Inflammation responsive logic gate nanoparticles for the delivery of proteins

Abstract

Oxidative stress and reduced pH are important stimuli targets for intracellular delivery and for delivery to diseased tissue. However, there is a dearth of materials able to deliver bioactive agents selectively under these conditions. We employed our recently developed dual response strategy to build a polymeric nanoparticle that degrades upon exposure to two stimuli in tandem. Our polythioether ketal based nanoparticles undergo two chemical transformations; the first is the oxidation of the thioether groups along the polymer backbone of the nanoparticles upon exposure to reactive oxygen species (ROS). This transformation switches the polymeric backbone from hydrophobic to hydrophilic and thus allows, in mildly acidic environments, the rapid acid-catalyzed degradation of the ketal groups also along the polymer backbone. Dynamic light scattering and payload release studies showed full particle degradation only in conditions that combined both oxidative stress and acidity, and these conditions led to higher release of encapsulated protein within 24 h. Nanoparticles in neutral pH and under oxidative conditions showed small molecule release and swelling of otherwise intact nanparticles. Notably, cellular studies show absence of toxicity and efficient uptake of nanoparticles by macrophages followed by cytoplasmic release of ovalbumin. Future work will apply this system to inflammatory diseases.

Figure 1

Degradation mechanism of poly thioether-ketal. Hydrogen peroxide and acidic pH stimulate the degradation of the polymeric nanoparticles in tandem.

Figure 2

Synthesis of poly thioether-ketal.

Figure 3

A SEM image of the nanoparticles showing a particle diameter of < 1μm.

Figure 4

Effect of pH and H₂O₂ on the Z-average of the nanoparticles. Triplicate measurements were taken using the DLS at a fixed attenuator of 7.

Figure 5

Release of Nile red from poly thioether-ketal nanoparticles. Decrease in nanoparticles suspension fluorescence was correlated to Nile red release in different conditions.

Figure 6

Degradation of polymer 5 at pH 5 in 1:1 deuterated acetonitrile and water by ¹HNMR. Hydrolysis of ketal is evidenced by the appearance of acetone peak(at δ 2.6ppm) and disappearance of the ketal peak( at δ 1.6ppm). There is also concomitant change in the peak at δ 3.8 ppm to δ 3.9 ppm due to the formation of alcohol upon degradation of the ketal.

Figure 7

Degradation of polymer 5 at pH 5 in 1:1 deuterated acetonitrile and water with addition of 20μl of 30% H₂O₂ by ¹HNMR. In addition to the changes noted in the Figure 6 upon addition of H₂O₂ we see that the peak at δ 3.6 ppm shifts to δ 3.7 ppm. This is consistent with the oxidation of sulfur in the backbone to a sulfoxide. These NMR studies establish that the sulfur moiety undergoes oxidation when subjected to H₂O₂.

Figure 8

Release of ovalbumin AlexaFluor® 488 from poly thio-ketal nanoparticles. Nanoparticles supernatants were tested for fluorescence to determine ovalbumin release profile over time (n=3).

Figure 9

Cytotoxicity of poly thioether-ketal nanoparticles at different concentrations in RAW 264.7 macrophage cells. The cells were incubated with the nanoparticles for 20hrs before performing the MTT assay.

Figure 10

Uptake of nanoparticles loaded with fluorescent ovalbumin. Raw 264.7 macrophage cells were treated for 8hrs with poly thioether-ketal or PLGA nanoparticles containing ovalbumin Alexa Fluor 594 (red) and stained with DAPI (blue).