Localized co-delivery of collagenase and trastuzumab by thermosensitive hydrogels for enhanced antitumor efficacy in human breast xenograft

Abstract

Modulation of the collagen-rich extracellular matrix (ECM) in solid tumors by the treatment with collagenase has been proved effective in enhancement of the interstitial transport and antitumor efficacy of antibodies. We, therefore, developed a PLGA-PEG-PLGA polymer-based thermosensitive hydrogel, which incorporated a HER2-targeted monoclonal antibody trastuzumab and collagenase (Col/Tra/Gel) for peritumoral administration. HER2-positvie BT474 tumor-bearing mice were selected as a model. The Col/Tra/Gel showed the continuous and biphasic release of protein drugs for 9 days in vitro. NIR imaging studies demonstrated a long-term retention of Col/Tra/Gel hydrogel in the peritumoral area for over 20 days. Treatment with Col/Tra/Gel reduced the collagen density and enhanced apoptotic cell death in tumor tissue, resulting in superior treatments with increased efficacy and reduced toxicity compared with other control groups. Moreover, a quarter-dose of Col/Tra/Gel exhibited a better antitumor efficacy than that of intravenous injection of clinical trastuzumab formulation. This localized co-delivery system offers a potential strategy for the modulation of dense ECM and enhancement of antibody efficacy.

Keywords: Thermosensitive hydrogel; antitumor efficacy; collagenase; sustained release; trastuzumab.

Figure 1.

A schematic of the preparation of Col/Tra/Gel, which can degrade ECM and enhance penetration of therapeutic antibody in tumor. (A) The chemical structure of PLGA-PEG-PLGA triblock copolymer (left) and the sol–gel phase transition in water (right). (B) The preparation of thermosensitive hydrogels incorporated trastuzumab and collagenase-I. (C) The antitumor procedures of Col/Tra/Gel. After peritumoral injection, a drug-loaded biodegradable hydrogel will form in situ. Both collagenase and Cy7-trastuzumab will be slowly and sustainably released from the hydrogel. The dense ECM will be degraded by the released collagenase, followed by the deep penetration of trastuzumab into the tumor tissue, thereby inducing the tumor cell apoptosis.

Figure 2.

Characterization of the thermosensitive hydrogel. (A) Storage modulus (G′) of the blank PLGA-PEG-PLGA hydrogel. (B) The SEM imaging of blank PLGA-PEG-PLGA hydrogel (scale bar =10 μm). (C) Release profiles of protein from the hydrogel. Inset is the release profile of the first 12 hours. (D) Storage stability of Col/Tra/Gel in 0 and 30 days at 4 °C observed by the CD spectrogram.

Figure 3.

In vivo sustained drug release by NIR imaging. (A) In vivo NIR images of BT474 tumor-bearing mice after i.v. injection of Cy7-Tra solution (upper) and s.c. injection of Cy7-Tra/Gel (middle) and Col/Cy7-Tra/Gel (lower) at 0, 1, 2, 4, 5, 7, 13, 18, 20 days (n = 3). (B) Quantification of the fluorescence intensity of in vivo NIR images as a function of time post-injection of Tra–Sol i.v. (red), Tra/Gel s.c. (green), and Col/Tra/Gel s.c. (blue) (n = 3).

Figure 4.

Anti-tumor efficacy and body weight change. Inhibition of tumor growth (A) and body weight (B) in BT474 tumor-bearing mice after treatment with different formulations at a total dose of 30 mg/kg of trastuzumab (n = 6). The tumor growth (C) and body weight (D) in BT474 tumor-bearing mice after treatment with Col/Tra/Gel at high dose (30 mg/kg) or low dose (10 mg/kg).

Figure 5.

In vivo collagen degradation and apoptosis staining. (A) Degradation of collagen in tumor tissues was analyzed by immunohistochemistry (red, collagen; blue, nuclei; scale bar, 100 µm). (B) Cell apoptosis in tumor tissues was analyzed by TUNEL assay (red, apoptotic cells; blue, nuclei; scale bar, 100 µm). (C) Quantitative assay of collagen staining. (D) Quantitative assay of apoptotic staining. *p < .05; **p < .01; ***p < .001, compared with the Col/Tra/Gel s.c. group.

Figure 6.

Comparison of the antitumor efficacy of Col/Tra/Gel with different clinical regimes in BT474 tumor-bearing mice. The tumor growth (A) and body weight (B) in BT474 tumor-bearing mice after treatment with different formulations (n = 6). *p < .05; **p < .01; ***p < .001, compared with the Col/Tra/Gel s.c. group.