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To investigate the sustainability of Robot-assisted upper limb therapy (RT-UL) as part of routine occupational therapy and physiotherapy clinical practice. Two separate audits, 12 months apart, of RT-UL computer data records were undertaken to determine sustainability in a subacute rehabilitation unit. Records of the two audits were compared in terms of the number of early subacute stroke survivors using RT-UL, the number of RT-UL sessions, duration of RT-UL sessions, and disciplines prescribing RT-UL. During Audit 1 58% (n = 18) of stroke survivors received RT-UL compared to 50% (n = 7) in Audit 2. The total number of RT-UL sessions reduced between audits (148 vs. 36 sessions) reflecting the overall reduction in admission rates for stroke survivors. There was no significant difference between audits in the average number of RT-UL sessions per patient (p = 0.203) nor the length of sessions (p = 0.762). Patients engaged in active therapy more than three-quarters of the time when on the robotic device. Physiotherapists were the primary prescribers of RT-UL when compared to occupational therapists. RT-UL was in continued and regular use with stroke survivors 2 years after initial implementation within an inpatient rehabilitation setting. RT-UL practice was intensive and used routinely with patients. RT-UL is a sustainable and intensive intervention for stroke survivors within an inpatient rehabilitative setting. The cost-benefits of RT-UL should be evaluated from the perspective of the whole rehabilitation service not just at an individual patient level. RT-UL may be considered a "bridging" form of UL practice for those with more limited active UL movement until there is sufficient UL movement and power for more complex real-world task-specific practice. [ABSTRACT FROM AUTHOR]

Robot assisted upper limb (UL) therapy has been identified as an intervention with the potential to help improve the amount of practice performed by stroke survivors. This study aimed to measure the amount of UL practice (i.e., repetitions, duration, intensity) performed by subacute stroke survivors, in particular those with severe UL impairment, pre and post implementation of robot assisted upper limb therapy (RT-UL) into an inpatient rehabilitation setting. Two observational study phases (pre-RT-UL and post-RT-UL) were undertaken of occupational therapy and physiotherapy sessions performed by subacute stroke survivors. Upper limb tasks observed and recorded in therapy were classified as either impairment-related therapy or activity-related. In the pre-RT-UL observational phase, 7 subacute stroke survivors were observed across 11 days involving 25 therapy sessions. Post-RT-UL, 12 subacute stroke survivors were observed across 12 days involving 29 therapy sessions. There were no significant differences in characteristics of patients observed in each phase (p >.05). The mean difference (95% CI) between pre and post RT-UL for repetitions (reps) (569 (1 to 1136) and intensity (7 (4–11)) reps/min of practice increased for all patients, including those with severe UL impairment (337 (37–638)) reps and 8 (2–14) reps/minute, with the duration of therapy unchanged. This is the first study to have observed an increase in UL practice with the inclusion of RT-UL as part of routine clinical practice. This increase in practice is considered to be due to RT-UL providing highly supportive and expeditious semi-supervised practice. Notably, RT-UL was able to be implemented within the existing organisational structures with only basic training of therapy staff. Robotics presents as a viable intervention to increase the amount and intensity of upper limb practice performed by stroke survivors in routine clinical practice Robotics were able to be implemented within the existing organisational structures with only basic training of therapy staff [ABSTRACT FROM AUTHOR]

