β-Cyclodextrin-Based Nanosponges Inclusion Compounds Associated with Gold Nanorods for Potential NIR-II Drug Delivery

This article describes the synthesis and characterization of two nanocarriers consisting of β-cyclodextrin-based nanosponges (NSs) inclusion compounds (ICs) and gold nanorods (AuNRs) for potential near-infrared II (NIR-II) drug-delivery systems. These nanosystems sought to improve the stability of two drugs, namely melphalan (MPH) and curcumin (CUR), and to trigger their photothermal release after a laser irradiation stimulus (1064 nm). The inclusion of MPH and CUR inside each NS was confirmed by field emission scanning electron microscopy (FE-SEM), Raman spectroscopy, Fourier transform infrared spectroscopy, (FT-IR) differential scanning calorimetry (DSC), transmission electron microscopy (TEM), and proton nuclear magnetic resonance (1H-NMR). Furthermore, the association of AuNRs with both ICs was confirmed by FE-SEM, energy-dispersive spectroscopy (EDS), TEM, dynamic light scattering (DLS), ζ-potential, and UV–Vis. Moreover, the irradiation assays demonstrated the feasibility of the controlled-photothermal drug release of both MPH and CUR in the second biological window (1000–1300 nm). Finally, MTS assays depicted that the inclusion of MPH and CUR inside the cavities of NSs reduces the effects on mitochondrial activity, as compared to that observed in the free drugs. Overall, these results suggest the use of NSs associated with AuNRs as a potential technology of controlled drug delivery in tumor therapy, since they are efficient and non-toxic materials.

As such, β-CDs have been widely investigated to increase drug bioavailability due to their well-defined structure, moderate toxicity when administered locally or orally, and their stability with crosslinking agents, such as carbonyl compounds, carboxylic acids, and epoxides to form nano-porous formulations [1][2][3][4][5][6]. In this context, β-CD-based nanosponges (NSs) emerge as carriers with a sponge-like morphology and lipophilic nanochannels,

Synthesis of AuNRs
The AuNRs were synthesized using a modified seed-mediated method [42]. The seed was prepared by mixing 4.915 mL of CTAB (0.1 M) with 85 μL of HAuCl4 (29.4 mM) on a small flask with constant stirring for 5 min; then, 460 μL of NaBH4/NaOH solution was added (10 mM/0.01 M) to the mix and stirred for 0.5 h. Preparation of the growth solution was performed by adding 170 μL of HAuCl4 (29.4 mM) to 9.83 mL of a CTAB solution (0.1 M) and stirring for 10 min. Then, 1000 μL of AgNO3 (10 mM) were added to the mixed solution, and, after stirring for 30 s, 500 μL of hydroquinone (100 mM) were added, stirring for another 30 s. Finally, 160 μL of the seed solution were mixed with the growth solution, stirred for 30 s, and left to rest overnight.
The AuNRs were centrifuged, resuspended in Milli-Q water, and stabilized with PEG to remove the CTAB from the nanoparticles. The synthesis was carried out at 27 °C on a water bath to prevent the CTAB from crystallizing.

Synthesis of the NSs
NSs were synthesized with minor modifications from previously reported methods [10,43], using β-CD and diphenyl carbonate (DPC) as precursors. Anhydrous β-CD (1.5 g) and DPC (0.856 g) were homogenized in a solid state, placed in a conical flask, and heated from 90 to 100 °C under constant stirring for 5 h. The reaction mixture was left at room temperature until it cooled down, and the obtained solid was ground with an agate mortar. Double distilled water and acetone separated the product from the unreacted precursors. Afterward, the solid was washed with Soxhlet extraction with ethanol and acetone for 48 h to remove phenol, which formed as a by-product of the cross-linking reaction.

Synthesis of AuNRs
The AuNRs were synthesized using a modified seed-mediated method [42]. The seed was prepared by mixing 4.915 mL of CTAB (0.1 M) with 85 µL of HAuCl 4 (29.4 mM) on a small flask with constant stirring for 5 min; then, 460 µL of NaBH 4 /NaOH solution was added (10 mM/0.01 M) to the mix and stirred for 0.5 h. Preparation of the growth solution was performed by adding 170 µL of HAuCl 4 (29.4 mM) to 9.83 mL of a CTAB solution (0.1 M) and stirring for 10 min. Then, 1000 µL of AgNO 3 (10 mM) were added to the mixed solution, and, after stirring for 30 s, 500 µL of hydroquinone (100 mM) were added, stirring for another 30 s. Finally, 160 µL of the seed solution were mixed with the growth solution, stirred for 30 s, and left to rest overnight.
The AuNRs were centrifuged, resuspended in Milli-Q water, and stabilized with PEG to remove the CTAB from the nanoparticles. The synthesis was carried out at 27 • C on a water bath to prevent the CTAB from crystallizing.

Synthesis of the NSs
NSs were synthesized with minor modifications from previously reported methods [10,43], using β-CD and diphenyl carbonate (DPC) as precursors. Anhydrous β-CD (1.5 g) and DPC (0.856 g) were homogenized in a solid state, placed in a conical flask, and heated from 90 to 100 • C under constant stirring for 5 h. The reaction mixture was left at room temperature until it cooled down, and the obtained solid was ground with an agate mortar. Double distilled water and acetone separated the product from the unreacted precursors. Afterward, the solid was washed with Soxhlet extraction with ethanol and acetone for 48 h to remove phenol, which formed as a by-product of the cross-linking reaction. Finally, the solid was dried at 100 • C for 48 h and stored at room temperature for further use. Figure 2 illustrates the synthetic route of NSs. Finally, the solid was dried at 100 °C for 48 h and stored at room temperature for further use. Figure 2 illustrates the synthetic route of NSs.

