Controlling and Predicting Nanoparticle Formation by Block Copolymer Directed Rapid Precipitations

Nanoparticles have shown promise in several biomedical applications, including drug delivery and medical imaging; however, quantitative prediction of nanoparticle formation processes that scale from laboratory to commercial production has been lacking. Flash NanoPrecipitation (FNP) is a scalable technique to form highly loaded, block copolymer protected nanoparticles. Here, the FNP process is shown to strictly obey diffusion-limited aggregation assembly kinetics, and the parameters that control the nanoparticle size and the polymer brush density on the nanoparticle surface are shown. The particle size, ranging from 40 to 200 nm, is insensitive to the molecular weight and chemical composition of the hydrophobic encapsulated material, which is shown to be a consequence of the diffusion-limited growth kinetics. In a simple model derived from these kinetics, a single constant describes the 46 unique nanoparticle formulations produced here. The polymer brush densities on the nanoparticle surface are weakly dependent on the process parameters and are among the densest reported in the literature. Though modest differences in brush densities are observed, there is no measurable difference in the amount of protein adsorbed within this range. This work highlights the material-independent and universal nature of the Flash NanoPrecipitation process, allowing for the rapid translation of formulations to different stabilizing polymers and therapeutic loads

Hydrophobic Ion Pairing of Peptide Antibiotics for Processing into Controlled Release Nanocarrier Formulations

Nanoprecipitation of active pharmaceutical ingredients (APIs) to form nanocarriers (NCs) is an attractive method of producing formulations with improved stability and biological efficacies. However, nanoprecipitation techniques have not been demonstrated for highly soluble peptide therapeutics. We here present a model and technique to encapsulate highly water-soluble biologic APIs by manipulating API salt forms. APIs are ion paired with hydrophobic counterions to produce new API salts that exhibit altered solubilities suitable for nanoprecipitation processing. The governing rules of ion pair identity and processing conditions required for successful encapsulation are experimentally determined and assessed with theoretical models. Successful NC formation for the antibiotic polymyxin B requires hydrophobicity of the ion pair acid to be greater than logP = 2 for strong acids and greater than logP = 8 for weak acids. Oleic acid with a logP = 8, and p<i>K</i>_a = 5, appears to be a prime candidate as an ion pair agent since it is biocompatible and forms excellent ion pair complexes. NC formation from preformed, organic soluble ion pairs is compared to in situ ion pairs where NCs are made in a single precipitation step. NC properties, such as stability and release rates, can be tuned by varying ion pair molecular structure and ion pair-to-API molar ratios. For polymyxin B, NCs ≈ 100–200 nm in size, displaying API release rates over 3 days, were produced. This work demonstrates a new approach that enables the formation of nanoparticles from previously intractable compounds

Formulation of pH-Responsive Methacrylate-Based Polyelectrolyte-Stabilized Nanoparticles for Applications in Drug Delivery

pH-responsive polyelectrolytes, including methacrylate-based anionic copolymers (MACs), are widely used as enteric coatings and matrices in oral drug delivery. Despite their widespread use in these macroscopic applications, the molecular understanding of their use as stabilizers for nanoparticles (NPs) is lacking. Here, we investigate how MACs can be used to create NPs for therapeutic drug delivery and the role of MAC molecular properties on the assembly of NPs via flash nanoprecipitation. The NP size is tuned from 59 to 454 nm by changing the degree of neutralization, ionic strength, total mass concentration, and the core-to-MAC ratio. The NP size is determined by the volume of hydrophilic domains on the surface relative to the volume of hydrophobic domains in the core. We calculate the dimensions of the hydrophobic NP core relative to the thickness of the polyelectrolyte layer over a range of ionizations. Importantly, the results are shown to apply to both high-molecular-weight polymers as core materials and small-molecule drugs. The pH responsiveness of MAC-stabilized NPs is also demonstrated. Future development of polyelectrolyte copolymer-stabilized nanomedicines will benefit from the guiding principles established in this study

Real-Time and Multiplexed Photoacoustic Imaging of Internally Normalized Mixed-Targeted Nanoparticles

