Formulation of Smart Hydrogels with Embedded Nanoparticles for Targeted Melanoma Treatment 

By: Emma Thiboutot

Background Information 

The problem discussed within this proposed project is melanoma skin cancer, with a focus on its impact and the current treatment options available. Every year, there are approximately 5.4 million cases of skin cancer diagnosed in the United States alone. This makes it the most common type of cancer (Skin Cancer Foundation, 2023). The default treatments for melanoma include, but are not limited to, chemotherapy, radiation, and surgical interventions. All of which are invasive and often significantly burden patients. These treatments are not limited to affecting the patient physically, but they can also impose financial strains during and after recovery, ultimately impacting the patient’s quality of life negatively beyond their diagnosis. Furthermore, the importance of addressing this problem goes beyond individual cases, but also affects families, the healthcare system, and society at large. Melanoma treatments are very expensive, and they can emotionally, physically, and financially drain the patients and their families. An effective treatment that manages skin cancer requires improvements in public health care policies, which would help to reduce the prevalence of skin cancer, enhance the current care options, and ensure equal access to prevention and treatment options. There are current innovations for the treatment of skin cancer that have included the development of hydrogels for localized and controlled drug delivery. This helps in minimizing side effects and improving the efficiency of the drugs that are delivered at the tumor site. There have been studies conducted by Ye et al. (2018) and Zheng et al. (2015) that have highlighted the use of injectables and pH-responsive hydrogels that have been shown to directly deliver multiple anti-cancer agents to affected tumors and sites. This would enhance therapeutic efficiency while sparing healthy tissues from the harmful effects of anti-cancer drugs. Additionally, there has been research conducted by Elhabal et al. (2024) and Ahmed et al. (2023) to support using heat-responsive and ROS-responsive hydrogels for localized cancer therapy. These studies highlight further advancements in responsive and targeted drug delivery systems. Although all these studies show research promise in optimizing anti-cancer drug delivery systems, there are challenges that still exist regarding biocompatibility and production scaling.

Research Question(s)/Thesis Statement/Aims

The proposed solution to this problem is to develop a novel hydrogel system loaded with an anti-cancer drug, doxorubicin, and an NIR responsive dye, indocyanine green, to treat melanoma skin cancer non-invasively with SMART-activated drug-releasing hydrogels. The project’s goal is to prove that these hydrogels can effectively target and eliminate melanoma cells with minimal impact on surrounding healthy tissues. The specific question that is being researched throughout this question is: Can PVA-chitosan hydrogel encapsulating doxorubicin and light-sensitive compounds demonstrate a significant cytotoxicity against melanoma cells? The research will focus on creating a PVA-chitosan crosslinked hydrogel loaded with an anti-cancer drug and SMART compounds.

Objectives of the Proposed Project

The completion of this project will contribute significantly to the field of skin oncology by providing a more targeted and potentially more effective treatment for melanoma with reduced side effects. Furthermore, this research could provide a foundation for future studies to explore the use of drug delivery in hydrogel formulations for various types of cancer. It could also influence the design of multifunctional biomaterials with engineered delivery systems.

Description of the Proposed Work

This project is an in vitro study, meaning it does not involve human participants but instead uses A375 cell lines, a type of skin cancer cell. These cells will be sourced from Dr. Tracie Ferreira at the University of Massachusetts–Dartmouth’s Bioengineering Department. Additional cells required will be purchased from Sigma-Aldrich. The research will be experimental, conducted in a controlled laboratory setting to test the effectiveness of different hydrogel formulations on melanoma cells. The experimental design for this project will include four treatment groups. First, there will be the formulated hydrogel without any loading, which will be the control. Then, there will be the formulated hydrogel loaded with indocyanine green for its activation due to near-infrared light. Then, there will be the hydrogel loaded with doxorubicin, a chemotherapy drug. Finally, the last formulation of the hydrogel will be the combination of doxorubicin and indocyanine green to offer a controlled drug release at the affected site. The main focus of this study is to observe the viability of melanoma cells after treatment with hydrogels. Cell viability, the dependent variable, will be assessed through the cell viability assay Alamar Blue, which measures metabolic activity, and live/dead cell assays. These tests help quantify data by measuring changes in fluorescence, which are then scored as a percentage of viability compared to untreated control groups. The research will unfold in two phases: 1. In the first phase, we will create light-responsive PVA-chitosan hydrogels containing the doxorubicin and/or the indocyanine green. We will evaluate the physical and chemical properties of these hydrogels, such as drug encapsulation efficiency and release kinetics, using several techniques: Scanning Electron Microscopy (SEM) to visualize the surface structure of the hydrogels, Thermogravimetric Analysis (TGA) to assess the thermal stability, Differential Scanning Calorimetry (DSC) to study the thermal properties, and Fourier-Transform Infrared Spectroscopy (FTIR) to identify the chemical composition. These characterizations will measure properties like light responsiveness, color changes, and drug delivery times. 2. In the second phase, we will test these hydrogels in vitro using A375 melanoma cell cultures in cell culture inserts. The response of these cells to the hydrogel treatment will be monitored by measuring cell viability using Alamar Blue and a live/dead cell assay conducted through Confocal Microscopy. Data collection will be electronic, ensuring accurate, real-time capture during experiments, and analyzed using software like GraphPad Prism.