Rapid clearance of thrombolytics from blood following intravenous injection is a major clinical challenge in cardiovascular medicine. To overcome this barrier, nanoparticle (NP) based drug delivery systems have been reported. Although superior than conventional therapy, a large proportion of the injected NP is still cleared by the reticuloendothelial system. Previously, we and others showed that ex vivo attachment of bioscavengers, thrombolytics, and nanoparticles (NPs) to glycophorin A receptors on red blood cells (RBCs) improved the blood half-life. This is promising, but ex-vivo approaches are cumbersome and challenging to translate clinically. Here, we developed a novel Ter119-polymeric NP containing tissue plasminogen activator for on-demand targeting of GPA receptors in vivo. Upon intravenous injection, the Ter119-NPs achieved remarkable RBC labeling efficiencies (>95%), resulting in marked enhancement of blood residence time of tPA from minutes to several days without any morphological, hematological, and histological complications. Our approach of RBC labeling with the NPs also prevented reticuloendothelial detections and the activations of innate and adaptive immune system. Data suggest that real-time targeting of therapeutics to RBC with NPs can potentially improve outcomes and reduce complications against a variety chronic disease. Unlabelled Image • Real-time targeting of circulatory erythrocytes with nanoparticles can be attained using the Ter119 antibody. • Ter119 nanoparticles prolong circulation half-life of thrombolytics from min to days. • Ter119 nanoparticles do not influence hematological parameters and are safe to use in murine models. [ABSTRACT FROM AUTHOR]

Introduction: Robot‐assisted therapy for the upper limb (RT‐UL) is an emerging form of intervention for stroke survivors with upper limb deficits. However, there is limited knowledge regarding therapists' perceptions of RT‐UL and the factors influencing the implementation of RT‐UL into the clinical setting. This is important when considering that therapists in Australia are primarily responsible for the prescription of RT‐UL in daily practice. This study aimed to explore occupational therapists' and physiotherapists' perceptions of RT‐UL and the perceived barriers and enablers influencing implementation. Methods: Two discipline‐specific focus groups were conducted involving occupational therapists (n = 6) and physiotherapists (n = 6). Participants were members of the same multidisciplinary team working in an Australian public health rehabilitation facility where RT‐UL (i.e. InMotion2) was being introduced for the first time. Focus groups explored therapist perceptions of the new RT‐UL as well as perceived barriers and enablers to implementation. Focus groups were recorded, transcribed and deductively analysed using the Theoretical Domains Framework (TDF). Results: Out of the 14 domains of the TDF, 7 were raised by participants during the focus groups: environmental context and resources, beliefs about consequences, optimism, knowledge, skills, social influences, and social and professional role and identity. Therapists' expressed their optimism towards the introduction of RT‐UL but believed successful implementation would be primarily dependent on the availability of clinical leadership, training and a suitable client mix. Conclusion: Therapists perceived that RT‐UL would provide opportunity for increased upper limb practice particularly for patients with severe upper limb impairment. To facilitate implementation, support of RT‐UL should come from both management and clinical leaders and training include RT‐UL efficacy, device functionality and patient suitability. The availability of a single RT‐UL device in a workplace may create unique interdisciplinary and logistical challenges. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate a library of poly- l -lysine (PLL)-graft (g)-polyethylene glycol (PEG) copolymers for the ability to encapsulate effectively a model protein, bovine serum albumin (BSA), and to characterize the stability and protein function of the resulting nanoparticle. A library of nine grafted copolymers was produced by varying PLL molecular weight and PEG grafting ratio. Electrostatic self-assembly of the protein and the grafted copolymer drove encapsulation. The formation of protein/polymer nanoparticles with a core/shell structure was confirmed using PAGE, dynamic light scattering, and electron microscopy. Encapsulation of the BSA into nanoparticles was strongly dependent on the copolymer-to-protein mass ratio, PEG grafting ratio, and PLL molecular weight. A copolymer-to-protein mass ratio of 7:1 and higher was generally required for high levels of encapsulation, and under these conditions, no loss of protein activity was observed. Copolymer characteristics also influenced nanoparticle resistance to polyanions and protease degradation. The results indicate that a copolymer of 15–30 kDa PLL, with a PEG grafting ratio of 10:1, is most promising for protein delivery. [ABSTRACT FROM AUTHOR]

