No abstract

Drug nanocarrier clearance by the immune system must be minimized to achieve targeted delivery to pathological tissues. There is considerable interest in finding in vitro tests that can predict in vivo clearance outcomes. In this work, we produce nanocarriers with dense PEG layers resulting from block copolymer-directed assembly during rapid precipitation. Nanocarriers are formed using block copolymers with hydrophobic blocks of polystyrene (PS), poly-ε-caprolactone (PCL), poly-D,L-lactide (PLA), or poly-lactide-co-glycolide (PLGA), and hydrophilic blocks of polyethylene glycol (PEG) with molecular weights from 1.5 kg/mol to 9 kg/mol. Nanocarriers with paclitaxel prodrugs are evaluated in vivo in Foxn1nu mice to determine relative rates of clearance. The amount of nanocarrier in circulation after 4 h varies from 10% to 85% of initial dose, depending on the block copolymer. In vitro complement activation assays are conducted in an effort to correlate the protection of the nanocarrier surface from complement binding and activation and in vivo circulation. Guidelines for optimizing block copolymer structure to maximize circulation of nanocarriers formed by rapid precipitation and directed assembly are proposed, relating to the relative size of the hydrophilic and hydrophobic block, the hydrophobicity of the anchoring block, the absolute size of the PEG block, and polymer crystallinity. The in vitro results distinguish between the poorly circulating PEG5k-PCL9k and the better circulating nanocarriers, but could not rank the better circulating nanocarriers in order of circulation time. Analysis of PEG surface packing on monodisperse 200 nm latex spheres indicates that the sizes of the hydrophobic PCL, PS, and PLA blocks are correlated with the PEG blob size, and possibly the clearance from circulation. Suggestions for next step in vitro measurements are made.

Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0-75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5 kg mol−1 methoxy-terminated PEG corona. While a 5 kg mol−1 methoxy-terminated PEG corona prevents non-specific uptake, a 1.8 kg mol−1 methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associate with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This characterization of nanocarrier uptake and targeting provides promise for optimizing drug delivery to macrophages for TB treatment and establishes a general route for optimizing targeted formulations of nanocarriers for specific delivery at targeted sites.

A principal advantage of magic angle spinning (MAS) NMR spectroscopy lies in its ability to determine molecular structure in a noninvasive and quantitative manner. Accordingly, MAS should be widely applicable to studies of the structure of active pharmaceutical ingredients (API) and formulations. However, the low sensitivity encountered in spectroscopy of natural abundance APIs present at low concentration has limited the success of MAS experiments. Dynamic nuclear polarization (DNP) enhances NMR sensitivity and can be used to circumvent this problem provided that suitable paramagnetic polarizing agent can be incorporated into the system without altering the integrity of solid dosages. Here, we demonstrate that DNP polarizing agents can be added in situ during the preparation of amorphous solid dispersions (ASDs) via spray drying and hot-melt extrusion so that ASDs can be examined during drug development. Specifically, the dependence of DNP enhancement on sample composition, radical concentration, relaxation properties of the API and excipients, types of polarizing agents and proton density, has been thoroughly investigated. Optimal enhancement values are obtained from ASDs containing 1% w/w radical concentration. Both polarizing agents TOTAPOL and AMUPol provided reasonable enhancements. Partial deuteration of the excipient produced 3× higher enhancement values. With these parameters, an ASD containing posaconazole and vinyl acetate yields a 32-fold enhancement which presumably results in a reduction of NMR measurement time by ∼1000. This boost in signal intensity enables the full assignment of the natural abundance pharmaceutical formulation through multidimensional correlation experiments.

Soluble peptides, susceptible to degradation and clearance in therapeutic applications, have been formulated into protected nanoparticles for the first time through the process of kinetically controlled, block copolymer directed rapid precipitation using Flash NanoPrecipitation. Complementary Brownian dynamics simulations qualitatively model the nanoparticle formation process. The simulations corroborate the hypothesis that the size of nanoparticles decreases with increasing supersaturation. Additionally, the influence of the polymer-peptide interaction energy on the efficiency of nanoparticle protection by polymer surface coverage is elucidated in both experiments and simulations.