Preparation of NSs-MPH and NSs-CUR Complexes
Each compound, namely, MPH and CUR, were loaded into the cavities of NSs using reported methods [29,44,45]. A total of 20 mg of NSs were immersed in 50 mL of doubledistilled water and kept under constant agitation. Afterward, 20 mL of MPH 0.1 mM or CUR 0.1 mM solution were added to the dispersed NSs. Both mixtures were sonicated for 10 min. and left under constant stirring for 1 day. The uncomplexed drugs were separated from the suspensions using centrifugation at 3000 rpm for 30 min. The obtained supernatants were freeze-dried at −81 °C and 0.001 mbar. The dried powders corresponding to the NSs-MPH and NSs-CUR complexes were stored in a desiccator for further use.

Association of AuNRs into the ICs
The association of AuNRs with the NSs-MPH or NSs-CUR complexes was carried out by immersing 30 mg of the ICs in 0.5 mL of AuNRs. After settling for 20 min., the mixture was centrifuged at 20.000 rpm for 30 min. The AuNRs associated with the ICs were separated from the supernatant and dried under vacuum. AuNRs concentration after association with the NSs drug complexes was determined using UV-Visible spectroscopy.

Proton Nuclear Magnetic Resonance ( 1 H-NMR) Spectroscopy
1 H-NMR characterization was performed using a Bruker Advance 400 MHz spectrometer (Bruker, Billerica, MA, USA) at 30 °C. Tetramethyl silane (TMS) was used as an internal standard. Stock solutions of NSs, the drugs, and the ICs were prepared using deuterated dimethyl sulfoxide (DMSO)-d6 as solvent due to the low solubility of NSs in deuterated water/chloroform, as reported previously [13,[46][47][48]. Data processing was carried out using the Mestre nova program.

Field Emission Scanning Electron Microscopy (FE-SEM)
The surface morphology features of NSs, MPH, CUR, and the ICs were analyzed using a Zeiss LEO Supra 35-VP scanning electron microscope equipped with EDS. Acceleration voltages of 2.0 and 5.0 kV were used. The samples were deposited onto a carbon tape stuck to an aluminum stub, following gold coating using a magneton sputtering (pressure of 0.5 mbar, argon atmosphere, and current of 25 mA, for 15 s) to minimize charging effects.

Preparation of NSs-MPH and NSs-CUR Complexes
Each compound, namely, MPH and CUR, were loaded into the cavities of NSs using reported methods [29,44,45]. A total of 20 mg of NSs were immersed in 50 mL of doubledistilled water and kept under constant agitation. Afterward, 20 mL of MPH 0.1 mM or CUR 0.1 mM solution were added to the dispersed NSs. Both mixtures were sonicated for 10 min. and left under constant stirring for 1 day. The uncomplexed drugs were separated from the suspensions using centrifugation at 3000 rpm for 30 min. The obtained supernatants were freeze-dried at −81 • C and 0.001 mbar. The dried powders corresponding to the NSs-MPH and NSs-CUR complexes were stored in a desiccator for further use.

Association of AuNRs into the ICs
The association of AuNRs with the NSs-MPH or NSs-CUR complexes was carried out by immersing 30 mg of the ICs in 0.5 mL of AuNRs. After settling for 20 min., the mixture was centrifuged at 20.000 rpm for 30 min. The AuNRs associated with the ICs were separated from the supernatant and dried under vacuum. AuNRs concentration after association with the NSs drug complexes was determined using UV-Visible spectroscopy.

Proton Nuclear Magnetic Resonance ( 1 H-NMR) Spectroscopy
1 H-NMR characterization was performed using a Bruker Advance 400 MHz spectrometer (Bruker, Billerica, MA, USA) at 30 • C. Tetramethyl silane (TMS) was used as an internal standard. Stock solutions of NSs, the drugs, and the ICs were prepared using deuterated dimethyl sulfoxide (DMSO)-d 6 as solvent due to the low solubility of NSs in deuterated water/chloroform, as reported previously [13,[46][47][48]. Data processing was carried out using the Mestre nova program.

Field Emission Scanning Electron Microscopy (FE-SEM)
The surface morphology features of NSs, MPH, CUR, and the ICs were analyzed using a Zeiss LEO Supra 35-VP scanning electron microscope equipped with EDS. Acceleration voltages of 2.0 and 5.0 kV were used. The samples were deposited onto a carbon tape stuck to an aluminum stub, following gold coating using a magneton sputtering (pressure of 0.5 mbar, argon atmosphere, and current of 25 mA, for 15 s) to minimize charging effects.

Ultraviolet and Visible Absorption (UV-Vis) Spectroscopy
UV-Visible spectra of the AuNRs and the ICs associated with AuNRs were measured using a Jasco V-760 UV-Visible spectrometer. Measurements were conducted in the range of 200-1100 nm, using deionized water as a reference. The UVProve 1.10 program was used for data processing.

Raman Spectroscopy
Raman spectra of the samples were acquired using a WI Tec SNOM/Raman microscopy model Alpha 300 equipped with a 785 nm laser line and employing the 50× objective. The Raman spectra (200-1700 cm −1 ) of the samples were registered setting the conditions as follows: 10 acquisitions with 10 s of integration time per spectrum. the intrinsic fluorescence of samples was quenched by using a thin sheet of gold prepared by metal sputtering method.

Fourier Transform Infrared Spectroscopy (FT-IR)
FT-IR spectra of the samples were acquired using a Jasco spectrometer model 4600 equipped with a Deuterated L-alanine Doped Triglycine Sulphate (DLATGS) detector. A total of 150 scans per sample (400-4000 cm −1 ) were performed by placing each sample on a micro-ATR (ATR pro one) accessory using a ZnSe crystal.

Transmission Electron Microscopy (TEM)
TEM analyses of AuNRs, NSs, ICs, and the ICs associated with the AuNRs were performed using a Hitachi model HT-7700 microscope, operating at 120 kV. The ICs associated with AuNRs were dispersed in ethanol (30% v/v). After sonication for 5 min., 10 µL of the formulations were deposited onto a copper grid with a Formvar film. In the case of AuNRs, 10 µL was deposited directly on a copper grid with a Formvar film. All samples were dried overnight for resolution enhancement.

