Photoreactive-proton-generating hyaluronidase/albumin nanoparticles-loaded PEG-hydrogel enhances antitumor efficacy and disruption of the hyaluronic acid extracellular matrix in AsPC-1 tumors

Abstract

Depletion of tumor extracellular matrix (ECM) is viewed as a promising approach to enhance the antitumor efficacy of chemotherapeutic-loaded nanoparticles. Hyaluronidase (HAase) destroys hyaluronic acid-based tumor ECM, but it is active solely at acidic pHs of around 5.0 and is much less active at physiological pH. Herein, we report the development of our novel UV-light-reactive proton-generating and hyaluronidase-loaded albumin nanoparticles (o-NBA/HAase-HSA-NPs). The method to prepare the nanoparticles was based on pH-jump chemistry using o-nitrobenzaldehyde (o-NBA) in an attempt to address the clinical limitation of HAase. When in suspension/PEG-hydrogel and irradiated with UV light, the prepared o-NBA/HAase-HSA-NPs clearly reduced the pH of the surrounding medium to as low as 5.0 by producing protons and were better able to break down HA-based tumor cell spheroids (AsPC-1) and HA-hydrogel/microgels, presumably due to the enhanced HA activity at a more optimal pH. Moreover, when formulated as an intratumor-injectable PEG hydrogel, the o-NBA/HAase-HSA-NPs displayed significantly enhanced tumor suppression when combined with intravenous paclitaxel-loaded HSA-NPs (PTX-HSA-NPs) in AsPC-1 tumor-bearing mice: The tumor volume in mice administered UV-activated o-NBA/HAase-HSA-NPs and PTX-HSA-NPs was 198.2 ​± ​30.0 ​mm³, whereas those administered PBS or non-UV-activated o-NBA/HAase-HSA-NPs and PTX-HSA-NPs had tumor volumes of 1230.2 ​± ​256.2 and 295.4 ​± ​17.1 ​mm³, respectively. These results clearly demonstrated that when administered with paclitaxel NPs, our photoreactive o-NBA/HAase-HSA-NPs were able to reduce pH and degrade HA-based ECM, and thereby significantly suppress tumor growth. Consequently, we propose our o-NBA/HAase-HSA-NPs may be a prototype for development of future nanoparticle-based HA-ECM-depleting tumor-ablating agents.

Keywords: Albumin nanoparticles; Extracellular matrix; Hyaluronic acid; Hyaluronidase; Photosensitive pH-jump; Tumor suppression.

Graphical abstract

Fig. 1

Schematic illustration of antitumor therapy based upon destruction of the HA extracellular matrix due to decreased acidity using PEG-hydrogel containing UV-responsive proton-generating o-NBA/HAase-HSA-NPs in conjunction with PTX-HSA-NPs.

Fig. 2

(A) Histograms of particle sizes of o-NBA-HSA-NPs, o-NBA/HAase-HSA-NPs and PTX-HSA-NPs (B) Zeta potentials of o-NBA-HSA-NPs and o-NBA/HAase-HSA-NPs. (C) Photographs of o-NBA in DW (left) and a 9:1 solution of chloroform and ethanol (right) and the lyophilized powder (left) and dispersed solution (right) of o-NBA/HAase-HSA-NPs. (D) Transmission electron microscopy (TEM; left) and field-emission scanning electron microscopy (FE-SEM) images of o-NBA/HAase-HSA-NPs. (E) Physical stability of o-NBA-HSA-NPs and o-NBA/HAase-HSA-NPs based on size changes (left) and polydispersity index (PDI; right).

Fig. 3

Monitoring of pH-dependent hyaluronidase activity based on FITC-HA microgel degradation. (A) Optical microscopic morphology of HA-microgel (5 ​mg/mL) incubated with naïve HAase (1 ​mg/mL) at a pH of 4.0, 5.0, 5.5, 6.0 or 7.4. (B) The relative fluorescence of the supernatants from the degraded FITC-HA-microgel. Inset: enlarged HA microgel as viewed in optical microscopic (left) and confocal microscopic images (right).