Clear Statement of the Originality of the Proposed Work

This research is original in its approach by incorporating cranberry extract, a natural substance, into hydrogel formulations for treating melanoma, an application not extensively explored in current literature, and comparing its efficacy to a chemotherapy drug, doxorubicin, with the same hydrogel delivery system. This novel approach could provide significant insights into the potential of natural compounds in cancer treatment, challenging traditional methods and potentially setting a precedent for future therapeutic developments. All laboratory procedures will adhere to standardized protocols for the ethical handling and treatment of cell cultures, data recording, and analysis.

Materials and Methods

Hydrogel Preparation

The PVA-chitosan hydrogels were prepared using a freeze-thaw and genipin crosslinking method to utilize both physical and chemical crosslinking methods. Medium molecular weight chitosan was dissolved in 9 mL of 2% (v/v). lactic acid, and subsequently 3 mL of 10% (w/v) poly(vinyl alcohol) (PVA) solution, prepared in distilled water, was added under continuous stirring. For crosslinking, 18 μL of genipin was incorporated into the mixture. Formulations were created into four groups: CP6 (control), CP6-DOX (loaded with 10 mg doxorubicin), CP6-ICG (loaded with 10 mg of indocyanine green), and CP6-DOX-ICG (co-loaded with 10 mg each of DOX and ICG). Drugs were added during the mixture stage and before the freeze-thaw cycles. The mixtures were cast into petri dishes and subjected to three freeze-thaw cycles (freezing at -20 °C for 18 hours and followed by 6 hours of thawing at room temperature). Additionally, the hydrogels were allowed to crosslink at room temperature on the bench for an additional 3 days.

Freeze-drying and Sample Preparation

All hydrogel samples were freeze-dried for 12 hours before thermal and morphological characterization tests. Freeze-dried samples were used directly for SEM imaging without sputter coating, due to equipment limitations.

Tensile Strength Testing

Mechanical testing was performed using a Shimadzu tensile tester to assess the tensile strength of each hydrogel formulation. The samples were tested after crosslinking.

Thermal Analysis

Thermogravimetric analysis and differential scanning calorimetry were done to evaluate the thermal stability and phase transitions of the hydrogel formulations. Differential Scanning Calorimetry (DSC) analysis was done using the Thermal Analyzer DC25. An average of 7 mg samples were used for the DSC, and an average of 15 mg samples were used for the TGA after freeze drying. TGA was performed from 30 °C to 500 °C under a nitrogen atmosphere, while DSC was carried out from 30 °C to 400 °C to capture water loss and the polymer transitions.

Scanning Electron Microscopy

The surface and cross-section areas of the hydrogels were analyzed using a HITACHI 3700nVP scanning electron microscope (SEM) at an accelerating voltage of 2 kV. Samples were mounted onto the stubs without sputter coating and imaged at magnifications x50, x150, and x250. Cross-section samples were prepared by cutting the freeze-dried hydrogels and positioning them vertically.

Swell Test

The swelling behavior of the hydrogel formulations CP6, CP6-ICG, and CP6-DOX was evaluated in three different solutions: ultra-pure water, Dulbecco’s Modified Eagle Medium (DMEM), and phosphate-buffered saline (PBS, pH 7.4). Freeze-dried hydrogel samples were individually weighed to obtain an initial dry weight and then submerged in 5 mL of the respective medium. Samples were incubated at 37 °C to mimic physiological conditions. At time intervals of 5 min, 10 min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 24 hr, 30 hr, 48 hr, 72 hr, 96 hr, 120 hr, 7 days, 8 days, 9 days, and 10 days. Hydrogels were gently removed from the medium, blotted with filter paper to remove surface moisture, and weighed. Between measurements, all samples were returned to the incubator in fresh medium to continue swelling under consistent conditions.

