Local triple-combination therapy results in tumour regression and prevents recurrence in a colon cancer model

Abstract

Conventional cancer therapies involve the systemic delivery of anticancer agents that neither discriminate between cancer and normal cells nor eliminate the risk of cancer recurrence. Here, we demonstrate that the combination of gene, drug and phototherapy delivered through a prophylactic hydrogel patch leads, in a colon cancer mouse model, to complete tumour remission when applied to non-resected tumours and to the absence of tumour recurrence when applied following tumour resection. The adhesive hydrogel patch enhanced the stability and provided local delivery of embedded nanoparticles. Spherical gold nanoparticles were used as a first wave of treatment to deliver siRNAs against Kras, a key oncogene driver, and rod-shaped gold nanoparticles mediated the conversion of near-infrared radiation into heat, causing the release of a chemotherapeutic as well as thermally induced cell damage. This local, triple-combination therapy can be adapted to other cancer cell types and to molecular targets associated with disease progression.

Figure 1 |. The rationale behind the design of local triple-combination therapy.

a, Drug-gold nanorods and siRNA-gold nanospheres doped in Implantable hydrogels for local drug/gene delivery and local hyperthermia. b,c, Transmission electron micrographs of gold nanorods with an aspect ratio of 4.1 (40 × 10 nm) (b) and gold nanospheres with 20 nm diameter (c). The red arrows point to the negative staining of the coating layer. d,e, High-resolution SEM (d) and SEM/energy-dispersive X-ray (e) images of dextran-dendrimer hydrogel scaffolds. The gold content embedded in the hydrogels can be better appreciated in the magnified image (e, inset). f, Cryosection of dextran-dendrimer adhesive hydrogel (12 μm thickness) where dextran aldehyde from the hydrogel is labelled with Alexa Fluor 405 (in blue), RNAi nanospheres are tagged with DY647 (in red) and drug gold nanorods are labelled with Alexa Fluor 555 (in green).

Figure 2 |. Functional characterization and in vitro performance.

a, Spectra for drug-gold nanorods (yellow) and siRNA-gold nanospheres (red) at equal gold concentrations. b, Rate of temperature escalation for drug-gold nanorod (yellow) and siRNA-gold nanosphere (red) solutions irradiated with a 808 nm laser at 1 W. c, Kras expression in LoVo-Luc CRC cells at 24, 48 and 72 h of incubation with 1 and 10 nM siRNA-gold nanospheres functionalized with and without the HA1 peptide (n = 3, statistical analysis was performed with a two-tailed Student’s f-test, **, P < 0.01; P < 0.05). Kras levels were normalized to the GAPDH reference gene. d, Avastin (that is, drug) release from gold nanorods following increasing exposure to laser (10–120 s). e, Confocal microscope images and analytical flow cytometry of cellular uptake of drug-gold nanorods (in green) and siRNA-gold nanospheres (in red) by human 3T3 fibroblasts and LoVo-Luc CRC cells. f, Live-dead staining of CRC cells after uptake with drug-gold nanorods, siRNA-gold nanospheres or the combination 24 h after the application of a 808 nm laser at 1 W for increasing irradiation duration (red, dead cells; green, live cells). All experiments were done in triplicates and errors are reported as standard deviation (s.d.).

Figure 3 |. Selective and efficient local triple therapy improves CRC therapeutic efficacy.

a, Development of a smart hydrogei-nanopartlcie patch as a prophylactic scaffold agent for in vivo local gene/drug delivery combined with phototherapy, before and after surgical removal of the tumour In an in vivo mouse model of CRC. A tunable hydrogel patch impregnated with drug-siRNA nanoparticle conjugates (drug-gold nanorods and siRNA-gold nanospheres) for local gene and drug release in colorectal tumoral cells was designed. b, Experiment flow: on day 0, CRC cells were injected into mice (n = 5 per group). Tumour volumes were monitored and on day 15 hydrogels were implanted. NIR treatment was applied at days 18,19, 20 and 25 using a 808 nm laser at 1 W for 120 s. Mice were monitored in the following weeks for survival and tumour profiling. c, Fluorescence and thermographic images of the hydrogels doped with drug-gold nanorods, siRNA-gold nanospheres or their combination (n = 5). d, Thermographic surveillance of photothermal heating in mice (n = 5) implanted with hydrogels doped with drug-gold nanorods, siRNA-gold nanospheres or their combination, 72 h after the first NIR treatment.

