Amphiphilic Nanoparticle-in-Nanoparticle Drug Delivery Systems Exhibiting Cross-Linked Inorganic Rate-Controlling Domains

Aiming to explore the potential of sol–gel chemistry to physically stabilize polymeric micelles and confer sustained release features, this work reports for the first time on the production of hybrid organic–inorganic multimicellar nanomaterials that, as opposed to the state-of-the-art materials, display cross-linked poly­(siloxane) rate-controlling domains. To achieve this goal, poly­(ethylene oxide)-<i>b</i>-poly­(propylene oxide) amphiphiles with different architectures (linear and branched) and hydrophilic–lipophilic balances were primarily modified with alkoxysilane moieties through the reaction of the terminal hydroxyl groups of the copolymer and 3-(triethoxysilyl)­propyl isocyanate. Then, ethoxysilane-modified polymeric micelles were prepared in water where hydrolysis resulted in a silanol-decorated surface that was cured by spray-drying. Because of the singular spraying mechanism of the Nano Spray-Dryer B-90 used in this work, which is based on a vibrating mesh spray with holes in the 4–7 μm size range that produce ultrafine droplets, a novel kind of hybrid amphiphilic nanoparticle-in-nanoparticle system with high physical stability was developed. Comprehensive microscopy studies demonstrated the multimicellar nature of these novel nanomaterials. Moreover, they hosted large payloads of the hydrophobic model drug tipranavir in the hydrophobic domains and sustained the release with a more controlled zero-order fashion compared to that of the pristine non-cross-linked counterparts that followed the classical biphasic release with an initial burst effect and a subsequent more moderate rate

Schematic of MET16 states and transitions.

<p>In buffered environment (pH 7) the peptide exists in two-state equilibrium between native (N) and unfolded (D) conformations. After ∼90 min a third, fibrillar aggregate conformation appears. The folded conformations appearing in the fibril need not be the same as N.</p

Analyses of CD-resolved kinetics.

<p>(<b>A</b>) RMS deviation of simulated CD spectra based on the derived basis set of P spectra (<b>B</b>) Simulated CD curves of the three basic structures calculated by CCA. These correspond to pure β-sheet (solid black line), amyloid (dashed) and unfolded (dot-dashed). The solid cyan line is the experimental spectrum of the fully folded peptide in the presence of 55% (w/w) MeOH. (<b>C</b>) Ratio of β-sheet (φ_f) over unfolded (φ_u) mole fraction as a function of time for MET16 in water (<i>squares</i>), and enough sorbitol (<i>circles</i>) or PEG 4000 (<i>triangles</i>) to induce a stabilization of ΔΔG = −1.5 kJ/mol to the β-sheet conformation. The dotted lines represent theoretical values for the equilibrium constant for folding for the reaction in aqueous media (ΔΔG = 0) and in the presence of the cosolutes (ΔΔG = −1.5 kJ/mol).</p

Effects of cosolute addition on ThT fluorescence.

<p>Ratio of ThT emission values at λ = 485 nm before and after cosolute addition. A value close to 1.0 represents no change in emission upon dilution. Inset shows ThT fluorescence emission vs. time, with the point of dilution at <i>t</i> = 2600 minutes. The value of signal at the plateau prior to dilution (<i>f^*</i>) (Eq. 2) was divided by the average emission value of the hour following ThT addition to obtain the relative deviation values of the fluorescence at peak emission as a result of cosolute addition. Circles (<i>green</i>) show the emission of buffered ThT without the addition of MET16.</p

Length distribution analysis of fibrils imaged using TEM.

<p>Fibril lengths were measured in the absence of cosolutes, (<b>A</b>) at <i>t</i> = 0 (average length, as calculated directly from measurements, 263±114 nm), (<b>B</b>) at <i>t</i> = 500 min, (average length 458±146 nm); and in presence of 30% (w/w) sorbitol, (<b>C</b>) at <i>t</i> = 0 min (average length is 142±64 nm), (<b>D</b>) at <i>t</i> = 500 min (average length 265±95 nm). Errors in average length are standard deviation of length measurements.</p

Aggregation lag times <i>t_lag</i> (in minutes) for different peptide stabilities ΔΔG (in kJ/mol).^a

a<p>value of <i>t_lag</i> in the absence of cosolutes was 340±80 min.</p

Kinetics of amyloid formation followed by CD spectroscopy.

<p>(<b><i>left column</i></b>) CD spectra measured at different times of the aggregation process in the absence (<i>top</i>) and presence of sorbitol (<i>center</i>) and PEG 4000 (<i>bottom</i>). (<b><i>right column</i></b>) Contribution of unfolded (<i>triangles</i>), β-sheet (<i>squares</i>) and amyloid (<i>circles</i>) formations to each of the CD spectra presented on the left column, as determined by CCA analysis, shown as a function of time for each of the aggregation reactions shown on the left. Lines are guides for the eyes.</p

Fluorescence emission plateau <i>f^*</i> (in AU) for different peptide stabilities ΔΔG (in kJ/mol).^a

a<p>value of <i>f</i> in the absence of cosolutes was 560±20.</p

Elongation lifetime <i>τ_el</i> (in minutes) for different peptide stabilities ΔΔG (in kJ/mol).^a

a<p>value of <i>τ_el</i> in the absence of cosolutes was 100±40 min.</p

TEM images of MET16 fibrils at different times and under different solution conditions.

<p>Negative stain TEM images taken from aggregation mixtures at different times: After 2 minutes, in the absence (<i>a</i>) and presence (<i>b</i>) of 1.5 M sorbitol, and after 500 minutes, in the absence (<i>c</i>) and presence (<i>d</i>) of 1.5 M sorbitol.</p