain and the scribed as “membrane fluidity” [48, 56]. Membrane fluidity N-terminal domain, and those are directly associated is controlled by a number of factors, including lipid com- with mechanically linking the extracellular matrix and position, sterol enrichment, and temperature. Fluidity is the cytoskeleton [40]. generally assessed using fluorescence polarization methods, Dystrophin is part of a large membrane-spanning com- electron spin resonance, and other spectroscopic methods plex of glycoproteins (dystrophin-glycoprotein complex or [59–62]. Along with membrane fluidity, the structure and DGC) that also include sarcoglycans (α, β, γ, δ), dystrogly- composition of the bilayer can be described by parameters cans (α and β), dystrobrevins, syntrophins, and sarcospan such as rigidity, elasticity, and tensile strength, all of which [38, 39, 41](Fig. 1a). This dystrophin-associated protein make up the membrane physical property known as plasma complex is found and enriched at the muscle costamere, a membrane order [58, 63]. Various studies have suggested network of proteins that physically connect the extracellu- that an optimal level of membrane order is essential for lar matrix to the cytoskeleton, through the muscle mem- normal myocyte function [57, 64]. Of particular interest to brane or sarcolemma, and as such orchestrates the lateral muscle, nicotinic acetylcholine receptors which are present force transmission [42–44]. As such, one essential func- at neuromuscular junctions of muscle cells can be allosteri- tion of dystrophin in striated muscle is to stabilize the cally modulated by surrounding lipids and thus require an muscle membrane against the forces associated with optimal membrane microenvironment to retain normal contraction thereby acting as a “molecular shock ab- function [65, 66]. Therefore, alterations to the muscle sorber” or molecular force dampener of the muscle mem- membrane surrounding these receptors, either during brane [45, 46]. The importance of dystrophin’sscaffolding mechanical stress or in diseased states, such as in DMD, support at the membrane is evident in studies showing have important ramifications for ion conductance and thus that dystrophin-deficient muscle fibers where the mem- ultimately affecting action potential generation and propa- brane was experimentally removed show no difference in gation during muscle contraction. contractile function compared to normal skeletal muscle From a structural perspective, the lipid bilayer alone is fibers, indicating a defect in the membrane-cytoskeleton not sufficient to counteract the significant forces placed linkage rather than in the contractile apparatus [47]. on the membrane during muscle contraction [67]. Mech- anical integrity of the sarcolemma is further supported by Striated muscle membrane fragility in DMD key cytoskeletal proteins, including dystrophin, spectrin, Biological membranes are asymmetrical bilayers approxi- and F-actin [68, 69]. Electron microscopy analysis of dys- mately 5–6 nm thick and comprised of various lipids, in- trophic muscle directly shows disruptions in the muscle cluding phospholipids, sphingolipids, glycolipids and membrane, termed delta lesions [70, 71]. This discovery sterols [48–51]. Phospholipid composition can vary sig- led to the theory that the loss of dystrophin and associated nificantly between different cell types and also in disease proteins at the sarcolemma renders the membrane leaky states [48, 49, 52, 53]. The eukaryotic cell membrane is and the muscle susceptible to contraction-induced injury. also typically composed of 20–30% proteins responsible Indeed, serum detection of the soluble enzyme creatine for ion conduction, various signaling pathways, and kinase as it is released from the injured muscle is a clinical structural integrity [53]. Irrespective of cell type and hallmark of the disease [72]. Membrane permeability is function, the primary role of the cellular membrane is to further exacerbated by mechanical stress, particularly with segregate the intracellular milieu from the outside envir- lengthening contractions of skeletal muscles such as dur- onment to actively preserve intracellular homeostasis. ing downhill walking/running [73]. Lengthening contrac- Transmembrane proteins are essential for normal con- tions occur when the force applied to the muscle exceeds duction of ions, allowing maintenance of physiological the force generated by the muscle, resulting in lengthening ionic gradients at affordable metabolic cost. Failure to of the muscle during active contraction. Repetitive length- maintain barrier function leads to exhaustion of the ening contractions cause significant damage to dystrophic metabolic energy of the cell, biochemical arrest, and muscle by injuring the membrane and downstream ele- eventual cellular demise. ments, including the EC coupling machinery [74, 75]. The membrane bilayer is held together via hydrophobic In DMD patients, muscle biopsies show active degen- effect among phospholipids and their interaction with the eration and regeneration of skeletal muscle fibers and surrounding polar solvent environment, involving van der creatine kinase is persistently elevated [18, 27, 76, 77]. Waals forces, hydrogen bonding, and electrostatic interac- Presently, it is unclear the precise nature of membrane tions [50, 51, 53, 54, 55, 56, 57]. Membrane constituents disruptions caused by lengthening contractions. How- are allowed various intra-bilayer motions, including lateral ever, the release of intracellular enzymes such as creatine diffusion, rotation of lipids around their major axes, and kinase and the uptake of large proteins such as albumin oscillations [56–58]. Intra-bilayer motion, as well as the and vital dyes like Procion orange [73] and Evans blue Houang et al. Skeletal Muscle (2018) 8:31 Page 4 of 19 [78] into non-necrotic muscle fibers indicate that the function and reduction of ATP production leading to cellu- membrane disruptions are sufficiently large to permit lar energy deprivation and cell death. Oxidative stress and 2+ the transmembrane passage of sizable macromolecules elevated intracellular Ca signaling are evident in hearts of which can be monitored as biomarkers of muscle injury mdx mice before pathological manifestations of cardiomy- [72]. Lengthening injury is also particularly apparent in opathy, and there is increasing evidence of mitochondrial the diaphragm which contracts to expand the lungs dur- dysfunction in dystrophic striated muscle [89]. Conse- 2+ ing breathing. Ventilatory muscles of DMD patients and quently, maintaining intracellular Ca homeostasis by pre- 2+ in animal models have impaired contractility and in- venting the deleterious influx of extracellular Ca is crucial creased fibrosis [79]. Dystrophin also plays a crucial role to the survival of dystrophic striated muscle. Moreover, 2+ in buffering against cardiac myocyte extension [80]. This another recent study indicates that Ca influx can progres- occurs when the ventricle fills with blood during diastole sively increase in dystrophic muscle and lead to mitochon- to cause passive lengthening of myocytes. In dystrophin drial dysfunction. This, in turn, further compromises the deficiency, this passive lengthening leads to membrane endogenous membrane repair ability of dystrophin- 2+ dysfunction as evidenced by Ca entry and uptake of deficient myofibers. This