Figure 4 |. Local triple-therapy combination results in complete tumour regression and recurrence elimination before and after tumour resection, respectively.

a, Live imaging of SCID hairless congenic mice with colorectal tumour xenografts implanted with hydrogels that are embedded with drug-gold nanorods and siRNA-gold nanospheres with NIR treatment, either with no tumour resection or after tumour resection (n = 5 per group). Ex vivo images of tumours and whole body organs (T, tumour; Lv, liver; K, kidneys; S, spleen; H, heart; Lu, lung; Int, intestines) are also depicted. b,c, Tumour burden following treatment as measured by luciferase activity, without tumour resection (b) and after tumour resection (c) (n = 5, statistical analysis was performed using two-way analysis of variance, ***, P < 0.001). Errors are reported as standard deviation (s.d.). d, Tumour burden of mice treated with gene therapy (siRNA-gold nanospheres), chemotherapy (drug-gold nanorods), phototherapy (gold nanorods) or double (chemo + gene, gene + photo, chemo + photo) and triple therapy (gene, chemo and phototherapy combination) (n = 5, statistical analysis performed with two-way analysis of variance, ***, P < 0.001), as measured by luciferase activity. Errors are reported as standard deviation (s.d.). e, Kaplan-Meier curves for mice treated with hydrogel scaffolds for gene therapy (siRNA-gold nanospheres), chemotherapy (drug-gold nanorods), phototherapy (gold nanorods) or triple therapy (gene therapy, chemotherapy and phototherapy combination). Statistical analysis (n = 5) was performed with a Log-Rank Mantel-Cox test (P = 0.008). Survival cutoff criteria included tumour ulceration or compassionate euthanasia, when the aggregate tumour burden >1 cm in diameter, or if the tumour impeded eating, urination, defecation or ambulation. f, Immunohistochemical evaluation of Ki67 for tumours treated with hydrogels alone or following triple therapy (gene therapy, chemotherapy and phototherapy combination). g, Histopathology and biodistribution analyses of tumour tissue from mice treated with triple-therapy combination for several time points (from 6 h to 15 days) (blue, nucleus, DAPI; red, RNAi nanospheres, DY647; green, antibody-drug nanorods, Alexa Fluor 555). Scale bars, 100 μm. Quantification of nanoparticle percentage distribution over time was calculated using unity-based normalization.

Figure 5 |. Altered tumour genetic profile in response to local therapy treatment.

a, Differential gene expression heat maps for GeneChip PrimeView Human Gene Expression Array of mice treated with gene therapy (siRNA-gold nanospheres) or chemotherapy (drug-gold nanorods) or phototherapy (gold nanorods) or triple therapy (gene, chemo and phototherapy combination), when compared with non-treated groups. b, Pathway analysis of significantly altered genes after single local therapy modalities: gene therapy, chemotherapy or phototherapy. c, Selection of potential gene targets (left, genes altered by phototherapy; right, genes altered by the three treatments) involved in tumour regression in response to local therapies. The whole-genome data are representative from three different tumours.

Figure 6 |. Canonical pathway kinetics in triple-therapy combination.

a, Differential gene expression heat maps for GeneChip PrimeView Human Gene Expression Array of mice treated with triple therapy (gene therapy, chemotherapy and phototherapy combination), for different time points of treatment: 6, 24, 48 and 72 h. b, Venn diagram from the differential gene expression heat maps of mice treated with triple therapy at different time points post-treatment. c, Pathway analysis of significantly altered genes after local combination triple therapy at different time points post-treatment. Selection of potential gene targets involved in tumour regression in response to local triple therapy over time. The whole-genome data are representative out of three different tumours.