Differential Scanning Calorimetry (DSC)
MPH, CUR, NSs, NSs-MPH, and NSs-CUR complexes were analyzed on a Differential Scanning Calorimeter DSC 8000 Perkin Elmer to obtain their respective DSC thermograms. Aluminum pans were used to place, weigh, and seal the samples. Measurements were carried out over a temperature range of 0-600 • C under a continuous nitrogen flow rate of 10 • C/min.

Determination of Drug Content in NSs
The encapsulation efficiency (EE) of NSs-MPH and NSs-CUR complexes can be defined as the concentration of the complexed drug over the initial concentration of the drug. EE values were obtained using Equation (1), as follows: The loading capacity (LC) of NSs-MPH and NSs-CUR was obtained from the total weight of NSs and the weight of entrapped drugs using Equation (2):

DLS and ζ-Potential
Size distribution, polydispersity index, and ζ-potentials were determined using a DLS Zetasizer NanoS series, Malvern. Proper dilution of all samples with double-distilled water was carried out before measurements were performed at 25 • C using disposable zeta cells. The size distribution and ζ-potentials were calculated using the cumulants fit and the Smoluchowsky approximation, respectively. A total of 12 measurements were acquired, expressing the results as their average. For NSs-based samples, measurement conditions were set as follows: refraction index: 1.49, k:0; whereas, for AuNRs, measurements were performed using a refraction index of 1.33 and k:0.

Laser Irradiation Assays
For laser irradiation assays, a laser at 1064 nm, with a light power of 150 mW and beam diameter of 1 mm, was used. A total of 200 µL of the NSs-MPH and NSs-CUR complexes conjugated with the AuNRs were added to a 500 µL Kahn test tube. The ternary systems were exposed to laser irradiation at different times (intervals of 1 min until reaching a maximum of 20 min). The release of both MPH and CUR was measured using UV-Vis spectroscopy. ICs without AuNRs were irradiated for control assays to determine whether the AuNRs were responsible for the release of the guest molecules.
Maximum absorbances of the released drugs were expressed as release percentages and then compared with the initial amount of the drug. All assays were carried out in triplicate. Percentages of the released drug (DR) were calculated using the following Equation (3):

Mitochondrial Activity Assays
Mitochondrial activity was measured by MTS using the CellTiter 96 AQueous one solution cell proliferation assay (Promega). The experiments were conducted as recommended by the manufacturer. In brief, 5000 cells per well were seeded on 96-well plates in 100 µL of complete Dulbecco's Modified Eagle Medium (DMEM). The medium was incubated at 37 • C and was subsequently removed after 1 day. Further, cells were incubated for another day with a titration (1 to 1 serial dilutions) of MPH, CUR, NSs-MPH, and NSs-CUR (all samples ranging from 0.1, 0.05, 0.025, and 0.01 mM in 1% DMSO, and then the volume was completed with DMEM medium). Afterward, phenol red-free DMEM medium (Gibco) containing the MTS/PMS reagent was added to replace the medium and incubated for 1 h at 37 • C. Absorbance measurements of all samples were carried out with a microplate reader at 490/655 nm (Synergy Mx, Biotek). For each experiment, fluorescence was corrected by subtracting the average fluorescence from a triplicate set of control wells without cells. Mitochondrial activity was calculated with respect to a non-treated control (medium). Each experiment was performed in 3 technical and 2 biological replicates.

Data Analysis
All the results are presented as mean ± SD, determined by at least three independent experiments. Statistical analyses were conducted using GraphPad Prism 9 Software Inc. (San Diego, CA, USA). A one-way ANOVA, followed by Tukey's Test, was performed to determine significance between results, which were considered as such if **** p < 0.0001, *** p < 0.001, and * p < 0.05.

1 H-NMR Spectra of the ICs
The inclusion of MPH and CUR inside the cavities of the NSs was confirmed using 1 H-NMR spectroscopy. The changes in the chemical shifts in the protons of both NSs and the drugs provided evidence for the formation of the NSs-MPH and NSs-CUR complexes. The acquired spectra of NSs, MPH, CUR, and the ICs are shown in Figure 3 (adapted from [36]) and Figure 4.   Proton signals of both guest molecules showed high-field chemical shifts, possibly due to screening effects caused by the change in the environment of the drugs, as they ended up entrapped inside the multiple interstitial sites of the NSs. The spatial restriction of MPH and CUR also contributed to the chemical shifts shown by the protons of both guests.
Notably, the protons within the hydrophobic cavities of the NSs (H3, H5 and H6) and the OH2 and OH3 hydroxyl groups displayed the most pronounced chemical shifts among the protons of NSs, which strongly suggests complexation of the drugs. Chemical shifts can also be observed for the protons located in the external cavities of the NSs (H1, H2, H4), implying that the complexation of MPH and CUR occurs in both the cavities of the β-CD monomers and the supramolecular sites that are produced in the polymerization, in accordance with previous studies of NSs inclusion compounds [10,36,[49][50][51][52]. The largest chemical shifts in both drug molecules correspond to the protons present in the benzyl ring structure, indicating their preferential inclusion inside the multiple β-CD cavities of NSs. Chemical shifts in MPH and CUR before and after inclusion are shown in Table 1 (adapted from [36]) and Table 2, respectively.   Proton signals of both guest molecules showed high-field chemical shifts, possibly due to screening effects caused by the change in the environment of the drugs, as they ended up entrapped inside the multiple interstitial sites of the NSs. The spatial restriction of MPH and CUR also contributed to the chemical shifts shown by the protons of both guests.
Notably, the protons within the hydrophobic cavities of the NSs (H3, H5 and H6) and the OH2 and OH3 hydroxyl groups displayed the most pronounced chemical shifts among the protons of NSs, which strongly suggests complexation of the drugs. Chemical shifts can also be observed for the protons located in the external cavities of the NSs (H1, H2, H4), implying that the complexation of MPH and CUR occurs in both the cavities of the β-CD monomers and the supramolecular sites that are produced in the polymerization, in accordance with previous studies of NSs inclusion compounds [10,36,[49][50][51][52]. The largest chemical shifts in both drug molecules correspond to the protons present in the benzyl ring structure, indicating their preferential inclusion inside the multiple β-CD cavities of NSs. Chemical shifts in MPH and CUR before and after inclusion are shown in Table 1 (adapted from [36]) and Table 2, respectively. Proton signals of both guest molecules showed high-field chemical shifts, possibly due to screening effects caused by the change in the environment of the drugs, as they ended up entrapped inside the multiple interstitial sites of the NSs. The spatial restriction of MPH and CUR also contributed to the chemical shifts shown by the protons of both guests.
Notably, the protons within the hydrophobic cavities of the NSs (H3, H5 and H6) and the OH2 and OH3 hydroxyl groups displayed the most pronounced chemical shifts among the protons of NSs, which strongly suggests complexation of the drugs. Chemical shifts can also be observed for the protons located in the external cavities of the NSs (H1, H2, H4), implying that the complexation of MPH and CUR occurs in both the cavities of the β-CD monomers and the supramolecular sites that are produced in the polymerization, in accordance with previous studies of NSs inclusion compounds [10,36,[49][50][51][52]. The largest chemical shifts in both drug molecules correspond to the protons present in the benzyl ring structure, indicating their preferential inclusion inside the multiple β-CD cavities of NSs. Chemical shifts in MPH and CUR before and after inclusion are shown in Table 1 (adapted from [36]) and Table 2, respectively.