negative feedback loop limits the extracellular molecules [80]. Moreover, the conse- cell intrinsic membrane repair machinery resulting in quences of membrane disruptions and increased perme- exacerbation of muscle deterioration in DMD [95]. ability are intrinsically different between cardiac and 2+ 2+ skeletal muscle as the process of Ca − induced Ca re- Current DMD therapeutic strategies: cell intrinsic/cell lease is predominant in the heart [81]. As such, with in- extrinsic strategies creases in contractility and larger passive extensions, There is no cure for DMD nor an effective treatment clin- 2+ subsequently more unregulated Ca entry into the cell ically demonstrated to halt, prevent, or reverse DMD stri- eventually results in terminal contracture of the dys- ated muscle deterioration. Glucocorticoids have been the trophic myocyte [80]. standard of care for DMD but are accompanied by several adverse effects such as excessive weight gain, behavioral Muscle membrane barrier function is severely disrupted issues, growth retardation, osteoporosis, and impairment in DMD of glucose metabolism, all associated with chronic 2+ Owing to membrane dysfunction, Ca homeostasis is per- long-term use [30, 96]. Prednisolone and deflazacort are 2+ turbed in dystrophic muscle (Fig. 1b). This Ca dysregula- regularly administered soon after diagnosis and have been tion is an important component of the pathological shown to slow the progression of the disease by improving processes leading to muscle cell death. Intracellular calcium muscle strength and exercise capacity thereby delaying levels are elevated in both mdx skeletal muscle fibers and loss of ambulation and improving both pulmonary and cardiac myocytes [80, 82–84]. It is still unclear what causes cardiac functions. Several ongoing experimental DMD 2+ this rise in intracellular Ca , with some studies suggesting therapeutics feature gene and cell-based strategies [97, 2+ Ca entering the cell due to increased membrane perme- 98], including exon-skipping strategies to restore dys- ability or “tears” [80], and other studies showing evidence trophin production [99–102]. Exon skipping strategies 2+ for the activation of Ca leak channels or stretch-activated using small molecules have been shown to ameliorate the channels [85]. Regardless of the initial mechanism of entry, severe dystrophic phenotype in both canine and murine 2+ this abnormal elevation in Ca has consequences to DMD models [99, 100, 102–104] while being well tolerated muscle structure and function due to activation of patho- and non-immunogenic. One significant caveat is that this 2+ logical Ca sensitive cellular pathways, including activation strategy is only applicable to the subset of DMD patients of the calpain proteases [86] and perturbation of calcium- with the corresponding targeted mutation. Additionally to activated signaling pathways including calmodulin [87], cal- date, most of these approaches have not yet been translated cineurin [88], and the mitochondrial permeability transition successfully in human patients [105, 106]. One exon pore [89]. Of importance, activation of calpains by extracel- skipping treatment, eteplirsen (Sarepta Therapeutics Inc.), 2+ lular Ca influx leads to cleavage of the transmembrane has recently been approved by the FDA through its acceler- protein dysferlin, a crucial mediator in the cell intrinsic ated approval pathway. A clinical trial in a small cohort of membrane repair machinery [90, 91]. A pathological rise in DMD patients resulted in a dose-dependent partial restor- 2+ cytosolic Ca also contributes to membrane damage via ation of dystrophin production with upregulation of other activation of phospholipase A2 and promotion of reactive dystrophin-associated proteins at the membrane, along oxygen species (ROS) production by the mitochondria [92]. with some improvement in patient walking ability com- ROS in turn leads to peroxidation of membrane lipids paredtoplacebocontrols[107, 108]. However, this 2+ [93, 94]. Additionally, mitochondrial Ca overload improvement was only observed in a small subset of the promotes irreversible opening of the mitochondrial patient group, with dystrophin levels observed to be highly permeability transition pore, aberration of mitochondrial variable among all patients, and a larger clinical trial is Houang et al. Skeletal Muscle (2018) 8:31 Page 5 of 19 currently underway to confirm these results across a larger topologically distinct hydrophilic and lipophilic compo- patient group. Unfortunately, eteplirsen is only targeted to nents. A wide range of block copolymers with distinct approximately 13% of DMD patients with a mutation physicochemical properties can be designed by varying the amenable to exon 51 skipping [108]leaving alarge popula- lengths of the PEO and PPO blocks. Poloxamers were the tion of DMD patients currently without treatment options. first commercially produced block copolymers, synthesized Many experimental therapeutic efforts preferentially tar- by Wyandotte Chemical Corporation in the late 1940s for get dystrophic skeletal muscles, leaving the diseased heart industrial purposes, and now widely found in both indus- untreated [29]. Skeletal muscle-centric strategies to im- trial and consumer products. Poloxamers span ~ 10–80% prove ambulation for DMD patients could lead to in- wt.% poly(ethylene oxide) and 1000 – 15000 g/mol molecu- creased stress on the untreated dystrophic myocardium as lar weight with complex interfacial behavior. Poloxamers a result of increased cardiac demands [29, 109, 110]. This have numerous biological applications, including as drug interplay between the progression of DMD cardiomyop- delivery adjuvants, enhancers of drug penetration in the athy and the skeletal myopathy as a pathophysiological treatment of multiple drug resistant tumors [113, 114], load on the heart underscores the importance of a thera- and membrane interacting agents, either as lysis deter- peutic strategy to effectively treat all striated muscles. In gents [115–117]or cellmembrane stabilizers [80, 118, this context, it is worth considering additional approaches 119] depending on structure. This latter feature is directly that target the primary defect of DMD: severe muscle attributed to poloxamers varying affinity for both the sur- membrane fragility. As the primary pathophysiological de- rounding solvent and with the similarly amphiphilic fect in DMD is the marked susceptibility to contraction- phospholipid membranes [120–123]. An excellent com- induced membrane stress, and the subsequent muscle prehensive review detailing copolymer physical and chem- damage and degeneration that occurs due to loss of ical properties, as well as safety, has been published [124]. muscle membrane barrier function, a unique therapeutic In the context of biomedical investigation, poloxamer approach is the use of synthetic membrane stabilizers to 188 (P188), with a PPO/PEO ratio of 0.20 and a molecu- prevent muscle damage by directly stabilizing the lar weight of 8400 Da, is the most widely studied tri- dystrophin-deficient muscle membrane (Fig. 1c). block copolymer (Table 1). P188’s earliest reported use was in 1952 as an additive to enhance blood oxygenation Copolymers as cell extrinsic muscle membrane [125]. It was found to reduce fat emboli and hemolysis stabilizers in patients under extended cardiopulmonary bypass The