Nearly 30 years ago, certain small, relatively nontoxic peptides were discovered to be capable of traversing the cell membrane. These cell-penetrating peptides, as they are now called, have been shown to not only be capable of crossing the cell membrane themselves but can also carry many different therapeutic agents into cells, including small molecules, plasmid DNA, siRNA, therapeutic proteins, viruses, imaging agents, and other various nanoparticles. Many cell-penetrating peptides have been derived from natural proteins, but several other cell-penetrating peptides have been developed that are either chimeric or completely synthetic. How cell-penetrating peptides are internalized into cells has been a topic of debate, with some peptides seemingly entering cells through an endocytic mechanism and others by directly penetrating the cell membrane. Although the entry mechanism is still not entirely understood, it seems to be dependent on the peptide type, the peptide concentration, the cargo the peptide transports, and the cell type tested. With new intracellular disease targets being discovered, cell-penetrating peptides offer an exciting approach for delivering drugs to these intracellular targets. There are hundreds of cell-penetrating peptides being studied for drug delivery, and ongoing studies are demonstrating their success both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Stability of therapeutic proteins is one of the most important obstacles in their development. We studied the effects of the zwitterionic amphiphile cocamidopropyl betaine (CAPB) on protein stability against elevated temperatures and shear. Bovine serum albumin (BSA) was selected as a model protein to study the interaction with CAPB. Techniques such as gel electrophoresis, fluorimetry, and surface tensiometry were utilized to study protein aggregation and protein/CAPB complex formation. Our results indicate that CAPB effectively decreased the concentration of BSA at the air/water interface and subsequently prevented aggregation of the protein at the interface. Furthermore, complexes formed by the association of BSA and CAPB were more stable against both temperature and shear. The increased stability was achieved without loss of enzymatic activity of the protein. The effects of zwitterionic CAPB on protein enzymatic activity and conformational (tertiary structure) stability were also compared with polysorbate 20 and sodium dodecyl sulfate. • CAPB prevents protein aggregation and denaturation at elevated temperatures. • The enzymatic activity/conformational stability of the protein is not diminished. • Zwitterionic CAPB associates with BSA, to form stable, less surface active complexes. [ABSTRACT FROM AUTHOR]

Erythrocytes have been extensively investigated as drug delivery vehicles. However, many existing methods for loading therapeutic biomolecules into erythrocytes perturb the cell membrane, resulting in a loss of erythrocyte structural integrity and recognition by the host immune system. To overcome this challenge, we developed a novel nanoparticle carrier for transporting proteins into intact erythrocytes. We designed and characterized a nanoparticle where a poly-L-lysine-grafted-poly(ethylene) glycol copolymer was used to encapsulate a model protein, bovine serum albumin. The nanoparticle was functionalized with either an erythrocyte targeting peptide ERY1, the cell-penetrating peptide low molecular weight protamine, or a combination of the two peptides. The three resulting constructs, ENP, LNP, and ELNP, had a core/shell structure and, depending on functionalization, were approximately 30–90 nm in diameter and had negative surface charge. Esterase-like activity of the encapsulated protein was not affected by encapsulation. Under physiological conditions, nanoparticles caused no significant hemolysis and were resistant to dissociation by surfactants. The three nanoparticle constructs were evaluated using confocal microscopy and flow cytometry for their ability to bind and internalize within intact erythrocytes. LNPs showed significant binding and internalization into intact erythrocytes, with ~90% of erythrocytes containing particles after 24 h of co-incubation. Internalization occurred without adversely affecting erythrocyte membrane structure. Together, these results demonstrate that low molecular weight protamine–functionalized nanoparticles may serve as a universal platform to deliver protein therapeutics into erythrocytes. [ABSTRACT FROM AUTHOR]

We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly- l -lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26–35 nm). The three copolymer-complexed enzymes exhibited reduced k cat (30–33% reduction), but no significant changes in K m . Inhibitory potency (as reflected by the bimolecular rate constant, k i ) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in k i (16–60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers. [ABSTRACT FROM AUTHOR]

