Glutathione-degradable drug-loaded nanogel effectively and securely suppresses hepatoma in mouse model

Abstract

The reduction-responsive polymeric nanocarriers have attracted considerable interest because of a significantly higher concentration of intracellular glutathione in comparison with that outside cells. The smart nanovehicles can selectively transport the antitumor drugs into cells to improve efficacies and decrease side effects. In this work, a facilely prepared glutathione-degradable nanogel was employed for targeting intracellular delivery of an antitumor drug (ie, doxorubicin [DOX]). DOX was loaded into nanogel through a sequential dispersion and dialysis approach with a drug loading efficiency of 56.8 wt%, and the laden nanogel (noted as NG/DOX) showed an appropriate hydrodynamic radius of 56.1±3.5 nm. NG/DOX exhibited enhanced or improved maximum tolerated dose on healthy Kunming mice and enhanced intratumoral accumulation and dose-dependent antitumor efficacy toward H22 hepatoma-xenografted mouse model compared with free drug. In addition, the upregulated antitumor efficacy of NG/DOX was further confirmed by the histopathological and immunohistochemical analyses. Furthermore, the excellent in vivo security of NG/DOX was confirmed by the detection of body weight, histopathology, and biochemical indices of corresponding organs and serum. With controllable large-scale preparation and fascinating in vitro and in vivo properties, the reduction-responsive nanogel exhibited a good prospect for clinical chemotherapy.

Keywords: antitumor efficacy; chemotherapy; controlled release; nanogel; reduction-responsiveness; security.

Figure 1

Synthetic pathway for mPEG–P(LP-co-LC) nanogel and schematic illustrations about circulation, intratumoral accumulation, endocytosis, and targeting intracellular DOX release of DOX-loaded nanogel after intravenous injection. Notes: mPEG–P(LP-co-LC) nanogel synthesis (A), DOX encapsulation and intravenous injection (B), in vivo circulation and intratumoral accumulation (C), and endocytosis and GSH-triggered intracellular DOX release (D). All the arrows indicate the preparation and in vivo metabolic process of DOX-loaded nanogel. Abbreviations: DOX, doxorubicin; GSH, glutathione; mPEG–P(LP-co-LC), methoxy poly(ethylene glycol)–poly(L-phenylalanine-co-L-cystine).

Figure 2

Typical TEM microimage and R_h of mPEG–P(LP-co-LC) nanogel. Abbreviations: TEM, transmission electron microscopy; R_h, hydrodynamic radius; mPEG–P(LP-co-LC), methoxy poly(ethylene glycol)–poly(L-phenylalanine-co-L-cystine).

Figure 3

Percent body weights, survival rates, and organ indices in MTD detections. Notes: Percent body weights, survival rates, and organ indices of Kunming mice after disposable intravenous injection of NG at doses of 100.0 mg (kg BW)⁻¹, 300.0 mg (kg BW)⁻¹, and 500.0 mg (kg BW)⁻¹, and DOX·HCl and NG/DOX at equivalent DOX·HCl doses of 5.0 mg (kg BW)⁻¹, 10.0 mg (kg BW)⁻¹, and 15.0 mg (kg BW)⁻¹. The statistical data are presented as a mean ± SD. Abbreviations: BW, body weight; DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; MTD, maximum tolerated dose; NG, nanogel; NG/DOX, DOX-loaded nanogel; SD, standard deviation.

Figure 4

Tissue distribution. Notes: Ex vivo DOX fluorescence images (A) and semi-quantitatively analyzed average fluorescence intensities (B) of tumors and major visceral organs (ie, kidney, heart, spleen, lung, and liver) isolated at 12 hours postinjection of free DOX·HCl or NG/DOX at a dose of 3.0 mg or 6.0 mg DOX·HCl equivalent per kilogram body weight toward BALB/c nude mice bearing HepG2 tumors. The statistical data are represented as mean ± SD (n=3; ^#P<0.001). Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; NG/DOX, DOX-loaded nanogel; SD, standard deviation.

Figure 5

In vivo antitumor efficacies. Notes: In vivo antitumor efficacies of NS as control, and free DOX·HCl and NG/DOX at a dosage of 3.0 and 6.0 mg DOX·HCl equivalent per kg body weight toward H22 hepatoma-xenografted BALB/c mouse models. Each set of data is represented as mean ± SD (n=10; *P<0.001). Abbreviations: DOX, doxorubicin; DOX·HCI, doxorubicin hydrochloride; NG/DOX, DOX-loaded nanogel; NS, normal saline; SD, standard deviation.