FE-SEM Analyses of the ICs
The formation of the NSs-MPH and NSs-CUR complexes can also be confirmed by FE-SEM analyses. Figure 5 shows SEM micrographs of NSs, MPH, CUR, and the ICs.
FE-SEM images of NSs confirm their highly rough surface and sponge-like morphology, which might be suitable for the inclusion of the guest molecules, in agreement with previous studies [13,29,32,36,53]. Both MPH and CUR show crystalline morphology. After drug loading, the ICs maintain the morphological features of native NSs, suggesting the formation of an inclusion compound rather than a physical mixture, as co-precipitation of the free drugs was not observed. FE-SEM images of NSs confirm their highly rough surface and sponge-like morphology, which might be suitable for the inclusion of the guest molecules, in agreement with previous studies [13,29,32,36,53]. Both MPH and CUR show crystalline morphology. After drug loading, the ICs maintain the morphological features of native NSs, suggesting the formation of an inclusion compound rather than a physical mixture, as co-precipitation of the free drugs was not observed.

TEM Analysis of the ICs
The morphology of free NSs, NSs-MPH and NSs-CUR can be elucidated using TEM analyses. As seen in Figure 6A,B, NSs have a spherical nature, with an average size of 90 nm. The NSs-MPH and NSs-CUR complexes also show a spherical structure, and an increase in size to 150 nm after drug loading, with respect to native NSs. This could probably be attributed to intermolecular interactions occurring in the NSs-drug complexes, as reported in previous studies [33,46,47].

TEM Analysis of the ICs
The morphology of free NSs, NSs-MPH and NSs-CUR can be elucidated using TEM analyses. As seen in Figure 6A,B, NSs have a spherical nature, with an average size of 90 nm. The NSs-MPH and NSs-CUR complexes also show a spherical structure, and an increase in size to 150 nm after drug loading, with respect to native NSs. This could probably be attributed to intermolecular interactions occurring in the NSs-drug complexes, as reported in previous studies [33,46,47].

Raman and FT-IR Spectra of the ICs
The obtained NSs were characterized by Raman and FT-IR spectroscopy, as can be seen in Figure 7, and the assignment and discussion of the signals were determined based on related published data [10,[54][55][56][57]. Spectrums were compared to observe their changes when the NSs were obtained.

Raman and FT-IR Spectra of the ICs
The obtained NSs were characterized by Raman and FT-IR spectroscopy, as can be seen in Figure 7, and the assignment and discussion of the signals were determined based on related published data [10,[54][55][56][57]. Spectrums were compared to observe their changes when the NSs were obtained. In the Raman spectral comparison, the disappearance and decrease in the relative intensity of some bands can be observed in the NSs profile, as a direct consequence of obtaining nanoparticles. The decrease in the relative intensity of the signal observed at 478 In the Raman spectral comparison, the disappearance and decrease in the relative intensity of some bands can be observed in the NSs profile, as a direct consequence of obtaining nanoparticles. The decrease in the relative intensity of the signal observed at 478 cm −1 in the NS profile allowed for us to infer a vibration of the compound's skeletal structure, which was now restricted because of the new structure; the same can be stated for the signal observed at 950 cm −1 . Related to this, the 576 cm −1 band is absent in the Raman profile of NSs, being consistent with the observed spectral facts explained above. Finally, significant spectral data were observed in the FT-IR spectrum of NSs as a new band at 1755 cm −1 , attributed to the C=O vibration. This signal confirms that we obtained the nanosponge because of the presence of the C=O stretching from the linker.
The ICs systems were also characterized by Raman and FT-IR. In this case, it is important to mention that FT-IR spectra gave us more molecular information than Raman because the latter has a low cross-section, even more so considering the low molar concentration of the CUR and MPH in each system. In the Raman profile of NSs-CUR (see Figure 8), a strong band observed at 672 cm −1 and another signal with medium relative intensity located at 703 cm −1 were ascribed to an out-of-plane deformation of the ring in CUR. However, the preparation of the NSs-CUR system was confirmed by the FT-IR spectrum (see Figure 8) from the signals observed at 1446, 1408, and 949 cm −1 , ascribed to in-plane deformation of −CH 3 groups, stretching of C=CH, and in-plane deformation of the ring, respectively. Furthermore, the interaction of CUR in the NSs is supported by additional signals observed in the FT-IR spectrum at 2912, 3009, 3263, and 3378 cm −1 .  Finally, the Raman and FT-IR profiles of the NSs-MPH system offered us information about the interaction of the species. The Raman spectrum (see Figure 9) displays two bands at 442 and 481 cm −1 , corresponding to the NNC and CCN bending mode in MPH, respectively. The FT-IR spectrum of NSs-MPH (see Figure 9) is dominated by the profile of the NSs; however, there are some shifts in specific signals. Some bands observed at 1027, 1099, 1074, 1644 and 1755 cm −1 show the mentioned shift because of the interaction between the species. A new band appeared at 1779 cm −1 and is ascribed to C=O vibrations from MPH. Finally, the Raman and FT-IR profiles of the NSs-MPH system offered us information about the interaction of the species. The Raman spectrum (see Figure 9) displays two bands at 442 and 481 cm −1 , corresponding to the NNC and CCN bending mode in MPH, respectively. The FT-IR spectrum of NSs-MPH (see Figure 9) is dominated by the profile of the NSs; however, there are some shifts in specific signals. Some bands observed at 1027, 1099, 1074, 1644 and 1755 cm −1 show the mentioned shift because of the interaction between the species. A new band appeared at 1779 cm −1 and is ascribed to C=O vibrations from MPH. about the interaction of the species. The Raman spectrum (see Figure 9) displays two bands at 442 and 481 cm −1 , corresponding to the NNC and CCN bending mode in MPH, respectively. The FT-IR spectrum of NSs-MPH (see Figure 9) is dominated by the profile of the NSs; however, there are some shifts in specific signals. Some bands observed at 1027, 1099, 1074, 1644 and 1755 cm −1 show the mentioned shift because of the interaction between the species. A new band appeared at 1779 cm −1 and is ascribed to C=O vibrations from MPH.