triblock copolymer class of membrane-interacting syn- [126–128] and as a priming agent in heart-lung bypass thetic molecules, known as poloxamers or pluronics, are [129]. P188 was also incorporated as a wetting agent linear structures comprised of a hydrophobic polypropylene [130, 131] and an emulsifier for clinically tested drug oxide (PPO) core block flanked on both sides by hydro- formulations [132, 133] as well as used as a solubilizing philic polyethylene oxide (PEO) chains (Fig. 2)(Table 1) agent of perfluorochemicals which have significant O [111, 112]. This constitutes the triblock copolymer A-B-A carrying capacity to create an emulsion used as an architecture. Poloxamers are non-ionic amphiphiles having artificial blood substitute [134]. P188 functions as a Fig. 2 Schematic representation of a triblock and diblock copolymers chemical structures. Chemical structures and representations of the triblock copolymer P188 (PEO –PPO –PEO ) and diblocks of P188 (PEO –PPO ) with differing end groups (–H and –C(CH ) ) where a and b represent 75 30 75 75 15 3 3 the number of repeating PEO and PPO group respectively Houang et al. Skeletal Muscle (2018) 8:31 Page 6 of 19 Table 1 Chemical properties of representative synthetic block copolymers a a b c d Architecture Polymer PEO PPO End group Mass PEO% Triblock copolymer/P188 PEO PPO PEO 150 30 – 8400 80 75 30 75 Triblock copolymer/P338 PEO PPO PEO 280 44 – 8400 84 140 44 140 Triblock copolymer/P331 PEO PPO PEO 14 54 – 3700 26 7 54 7 Diblock copolymer PEO PPO −H75 15 −H 4200 80 75 15 PEO PPO − C4 75 15 −C(CH ) 4430 77 75 15 3 3 Homopolymer PEO 198 0 – 8700 100 Total number of EO or PO monomer units Chemical end group at terminal PO c 1 Average molecular weight in g/mol by H NMR end-group analysis PEO weight percent to total molecular weight Manufacturer BASF Number average molecular weight rheological agent to reduce blood viscosity and platelet Following these reports, a seminal study by Yasuda aggregation [135–138]. It was also reported that P188 et al. [80] demonstrated that the acute application of reduces membrane fluidity and improves cell survivabil- P188 to isolated dystrophic mdx cardiac myocytes re- ity during shear stress in HB-32 hybridoma cell lines, stored myocyte cellular compliance to wild-type levels presumably through direct membrane interaction [61]. by blocking passive stretch-mediated calcium overload. P188 was subsequently widely deployed as a shear pro- Dystrophic mdx cardiac myocytes demonstrated in- tective agent used in cell bioreactors [139]. Additionally, creased passive tension during extension, resulting, in 2+ P188 was determined to reduce endothelial adherence part, by the influx of extracellular Ca during physio- and improves the rheology of sickled red blood cells logical passive myocyte lengthening. P188 fully normal- [140], leading to P188 in clinical trial as a therapeutic ized myocyte passive compliance to normal levels [80]. agent for sickle cell anemia [141–143]. A main outcome At the level of the whole organ, P188 decreased passive of a ~ 350 patient sickle cell anemia trial was its safety tension and thereby improved myocardial relaxation, profile in long-term use. P188’s first FDA approved use allowing for complete filling of the ventricles and return in humans was as a skin wound cleanser that has dem- to normal working end diastolic and end systolic onstrated lack of toxicity to the cellular components of volumes [29]. blood and lack of interference to the wound’s ability to heal and resist infection after being tested in more than Copolymer-based membrane stabilizers in vivo 1000 patients [144, 145]. Yasuda et al. further showed that in vivo systemic ad- ministration of P188 to mdx mice improved ventricular Copolymer-based muscle membrane stabilization: cellular geometry and prevented acute cardiac failure during a studies dobutamine cardiac stress test protocol [80]. In the The first applications of P188 in muscle demonstrated sig- golden retriever dystrophic canine model, chronic P188 nificant reduction in electroporation-induced leakage of administration prevented left-ventricular remodeling, re- carboxyfluorescein dye from isolated skeletal muscle cells duced myocardial fibrosis, and blocked cardiac troponin [118]. In parallel experiments, the hydrophilic control mol- I release [148]. In addition, long-term intermittent ad- ecule Dextran showed no membrane protective effect ministration of P188 was shown to confer protection [118], suggesting that P188 interacts with the damaged during isoproterenol-induced cardiomyopathy in mdx membrane in a way that alters membrane properties and mice [149]. promotes stability. Other reports produced similar results The ability of synthetic membrane stabilizers to protect in in vitro models of acute radiation injury which involves fragile DMD skeletal muscles had, up until recently, been the generation of reactive oxygen species which can rapidly less clear. Early investigations with P188 showed little to alter the structure and organization of the cell membrane no efficacy in protecting dystrophic limb skeletal muscle leading to cell necrosis. In a study by Hannig et al. [146], function in vivo [150, 151], even though P188 had been P188 was shown to retard cytoplasmic calcein leakage from shown effective in protecting hindlimb skeletal muscle in isolated rat skeletal muscle cells undergoing radiopermeabi- a range of other conditions, including electrocution injury lization. Greenebaum et al. [147] further showed that skel- [118, 152], hindlimb ischemia-reperfusion injury [153, etal muscle cells treated with P188 manifested enhanced 154], and in a model of dysferlin-deficiency [155]. Interest- viability and survival following high-dose irradiation. ingly, a recent study evaluating the pharmacodynamics of Houang et al. Skeletal Muscle (2018) 8:31 Page 7 of 19 P188 demonstrated P188 can fully protect dystrophic skel- measurable changes in membrane dynamics or structure, etal muscle against mechanical stress in vivo [156]. This as detected by electron paramagnetic resonance and iso- study showed how in vivo membrane protection is critic- thermal calorimetry techniques. Collectively, this is evi- ally dependent on delivery route [156] wherein subcutane- dence that P188 does not fully insert in the intact bilayer ous delivery of P188 led to dramatic improvement in mdx interior nor does it affect overall lipid packing [123]. hindlimb muscle function during lengthening contractions As DMD pathophysiology is exacerbated by lengthening and decreased uptake of Evans blue dye in vivo. In con- contractions, it is important to compare results from trast, in this model, neither intraperitoneal nor intraven- non-stressed membranes to mechanically stressed mem- ous delivery, which were routes used in previous studies, branes. To mimic bilayer mechanical stress using artificial led to improvement in muscle function [156]. Thus, the membranes in vitro, studies have used Langmuir troughs. lack of skeletal muscle efficacy reported in previous stud- This approach permits fine control of the surface area and ies using P188 [150, 151] could be attributed to subopti- therefore lipid packing density of supported phospholipid mal mode of delivery of P188, rather than a fundamental monolayers at the air/water interface [121, 158]. Maskari- limitation in the mechanism by which the block copoly- nec et