Construction and application of a liver cancer-targeting drug delivery system based on core-shell gold nanocages

Abstract

Background: In order to achieve drug targeting and controlled release, we have successfully developed a novel drug release system DOX/AuNCs-PM-HA with gold nanocages (AuNCs) as photothermal cores, thermally responsive copolymer P(NIPAM-co-Am) (PM) as the near-infrared (NIR) stimuli gatekeeper and hyaluronic acid as a targeting ligand as well as a capping agent.

Methods: Cell uptake and cell viability were investigated. In vivo photoacoustic tomography imaging in H22 tumor bearing mice was analyzed for the tumor targeting effect of the nanocomplexes. Antitumor efficacy and the tissue distribution in vivo were investigated.

Results: In vitro results demonstrated that the DOX/AuNCs-PM-HA had significant anticancer activity against SMMC-7721 cells under NIR irradiation. Furthermore, in vivo photoacoustic tomography imaging of the nanocomplexes in H22 tumor bearing mice could indicate effective tumor targeting. Our studies on antitumor efficacy and the tissue distribution in vivo showed that many DOX/AuNCs-PM-HA nanocomplexes could efficiently accumulate at the tumor site so that they could inhibit the tumor growth effectively with limited side effects. The in vitro and in vivo results confirmed that the tumor-targeting and controlled-release drug system DOX/AuNCs-PM-HA with the combination of chemotherapy and photothermal therapy showed strong anti-tumor effect and would have great potential for future cancer therapy.

Conclusion: This tumor targeting DOX/AuNCs-PM-HA nanocomplex responded not only to the external stimuli of NIR, but also the internal stimuli of hyaluronidase, providing the potential for pinpointed and multi-stimuli responsive intracellular drug release.

Keywords: chemotherapy; drug delivery; hyaluronic acid; photoacoustic imaging; photothermal therapy; temperature-responsive polymers.

Figure 1

The temperature sensibility of PNIPAM and PM. Notes: (A) Different solution states of P(NIPAM-co-Am) at room temperature (a) and 42°C (b). (B) The LCST detected spectrophotometrically with the solution being heated at a speed of 1°C/min. The temperature of the initial polymer solution with 90% of initial transmittance (at 460 nm) was defined as the LCST. The red curve is the transmittance curve of PNIPAM and the black curve is the transmittance curve of PM. Abbreviations: Am, acrylamide; LCST, lower critical solution temperature; PM, copolymer of N-isopropylacrylamide and acrylamide; PNIPAM, poly(N-isopropylacrylamide).

Figure 2

Characterization of AuNCs-PM and DOX/AuNCs-PM. Notes: (A) TEM images of the AuNCs-PM. A magnified TEM picture of the AuNCs-PM is shown in the inset. (B) UV–Vis absorption spectra of AuNCs and AuNCs-PM. (C) Mean hydrodynamic diameters of AuNCs and AuNCs-PM plotted as a solution temperature function. (D) UV–Vis absorption spectra of AuNCs, DOX solution, AuNCs-PM and DOX/AuNCs-PM. Abbreviations: AuNCs, gold nanocages; AuNCs-PM, PM-grafted AuNCs; DOX, doxorubicin; DOX/AuNCs-PM, DOX-loaded and PM-grafted AuNCs; DOX/AuNCs-PM-HA, DOX-loaded, PM-grafted and HA-modified AuNCs; HA, hyaluronic acid; PM, copolymer of N-isopropylacrylamide and acrylamide; TEM, transmission electron microscopy; UV–Vis, ultraviolet–visible.

Figure 3

Characterization, photothermal conversion and the release profile of functionalized AuNCs. Notes: (A) Sizes of AuNCs, AuNCs-PM, DOX/AuNCs-PM and DOX/AuNCs-PM-HA. (B) Zeta potentials of AuNCs, AuNCs-PM, DOX/AuNCs-PM and DOX/AuNCs-PM-HA. (C) Photothermal curves of the AuNCs-PM-HA (AuNCs content at 40 μg/mL) and H₂O irradiated by NIR laser (808 nm, 1.5 W/cm²). (D and E) DOX release curves of DOX/AuNCs-PM-HA at pH 7.4 and pH 4.5, with or without HAase. (F) DOX release curves of DOX/AuNCs-PM-HA at pH 7.4 and pH 4.5 with NIR laser irradiation (808 nm, 1.5 W/cm², 7 min), with or without HAase. Abbreviations: AuNCs, gold nanocages; AuNCs-PM, PM-grafted AuNCs; DOX, doxorubicin; DOX/AuNCs-PM, DOX-loaded and PM-grafted AuNCs; DOX/AuNCs-PM-HA, DOX-loaded, PM-grafted and HA-modified AuNCs; HA, hyaluronic acid; HAase, hyaluronidase; NIR, near-infrared irradiation; PM, copolymer of N-isopropylacrylamide and acrylamide.

Figure 4

Cellular uptake of SMMC-7721 cells. SMMC-7721 cells stained by DAPI and cultured with DOX/AuNCs-PM and DOX/AuNCs-PM-HA (DOX and AuNCs content at 1.68 and 40 μg/mL, respectively) for 4 h at 37°C; magnification ×400. Notes: (A) DOX/AuNCs-PM, (B) DOX/AuNCs-PM-HA, (C) DOX/AuNCs-PM-HA+NIR (808 nm, 1.5 W/cm², 7 min). Blue fluorescence: nuclei stained by DAPI, red fluorescence: DOX in cells. Abbreviations: AuNCs, gold nanocages; DAPI, 4,6-diamidimo-2-phenylindole; DOX, doxorubicin; DOX/AuNCs-PM, DOX-loaded and PM-grafted AuNCs; DOX/AuNCs-PM-HA, DOX-loaded, PM-grafted and HA-modified AuNCs; DOX/AuNCs-PM-HA+NIR, DOX/AuNCs-PM-HA with NIR laser; HA, hyaluronic acid; NIR, near-infrared irradiation; PM, copolymer of N-isopropylacrylamide and acrylamide.