Reducing the promiscuous tropism of native adenovirus by using fiberless adenovirus is advantageous toward its use as a gene therapy vector or vaccine component. The removal of the fiber protein on native adenovirus abrogates several undesirable interactions; however, this approach decreases the particle's physical stability. To create stable fiberless adenovirus for pharmaceutical use, the effects of temperature and pH on the particle's stability profile must be addressed. Our results indicate that the stability of fiberless adenovirus is increased when it is stored in mildly acidic conditions around pH 6. The stability of fiberless adenovirus can be further enhanced by using excipients. Excipient screening results indicate that the nonionic surfactant Pluronic F-68 and the amino acid glycine are potential stabilizers because of their ability to increase the thermal transition temperature of the virus particle and promote retention of biological activity after exposure to prolonged thermal stress. Our data indicate that the instability of fiberless adenovirus can be ameliorated by storing the virus in the appropriate environment, and it should be possible to further optimize the virus so that it can be used as a biopharmaceutical. [ABSTRACT FROM AUTHOR]

Purpose: Nanoparticle (NP) attachment to biocompatible secondary carriers such as red blood cell (RBC) can prolong blood residence time of drug molecules and help create next-generation nanotherapeutics. However, little is known about the impact of RBC-targeted NPs on erythrocyte function. Methods: The objectives of this study were to develop and characterize in vitro a novel poly-L-lysine (PLL) and polyethylene glycol (PEG) copolymer-based NP containing fluorescent-tagged bovine serum albumin (BSA), and conjugated with ERY1, a 12 amino acid peptide with high affinity for the RBC membrane protein glycophorin A (ENP). Results: Confocal and flow cytometry data suggest that ENPs efficiently and irreversibly bind to RBC, with approximately 70% of erythrocytes bound after 24 h in a physiologic flow loop model compared to 10% binding of NPs without ERY1. Under these conditions, synthesized ENPs were not toxic to the RBCs. The rheological parameters at the applied shear. (0-15 Pa) were not influenced by ENP attachment to the RBCs. However, at high concentration, the strong affinity of ENPs to the glycophorin-A reduced the deformability of the RBC. Conclusions: ENPs can be efficiently attached to the RBCs without adversely affecting cellular function, and this may potentially enhance circulatory half-life of drug molecules. [ABSTRACT FROM AUTHOR]

Effective use of exogenous human BChE as a bioscavenger for organophosphorus toxicants (OPs) is hindered by its limited availability and rapid clearance. Complexes made from recombinant human BChE (rhBChE) and copolymers may be useful in addressing these problems. We used in vitro approaches to compare enzyme activity, sensitivity to inhibition, stability and bioscavenging capacity of free enzyme and copolymer-rhBChE complexes (C-BCs) based on one of nine different copolymers, from combinations of three molecular weights (MW) of poly- l -lysine (PLL; high MW, 30–70 kDa; medium MW, 15–30 kDa; low MW, 4–15 kDa) and three grafting ratios of poly(ethylene glycol) (PEG; 2:1, 10:1, 20:1). Retarded protein migration into acrylamide gels stained for BChE activity was noted with all copolymers as the copolymer-to-protein ratio was increased. BChE activity of C-BCs was lower relative to free enzyme, with the 2:1 grafting ratio showing generally greater reduction. Free enzyme and C-BCs showed relatively similar in vitro sensitivity to inhibition by paraoxon, but use of the 20:1 grafting ratio led to lower potencies. Through these screening assays we selected three C-BCs (high, medium and low MW; 10:1 grafting) for further characterizations. BChE activity was higher in C-BCs made with the medium and low compared to high MW-based copolymer. C-BCs generally showed higher stability than free enzyme when maintained for long periods at 37 °C or following incubation with chymotrypsin. Free enzyme and C-BCs were similarly effective at inactivating paraoxon in vitro . While these results are promising for further development, additional studies are needed to evaluate in vivo performance. [ABSTRACT FROM AUTHOR]