Cell Viability Assay (alamarBlue™)

Cell viability was assessed using the alamarBlue™ assay to evaluate the cytotoxic effects of CP6 hydrogel formulations. A375 human melanoma cells were seeded in 96-well plates at a density of 25,000 cells per well and incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 hours to allow cell attachment. Treatment groups included direct contact and cell culture inserts of each of the formulations: CP6, CP6-ICG, CP6-DOX, and CP6-DOX-ICG. Additional groups were exposed to near-infrared (NIR) laser irradiation (808 nm) for photothermal activation when applicable.

For insert-based experiments, the sterile hydrogels were prepared and placed into inserts suspended above seeded cells to simulate indirect drug exposure. NIR-activated groups were exposed for 10 minutes before incubation. Alamar Blue reagent was added to each well at 24, 48, and 72 hours post-treatment. After 2 hours of incubation with the reagent, absorbance was measured at 570 nm and 600 nm using a microplate reader. Cell viability was calculated and normalized to untreated control cells and the working solution of Alamar Blue.

Results and Discussion

Tensile Strength

Tensile strength testing and data were collected using the Shimadzu machine that has been provided for use in the biomechanics laboratory. The tensile strength testing was conducted on three different formulations of the PVA-chitosan hydrogels. There was the CP formulation, the ICG incorporated CP formulation, and the NPC incorporated hydrogel, which includes the ICG and cranberry nanoparticles. The objective of this testing was to evaluate how the baseline hydrogel performs, while also evaluating how the hydrogel performs when there are added compounds into it under induced tensile load for real-time applications in the future. These tests have determined maximum and minimum force with stained, average tensile strength, and standard deviation to assess the strength and the consistency based on all different types of hydrogel formulation.

Force	N
Average	0.409954
SD	0.297193
Max	1.038734
Min	0.05095

Table 1: A table representing the CP hydrogel formation tensile data, including the average, standard deviation, maximum, and minimum forces in Newton values.

 

Displacement	mm
Average	20.6691
SD	11.93479
Max	41.32924
Min	0.0021

Table 2:A table representing the CP hydrogel formation tensile data, including the average, standard deviation, maximum, and minimum displacements in millimeter values.

 

Figure 1: This is the force versus displacement curve for the CP hydrogel. This graph displays the typical nonlinear tensile behavior with minimal outliers and a linear trendline. The trendline equation of y=0.051x-0.0959 represents the best-fit linear relationship approximation of the sample’s tensile behavior.

 

Force	N
Average	0.368162
SD	0.323111
Max	1.173194
Min	-0.03352

Table 3: A table representing the ICG hydrogel formation tensile data, including the average, standard deviation, maximum, and minimum forces in Newton values. 

 

Displacement	mm
Average	20.6691
SD	11.93479
Max	41.32924
Min	0.0021

Table 4: A table representing the ICG hydrogel formation tensile data, including the average, standard deviation, maximum, and minimum displacements in millimeter values.

 

Figure 2: This is the force versus displacement curve for the ICG hydrogel. This graph displays the typical nonlinear tensile behavior with minimal outliers and a linear trendline. The trendline equation of y=0.0258x-0.1655 represents the best fit linear relationship approximation of the sample’s tensile behavior.

 

Force	mm
Average	0.301788
SD	0.228036
Max	0.798583
Min	-0.04087

Table 5: A table representing the DOX hydrogel formation tensile data, including the average, standard deviation, maximum, and minimum forces in Newton values.

 

Displacement	mm
Average	12.21906
SD	7.05618
Max	24.42903
Min	0.001833

Table 6: A table representing the NPC hydrogel formation tensile data, including the average, standard deviation, maximum, and minimum displacements in millimeter values.

 

Figure 3: This is the force versus displacement curve for the DOX hydrogel. This graph displays the typical nonlinear tensile behavior with minimal outliers and a linear trendline. The trendline equation of y=0.0317x-0.0851 represents the best fit linear relationship approximation of the sample’s tensile behavior.

 

CP6 (1)		ICG (1)		DOX (1)
Force	N	Force	N	Force	N
Average	0.409954	Average	0.368162	Average	0.301788
SD	0.297193	SD	0.323111	SD	0.228036
Max	1.038734	Max	1.173194	Max	0.798583
Min	-0.05095	Min	-0.03352	Min	-0.04087

CP6 (1)		ICG (1)		PCN (1)
Displacement	mm	Displacement	mm	Displacement	mm
Average	9.922375	Average	20.6691	Average	12.21906
SD	5.730193	SD	11.93479	SD	7.05618
Max	19.83097	Max	41.32924	Max	24.42903
Min	0.0018	Min	0.0021	Min	0.001833

Table 7: Combined summary of tensile force and displacement data collected from all hydrogel groups: CP, ICG, and DOX. This table shows a direct comparison of the mechanical performance between the three formulations.