al. [159] focused on P188 insertion as a function of mer stabilizes fragile dystrophic skeletal muscle mem- surface pressure, which directly correlates to lipid packing branes. This was further supported by another recent density. Here, using either anionic dipalmitoylphosphati- study showing that chronic dosing of P188 using subcuta- dylglycerol (DPPG) or zwitterionic dipalmitoylphosphati- neous delivery improves diaphragm function in mdx and dylcholine (DPPC) monolayers, results showed P188 −/− mdx:utr mouse models in vivo [157]. In that study, inserts into both lipid types at a surface pressure (π) ≤ P188 improved dystrophic mouse respiratory parameters 22 mN/m, which is lower than that of a healthy cell mem- in vivo, including tidal volume/body weight and minute brane (~ 30–35 mN/m) [160, 161]. P188 was found to re- volume/body weight, as well as decreased central nucle- main inserted until the surface pressure increased back to ation and decreased collagen deposition in treated dia- threshold surface pressure equivalent to that of an intact phragm muscle fibers [157]. These results are promising membrane [158, 159]. X-ray reflectivity results further in indicating that chronic P188 treatment may be benefi- showed that at high surface pressure lipid films, in the cial in preserving respiratory and limb muscle functions. presence and absence of P188 in the subphase, exhibit Taken together, these findings are evidence that synthetic similar electron density profiles [121, 162]. membrane stabilizers provide a unique first-in-class treat- Morphologically, P188 insertion appears to tighten lipid ment strategy for simultaneously treating all affected stri- packing via physical occupation of surface area in localized ated muscles in DMD. A summary of in vivo studies patches rather than uniformly across the whole membrane testing block copolymers as a therapeutic strategy in [121, 159]. The hypothesis follows that only when lipid DMDmodelsispresented in Table 2. packing density is low, and the hydrophobic core of the monolayer is exposed, that P188 partitions to the mem- Elucidating the copolymer-muscle membrane interface brane via hydrophobic interactions between the acyl The mechanism underlying copolymer-lipid bilayer inter- chains of the bilayer and the copolymer hydrophobic PPO action has not been delineated. Elucidating copolymer block. Inability to remain inserted above a threshold sur- chemical and structural characteristics are essential to de- face pressure suggests that P188 does not insert into nor- termine membrane stabilizer function, under both normal mal intact cell membranes and only inserts once lipid and disease conditions. Because biological membranes are density is decreased. This leads to a dynamic interaction, structurally complex, artificial phospholipid-based mem- wherein P188 is “squeezed out” from the cell bilayer when branes are an invaluable model to study the biophysical normal membrane structure is restored (Fig. 3). Copoly- basis of copolymer-membrane interactions. To investigate mer “squeeze out” upon normalization of membrane lipid the physical nature of P188-membrane interactions, packaging density is an important concept driving thera- Cheng et al. employed H Overhauser dynamic nuclear peutic applications. In this context, copolymers only insert polarization/Nuclear Magnetic Resonance spectroscopy to into areas of the membrane that are damaged. This work- determine local hydration dynamics at the P188-lipid ing model hypothesizes that when copolymer insertion membrane interface [123]. The high spatial resolution re-establishes membrane barrier function and prevents 2+ afforded by this technique allows for probing the local Ca overload during muscle contraction, the endogenous water diffusivity in lipid bilayer systems. Here, P188 cell membrane repair response would be able to patch the weakly adsorbed to the intact vesicle membrane surface. membrane [1]. Upon repair, the copolymer would then This was shown by membrane hydration dynamics and disengage from the membrane (Fig. 3). This copolymer intra-bilayer water diffusivity, both at the membrane sur- squeeze out at normal surface pressure would be benefi- face and bilayer interior. Furthermore, P188 weakly cial in the context of biomedical applications of damaged adsorbed at the membrane surface and produced no cellular membranes where copolymers selectively insert Houang et al. Skeletal Muscle (2018) 8:31 Page 8 of 19 Table 2 Summary of studies using block copolymers as a treatment in DMD models in vivo Copolymer Pathophysiology DMD Treatment Dosage Delivery Results References model time route P188 Cardiomyopathy mdx Pre-treatment 460 mg/kg i.v. P188 significantly improved cardiac hemodynamic response and Yasuda et al. (30 min) animal survival during cardiac stress testing (2005) [80] P188 Skeletal muscle mdx Pre-treatment 600–1800 mg/kg i.p. No significant difference in % EBD penetration in rectus femoris Quinlan et al. (30 min) muscle fibers in P188 treated mdx mice exercised by downhill (2006) [150] treadmill running P188 Cardiomyopathy GRMD 8 weeks 60 mg/kg/hr i.v. Chronic P188 treatment normalized serum cTnI levels, blocked Townsend et al. increases in heart failure marker BNP, significantly decreased (2010) [148] cardiac fibrosis, and prevented dilated cardiomyopathy. Cardiac hemodynamic function in response to dobutamine stress was significantly improved compared to saline treatment. Serum CK levels were not affected. P188 Cardiomyopathy mdx 2–4 weeks 460 mg/kg i.p. P188 treatment prevented a decrease in cardiac function in Spurney et al. response to isoproterenol stress testing. Treated mice did (2010) [149] not show significant differences in cardiac fibrosis but had increase in EBD positive fibers, these hearts showed increased systolic function compared to untreated hearts. P188 Skeletal muscle mdx Pre-treatment 30 mg/kg, 460 mg/ i.p. Single dose P188 treatment induced an increase in specific force Terry et al. 2-week daily kg and decreased the number of IgG positive fibers in both non-stressed (2014) [151] and stressed muscles. P188 treatment improved the histological appearance in TA muscles under some conditions. 2-week P188 did not affect TA force. During lengthening contraction injury, it was reported that in a subset of contractions the P188 treatment group had slightly but statistically significant lower force than saline control. P188, P338 Skeletal muscle mdx Pre-treatment 60–460 mg/kg i.p., i.v., s.c., Subcutaneous but not intravenous nor intraperitoneal injection of P188 Houang et al. (0.5–3h) i.m. significantly decreased the force loss during and after lengthening (2015) [156] contractions of hindlimb mdx muscle and significantly decreased EBD uptake into TA myofibers post-injury. Subcutaneous delivery of PEO8000 had no protective effect. Lower dosage of intraperitoneal and intramuscular but not subcutaneous or intravenous injections of P338 shows significant protective effect. P188 Respiratory mdx Q.D., 22 weeks 3 mg/kg s.c. Chronic delivery of P188 had significant positive effects on respiratory Markham et al. function parameters and improved diaphragm histological parameters (2015) [217] and caused improvement in cardiac hemodynamics of treated mdx mice −/− Cardiomyopathy mdx/utr Q.D., 8 weeks 1 mg/kg s.c. P188 treatment slowed the loss of respiratory function and improved diaphragm histological