DSC Thermograms of the ICs
The NSs-MPH and NSs-CUR complexes were characterized through DSC. Thermal analyses of NSs, the ICs, and the free drugs were performed to confirm the formation of an inclusion compound between the guests and the NSs matrix rather than a physical mixture. Figure 10 depicts the thermograms of MPH, CUR, NSs, NSs-MPH, and NSs-CUR. NSs exhibit an endothermic peak at 350 • C, representing the melting point of the crosslinked polymer, as reported previously [43,58,59]. DSC thermogram of MPH shows a sharp endothermic peak at around 203 • C, corresponding to the melting point of the free drug [60]. The NSs-MPH and NSs-CUR complexes were characterized through DSC. Thermal analyses of NSs, the ICs, and the free drugs were performed to confirm the formation of an inclusion compound between the guests and the NSs matrix rather than a physical mixture. Figure 10 depicts the thermograms of MPH, CUR, NSs, NSs-MPH, and NSs-CUR. NSs exhibit an endothermic peak at 350 °C, representing the melting point of the crosslinked polymer, as reported previously [43,58,59]. DSC thermogram of MPH shows a sharp endothermic peak at around 203 °C, corresponding to the melting point of the free drug [60].
Free CUR showed an endothermic peak at 183 °C, which, according to the literature, corresponds to its intrinsic melting point [25,61]. Furthermore, the thermograms of the ICs indicate the disappearance of the characteristic peaks in the drugs, suggesting the complexation of the guests inside the cavities of NSs, while excluding the possibility of a physical mixture. Similar results have been observed for other NSs-drug complexes [43,45,[62][63][64][65][66]. The characterization of AuNRs was carried out by TEM, UV-Vis, DLS and ζ-potential. Figure 11A shows the TEM micrograph of the AuNRs, and its size distribution was estimated. Both the length and width of the nanoparticles were plotted (Figure 11C,D) and their average sizes were 55 nm and 8.6 nm, respectively. The UV-Vis spectra of AuNRs confirmed the presence of its characteristic plasmon band in the NIR-II window, showing a maximum absorbance for the longitudinal plasmon at 1070 nm ( Figure 11B), Free CUR showed an endothermic peak at 183 • C, which, according to the literature, corresponds to its intrinsic melting point [25,61]. Furthermore, the thermograms of the ICs indicate the disappearance of the characteristic peaks in the drugs, suggesting the complexation of the guests inside the cavities of NSs, while excluding the possibility of a physical mixture. Similar results have been observed for other NSs-drug complexes [43,45,[62][63][64][65][66].

TEM, UV-Vis, DLS, and ζ-Potential of AuNRs
The characterization of AuNRs was carried out by TEM, UV-Vis, DLS and ζ-potential. Figure 11A shows the TEM micrograph of the AuNRs, and its size distribution was estimated. Both the length and width of the nanoparticles were plotted (Figure 11C,D) and their average sizes were 55 nm and 8.6 nm, respectively. The UV-Vis spectra of AuNRs confirmed the presence of its characteristic plasmon band in the NIR-II window, showing a maximum absorbance for the longitudinal plasmon at 1070 nm ( Figure 11B), making them suitable for biological and photothermal applications [67]. The aspect ratio (length/width) for the synthesized AuNRs was 6.4. This agrees with the reported values, which indicate that AuNRs absorb in the second biological window [42,68,69]. The hydrodynamic diameter (Dh) of AuNRs provided by DLS is shown in Table 3, along with the ζ-potential. The Dh was 9.02 ± 4.1 nm and 84.7 ± 46.7 nm, which can be attributed to the rotational and translational light dispersed by the nanoparticles, respectively. The polidispersity index (PDI) was 0.47, thus indicating that the nanoparticles exhibited good monodispersity (PDI < 0.7). Furthermore, the ζ-potential was estimated to be −30 ± 3.9 mV, suggesting good colloidal stability and that the AuNRs would not undergo aggregation over time. The AuNRs attached to the ICs are shown in Figure 12. The ICs retained their porous morphology after their association with the AuNRs. The FE-SEM images indicate that the AuNRs are homogeneously distributed all over the organic matrix, with no evident  Table 3, along with the ζ-potential. The D h was 9.02 ± 4.1 nm and 84.7 ± 46.7 nm, which can be attributed to the rotational and translational light dispersed by the nanoparticles, respectively. The polidispersity index (PDI) was 0.47, thus indicating that the nanoparticles exhibited good monodispersity (PDI < 0.7). Furthermore, the ζ-potential was estimated to be −30 ± 3.9 mV, suggesting good colloidal stability and that the AuNRs would not undergo aggregation over time. Table 3. DLS, ζ-potential and PDI of AuNRs before and after stabilization with PEG.