 

Figure 4: A bar graph comparing the average tensile force in newtons of all three hydrogel formulations, labeled respectively with a 5% error bar for each, indicating the relative variability. Hydrogel CP shows the highest average force, suggesting the strongest mechanical integrity, while DOX shows the lowest average force, suggesting the weakest mechanical integrity. Average values are listed on top of the bar graph.

 

Figure 5: A bar graph comparing the average displacement in millimeters of all three hydrogel formulations, labeled respectively with a 5% error bar for each, indicating the relative variability. The ICG hydrogel shows the greatest deformation before failure, suggesting that it has the highest elasticity of all of the hydrogel formations. Contrary, hydrogel CP displayed the least elasticity. Average values are listed on top of the bar graph.

The tensile strength testing data that have been collected provide valuable insights into how the formulation modification of the PVA-chitosan hydrogel affects its mechanical integrity, specifically in terms of the tensile strength and elasticity. When comparing the hydrogel formulations CP, ICG, and DOX, there were clear trends that can be observed regarding the average force, maximum displacement, and the variability between all three hydrogels.

The CP hydrogel was observed to have an average tensile strength of 0.41 N with a relatively low standard deviation. This supports the idea that the original hydrogel formulationwith no extra additions, forms a well-structured and dense hydrogel. The CP hydrogel does have a relatively lower average displacement, which further supports its classification as a stiffer material. The CP hydrogel is concluded to be a stiffer material and the best option for structural support applications compared to the other formulations.

The ICG hydrogel that incorporates the indocyanine dye shows the highest maximum tensile force of 1.17 N, but it also has the highest variability. This suggests that although the ICG hydrogel can withstand a higher maximum tensile force, due to perhaps a localized stiffness, its overall impact is less consistent due to its lower average of 0.37 N than the CP hydrogel. Furthermore, the ICG hydrogel had the greatest displacement with a value of 20.67 mm, which indicates a higher elasticity. To point out a balance between the two measurements, the ICG hydrogel has a mechanical consistency and responsiveness, making it potentially useful for drug delivery applications in the future.

The DOX hydrogel has both ICG and cranberry nanoparticles incorporated into it. These hydrogels displayed the lowest average force of 0.3 N and a median average displacement of 12.22mm. However, it did have the lowest standard deviation, which suggests that the mechanical testing was the most consistent across all of the differently formulated hydrogels. The hypothesis is that the nanoparticles that are embedded in the hydrogel affect the crosslinking density, which, as a result, weakens the tensile strength. Alternatively, this shows the potential for the improvement of uniformity if well dispersed. This makes the DOX hydrogel the best fit for a controlled delivery system, with emphasis on when the mechanical load is minimal.

Furthermore, the trendlines that are displayed respectively to each hydrogel formation quantify the slope of the force versus displacement relationship. A steeper slope, like the CP hydrogel, corresponds to the higher stiffness, while a shallower slope, like the ICG and DOX hydrogels, corresponds to a more elastic behavior. These equations are represented in the form of y=mx+b, where the slope is the rate of increase in force with respect to the displacement, which means how much force is required to stretch the material. For the CP formulation, the equation is:

= 0.051 − 0.0959

The slope of 0.51 shows that the CP hydrogel needs a higher amount of force per millimeter of stretch, which is supported by the high tensile strength average and low displacement average, and confirms the stiffness of the CP hydrogel. In contrast, the ICG hydrogel displayed an equation of:

= 0.0258 − 0.1655

This shallower slope of 0.0258 shows that the ICG hydrogel is more elastic and displays a more stretchable nature, which requires less force per millimeter. For the DOX hydrogel that contains the ICG and cranberry nanoparticles, the equation that is displayed is:

= 0.0317 − 0.0851

The slope of 0.0317 demonstrates that this hydrogel is more deformable than CP6, but it is more resistant than the ICG hydrogel. The PCN hydrogel displays the lowest average force of 0.302 N but has a moderate displacement of 12.22 mm. However, the PCN hydrogel’s trendline displays a relatively uniform response to force loading and is supported further by its low standard deviation. It is important to note that these slope values are representative of Young’s Modulus.

It is also important to note that only one sample from each hydrogel formulation was successfully tested due to the time constraints and issues with sample integrity. Several other samples of each formulation were mechanically damaged before testing, or they could not be tested in time for the submission of this report. This limits the statistical prevalence of the findings. For the future, results should include multiple test samples of each hydrogel formulation.

Fourier Transform Infrared Analysis

Figure 6: The FTIR results are shown above, visually compared. CP6 is the unloaded hydrogel, CP6-DOX is a doxorubicin-loaded hydrogel, CP6-ICG is the indocyanine green-loaded hydrogel, and CP6-DOX-ICG is the doxorubicin and indocyanine green-loaded hydrogel. The plot visually shows absorption peaks for each formulation.