parameters in double knockout mice diP188 diP188-CH Skeletal muscle mdx Pre-treatment 1000 mg/kg i.p. A diblock copolymer architecture confers membrane stabilization. Houang et al. diP188-(CH ) (0.5–3h) The addition of a single hydrophobic tert-butoxy end-group to the (2017) [183] 3 3 PPO core significantly enhanced membrane protection against lengthening contractions. The less hydrophobic methoxy and hydrophilic hydroxyl end groups did not confer membrane protection in vivo. i.v., intravenous; i.p., intraperitoneal; s.c., subcutaneous; i.m., intramuscular; EBD, Evans blue dye; GRMD, golden retriever muscular dystrophy; cTnI, cardiac troponin I; BNP, brain natriuretic peptide; CK, creatine kinase; TA, tibialis anterior; Q.D., daily; diP188, diblock P188 Houang et al. Skeletal Muscle (2018) 8:31 Page 9 of 19 Fig. 3 Model of copolymer-based membrane stabilization. a In DMD, susceptibility to sarcolemmal damage from lengthening muscle contraction 2+ 2+ renders the muscle cell membrane leaky to extracellular Ca (pink circles). Subsequent intracellular Ca overload leads to activation of pathological cellular pathways. Further membrane damage overloads the repair capacity of endogenous cell membrane repair mechanisms and ultimately leads to cell death. b Copolymer insertion driven by hydrophobic interactions (red PPO block of the copolymer with the hydrophobic part of the membrane 2+ that is now exposed due to instability). Membrane stabilization prevents pathological Ca entry into the cell and prevents activation of cellular death pathways. c While the copolymer stabilizes the membrane and prevents further damage, intrinsic cell membrane repair mechanisms can repair lesions at damaged sites [215]. d Once the membrane integrity is restored, the copolymer membrane stabilizer is “squeezed out” of the membrane. Here, the membrane is resealed, its lipid packing density is restored, and its hydrophobic portion is now enclosed [159, 216] only onto localized areas of the membrane where the local or PEG), the hydrophilic constituent of poloxamers, has been lipid density is reduced, and thus only where the mem- well investigated in the fusion of model membranes and for brane is structurally impaired, and not interact with intact its ability to lower water molecule activity at the membrane- with healthy areas of the membrane. solvent interface [165]. While PEO-mediated membrane stabilization has been shown to be effective, the very high Copolymer structure-function analysis concentrations (mM-M) required for effectiveness indicate Mechanistic investigation via the structure−function relation- that the hydrophobic block plays an essential role in ship of block copolymer chemistry is required to define the copolymer-membrane interactions [166]. basis of copolymer-based membrane interaction. This is The relationship between copolymer chemical struc- crucial in the long-term to guide the design of an optimal ture and the kinetics of adsorption, insertion, and subse- membrane stabilizer. There is considerable interest in block quent squeeze out from lipid monolayers has been copolymers as membrane stabilizers due to their overall sur- investigated by Frey et al. via Langmuir trough experi- face active and solvent-selective characteristics and intrinsic ments and Monte Carlo simulations [120]. Here, upon thermodynamic properties and architectures [163, 164]. compression of the monolayer, copolymers with higher P188 is part of a large family of poloxamers, each with dis- PPO/PEO ratio favored a higher squeeze out pressure. tinct physicochemical properties. Polyethylene glycol (PEO Moreover, higher molecular weight copolymers were Houang et al. Skeletal Muscle (2018) 8:31 Page 10 of 19 observed to squeeze out at higher surface pressures, pressures before being squeezed out [120, 158]. More- while at constant PPO/PEO ratios smaller copolymers over, hydrophobic copolymers with bulkier PPO blocks squeezed out at lower pressures. Results showed that the were found to increase flippase activity compared to co- ratio dictates the equilibrium spreading pressure of polymers with shorter PPO blocks [117]. Copolymer- copolymers at the phospholipid interface. Hydrophobic bilayer interactions have been investigated using pulse copolymers were less soluble resulting in a higher field gradient nuclear magnetic resonance to quantify proportion of adsorption at the monolayer interface and copolymer diffusion in the presence and absence of uni- thus higher equilibrium spreading pressure [120]. These lamellar liposomes [171]. Here, the binding percentage findings demonstrate the relationship between the PPO/ of copolymers to liposomes was quantified, and results PEO ratio and molecular weight in determining further confirmed that increased copolymer molecular copolymer-membrane interactions. weight and increased relative hydrophobicity cause in- Overall, copolymer hydrophobicity has a principal role creased binding and liposome coverage relative to in affecting membrane bilayer physical structure. Thus, smaller, more hydrophilic copolymers. Another recent more hydrophobic copolymers decrease membrane study using surface plasmon resonance to probe and microviscosity [117, 167] and increase the rate of lipid compare binding of P188 and a PEO homopolymer of motion across the outer and inner leaflets of vesicular similar size provides direct evidence of binding onto membranes [117], causing membrane leakiness [115, 168]. supported intact lipid bilayers with comparable binding Chang et al. [169] showed that surface pressure-area kinetics. Moreover, this study provides biophysical isotherms exhibited by P188 (PEO –PPO –PEO )com- evidence that copolymer adsorption alone does not fully 75 30 75 pared to the highly hydrophobic P181 (PEO –PPO – account for membrane protection efficacy. [172]A 2 30 PEO ) are significantly different. P181 exhibits condensed- schematic summary of structure-function of copolymer- film-like surface behavior whereas P188 exhibits an based membrane stabilization is presented in (Fig. 4). expanded-like behavior. This was confirmed by Cheng et al. [123] using dynamic light scattering, isothermal cal- Molecular dynamics analysis of copolymer- orimetry, and small molecule-directed lipid peroxidation membrane interactions of liposomes. The PPO/PEO ratio was shown to be a key Mechanistic insights into copolymer-membrane interaction feature in effectively protecting intact liposomes from are aided by studies pursued at the atomistic level. Molecular peroxidation. Copolymers that adsorb at the membrane dynamics (MD) simulations have been recently developed to surface, without penetration into the bilayer core, such as investigate copolymer-phospholipid bilayer interactions P188 and PEG8000, presumably affect the hydration shell [173, 174]. MD simulations are physics-based computa- of the bilayer. This would suppress the diffusion of the tional methods to simulate and observe the interactions free radical lipid peroxidation initiator into the lipid bi- of atoms and molecules at resolutions that are currently layer, thereby preventing the initiation of lipid peroxida- hard or impossible to probe experimentally. In general, tion. The more hydrophobic poloxamers, for example, MD simulations of large macromolecules, such as P335 (PEO –PPO –PEO ), P333 (PEO –PPO – copolymers, are computationally challenging to per- 38 54 38 20 54 PEO ), and P181 (PEO –PPO –PEO ), have significant form. Past MD efforts have focused on coarse-grained 20 2 30 2 heat of partitioning indicative of insertion into the liposo- [120, 175, 176] and united atom [168, 177, 178]models, mal membrane [123]. These hydrophobic copolymers do which are models that reduce the total number of de- not prevent initiation of lipid peroxidation [170] indicating grees of freedom in the system by representing mole- that copolymer hydrophobicity affects kinetics of inser- cules and their interactions at lower resolution. This tion. More hydrophobic copolymers insert at faster rates allows for significantly increased simulation timescale by initially embedding below the lipid head group region, at lower computational cost but in exchange for the opening up the packing of acyl chains and accelerating the loss of atomistic level details. passage of water across the membrane, thus increasing An in silico model of copolymer adsorption using permeability [123, 166]. coarse-grained force field showed copolymer-membrane The size of the hydrophobic PPO block influences in- insertion, followed by percolation across the unstressed sertion of the copolymer into lipid films. Poloxamers at lipid bilayers [179]. Here, copolymers containing a PPO fixed 80% PEO composition and different molecular block with a length comparable to that of the bilayer weights (P108, P238, P188, and P338) have been tested thickness tended to span across, or percolate across, the for their relative ability to insert into lipid monolayers lipid bilayer. In comparison, copolymers with shorter [158]. Copolymers with high PPO content required PPO blocks inserted partially, with the PEO blocks lower surface pressure for insertion. Additionally, once remaining in water on one side of the bilayer. Moreover, inserted, high mass copolymers are able to retain pos- total percolation of copolymers across the bilayer led to ition within the monolayer at much higher surface reduction in membrane thickness and an increase in the Houang et al. Skeletal Muscle (2018) 8:31 Page 11 of 19 Fig. 4 Schematic representation of structure-function of copolymer-membrane interaction. Triblock copolymer membrane stabilization occurs via insertion of the hydrophobic PPO core block (red) and balanced by flanking of the two hydrophilic PEO blocks (blue) that are required to prevent complete translocation across the membrane. Without a second flanking PEO chain, diblock copolymers can also insert into the membrane, but insertion is at least in part dictated by the PPO end group. Here, the more hydrophobic end group, such as –C(CH ) (†), driving insertion and 3 3 anchoring and the more hydrophilic end groups, such as –OH, retained at the solvent-polar head group interface. Variation in PEO (blue) and PPO (red) block lengths alters the hydrophobic/hydrophilic balance that is required for optimal membrane insertion and stabilization. Too high a PPO/PEO ratio and large size PPO group drives the copolymer deeper into the membrane and further exacerbates damage to the membrane area per lipid. Goliaei et al. [177] used an united-atom molecule (A ) to model bilayer mechanical stress [181]. force field-based MD model to show that P188 can pas- P188 interaction with lipid bilayers was demonstrated to sively insert into the 1,2-dilauroyl-sn-glycero-3-phospho- be dependent on A with insertion of the PPO block oc- 0, choline (DLPC) lipid bilayer under non-stressed curring at a ~ 15–20% increase in A Additionally, P188 conditions after extensive simulation time (> 500 ns). insertion into the membrane significantly increased the Here, the PPO block inserted into the hydrophobic part lateral pressure required for membrane rupture under of the bilayer and the PEO chains remained solvated mechanical stress [181]. Further, membrane insertion and outside the membrane [177]. Moreover, using a 3 nm stabilization efficacy appeared dependent on the PPO/ water pore model to simulate a damaged lipid bilayer, PEO ratio. MD simulations of hydrophobic copolymers, the PPO block of P188 inserted adjacent to the water such as P331 (PEO –PPO –PEO ), inserted at signifi- 7 54 7 pore and “pushed” water molecules out of the pore to cantly lower A , as well as decreased the lateral pressure reduce pore size. required to rupture the membrane. This is consistent with Simplified force field models allow for larger timescale the results of Nawaz et al. [168] who demonstrated perco- simulation; however, they yield only a partial view of lation across the bilayer of highly hydrophobic copolymers membrane structural properties and limit atomic reso- causing membrane bending and an increase in local per- lution insights [180]. Importantly, previous MD studies meability allowing water molecule penetration into the have focused on copolymer-bilayer interactions under hydrophobic region of the membrane. The timescale for constant pressure and temperature (NPT) and constant percolation was inversely proportional to the PEO block area and temperature conditions (NPAT), and thus are length [168]. Moreover, another all-atom MD study by computational models of membranes under normal non- Zaki and Carbone showed that incorporation of multiple stressed conditions. Recently, an all-atom MD simulation copolymer units within the bilayer hinders lipid diffusion model was developed to investigate copolymer-lipid mem- and forced nearby lipids to remain closely packed, even brane interaction under conditions of varied lateral mech- during lateral mechanical stress [182]. anical stress. This in silico approach correlates to the Overall, the results from MD studies are consistent physiological state to lengthening contraction muscle in- with experimental observations from Langmuir trough jury in DMD. Here, an increase in surface tension (γ) was studies in that P188 inserts into areas of low lipid dens- applied to induce expansion in the bilayer area per lipid ity and at low surface pressures [158, 159]. MD studies Houang et al. Skeletal Muscle (2018) 8:31 Page 12 of 19 feature a simplified phospholipid bilayer as a basic for sensitive modulation of the diblock PPO block hydropho- model of the biological membrane, which is comprised bicity. This strategy has precedence in the surfactancy litera- of proteins, complex mixtures of lipid types, and other ture where novel terminal functional groups have been macromolecules, all organized in a tightly regulated shown to influence solution and bulk phase behavior [187, manner. Nonetheless, all atom MD results are qualita- 188]. Diblock copolymers have never been investigated for tively comparable to results derived in cells and animals biological membrane stabilization until a recent report dem- [156, 183, 184]. Complementation of findings from in onstrating that diblock PEO–PPO architectures can confer silico to in vivo methods underscores MD simulations as membrane stabilization in both in vitro and in vivo DMD a powerful tool to further mechanistic understanding of models [156, 171, 183, 184]. This establishes that specific copolymer-bilayer interactions and to ultimately guide PPO end group chemistries play a critical role in defining design and optimization of copolymers for physiological muscle membrane stabilization [183, 184]. membrane stabilization. Recent diblock studies have advanced an “anchor and chain” model of membrane stabilization (Fig. 4)[156, Copolymer