FE-SEM and EDS Mapping Analyses of the ICs Associated with the AuNRs
The AuNRs attached to the ICs are shown in Figure 12. The ICs retained their porous morphology after their association with the AuNRs. The FE-SEM images indicate that the AuNRs are homogeneously distributed all over the organic matrix, with no evident changes in their aspect ratio nor morphology, thus confirming that the NSs-drug complexes are optimum substrates for stabilizing the AuNRs.

TEM Analyses of the ICs Associated with the AuNRs
TEM micrographs of the ICs associated with AuNRs are shown in Figure 14. Immobilization of AuNRs in the polymeric matrix does not seem to affect their integrity and aspect ratio, indicating that the formation of the AuNRs-NSs-MPH and AuNRs-NSs-CUR systems contribute to the nanoparticles' stability, which is consistent with the FE-SEM  EDS analysis provided information of the ICs associated with the AuNRs regarding their elemental composition, as seen on Figure 13. The elemental mapping shows the presence of C, O, N, and Cl in the AuNRs-NSs-MPH complex, where N and Cl can be attributed to the amine and chloroethylamine functional groups of MPH. Elemental mapping of AuNRs-NSs-CUR evidenced the presence of C and O on the supramolecular matrix, corresponding to the functional groups of both NSs and CUR. The EDS analyses also showed the detection of Au, thus confirming the association of AuNRs and the ICs.  Figure 14. Immobilization of AuNRs in the polymeric matrix does not seem to affect their integrity and

TEM Analyses of the ICs Associated with the AuNRs
TEM micrographs of the ICs associated with AuNRs are shown in Figure 14. Immobilization of AuNRs in the polymeric matrix does not seem to affect their integrity and aspect ratio, indicating that the formation of the AuNRs-NSs-MPH and AuNRs-NSs-CUR systems contribute to the nanoparticles' stability, which is consistent with the FE-SEM images shown in Section 3.2.2. The micrographs also show that the impurities assigned to spherical gold nanoparticles are minimal.

UV-Vis Spectra of the ICs Associated with the AuNRs
Deposition of AuNRs in the NSs-drug complex can also be confirmed using UV-Vis spectroscopy. Absorption UV-Vis spectra of the ICs associated with AuNRs are shown in Figure 15. The characteristic plasmonic bands of AuNRs were observed at 520 nm and 1060 nm, which correspond to the transversal and longitudinal absorption peaks, respectively.
A hypsochromic shift in the plasmon bands from 1070 nm to 1060 nm was observed, due to the proximity and environmental changes around the AuNRs, after their deposition in the NSs-drug complexes. Notably, the shift of the plasmon resonance peaks increases when the AuNRs concentration decreases. The presence of the plasmonic bands in the ICs-AuNRs provided evidence that the polymeric matrix provides stability to the nanoparticles, as reported by previous studies [36,70,71].

UV-Vis Spectra of the ICs Associated with the AuNRs
Deposition of AuNRs in the NSs-drug complex can also be confirmed using UV-Vis spectroscopy. Absorption UV-Vis spectra of the ICs associated with AuNRs are shown in Figure 15. The characteristic plasmonic bands of AuNRs were observed at 520 nm and 1060 nm, which correspond to the transversal and longitudinal absorption peaks, respectively.
A hypsochromic shift in the plasmon bands from 1070 nm to 1060 nm was observed, due to the proximity and environmental changes around the AuNRs, after their deposition in the NSs-drug complexes. Notably, the shift of the plasmon resonance peaks increases when the AuNRs concentration decreases. The presence of the plasmonic bands in the ICs-AuNRs provided evidence that the polymeric matrix provides stability to the nanoparticles, as reported by previous studies [36,70,71]. Pharmaceutics 2022, 14, x FOR PEER REVIEW 17 of 29 Figure 15. UV-Vis NIR-II spectra of the ICs associated with the AuNRs. Table 4 depicts the hydrodynamic diameters (DLS), ζ-potentials, and polydispersity indexes (PDI) of AuNRs, the NSs-drug complexes and the ICs associated with AuNRs. DLS provided information about the hydrodynamic diameters of the ICs and the ICs-AuNRs. The NSs, NSs-MPH, NSs-CUR and the ternary systems depicted values over 200 nm, thus confirming the nanometric size of the supramolecular systems. Upon immobilization with the ICs, the ζ-potential of the AuNRs changed due to their stabilization by the NSs-drug complexes. All nano-formulations showed ζ-potentials ranging from −21 to −35, confirming their stability (for further information see Figure A1 and Table A1). The PDI values of all samples indicated that the NSs, the NSs complexes, and the ternary systems are stable and homogeneous in nature (PDI < 0.7).

Encapsulation Efficiencies and Loading Capacities
The encapsulation efficiencies (EE%) and loading capacities (LC%) of the NSs-MPH and NSs-CUR complexes were determined using Equations (1) and (2), respectively. As described in Table 5, MPH showed a higher encapsulation efficiency and loading capacity than CUR, indicating that the structure of the guest strongly influences the complexation  Table 4 depicts the hydrodynamic diameters (DLS), ζ-potentials, and polydispersity indexes (PDI) of AuNRs, the NSs-drug complexes and the ICs associated with AuNRs. DLS provided information about the hydrodynamic diameters of the ICs and the ICs-AuNRs. The NSs, NSs-MPH, NSs-CUR and the ternary systems depicted values over 200 nm, thus confirming the nanometric size of the supramolecular systems. Upon immobilization with the ICs, the ζ-potential of the AuNRs changed due to their stabilization by the NSs-drug complexes. All nano-formulations showed ζ-potentials ranging from −21 to −35, confirming their stability (for further information see Figure A1 and Table A1).

DLS and ζ-Potential of the ICs Associated with the AuNRs
The PDI values of all samples indicated that the NSs, the NSs complexes, and the ternary systems are stable and homogeneous in nature (PDI < 0.7).