Fig. 4

HA matrix-breaking activity of o-NBA/HAase-HSA-NPs exposed to UV light (2.7 ​mW/cm² at 5 ​cm distance). The HA-hydrogel disc was approximately 8.5 and 2.0 ​mm in diameter and thickness, respectively. An 1 ​mL aliquot of 5 ​mM PBS (pH 7.4) was used as a media. (A) Photographs of macroscopic HA-hydrogel breakdown after 24 ​h incubation with different formulations after 2-h UV irradiation of the NPS: F1 ​= ​5 ​mM PBS; F2 ​= ​o-NBA/HSA-NPs and no UV; F3 ​= ​o-NBA/HSA-NPs with UV; F4 ​= ​o-NBA/HAase-HSA-NPs and no UV; F5 ​= ​o-NBA/HAase-HSA-NPs with UV. (B) The relative fluorescence of the supernatants from the degraded FITC-HA-hydrogel incubated with F1 to F5 formulas. (C) Monitoring of pH change induced by o-NBA/HAase-HSA-NPs with or without UV light. (D) Mass ratios of F1 to F5 hydrogels versus their initial weight. ∗P ​< ​0.001 over no-UV group (F4). (E) SEM images of F1, F3 and F5 after 24 h-incubation (top) and photographs of PBS-washed HA-hydrogels (F1∼F5) after 24 h-incubation.

Fig. 5

HA matrix-breaking activity of o-NBA/HAase-HSA-NPs exposed to UV light (2 ​h, 2.7 ​mW/cm² at 5 ​cm distance). (A) Optical images of HA-microgel incubated with different formulas over 24 ​h: 5 ​mM PBS (F1); o-NBA/HSA-NPs ​+ ​UV(−) (F2); o-NBA/HSA-NPs ​+ ​UV(+) (F3); o-NBA/HAase-HSA-NPs ​+ ​UV(−) (F4); o-NBA/HAase-HSA-NPs ​+ ​UV(+) (F5). (B) Monitoring of microgel size change at each formula of F1∼F5. (C) Monitoring of color change of phenol red at different pHs or o-NBA/HAase-HSA-NPs with UV light. (D) Monitoring of pH change induced by o-NBA/HAase-HSA-NPs with or without UV light exposure. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Cytotoxic effects of o-NBA at various concentrations with or without UV light in HepG2 and AsPC-1 ​cells as measured using the MTT assay.

Fig. 7

(A) Monitoring of morphology of HepG2 cell spheroids incubated with 5 ​mM PBS (pH 7.4) and o-NBA/HAase-HSA-NPs (with or without UV exposure). Enlarged surface analysis of HepG2 cell spheroids incubated with o-NBA/HAase-HSA-NPs (with UV). (B) Optical and CLSM images of HepG2 cell spheroids embedded with FITC-conjugated low molecular weight HA (Mw ∼8 ​kDa).

Fig. 8

(A) Optical and CLSM fluorescence images of hyaluronic acid development in AsPC-1 ​cell spheroids using HABP-1/Alexa-488 dye staining (bottom: z-stack images of nine 18-μm slices obtained). (B) Monitoring of morphology of AsPC-1 ​cell spheroids incubated with the F1 to F5 formulations over 3 ​h.

Fig. 9

(A) Photographs of in situ gelling of the PEG-hydrogel formula o-NBA/HAase-HSA-NPs and phenol red with or without UV light. (B) In vivo visualization of AsPC-1-tumor bearing mouse treated with PEG-hydrogel loaded with o-NBA/Cy5.5-HAase-HSA-NPs (intratumor injection) and PTX- Cy5.5-HSA-NPs (tail vein injection). (C) Antitumor therapy plan for combination of intravenous PTX-HSA-NPs (200 ​μg PTX/mouse) and intratumoral o-NBA/HAase-HSA-NPs (500 ​μg HAase and UV). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

In vivo antitumor efficacy of PEG-hydrogel containing UV-responsive o-NBA/HAase-HSA-NPs in conjunction with PTX-HSA-NPs in five mouse treatment groups: (I) PBS (i.v.); (II) PBS ​+ ​naive HAase (intratumor; 500 μg/0.1 ​mL); (III) PTX-HSA-NPs (200 ​μg PTX/mouse); (IV) PTX-HSA-NPs (200 ​μg PTX/mouse) ​+ ​o-NBA/HAase-HSA-NPs (500 ​μg HAase and UV(−)); (V) PTX-HSA-NPs (200 ​μg PTX/mouse) ​+ ​o-NBA/HAase-HSA-NPs (500 ​μg HAase and UV(+)). (A) Tumor volumes in each group over 24 days. (B) Focused tumor volume profiles of G3, G4 and G5 over 24 days (⁺P ​< ​0.009 over G3 and ∗P ​< ​0.008 over G4). (C) Representative photographs of AsPC-1 tumor-bearing mice at the indicated days after treatment. (D) Photographs of tumors excised from each treatment group. (E) Body weight change of AsPC-1 tumor-bearing mice in the different treatment groups over 24 days post-treatment.

Fig. 11

(A) Microscopic images showing H&E, Alcian blue, HABP-1, DAPI and TUNEL staining of tumor slices from mice of each treatment group of G1∼G5. blue and HA-specific staining of tumor slices from mice of each treatment group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)