The FTIR results confirm successful crosslinking and a successful functional integration of the ICG and Doxorubicin within the CP6 base hydrogel. All of the different formulations do show a similar baseline upon further analysis, suggesting that the chemical backbone of the hydrogels remains stable throughout different variations of loading withstood.

For the CP6 hydrogel, the absorption span was observed to be around 3200-3400 cm-1. This observation corresponds to the PVA and chitosan’s hydrogen bond interactions and the presence of the hydroxyl and amine groups. The peak is around 1640 cm-1, which is attributed to double-bonded oxygen and carbon, which confirms the genipin crosslinking with chitosan’s primary amine groups. Other peaks observed range around 1050-1150 cm-1 and correspond to C-O-C stretching, indicating the polysaccharide backbone.

For the CP6-Doxorubicin hydrogel, there are observed subtle increases in peak intensities near 1600 cm-1 and 1250 cm-1, which could indicate possible hydrogen bonding between the doxorubicin and hydrogel network. For the CP6-ICG hydrogel, the integration of the ICG is observed to shift towards higher wavenumbers, which is potentially due to the dispersion of ICG ’sulfonate groups within the hydrogel.

For the CP6-DOX-ICG hydrogel, both features unique to each compound, as observed above, are present, but they do not introduce any significant peaks outside of the expected functional group regions. This would suggest that the doxorubicin and the indocyanine green are physically entrapped within the hydrogel rather than chemically bonded to the hydrogel structure.

Thermogravimetric Analysis

Figure 7: TGA analysis of the CP6 hydrogel formulations. The CP6 base hydrogel (top left), CP6-ICG (top right), CP6-DOX (bottom left), and CP6-DOX-ICG (bottom right) were subjected to the TGA to assess the thermal stability. All samples did display degradation with initial water loss and were then followed by polymer decomposition. The doxorubicin-loaded and the indocyaninegreen-loaded hydrogel exhibited lower thermal stability compared to the base CP6 hydrogel, while CP6-DOX-ICG showed the greatest total weight loss.

The thermogravimetric analysis was performed to evaluate the thermal stability and decomposition behavior of the CP6 base hydrogel and its indocyanine green and doxorubicin-loaded formulations. All of the samples exhibited a multi-step thermal degradation pattern, visually showing the composition of the PVA-chitosan crosslinked hydrogel network and the presence of two drug molecules.

The CP6 hydrogel showed an initial weight loss below 150 °C, which corresponds to the evaporation of physically bound water. The major weight loss occurred between 200-350 °C, representing the thermal composition of the polymer’s backbone, specifically regarding the breakdown of PVA and chitosan chains, as well as the cleavage of genipin crosslinks.

The drug incorporation was shown to have altered the degradation profile. CP6-ICG displayed a slightly earlier onset of degradation, potentially due to the light sensitivity of the ICG and its destabilizing effect on the hydrogel’s structure at elevated temperatures. Similarly, the CP6-DOX showed an increased weight loss in to 20-250 °C range, which suggests that the interactions between the doxorubicin in the hydrogel may have influenced the crosslink density or stability.

The CP6-DOX-ICG formulation showed the greatest mass loss in the higher temperature range above 350 °C. The lower residual weight observed in CP6-DOX-ICG that’s relative to CP6, supports the hypothesis that drug incorporation decreases the thermal stability of the hydrogel, possibly due to the disruption of hydrogen bonds.

Differential Scanning Calorimetry

Figure 8: The DSC thermograms of the CP6 hydrogel formulations. The CP6 base hydrogel (topleft), CP6-ICG (top right), CP6-DOX (bottom left), and CP6-DOX-ICG (bottom right). All samples display endothermic transitions associated with water evaporation and polymer relaxation. Drug loading does alter the thermal transitions, which suggests changes in crystallinity and crosslinking within the hydrogel.

The DSC was used to show the thermal transitions and the energetic behavior of the CP6 hydrogel and its loaded drug formulations. The DSC curves of all of the formulations display multiple endothermic events and indicate the different thermal transitions.

The first large endothermic peak below about 150 °C observed in all of the formulations does correspond to the evaporation of bound and unbounded water molecules, which is to be expected of the hydrophilic nature of PVA-chitosan hydrogels. Additionally, the thermal transitions between 150 C-300 °C reflect the disruption of crystalline regions. In the CP6 hydrogel, this is likely associated with the breakdown of hydrogen bonding and the crystalline area in PVA and chitosan, as well as the genipin crosslinking sites.