Encapsulation Efficiencies and Loading Capacities
The encapsulation efficiencies (EE%) and loading capacities (LC%) of the NSs-MPH and NSs-CUR complexes were determined using Equations (1) and (2), respectively. As described in Table 5, MPH showed a higher encapsulation efficiency and loading capacity than CUR, indicating that the structure of the guest strongly influences the complexation efficiency and molecular binding [72]. This suggests that MPH might be more aptly sized than CUR to be included in the supramolecular sites of NSs. The encapsulation efficiency and loading capacity values for NSs-CUR and NSs-MPH complexes increased, compared to native β-CD, where the calculated values were 30% (EE%) and 20% (LC%) for β-CD-CUR [65,73], and 70% (EE%) and 61% (LC) for β-CD-MPH [74].

Laser Irradiation Assays
The ICs were associated with the AuNRs to form a ternary system capable of inducing the release of both MPH and CUR by means of local photothermia using NIR-II irradiation. To achieve this objective, drug released phenomena were studied by adding the ICs conjugated to the AuNRs in a Kahn test tube. Then, the systems were irradiated using a continuous laser of 1064 nm for 20 min. The drug release percentages (%) were determined using the Lambert-Beer equation and Equation (3). The molar attenuation (ε) of both drugs was calculated with UV-Vis spectroscopy using a set of MPH and CUR solutions. Molar attenuation was 10.15 mM −1 cm −1 for MPH and 4.37 mM −1 cm .1 for CUR. Drug release percentages in the ternary systems were compared with those calculated in the control systems: ICs without AuNRs, ICs-AuNRs without irradiation, and physiological temperature (37-42 • C) to determine if the guests migrated from the supramolecular sites through diffusion. Figure 16 shows the percentages of released drug at 20 min. efficiency and molecular binding [72]. This suggests that MPH might be more aptly sized than CUR to be included in the supramolecular sites of NSs. The encapsulation efficiency and loading capacity values for NSs-CUR and NSs-MPH complexes increased, compared to native β-CD, where the calculated values were 30% (EE%) and 20% (LC%) for β-CD-CUR [65,73], and 70% (EE%) and 61% (LC) for β-CD-MPH [74].

Laser Irradiation Assays
The ICs were associated with the AuNRs to form a ternary system capable of inducing the release of both MPH and CUR by means of local photothermia using NIR-II irradiation. To achieve this objective, drug released phenomena were studied by adding the ICs conjugated to the AuNRs in a Kahn test tube. Then, the systems were irradiated using a continuous laser of 1064 nm for 20 min. The drug release percentages (%) were determined using the Lambert-Beer equation and Equation (3). The molar attenuation (ε) of both drugs was calculated with UV-Vis spectroscopy using a set of MPH and CUR solutions. Molar attenuation was 10.15 mM −1 cm −1 for MPH and 4.37 mM −1 cm .1 for CUR. Drug release percentages in the ternary systems were compared with those calculated in the control systems: ICs without AuNRs, ICs-AuNRs without irradiation, and physiological temperature (37-42 °C) to determine if the guests migrated from the supramolecular sites through diffusion. Figure 16 shows the percentages of released drug at 20 min. After irradiation, the ternary systems showed the highest drug release percentages of MPH and CUR (about 80% and 60%, respectively). In contrast, the amount of drug released from the AuNRs-ICs at physiological temperature (37-42 °C) was less than 10%. AuNRs exhibit high photothermal efficiencies and effectively diffuse heat to the surrounding media upon exposure to a laser tunable with the AuNRs NIR-II surface plasmon resonance [42,67,68,75]. AuNRs absorb photons, which exchange energy in the metal lattice through electron-phonon coupling and phonon-to-phonon relaxation. This produces an increase in the temperature of the metal surface while also increasing the surrounding After irradiation, the ternary systems showed the highest drug release percentages of MPH and CUR (about 80% and 60%, respectively). In contrast, the amount of drug released from the AuNRs-ICs at physiological temperature (37-42 • C) was less than 10%. AuNRs exhibit high photothermal efficiencies and effectively diffuse heat to the surrounding media upon exposure to a laser tunable with the AuNRs NIR-II surface plasmon resonance [42,67,68,75]. AuNRs absorb photons, which exchange energy in the metal lattice through electron-phonon coupling and phonon-to-phonon relaxation. This produces an increase in the temperature of the metal surface while also increasing the surrounding local temperature [37,38,42,67]. Thus, the 1064 nm-red laser, with a light power of 150 mW, generated enough local heat in the supramolecular systems to disassemble the AuNRs-ICs complexes and, subsequentially, trigger drug release from the NSs cavities. This confirms that the local photothermal effect produced by AuNRs in the NIR-II window promotes the release of both guests. On the other hand, the drug release percentages were drastically reduced in the control systems, compared to the ICs-AuNRs systems after irradiation. Both anti-tumor drugs migrated from all systems by means of passive diffusion. In summary, the irradiation of the ICs-AuNRs systems allowed a faster and more efficient release of the guests, proving that NIR-II irradiation of the ternary systems may open many opportunities for biological and tumor therapy applications.

MTS Assays
MTS assays were conducted to evaluate the effects of MPH, CUR and the NSs-MPH and NSs-CUR complexes on mitochondrial activity. The aim of this experiment was to evaluate if the inclusion of the drugs inside the NSs reduced their effects on cell viability, since the formulations should not produce cellular toxic effects if they are to be used as therapeutic agents.
The effects of MPH, CUR, and their corresponding ICs on the mitochondrial activity of HeLa cells were compared at equivalent concentrations ( Figure 17). In the case of MPH, both the free drug and its ICs did not show significant effects up to 0.05 mM of drug. local temperature [37,38,42,67]. Thus, the 1064 nm-red laser, with a light power of 150 mW, generated enough local heat in the supramolecular systems to disassemble the AuNRs-ICs complexes and, subsequentially, trigger drug release from the NSs cavities. This confirms that the local photothermal effect produced by AuNRs in the NIR-II window promotes the release of both guests. On the other hand, the drug release percentages were drastically reduced in the control systems, compared to the ICs-AuNRs systems after irradiation. Both anti-tumor drugs migrated from all systems by means of passive diffusion. In summary, the irradiation of the ICs-AuNRs systems allowed a faster and more efficient release of the guests, proving that NIR-II irradiation of the ternary systems may open many opportunities for biological and tumor therapy applications.