Photoacoustic (PA) imaging is a developing diagnostic technique where multiple species can be simultaneously imaged with high spatial resolution in 3D if the absorbance spectrum of each species is distinct and separable. However, multiplexed PA imaging has been greatly limited by the availability of spectrally separable contrast agents that can be used in vivo. Toward this end, we present the formation and application of a series of poly ethylene glycol (PEG)-coated nanoparticles (NPs) with unique separable absorbance profiles suitable for simultaneous multiplexed imaging. As a proof-of-concept, we demonstrate this form of mixed-sample multiplexed imaging, using cRGD peptide surface-modified NPs with nonmodified NPs in a murine subcutaneous Lewis lung carcinoma tumor model. The simultaneous imaging of nonmodified NPs provides an “internal standard”, to deconvolute the contributions of active-ligand and passive-NP targeting effects. Particles with 25% surface cRGD modification display 52 ± 22 fold higher liver to tumor ratio accumulation levels, while the same set of particles display only 9.8 ± 4 fold accumulation levels when internally normalized. The pharmacokinetic profiles of targeted and nontargeted NPs can be simultaneously tracked in real-time to study how biodistribtions of particles are affected by ligand modification. The internal normalization of control particles greatly enhances the precision and decreases the number of animals needed in studies of nanoparticle targeting. These new dyes are an enabling technology for PA imaging of NP fate and targeting. This is the first demonstration of real-time multiplexed PA imaging of mixed-targeted samples in vivo

Polymer Directed Self-Assembly of pH-Responsive Antioxidant Nanoparticles

We have developed pH-responsive, multifunctional nanoparticles based on encapsulation of an antioxidant, tannic acid (TA), using flash nanoprecipitation, a polymer directed self-assembly method. Formation of insoluble coordination complexes of tannic acid and iron during mixing drives nanoparticle assembly. Tuning the core material to polymer ratio, the size of the nanoparticles can be readily tuned between 50 and 265 nm. The resulting nanoparticle is pH-responsive, i.e., stable at pH 7.4 and soluble under acidic conditions due to the nature of the coordination complex. Further, the coordination complex can be coprecipitated with other hydrophobic materials such as therapeutics or imaging agents. For example, coprecipitation with a hydrophobic fluorescent dye creates fluorescent nanoparticles. <i>In vitro</i>, the nanoparticles have low cytotoxicity and show antioxidant activity. Therefore, these particles may facilitate intracellular delivery of antioxidants

A Computational Study of the Ionic Liquid-Induced Destabilization of the Miniprotein Trp-Cage

Fundamental understanding of protein stability away from physiological conditions is important due to its evolutionary implications and relevance to industrial processing and storage of biological materials. The molecular mechanisms of stabilization/destabilization by environmental perturbations are incompletely understood. We use replica-exchange molecular dynamics simulations and thermodynamic analysis to investigate the effects of ionic liquid-induced perturbations on the folding/unfolding thermodynamics of the Trp-cage miniprotein. We find that ionic liquid-induced denaturation resembles cold unfolding, where the unfolded states are populated by compact, partially folded structures in which elements of the secondary structure are conserved, while the tertiary structure is disrupted. Our simulations show that the intrusion of ions and water into Trp-cage’s hydrophobic core is facilitated by the disruption of its salt bridge and 3₁₀-helix by specific ion–residue interactions. Despite the swelling and widening of the hydrophobic core, however, Trp-cage’s α-helix remains stable. We further show that ionic liquid disrupts protein–protein and protein–water hydrogen bonds while favoring the formation of ion–protein bonds, shifting the equilibrium of conformational states and promoting denaturation near room temperature

Adsorption and Denaturation of Structured Polymeric Nanoparticles at an Interface

Nanoparticles (NPs) have been widely applied in fields as diverse as energy conversion, photovoltaics, environment remediation, and human health. However, the adsorption and trapping of NPs interfaces is still poorly understood, and few studies have characterized the kinetics quantitatively. In many applications, such as drug delivery, understanding NP interactions at an interface is essential to determine and control adsorption onto targeted areas. Therapeutic NPs are especially interesting because their structures involve somewhat hydrophilic surface coronas, to prevent protein adsorption, and much more hydrophobic core phases. We initiated this study after observing aggregates of nanoparticles in dispersions where there had been exposure of the dispersion to air interfaces. Here, we investigate the evolution of NP attachment and structural evolution at the air–liquid interface over time scales from 100 ms to 10s of seconds. We document three distinct stages in NP adsorption. In addition to an early stage of free diffusion and a later one with steric adsorption barriers, we find a hitherto unrealized region where the interfacial energy changes due to surface “denaturation” or restructuring of the NPs at the interface. We adopt a quantitative model to calculate the diffusion coefficient, adsorption rate and barrier, and extent of NP hydrophobic core exposure at different stages. Our results deepen the fundamental understanding of the adsorption of structured NPs at an interface

Narrow Absorption NIR Wavelength Organic Nanoparticles Enable Multiplexed Photoacoustic Imaging

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