Figure 5

Cell viability of SMMC-7721 cells. Notes: (A) Cell viability of SMMC-7721 cells incubated for 24 h with different concentrations of AuNCs-PM-HA with or without NIR irradiation (808 nm, 1.5 W/cm², 7 min). (B) Viability of SMMC-7721 cells incubated for 24 h with DOX/AuNCs-PM-HA with or without NIR, free DOX (DOX content at 0, 0.42, 0.84, 1.26, 1.68 μg/mL) and AuNCs-PM-HA with NIR irradiation (808 nm, 1.5 W/cm², 7 min). *p<0.05, **p<0.01, and ***p<0.001 indicate significant difference between two groups. Abbreviations: AuNCs, gold nanocages; DOX, doxorubicin; DOX/AuNCs-PM-HA, DOX-loaded, PM-grafted and HA-modified AuNCs; HA, hyaluronic acid; NIR, near-infrared irradiation; PM, copolymer of N-isopropylacrylamide and acrylamide.

Figure 6

Fluorescence microscopy images of DOX in slices of tumors and other organs after injection (0.5 and 6 h) with normal saline (control), DOX solution (free DOX), DOX/AuNCs-PM-HA (200×). Notes: The scale bars represent 500 μm. The red fluorescence is expressed by released DOX. The tumors of DOX/AuNCs-PM-HA group were illuminated by NIR laser (1.5 W/cm², 808 nm, 7 min) at 0.5 and 6 h postinjection, respectively. Abbreviations: AuNCs, gold nanocages; DOX, doxorubicin; DOX/AuNCs-PM-HA, DOX-loaded, PM-grafted and HA-modified AuNCs; HA, hyaluronic acid; NIR, near-infrared irradiation; PM, copolymer of N-isopropylacrylamide and acrylamide.

Figure 7

In vivo photoacoustic tomography image of H22 tumor (marked as green)-bearing mice treated with normal saline, AuNCs, DOX/AuNCs-PM-HA (AuNCs content at 23.78 mg/kg). Note: The scale bars represent 3 mm. The tumor areas are marked by red ellipses. Abbreviations: AuNCs, gold nanocages; DOX, doxorubicin; DOX/AuNCs-PM-HA, DOX-loaded, PM-grafted and HA-modified AuNCs; H22, hepatocarcinoma 22; HA, hyaluronic acid; PM, copolymer of N-isopropylacrylamide and acrylamide.

Figure 8

In vivo treatment on H22 tumor-bearing mice with the DOX/AuNCs-PM-HA. Notes: (A) Body weight changes with times of H22 tumor-bearing mice after first treatment with different formulations (n=5, mean ± SD). (B) Hematoxylin and eosin staining of tumor sections collected from different treatment groups (normal saline [control], DOX solution, DOX/AuNCs-PM-HA, DOX/AuNCs-PM-HA+NIR [1.5 W/cm², 808 nm, 7 min] [DOX content at 2 mg/kg]). The scale bars represent 500 μm. Abbreviations: AuNCs, gold nanocages; DOX, doxorubicin; DOX/AuNCs-PM-HA, DOX-loaded, PM-grafted and HA-modified AuNCs; DOX/AuNCs-PM-HA+NIR, DOX/AuNCs-PM-HA with NIR laser; H22, hepatocarcinoma 22; HA, hyaluronic acid; NIR, near-infrared irradiation; PM, copolymer of N-isopropylacrylamide and acrylamide.

Figure 9

Histological observation of major organs (hearts, livers, spleens, lungs and kidneys) and tumor sections. The organs were collected from different groups after treatment with different formulations and were stained by H&E. Notes: (A) Normal saline (control), (B) DOX solution, (C) DOX/AuNCs-PM-HA and (D) DOX/AuNCs-PM-HA+NIR (1.5 W/cm², 808 nm, 7 min) (DOX content at 2 mg/kg). The scale bars represent 500 μm. Abbreviations: AuNCs, gold nanocages; DOX, doxorubicin; DOX/AuNCs-PM-HA, DOX-loaded, PM-grafted and HA-modified AuNCs; DOX/AuNCs-PM-HA+NIR, DOX/AuNCs-PM-HA with NIR laser; HA, hyaluronic acid; NIR, near-infrared irradiation; PM, copolymer of N-isopropylacrylamide and acrylamide.

Scheme 1

Schematic illustration for the preparation of the tumor-targeted, multi-stimuli responsive drug delivery system (DOX/AuNCs-PM-HA) as a combination of photoacoustic tomography and photothermal-chemotherapy. Note: **p<0.01 and ***p<0.001 represent significant difference between two groups. Abbreviations: DOX, doxorubicin; AuNCs, gold nanocages; P(NIPAM-co-Am), copolymer of N-isopropylacrylamide and acrylamide; PM, copolymer of N-isopropylacrylamide and acrylamide; HA, hyaluronic acid; DOX/AuNCs-PM-HA, DOX-loaded, PM-grafted and HA-modified AuNCs; IV, intravenous; NIR, near-infrared irradiation; photothermal-chemotherapy, combination of photothermal therapy and chemotherapy.