Figure 6

Histopathological and immunohistochemical analyses. Notes: Histopathological (ie, H&E) and immunohistochemical (ie, caspase-3, survivin, Bax, and Bcl-2) analyses of tumor tissues sections after treatment of NS as control, or free DOX·HCl or NG/DOX at a dosage of 3.0 mg or 6.0 mg DOX·HCl equivalent per kilogram body weight. The arrows indicate the typical necrotic area. Magnification: 200×. Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; H&E, hematoxylin and eosin; NG/DOX, DOX-loaded nanogel; NS, normal saline.

Figure 7

Semi-quantitative analyses of histopathological and immunohistochemical results. Notes: Tumor necrotic area from H&E (A), and relative optical densities of tumor sections from caspase-3 (B), survivin (C), Bax (D), and Bcl-2 (E) after treatment with NS as control, or free DOX·HCl or NG/DOX at a dosage of 3.0 mg or 6.0 mg DOX·HCl equivalent per kg body weight. Each set of data is represented as mean ± SD (n=10; ^#P<0.05, ^&P<0.01, *P<0.001). Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; H&E, hematoxylin and eosin; NG/DOX, DOX-loaded nanogel; NS, normal saline; SD, standard deviation.

Figure 8

Body weight changes. Notes: Body weight changes of H22 hepatoma-bearing BALB/c mice in the course of treatment with NS as control, or free DOX·HCl or NG/DOX at a dosage of 3.0 mg or 6.0 mg DOX·HCl equivalent per kg body weight. Each set of data is represented as mean ± SD (n=10; *P<0.001). Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; NG/DOX, DOX-loaded nanogel; NS, normal saline; SD, standard deviation.

Figure 9

Histopathology analyses of visceral organ sections. Notes: Histopathology analyses of visceral organ sections, that is, heart, liver, spleen, lung, kidney, thymus, and marrow, from normal mice, or H22 hepatoma-xenografted BALB/c mice after treatment with NS as control, or free DOX·HCl or NG/DOX at a dosage of 3.0 mg or 6.0 mg DOX·HCl equivalent per kg body weight. The arrows in the heart, liver, and kidney sections indicate a certain myocardial damage and fracture, the blood oozing and nucleus shrinking of liver tissue, and nephrotoxicity judged from the glomerular mesangial cell proliferation, respectively. The virtual coil indicates the tumor metastasis in thymus. Magnification: 200×. Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; NG/DOX, DOX-loaded nanogel; NS, normal saline.

Figure 10

MMCR and WBC count. Notes: MMCR (A) and WBC count (B) of normal mice, or H22 hepatoma-xenografted BALB/c mice after treatment with NS as control, or free DOX·HCl or NG/DOX at a dosage of 3.0 mg or 6.0 mg DOX·HCl equivalent per kg body weight. Each set of data is represented as mean ± SD (n=10, *P<0.001). Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; NG/DOX, DOX-loaded nanogel; MMCR, marrow micronucleus cell rate; NS, normal saline; WBC, white blood cell; SD, standard deviation.

Figure 11

Biochemical analyses of corresponding organs. Notes: Evaluations of CK-MB (A, heart), LDH (B, heart), ALT (C, liver), AST (D, liver), BUN (E, kidney), and Cr (F, kidney) in corresponding internal organs of normal mice or H22 hepatoma-xenografted BALB/c mice after treatment with NS as control, or free DOX·HCl or NG/DOX at a dosage of 3.0 mg or 6.0 mg DOX·HCl equivalent per kg body weight. Each set of data is represented as mean ± SD (n=10, *P<0.001). Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; NG/DOX, DOX-loaded nanogel; NS, normal saline; CK-MB, creatine kinase isoenzyme; LDH, lactate dehydrogenase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine; SD, standard deviation.

Figure 12

Blood biochemical analyses. Notes: Evaluations of CK-MB (A), LDH (B), ALT (C), AST (D), BUN (E), and Cr (F) in serum of normal mice or H22 hepatoma-xenografted BALB/c mice after treatment with NS as control, or free DOX·HCl or NG/DOX at a dosage of 3.0 mg or 6.0 mg DOX·HCl equivalent per kg body weight. Each set of data is represented as mean ± SD (n=10, *P<0.001). Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; NG/DOX, DOX-loaded nanogel; NS, normal saline; CK-MB, creatine kinase isoenzyme; LDH, lactate dehydrogenase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine; SD, standard deviation.