architecture: diblock copolymers as 171, 183, 184]. Here, the addition of a small hydrophobic membrane stabilizers end group “anchor,” as demonstrated by tert-butoxy to Block copolymers can be designed with two or more dis- the PPO block, discretely increases the hydrophobic tinct polymer blocks covalently bonded together. These character of the end of the PPO block, without signifi- can exist in a variety of molecular sizes, relative degree of cantly increasing the overall mass of the copolymer. polymerization of each block (composition), hydrophobi- From these results, it is hypothesized that discrete alter- city, chemical moieties, and architectures, from diblock ations in the structure of the PPO terminal functional and triblock to multi-blocks. This broad landscape leads group, such as replacing tert-butoxy with n-butoxy or to a nearly infinite number of possible distinct chemical other non-polar end groups, will further influence the configurations [112]. Previously, from a practical perspec- packing and interaction strength with the lipid core. The tive, the use of poloxamers has been generally constrained PEO chain appears to be required to preserve the to those made available commercially. This limitation pro- amphiphilic behavior of the copolymer and to maintain vides an impetus for advancing discovery of the copolymer the copolymer at the solvent-membrane interface. De- chemical landscape beyond that of the triblock architec- tailed structure-function analysis of the PEO block, in- ture. As above, P188 is reported to be weakly adsorbed to cluding length, structure, and chemical characteristics, the lipid bilayer [123, 170] and it is hypothesized that this has not yet been initiated and this will be important to weak association is due to steric constraints imposed by determine in further experimentation. Taken together, the flanking PEO chains [162]. The removal of one of the these proof-of-principle results establish physiological flanking PEO chains to form the diblock PEO–PPO archi- relevance to diblock copolymers and support further in- tecture (Fig. 2) allows for facile assessment of the associ- vestigation of this expansive copolymer chemical space. ation of the hydrophobic PPO core with the lipid bilayer. Firestone et al. employed small- and wide-angle X-ray scattering techniques to examine the structure of a lipid Clinical applications, challenges, and ongoing bilayer and the phase produced by either the triblock developments P188 or a PEO–PPO diblock with an equivalent PPO P188 was first approved by the FDA as an anti-viscosity block length [185]. P188-synthetic lipid bilayer interaction agent added to blood before transfusions [135, 189]. P188 produced an aggregate phase structure suggesting limited (labeled as RheothRx, Glaxo Wellcome Inc.) has been previ- insertion of the copolymer into the lipid bilayer. On the ously tested in clinical trials for both sickle cell anemia [141, other hand, the PEO–PPO diblock produced a well- 142] and myocardial infarction [190, 191]. Due to its nature ordered lamellar phase suggesting enhanced interfacing as a nonionic surfactant and demonstrated hemorrheologic within the bilayer [185]. This suggests that removing one properties, a randomized, double-blind, placebo-controlled of the flanking PEO chains facilitates PPO block inter- pilot study in 50 patients was initiated in the early 1990s to action with the hydrophobic acyl chain region of the lipid determine the safety and efficacy of P188 in treating acute bilayer to strengthen copolymer-bilayer interaction. vaso-occlusive crises in sickle cell anemia disease. Treated The PEO–PPO diblock architecture offers several advan- patients showed a significant decrease in painful episodes, re- tages for advancing copolymer-membrane structure-function duced hospital stay, requirement of analgesics, and reported studies. These include an easier and more controlled chem- pain [142]. Moreover, continuous RheothRx intravenous in- ical synthetic process [186], the more precise control of PPO fusion over 48 h (60-min loading dose of 300 mg/kg and PEO block sizes and the ability to design specific func- followed by a 47-h maintenanceinfusion of 30 mg/kg) was tional end groups to the hydrophobic PPO core to finely well tolerated with the exception of a mild increase in serum tune membrane interactions. This latter modification allows creatinine in one patient with underlying renal dysfunction. Houang et al. Skeletal Muscle (2018) 8:31 Page 13 of 19 Pharmacokinetic study of P188 injection in healthy Conclusions males has been conducted in a cohort of volunteers and From a conceptual perspective for clinical application, determined that elimination occurs primarily through synthetic muscle membrane stabilizers for treating DMD renal clearance [192]. RheothRx (P188) clinical trial in patients have several attractive features. These include (1) patients tested adjunctive therapy during thrombolytic treatment strategy targeting the primary defect in DMD— therapy for acute myocardial infarction at time of severe muscle membrane instability causing muscle de- hospitalization. Initial reports showed P188 resulted in terioration and cell death, (2) copolymers as muscle mem- significantly smaller-sized infarcts, greater myocardial brane interfacing molecules could in principle treat all salvage, and improved median ejection fraction [191]. DMD patients regardless of their genetic lesion, (3) However, in follow up large-scale clinical studies, pre-clinical studies provide evidence of copolymer protec- Rheothrx administration did not significantly decrease tion in other applications, (4) first-in-class membrane infarct size or favorably alter outcome [193]. Moreover, stabilizer P188 NF has a favorable safety profile in cardiac, in a subset of elderly patients with pre-existing renal dis- respiratory, and limb striated muscles, as derived from hu- ease increased renal dysfunction was reported. This ad- man clinical trial data in humans. The inherent limitation verse effect was later determined to be due to small with membrane stabilizers as a potential therapy for DMD molecular weight impurities in the P188 formulation, is that this approach is not a cure and would necessitate which was manufactured as an excipient-grade product chronic treatment for DMD patients. following National Formulary specifications [194]. Sub- The ultimate goal for membrane stabilizing therapy is to sequent clinical studies using the purified formulation of significantly improve and prolong patient quality of life P188 significantly improved the renal safety profile and while awaiting a potential effective cure for DMD. As tolerability [194]. DMD is a chronic progressive disease, membrane Purified formulation of P188 was repackaged as MST-188 stabilization treatment would require life-long administra- or vepoloxamer (Mast Therapeutics, Inc.) which was then tion. In the best case scenario, this clinical treatment further evaluated in another interventional clinical trial would effectively manage the disease, analogous, for ex- (EPIC trial) in children with sickle cell disease. In a recent ample, to the highly effective life-long daily insulin treat- large-scale phase 3 clinical trial, vepoloxamer did not meet ment used by type I diabetic patients. One could envision primary efficacy endpoints of demonstrating a statistically chronic copolymer