Drug incorporation did influence the transitions. The CP6-DOX and the CP6-ICG show shifts in the second endothermic peak, which suggests that the doxorubicin and indocyanine green interfered with the crystallinity or the crosslinking density. The CP6-DOX-ICG formulations show the most evident intensity change over time, showing reduced crystallinity and increased amorphous character due to the entrapment of the drugs.

These results support the hypothesis that the incorporation of indocyanine green and doxorubicin disrupts the physical formation of the hydrogel, which can influence swelling behaviors, degradation rates, and drug release kinetics. The decreased sharpness and intensity of thermal transitions in the CP6-DOX-ICG formulation indicate an enhanced molecular dispersion of the drugs.

Scanning Electron Microscopy

Figure 9: SEM images of the CP6 hydrogel. The micrographs show the top surface (left) and cross-section (right) morphology at different magnifications. The top view reveals a fibrous and interconnected surface, while the cross-sectional images display a porous internal structure with well-defined pores.

The SEM analysis of the CP6 hydrogel revealed a porous network at both the surface and within the cross-section, which is a characteristic of the freeze-thaw method. The top-view viewimages display the interconnected surface with micro- and nano-scale features, while the cross-section views show the well-distributed pores ranging from approximately 100-300 μm. These pores were supported by thin polymer walls, which indicates a stable structure but a highly permeable scaffold. The observed porosity and interconnected structure are ideal for facilitating drug diffusion and nutrient transport. This confirms the hydrogel’s suitability for localized drug delivery.

Figure 10: SEM image of the CP6-DOX hydrogel. The micrographs show the surface (left column)and cross-section (right column) views at magnifications x50, x150, and x250. The images reveal a porous and irregular structure with increased pore density and variability compared to the base CP6 hydrogel. Pores range from 100 to 300 micrometers and appear more disorganized, which suggests that doxorubicin incorporation alters the internal structure.

SEM imaging of the CP6-DOX hydrogel reveals a porous and heterogeneous microstructure. The top-view images show a higher density of rounded and irregular pores, suggesting that doxorubicin incorporation disrupted the uniformity of the hydrogel structure and potentially interfered with the crosslinking and an increase in free volume. Cross-section images revealed more interconnected pores with thinner walls and some collapsed areas, as noted when the magnification increased. This indicates a softer internal structure. The pore sizes ranged from 100 to 300 micrometers, with increased frequency and irregularity. This may enhance drug diffusion but could also impact mechanical stability. These structural modifications are consistent with the enhancement of drug loading capacity and altered swelling or degradation behaviors that are associated with doxorubicin integration.

Figure 11: SEM image of the CP6-ICG hydrogel. The surface (left) and cross-section (right)images at x50, x150, and x250 magnifications illustrate a more compact and less porous structure compared to the CP6 and CP6-DOX. The Cp6_ICG shows reduced pore size and density, showing structural tightening.

The SEM imaging of the CP6-ICG hydrogel revealed a denser and less porous morphology compared to the CP6 and CP6-DOX formulations. The top-view images showed a compact surface with limited pore openings, which shows that indocyanine green may have disrupted the formation of larger pores or caused partial pore collapse during the freeze-thaw cycles. The cross-section images at x150 and x250 magnifications do show the presence of layers and elongated pores. This could suggest that there is a tighter internal structure that could slow down water uptake and drug diffusion. The reduced porosity and thicker pore walls do show a stabilizing effect of ICG on the polymer, which could enhance structural integrity and potentially limit diffusivity.

Figure 12: SEM image of the CP6-DOX-ICG hydrogel. Surface (left) and cross-sections (right)views at x50, x150, and x250 magnifications show a rough and heterogeneous morphology. The dual-drug formulation exhibits large and irregular pores, indicating disruption of the internal structure and suggesting a disruption of the structure from the incorporation of the ICG and doxorubicin.

The SEM images of the CP6-DOX-ICG hydrogel revealed a heterogeneous and highly porous structure as a result of the combined incorporation of the doxorubicin and ICG. The top-view micrographs show a rough surface with visible grooves and variable pore openings, which indicates the disrupted polymer packing. Cross-section images displayed a well-developed internal structure with large and irregularly shaped pores ranging from 100-300 micrometers in size with thin polymer walls. This structure suggests enhanced porosity and diffusion paths that could facilitate controlled delivery of both agents. However, the uneven pore distribution could lead to a possible compromise in the structure due to the competing interactions of the indocyanine green and the doxorubicin.

