Stabilizing proteins at high concentration is of broad interest in drug delivery, for treatment of cancer and many other diseases. Herein, we create highly concentrated antibody dispersions (up to 260 mg/mL) comprising dense equilibrium nanoclusters of protein (monoclonal antibody 1B7, polyclonal sheep immunoglobulin G, and bovine serum albumin) molecules which, upon dilution in vitro or administration in vivo, remain conformationally stable and biologically active. The extremely concentrated environment within the nanoclusters (∼700 mg/mL) provides conformational stability to the protein through a novel self-crowding mechanism, as shown by computer simulation, while the primarily repulsive nanocluster interactions result in colloidally stable, transparent dispersions. The nanoclusters are formed by adding trehalose as a cosolute which strengthens the short-ranged attraction between protein molecules. The protein cluster diameter was reversibly tuned from 50 to 300 nm by balancing short-ranged attraction against long-ranged electrostatic repulsion of weakly charged protein at a pH near the isoelectric point. This behavior is described semiquantitatively with a free energy model which includes the fractal dimension of the clusters. Upon dilution of the dispersion in vitro, the clusters rapidly dissociated into fully active protein monomers as shown with biophysical analysis (SEC, DLS, CD, and SDS-PAGE) and sensitive biological assays. Since the concept of forming nanoclusters by tuning colloid interactions is shown to be general, it is likely applicable to a variety of biological therapeutics, mitigating the need to engineer protein stability through amino acid modification. In vivo subcutaneous injection into mice results in indistinguishable pharmacokinetics versus a standard antibody solution. Stable protein dispersions with low viscosities may potentially enable patient self-administration by subcutaneous injection of antibody therapeutics being discovered and developed.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram-and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of ± 6%. Video Link The video component of this article can be found at https://www.jove.com/video/58757/ 13. Nucleation occurs uniformly in the mixing chamber and particle growth proceeds until halted by the assembly of the BCP onto the surface 9,14. The mixed stream containing stable particles may then be diluted with additional antisolvent to suppress Ostwald ripening of the particles 15,16,17 .

Practice research networks may be one way of advancing knowledge translation and exchange (KTE) in psychotherapy. In this study, we document this process by first asking clinicians what they want from psychotherapy research. Eighty-two psychotherapists in 10 focus groups identified and discussed psychotherapy research topics relevant to their practices. An analysis of these discussions led to the development of 41 survey items. In an online survey, 1,019 participants, mostly practicing clinicians, rated the importance to their clinical work of these 41 psychotherapy research topics. Ratings were reduced using a principal components analysis in which 9 psychotherapy research themes emerged, accounting for 60.66% of the variance. Two postsurvey focus groups of clinicians (N = 22) aided in interpreting the findings. The ranking of research themes from most to least important were-Therapeutic Relationship/Mechanisms of Change, Therapist Factors, Training and Professional Development, Client Factors, Barriers and Stigma, Technology and Adjunctive Interventions, Progress Monitoring, Matching Clients to Therapist or Therapy, and Treatment Manuals. Few differences were noted in rankings based on participant age or primary therapeutic orientation. Postsurvey focus group participants were not surprised by the top-rated items, as they were considered most proximal and relevant to therapists and their work with clients during therapy sessions. Lower ranked items may be perceived as externally imposed agendas on the therapist and therapy. We discuss practice research networks as a means of creating new collaborations consistent with KTE goals. Findings of this study can help to direct practitioner-researcher collaborations.

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Antimicrobial resistance is a healthcare problem of increasing significance, and there is increasing interest in developing new tools to address bacterial infections. Bacteria-targeting nanoparticles hold promise to improve drug efficacy, compliance, and safety. In addition, nanoparticles can also be used for novel applications, such as bacterial imaging or bioseperations. We here present the use of a scalable block-copolymer-directed self-assembly process, Flash NanoPrecipitation, to form zinc(II)-bis(dipicolylamine) modified nanoparticles that bind to both Grampositive and Gram-negative bacteria with specificity. Particles have tunable surface ligand densities that change particle avidity and binding efficacy. A variety of materials can be encapsulated into the core of the particles, such as optical dyes or iron oxide colloids, to produce imageable and magnetically active bacterial targeting constructs. As a proof-of-concept, these particles are used to bind and separate bacteria from solution in a magnetic column. Magnetic manipulation and separation would translate to a platform for pathogen identification or removal. These magnetic and targeted nanoparticles enable new methods to address bacterial infections.

Photoacoustic (PA) imaging is an emerging hybrid optical-ultrasound based imaging technique that can be used to visualize optical absorbers in deep tissue. Free organic dyes can be used as PA contrast agents to concurrently provide additional physiological and molecular information during imaging, but their use in vivo is generally limited by rapid renal clearance for soluble dyes and by the difficulty of delivery for hydrophobic dyes. We here report the use of the block copolymer directed self-assembly process, Flash NanoPrecipitation (FNP), to form series of highly hydrophobic optical dyes into stable, biocompatible, and water-dispersible nanoparticles (NPs) with sizes from 38 to 88 nm and with polyethylene glycol (PEG) surface coatings suitable for in vivo use. The incorporation of dyes with absorption profiles within the infrared range, that is optimal for PA imaging, produces the PA activity of the particles. The hydrophobicity of the dyes allows their sequestration in the NP cores, so that they do not interfere with targeting, and high loadings of >75 wt % dye are achieved. The optical extinction coefficients (ε (mL mg(-1) cm(-1))) were essentially invariant to the loading of the dye in NP core. Co-encapsulation of dye with vitamin E or polystyrene demonstrates the ability to simultaneously image and deliver a second agent. The PEG chains on the NP surface were functionalized with folate to demonstrate folate-dependent targeting. The spectral separation of different dyes among different sets of particles enables multiplexed imaging, such as the simultaneous imaging of two sets of particles within the same animal. We provide the first demonstration of this capability with PA imaging, by simultaneously imaging nontargeted and folate-targeted nanoparticles within the same animal. These results highlight Flash NanoPrecipitation as a platform to develop photoacoustic tools with new diagnostic capabilities.

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