Background Adenovirus (Ad) is one of the viral vectors most widely used for gene delivery. The virus, however, has serious shortcomings such as immunogenicity, promiscuous tropism, and the inability to efficiently infect certain types of cells. The goal of this study was to improve the ability of an Ad-based vector to efficiently transform cells that lack the native coxsackie-adenovirus receptor (CAR − ) by modifying the virus with CPP-PEG conjugates. Methods The vector was produced by PEGylating Ad, which packages a lacZ reporter gene, and then conjugating CPPs to form CPP-PEG-Ad particles. The study compared the effectiveness of four different CPPs: Pen, Tat, Pep1, and pArg. The effects of CPP amount per virus, degree of PEGylation, and PEG molecular weight on transduction efficiency were studied on CAR − NIH/3T3 cells. Results CPP-PEG-Ad particles transduced CAR − cells significantly better than unmodified Ad. Pen, the most effective CPP, produced an 80-fold improvement in transduction compared to the unmodified virus. The Pen peptide utilized a combination of electrostatic and hydrophobic interactions with the cell membrane to maximize cellular association while the other CPPs used only electrostatic or hydrophobic interactions but not both. Lastly, higher degrees of PEGylation, which prompted PEG to adopt a “brush” conformation, resulted in more efficient CPP-PEG-Ad particles because of both better conjugation of CPPs to the PEGylated virus and better exposure of the conjugated CPPs on the surface of the particle. Conclusions CPP-PEG-Ad particles efficiently deliver genes to cells that Ad alone would not efficiently infect, thereby extending potential gene therapy treatments to a much broader range of cell types and diseases. [ABSTRACT FROM AUTHOR]

Adenovirus (Ad) is a promising gene therapy vector, and is used currently in more than 23% of clinical gene therapy trials. The viral vector, however, has drawbacks such as immunogenicity, promiscuous tropism, and the inability to infect certain types of cells. The focus of this work was to develop an improved vector through electrostatic formation of a complex between negatively charged Ad and positively charged cell-penetrating peptides (CPPs), including Tat, Penetratin, polyarginine, and Pep1. The resulting complexes were demonstrated to be capable of transducing cells that lack the coxsackie-adenovirus receptor (CAR), and are otherwise difficult to infect with native Ad. The transduction efficiency of the complexes was optimized by varying the multiplicity of infection, complex formation time, and ratio of CPPs to Ad, which improved the transduction efficiency of CPP/Ad on CAR-negative cells more than 100-fold compared with unmodified Ad. The size of the CPP/Ad complex was initially less than 300 nm, but stability studies performed in the presence of serum indicate that the complex aggregates with serum after an extended period of time. The results of the current study indicate that electrostatic modification of Ad with CPPs provides a relevant platform for developing effective Ad-based gene therapy vectors. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1981-1993, 2013 [ABSTRACT FROM AUTHOR]

The interfacial adsorption and denaturation of proteins is a significant concern for the loss of protein structure and function during protein processing or storage. Surfactants can be used to prevent interfacial damage to proteins. Zwitterionic surfactants have shown intriguing effects on stability and activity of proteins. The aim of this research was to study the effect of cocamidopropyl betaine (CAPB), a non-denaturing zwitterionic carboxy betaine containing surfactant, on the air/water interfacial adsorption of a model protein bovine serum albumin (BSA). Dynamic and equilibrium surface tensions were measured using the pendant drop method. The results of surface tension measurements were coupled with a thermodynamic analysis, based on the interface and bulk chemical potentials, to estimate the surface coverage of the components and to calculate surface adsorption. Our results indicated that CAPB can effectively associate with the protein at different concentrations, even at concentrations lower than the critical micelle concentration (CMC). Furthermore, the addition of the protein significantly increased the CMC of the CAPB form 45 ppm to 407 ppm. Most importantly, CAPB prevented the adsorption of the protein at the interface. The interface of aqueous solution of CAPB+BSA was also compared with the Triton X-100 + BSA. Results of this study will help us to understand surfactant+protein interactions at interfaces. Unlabelled Image • Interfacial behavior of zwitterionic surfactant+protein is experimentally investigated. • Protein and surfactant surface coverages are estimated using thermodynamic modeling. • Spontaneous self-assembly of zwitterionic surfactant-albumin reduced system surface activity. • Zwitterionic surfactant interacts with the protein more strongly than Triton X-100. [ABSTRACT FROM AUTHOR]