Swell Test

Ultra Pure Water

	Initial Wt, (g)	5m(g) 300 sec	10m(g) 600 sec	30m(30g) 1800 sec	1hr (g) 3600 sec	2hr(g) 7200 sec	4hr(g) 14400 sec	6hr(g)	8hr(g)	24hr(g)	30hr(g)	48hr(g)	72hr(g)	96hr(g)	120hr(g)	144hrs	7days	8days	9days	10days
CP6	0.0197	0.1168	0.1582	0.2000	0.2145	0.2508	0.2689	0.2975	0.3188	0.3647	0.3797	0.4095	0.5245	0.5500	0.5735	0.6086	0.6357	0.6423	0.6077	0.5677
CP6 ICG	0.0202	0.1611	0.2007	0.2465	0.2632	0.2972	0.3042	0.3215	0.3487	0.4208	0.4316	0.4651	0.5059	0.5297	0.4850	0.4644	0.5181	0.5179	0.5175	0.5056
CP6 DOX	0.0184	0.1309	0.1634	0.2055	0.2182	0.2366	0.2523	0.2753	0.2825	0.3455	0.3528	0.3842	0.4439	0.4626	0.4816	0.4798	0.4845	0.4700	0.4782	0.4642

Figure 13: Swelling behavior of the CP6 hydrogel formulations in ultra-pure water over 10 days. Initial dry weights were recorded, and hydrogels were submerged in ultra-pure water at multiple time intervals from 5 minutes to 10 days. The base CP6 hydrogel exhibits the highest overall swelling capacity.

Swelling analysis in ultra-pure water shows the absorption behaviors among three different formulations. CP6 displays the greatest swelling capacity, increasing from an initial weight of 0.0197 g to a peak of 0.6423 g by day 8, indicating a high water affinity and extensive pore volume.CP6-ICG also showed significant swelling, but it plateaued earlier and reached a slightly lower maximum (0.581g at day 7), suggesting that ICG loading may have partially reduced free hydrophilic sites. CP6-DOX showed the lowest swelling capacity with a maximum of 0.4845 g, potentially due to an increased crosslink density. All formulations showed a rapid initial uptake within the first few hours, followed by a slower equilibration phase, consistent with typical swelling kinetics in porous hydrogels.

Pure DMEM

	Initial Wt, (g)	5m(g) 300 sec	10m(g) 600 sec	30m(30g) 1800 sec	1hr (g) 3600 sec	2hr(g) 7200 sec	4hr(g) 14400 sec	6hr(g)	8hr(g)	24hr(g)	30hr(g)	48hr(g)	72hr(g)	96hr(g)	120hr(g)	144hrs	7days	8days	9days	10days
CP6	0.0197	0.1168	0.1582	0.2000	0.2145	0.2508	0.2689	0.2975	0.3188	0.3647	0.3797	0.4095	0.5245	0.5500	0.5735	0.6086	0.6357	0.6423	0.6077	0.5677
CP6 ICG	0.0202	0.1611	0.2007	0.2465	0.2632	0.2972	0.3042	0.3215	0.3487	0.4208	0.4316	0.4651	0.5059	0.5297	0.4850	0.4644	0.5181	0.5179	0.5171	0.5056
CP6 DOX	0.0184	0.1309	0.1634	0.2055	0.2182	0.2366	0.2523	0.2753	0.2825	0.3455	0.3528	0.3842	0.4439	0.4626	0.4816	0.4798	0.4845	0.4700	0.4782	0.4642

Figure 14: Swelling behavior of CP6 hydrogel formulations in pure DMEM over 10 days. CP6, CP6-ICG, and CP6-DOX hydrogels were submerged in Dulbecco’s Modified Eagle Medium(DMEM), and swelling was monitored over 10 days. Unlike in ultra-pure water, all formulations exhibited limited and declining swelling trends after initial uptake, with CP6 showing slightly higher absorption than the drug-loaded variations. The presence of salts and proteins in DMEM may have influenced osmotic balance.

In contrast to the swelling observed in ultra-pure water, all hydrogel formulations showed reduced swelling in pure DMEM, with peak values within the first hour and subsequent weight loss over time. CP6 absorbed the most overall, increasing from 0.0234 g to an early maximum of 0.1276 g at 10 minutes before gradually declining. CP6-DOX followed a similar trend, reaching a peak of 0.1063 g, while CP6-ICG demonstrated the lowest and most rapid deswelling since it never exceeded 0.0876 g. This behavior is due to the ionic crosslinking and osmotic imbalances due to the presence of salts, amino acids, and glucose in DMEM. These findings suggest the physiological environment significantly alters hydrogel swelling.