treatment starting soon after diagnosis significant reduction in the mean duration of vaso-occlusive with the aim to preserve striated muscle function before crisis events. However, this clinical trial did show that vepo- muscle degeneration and wasting occurs. Membrane sta- loxamer was generally well tolerated with no statistically sig- bilizers may also be envisioned in acute settings for DMD nificant differences in treatment-related adverse events in the patients, for example, during orthopedic surgery or other vepoloxamer group compared to the placebo group (https:// stress-inducing events [148]. Another setting where co- clinicaltrials.gov/ct2/show/NCT01737814). polymer administration could make a significant positive For membrane stabilizers in DMD, Phrixus Pharmaceuti- impact is during exercise training protocols for DMD pa- cals, Inc. has initiated a Phase 2 single site, open-label trial tients implemented to oppose the loss of functional abil- for respiratory, cardiac and skeletal limb muscle end points ities as a result of muscle disuse [195]. It is still unclear in non-ambulatory DMD patients (ClinicalTrials.gov Iden- whether exercise training and which exercise protocols tifier: NCT03558958). Drug P-188 NF (Carmeseal-MD) is could be beneficial to DMD patients or other patients with directed toward DMD patients with early heart failure and myopathic disorders, at least in part due to the potential respiratory dysfunction who are currently on stable regi- detrimental effects of strenuous exercise and muscle con- men of background therapies. Phrixus Pharmaceuticals traction on the muscle membrane [196]. Treating DMD and Ethicor Pharma Ltd. have made Carmeseal-MD avail- patients with membrane stabilizers prior to an exercise able in 2015 as a “special” or unlicensed medicinal product training bout may support striated muscle membranes in the European Union prior to regulatory approval. This during strength exercise and abrogate deleterious effects allows access to Carmeseal-MD to DMD patients with re- that would occur while supporting muscle repair and spiratory and cardiac deficits through physician request. As strength building. of the end of 2017, one patient under the Expanded Access It is also likely that effective DMD treatment will ultim- Program has been reported to have met the 15-month ately require a combination of approaches to achieve opti- treatment mark with treatment reported to have been well mal outcomes. One example where bundled therapies tolerated and reductions in creatine kinase and cardiac containing P188 has already shown promise is cardiac ar- troponin I observed (Phrixus Pharmaceuticals). Moving rest and resuscitation [197]. Block copolymers have been in forward, larger scale human clinical data will be required use as vehicles for enhanced gene delivery in other applica- to fully evaluate membrane stabilizer treatment efficacy in tions [198, 199], and the prospect of bundled therapies of DMD patients. block copolymers and gene-directed strategies would be of Houang et al. Skeletal Muscle (2018) 8:31 Page 14 of 19 significant interest to pursue in future works. Another strat- Abbreviations DGC: Dystrophin-glycoprotein complex; DLPC: 1,2-dilauroyl-sn-glycero-3- egy where copolymer-based membrane stabilizers could be phosphocholine; DMD: Duchenne muscular dystrophy; combined would be stem cell therapy to regenerate muscle. DPPC: Dipalmitoylphosphatidylcholine; DPPG: Dipalmitoylphosphatidylglycerol; Induced pluripotent stem cell (iPSC) technology allows MD: Molecular dynamics; NPAT: Constant pressure, area, and temperature; NPT: Constant pressure and temperature; NPγT: Constant pressure, surface derivation of patient-derived stem cells which obviates tension, and temperature; PEG: Polyethylene glycol; PEO: Polyethylene oxide; immunological concerns. One recent study showed PPO: Polypropylene oxide; ROS: Reactive oxygen species proof-of-principle application of ex vivo genetic correction Acknowledgements of dystrophic iPS cells with a micro-utrophin transgene be- We thank our colleagues at the University of Minnesota, in particular the fore transplantation back into dystrophin/utrophin double UMN Biomedical Block Copolymer Research Consortium for dynamic knockout mice [200]. They observed that engrafted muscle interactions and discussions. had large numbers of corrected myofibers, restoration of Funding the dystrophin and associated proteins complex and im- This work was supported by grants from the National Institutes of Health proved contractile strength. While these results are positive (J.M.M.), the Lillehei Heart Institute, the Muscular Dystrophy Association (J.M.M.), and the American Heart Association Predoctoral Fellowship (E.M.H.). and exciting, this strategy still has to overcome multiple im- portant hurdles, such as improved survival of the cells Authors’ contributions post-injection, effective migration to the compromised EMH, FSB, YYS, and JMM all contributed to the writing of this manuscript. All muscles, and successful engraftment. Copolymer-based authors read and approved the final manuscript. membrane stabilizers injected alongside iPS-derived myo- Ethics approval and consent to participate cytes may help improve survival of these cells post- Not applicable injection. Synthetic membrane stabilizers may ultimately extend to Consent for publication Not applicable numerous other inherited or acquired diseases in which cell membrane integrity is compromised. In the last few years, Competing interests many preclinical studies using P188 as cell membrane stabi- The authors declare the following potential conflict of interest: J.M.M. is on the scientific advisory board of and holds zero value equity shares in Phrixus lizers have been published in a variety of pathological set- Pharmaceuticals Inc., a company developing novel therapeutics for heart tings, including amyotrophic lateral sclerosis [201], traumatic failure and DMD, and this is actively managed by the UMN Office of brain injury [202], aggregation of unfolded protein [203– Institutional Compliance. 207], hypoxia and ischemia-reperfusion injury [154, 208, 209], irradiation and burn injury [152, 210, 211], cartilage Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in damage, and joint degeneration following blunt impact published maps and institutional affiliations. [212–214]. Based on the potential novel uses of copolymer- based membrane stabilizers in various other diseases where Author details Department of Integrative Biology and Physiology, University of Minnesota the cell membrane is damaged, one could anticipate that in- Medical School, 6-125 Jackson Hall, 321 Church Street SE, Minneapolis, MN creased academic and clinical interest in this therapeutic 55455, USA. University of Minnesota Informatics Institute, MN, USA. strategy will help promote faster translation to human clin- Bioinformatics and Computational Biology Program, University of Minnesota, MN, USA. Department of Chemical Engineering and Materials Science, ical applications. University of Minnesota, MN, USA. Finally, as detailed in this review, first-in-class copolymer-based membrane stabilizer P188 has a long Received: 30 May 2018 Accepted: 13 September 2018 history. 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Skeletal MuscleSpringer Journals

Published: Oct 10, 2018

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