Fig. 8.

a Schematic illustration of NP fabrication and penetration in tumors via the degradation of hyaluronic acid (HA). The NPs were fabricated by conjugating thiolated rHuPH20 on the first PEG layer followed by anchoring the second PEG layer. b NP diffusion in ECM-mimicking gels. (Scale bar: 200 μm). The gels composed of 6.5 mg mL⁻¹ of rat collagen I and 1 mg/mL of HA in capillary tubes. Ten microliters of 1 mg mL⁻¹ of NPs (green) were added on the top of gels and incubated at 37 °C for 1.5 h before being imaged. The activity of free or conjugated rHuPH20 was 500 U mL⁻¹ and normalized NP fluorescence with diffusion distance in gels. Images were analyzed via ImageJ. Diffusion coefficients were obtained by fitting the data to a one-dimensional diffusion model in MATLAB. Black lines display theoretical intensity profiles for particles with diffusion coefficients of 1.66 × 10⁻⁷, 7.17 × 10⁻⁸, and 1.11 × 10⁻⁸ cm² s⁻¹. c Confocal microscopy images of 4T1 cells treated with 0.02 mg mL⁻¹ of either HPEG-NPs or HPEG-PH20-NPs with enzyme activity of 10 U mL⁻¹ for 0, 1, 2, and 4 h. HA, nuclei, and DiD-labeled NPs were shown in green, blue, and red, respectively. The signal included both internalized particles and particles bound on cell surfaces (Scale bar: 50 μm). d In vivo tumor growth inhibition curves for 4T1 tumor-bearing mice that were treated with either saline, free DOX, DOX-HPEG-NPs or DOX-HPEG-PH20-NPs. The dose of DOX was 2 mg kg⁻¹ for free DOX and DOX-encapsulated NPs. Values indicate mean ± SD (n = 6). **P < 0.01. Black arrows indicate the time of injection. e Survival rate plots show the percentage of animals remained alive in the study. Mice were sacrificed and were no longer counted for survival rate when their tumor size exceeded 2000 mm³. **P < 0.01. f TUNEL staining of sectioned tumor tissues that were collected after the completion of all doses for the groups treated with saline, free DOX, DOX-HPEG-NPs, and DOX-HPEG-PH20-NPs. Green, TUNEL; blue, nuclei. Scale bar: 100 μm. g Staining of sectioned tumor trusses that were collected 24 h post the administration of saline or NPs. The mice were iv injected with saline or DiD-labeled NPs on day 9 post 4T1 cell inoculation. Left column showed the staining of CD31 (green, representing blood vessels) and the distribution of NPs (red). The middle column showed the HA staining and the right column showed the α-SMA staining for the four study groups. Scale bar: 50 μm (left); 200 μm (middle); 100 μm (right).

Reproduced with permission from Ref. [187]