Novel Photo- and Thermo-Responsive Nanocomposite Hydrogels Based on Functionalized rGO and Modified SIS/Chitosan Polymers for Localized Treatment of Malignant Cutaneous Melanoma

Abstract

Melanoma is an aggressive type of skin cancer that accounts for over 75% of skin cancer deaths despite comprising less than 5% of all skin cancers. Despite promising improvements in surgical approaches for melanoma resection, the survival of undetectable microtumor residues has remained a concern. As a result, hyperthermia- and drug-based therapies have grown as attractive techniques to target and treat cancer. In this work, we aim to develop a stimuli-responsive hydrogel based on chitosan methacrylate (ChiMA), porcine small intestine submucosa methacrylate (SISMA), and doxorubicin-functionalized reduced graphene oxide (rGO-DOX) that eliminates microtumor residues from surgically resected melanoma through the coupled effect of NIR light-induced photothermal therapy and heat-induced doxorubicin release. Furthermore, we developed an in silico model to optimize heat and mass transport and evaluate the proposed chemo/photothermal therapy in vitro over melanoma cell cultures.

Keywords: COMSOL multiphysics; melanoma; methacrylation; photothermal therapy; reduced graphene oxide.

FIGURE 1

Proposed approach: a SISMA/ChiMA/rGO composite hydrogel employed as a platform for chemo/photothermal therapy to target microtumor residues that remain after the surgical resection of cutaneous melanoma. When stimulated with a NIR laser, the rGO in the hydrogel generates local hyperthermia by converting light into heat. Subsequently, DOX is released from the hydrogel, as the increasing temperature breaks the azo bonds that link the anticancer drug to the GO vehicle.

FIGURE 2

Methacrylation of SIS and chitosan. (A) Activation of methacrylic acid mediated by EDC and NHS. (B) Chemical conjugation of MA’s carboxyl groups to free primary amines of collagen in SIS. (C) Chemical conjugation of MA’s carboxyl groups into the free amines of the glucosamine units of chitosan. (D) Methacryloyl group destabilization and crosslinking are induced by the light-directed degradation of a photoinitiator.

FIGURE 3

Loading of DOX into a GO vehicle. (A) Activation of graphene oxide (GO) mediated by EDC and NHS. (B) Chemical conjugation of the v50 thermo-linker to GO. (C) Conjugation of doxorubicin (DOX) to the GO-v50 nanoconjugate using glutaraldehyde as a crosslinker.

FIGURE 4

Geometry and boundary conditions for the NIR-induced heating and doxorubicin-controlled release model involving tumor residual cells after the removal of malignant cutaneous melanoma and the surrounding skin layers of healthy tissue.

FIGURE 5

Thermal and spectroscopic analyses of pristine and modified GO. (A) TGA of GO. (B) FTIR spectra of graphite, pristine GO, GO-v50 nanoconjugate, and GO-v50-DOX nanoconjugate. (C,D) Raman spectra of graphite, pristine GO, GO-v50 nanoconjugate, and GO-v50-DOX nanoconjugate.

FIGURE 6

Microscopy of pristine and modified GO. (A) AFM images of GO, (B) Sheet height of pristine GO, and TEM images of (C) pristine GO, (D) GO-v50 nanoconjugate, and (E) GO-v50-DOX nanoconjugate.

FIGURE 7

Rheology of the ChiMA 1% (C1), SISMA 2% (S2), SISMA 1%/ChiMA 1% (S1C1), SISMA 2%/ChiMA 1% (S2C1), and SISMA 2%/ChiMA 1%/graphene oxide (S2C1GO) hydrogels. (A) Manual injection of the hydrogels through a 21-gauge needle. (B) Flow sweeps of photocrosslinked samples. (C) Time sweeps before and after blue-light photocrosslinking. (D) Temperature sweeps of photocrosslinked samples between 5 and 37°C. (E) Frequency sweep of photocrosslinked samples.

FIGURE 8

Dispersion of GO-DOX nanoconjugates and morphological features of the nanocomposite hydrogel. Dispersion of (A) pristine GO, (B) DOX, and (C) GO-DOX nanoconjugates in the SISMA/ChiMA matrix. (D) Particle size distribution of pristine GO, seen as a right tail distribution centered at 0.096 µm2 (E) SEM images of the microporous structure of the SISMA 2%/ChiMA 1%/graphene oxide (S2C1GO) formulation prior to and after photocrosslinking.

FIGURE 9

Mechanical and texture evaluation of the SISMA 2%/ChiMA 1% (S2C1) and SISMA 2%/ChiMA 1%/graphene oxide (S2C1GO) nanocomposite hydrogels. (A) Qualitative adhesion of skin cut wounds at 0, 1, and 3 days after application of the hydrogels. (B) Hardness, (C) compressibility, (D) cohesiveness, and (E) adhesiveness of the hydrogels before (w/o) and after (w/) photocrosslinking (pcl) and 2-weeks incubation (r) at 37°C.

FIGURE 10

Biocompatibility evaluation of the pristine graphene oxide (GO), graphene oxide/v50 (GO-v50) and graphene oxide/v50/doxorubicin (GO-v50-DOX) nanoconjugates and the ChiMA 1% (C1), SISMA 2% (S2), SISMA 2%/ChiMA 1% (S2C1), and SISMA 2%/ChiMA 1%/graphene oxide (S2C1GO) hydrogels. Hemolytic activity of the (A) nanoconjugates and the (B) composite hydrogels, using triton and phosphate-buffered saline (PBS) as positive and negative controls. Platelet aggregation of the (C) nanoconjugates and the (D) composite hydrogels using platelet-rich plasma (PRP) and PBS as positive and negative controls. A-375 melanocytes’ viability evaluation after 24 h of exposure to the (E) GO-v50 and GO-v50-DOX nanoconjugates and the (F) nanocomposite hydrogels. Cell viability after 72 h of exposure to the (G) GO-v50 and GO-v50-DOX nanoconjugates and the (H) composite hydrogels.

FIGURE 11

Heat transfer and transport of diluted species results for the chemo/photothermal therapy. (A) Temperature profile due to NIR-induced heating in the skin multilayer model. (B) Heat transport with respect to time at locations of interest along with the different components of the computational domain. (C) Doxorubicin release profile and streamlines. (D) Time evolution of doxorubicin mass diffusion after thermo-linker breakup. (E) Degree of tissue injury due to NIR-induced injury as calculated by the Henriques-Moritz equation. (F) Optimization function for heating the tumor tissue relative to the healthy tissue.

FIGURE 12

In vitro evaluation of the chemo/photothermal therapy. The temperature profile of (A) SISMA 2%/ChiMA 1% (S2C1) and (B) SISMA 2%/ChiMA 1%/graphene oxide (S2C1GO) hydrogels after 3 min irradiation with NIR. Photothermal heating of S2C1 and S2C1GO during (C) a first therapy cycle (T1) and (D) subsequent second (T2) and third (T3) therapy cycles. (E) Concentration of DOX released from S2C1GO for the different therapies using simulations (in silico) and unirradiated hydrogels with (w/) and without (w/o) ascorbic acid (aa) as positive and negative controls.