PBS 7.4

	Initial Wt, (g)	5m(g) 300 sec	10m(g) 600 sec	30m(30g) 1800 sec	1hr (g) 3600 sec	2hr(g) 7200 sec	4hr(g) 14400 sec	6hr(g)	8hr(g)	24hr(g)	30hr(g)	48hr(g)	72hr(g)	96hr(g)	120hr(g)	144hrs	7days	8days	9days	10days
CP6	0.0218	0.0857	0.1032	0.1338	0.1441	0.1591	0.1668	0.1722	0.1806	0.1994	0.1938	0.2102	0.1963	0.2047	0.1980	0.1254	0.1336	0.1343	0.1332	0.1360
CP6 ICG	0.0170	0.0915	0.1131	0.1355	0.1489	0.1600	0.1617	0.1685	0.1820	0.1993	0.2038	0.2116	0.1235	0.1271	0.1296	0.1359	0.1300	0.1297	0.1444	0.1394
CP6 DOX	0.0153	0.0355	0.0523	0.0378	0.0478	0.0381	0.0371	0.0403	0.3520	0.0386	0.0449	0.0347	0.0378	0.0383	0.0418	0.0427	0.0413	0.0394	0.0420	0.0402

Figure 15: Hydrogels CP6, CP6-ICG, and CP6-DOX were immersed in PBS and monitored at regular intervals for 10 days. CP6 and CP6-ICG exhibited gradual and stable swelling, reaching maximum uptake between 24-72 hours. CP6-DOX showed significantly reduced swelling behaviors, indicating altered structural response in a buffered ionic environment.

Swelling analysis in PBS (pH 7.4) revealed that both CP6 and the CP6-ICG hydrogels maintained moderate and consistent water uptake, peaking around 72 hours (0.2102 g and 0.2116 g) before stabilizing with minor fluctuations. This suggests that the ionic strength of PBS did not strongly inhibit swelling for these formulations. The CP6-DOX shows reduced swelling throughout the entire duration, with irregular absorption and values remaining below 0.045 g, except for a single outlier at the 8-hour mark. The suppressed swelling could be due to interactions between the doxorubicin and the chitosan backbone, additionally leading to reduced porosity. These findings show that buffer composition and drug loading significantly affect hydrogel swelling dynamics.

 

Alamar Blue

Figure 16: Bar graph displays the percentage of viable cells following the treatment with unloaded and loaded hydrogels: CP6, CP6-ICG, CP6-DOX, CP6-DOX-ICG, with or without near-infrared irradiation exposure, as well as controls and insert-based delivery systems. Untreated control samples show consistent high viability, while free DOX and DOX-ICG formulations significantly reduced cell viability, especially at 48 and 72 hours. CP6 alone exhibited minimal cytotoxicity, while insert-based and NIR assisted formulations demonstrated time-dependent effects on metabolic activity, which indicates enhanced photothermal or chemotherapeutic impact in the CP6-DOX-ICG.

Alamar Blue assay reveals distinct cytotoxicity properties amongst the various hydrogel formulations and treatment conditions over time. The CP6 hydrogel showed minimal cytotoxicity, maintained cell viability above 30% at 24 hours, and returned to baseline at later time points. This confirms its biocompatibility. In contrast, the formulations containing doxorubicin showed pronounced cytotoxic effects, with viability dropping below 1% by 48 hours, which highlights doxorubicin’s chemotherapeutic activity. Interestingly, ICG alone had relatively mild effects on cell viability, while the NIR exposure did slightly enhance the cytotoxicity in the ICG and DOX-formulated samples, likely due to the photothermal effects. The cell culture insert samples showed a more gradual and sustained viability reduction, specifically with the ICG and DOX formulations, which suggests prolonged release and localized accumulation. The most significant reduction in inviability occurred in DOX-ICG NIR insert groups. These findings demonstrate that the CP6-DOX-ICG hydrogels, especially under NIR activation, are highly effective in reducing cell viability, while CP6 alone remains non-toxic and suitable to be a carrier for controlled drug delivery.

Conclusion

This study demonstrates the successful development and characterization of genipin-crosslinked PVA-chitosan hydrogels for the potential application in localized melanoma therapy. Structural analysis via SEM confirmed the presence of pores, with the drug incorporation controlling the distribution. TGA and DSC revealed that drug loading altered the thermal stability and phase behavior of the hydrogels, while the tensile testing confirmed that the mechanical integrity was retained after crosslinking. Swelling studies conducted in ultra-pure water, PBS, and DMEM highlighted that CP6-DOX-ICG and CP6-DOX have the lowest swelling capacity, likely due to the drug and polymer interactions. Importantly, the Alamar Blue assays confirmed the biocompatibility of CP6 alone, while DOX and ICG-loaded formulations, especially the samples exposed to NIR, showed significant cytotoxicity toward melanoma cells, which supports therapeutic potential. While the results of this study are promising, the research does remain ongoing, and further in vitro assays are currently being on to assess biocompatibility and drug diffusion.

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