MTS Assays
MTS assays were conducted to evaluate the effects of MPH, CUR and the NSs-MPH and NSs-CUR complexes on mitochondrial activity. The aim of this experiment was to evaluate if the inclusion of the drugs inside the NSs reduced their effects on cell viability, since the formulations should not produce cellular toxic effects if they are to be used as therapeutic agents.
The effects of MPH, CUR, and their corresponding ICs on the mitochondrial activity of HeLa cells were compared at equivalent concentrations ( Figure 17). In the case of MPH, both the free drug and its ICs did not show significant effects up to 0.05 mM of drug. However, at the highest concentration assayed, the free drug (**** p < 0.0001) and NSs-MPH (* p < 0.05) presented a significant difference compared to the medium control. Despite this, it is important to note that there was a significant difference in the mitochondrial activity observed between free MPH and NSs-MPH at 1 mM (*** p < 0.001), with the being former 25-fold lower than the latter (2.4% vs. 59.1%). However, at the highest concentration assayed, the free drug (**** p < 0.0001) and NSs-MPH (* p < 0.05) presented a significant difference compared to the medium control. Despite this, it is important to note that there was a significant difference in the mitochondrial activity observed between free MPH and NSs-MPH at 1 mM (*** p < 0.001), with the being former 25-fold lower than the latter (2.4% vs. 59.1%). This is relevant because most alkylating agents, such as MPH, have shown to produce adverse side effects when they are used as free drug, despite their uses as anti-tumoral drugs [20,27,76].
On the other hand, free CUR depicted low cytotoxic effects, being only significantly different from the medium at 1 mM. Moreover, the effects of CUR on mitochondrial activity were reduced when the drug was encapsulated (NSs-CUR), showing no significant differences at any assayed concentration.
Furthermore, we observed that there were not significant effects on HeLa cells when lower concentrations were assayed both for free drugs and their respective complexes forms ( Figure A2). These results are very promising because they indicated that the incorporation of MPH or CUR into the NSs significantly reduced their toxic effects on HeLa cells.
Finally, considering the possibility of controlling drug release from the NSs through irradiation (Figure 16), and taking into consideration that the developed nano-formulations proved to be safer in comparison to the free guests (Figure 17), the potential applications of these drug delivery systems are auspicious.

Conclusions
We successfully included both MPH and CUR inside the cavities of NSs, as proven by 1 H-NMR, FE-SEM, TEM, DSC, FT-IR, and Raman characterization. The encapsulation percentages were 89% for MPH and 63% for CUR, confirming that NSs can efficiently form an inclusion complex with the drugs. The synthesized AuNRs showed an aspect ratio (length/width) of 6.4, which is consistent with the UV-Vis absorption band in the NIR-II optical window. FE-SEM, EDS, UV-Vis, TEM, DLS and the ζ-potential provided evidence that the NSs-MPH and NSs-CUR systems are appropriate substrates to stabilize AuNRs nanoparticles, as the latter retained their characteristic absorption band in the second biological window (1000-1300 nm), making them suitable for biological and photothermal drug release studies. Cellular studies performed through MTS assays were used to evaluate the inherent cytotoxicity of the drugs before and after encapsulation inside the supramolecular sites of NSs. The mitochondrial activity assays confirmed that the NSs-MPH and NSs-CUR complexes are safer formulations than the free drugs, which is promising in terms of potential biological applications in drug delivery. Finally, via plasmonic heating of the AuNRs associated with the ICs, the ternary systems easily outperformed all the control systems regarding the controlled release of the guests. Drug release percentages were drastically reduced in the control systems in comparison to the ICs-AuNRs after NIR-II irradiation. NSs conjugated to anisotropic gold nanoparticles have been considered in future perspectives, as they are safe, efficient, and non-toxic materials.

Stability of the Ternary Systems after 2 Months of Storage
Colloidal stability of NSs-MPH, NSs-CUR, and the AuNRs-ICs was evaluated after 2 months of storage at 4 • C by means of UV-Vis, DLS, ζ-potential, and PDI. The characteristic plasmon bands, the hydrodynamic diameters and surface charges of the supramolecular systems did not show any significant changes, demonstrating that the formulations maintained their nanometric size, negative ζ-potential and PDI > 0.7 following storage at 4 • C for 2 months, as depicted in Figure A1 and Table A1. research costs. We would also like to express our gratitude to Ana Riveros for carrying out S FE-SEM measurements.

Conflicts of Interest:
The authors declare no conflict of interest.

Stability of the Ternary Systems after 2 Months of Storage
Colloidal stability of NSs-MPH, NSs-CUR, and the AuNRs-ICs was evaluate 2 months of storage at 4 °C by means of UV-Vis, DLS, ζ-potential, and PDI. The cha istic plasmon bands, the hydrodynamic diameters and surface charges of the supra ular systems did not show any significant changes, demonstrating that the formu maintained their nanometric size, negative ζ-potential and PDI > 0.7 following sto 4 °C for 2 months, as depicted in Figure A1 and Table A1.   Appendix C Appendix C.1. 1

H-NMR of NSs, DPC, and Phenol
The removal of by-product phenol and the cross-linker diphenyl carbonate w lyzed using 1 H-NMR. Figure A3 shows the 1 H-NMR spectra of NSs, DPC, and None of the characteristic peaks in DPC (H1, 7.40 ppm; H2, 7.29 ppm; H3, 7.25 pp phenol (−OH, 5.35 ppm; H1, 7.20 ppm; H2, 6.93 ppm; H3, 6.83 ppm) were observe 1 H-NMR spectra of NSs, thus confirming the elimination of by-products and r cross-linker. Table A2 shows the proton assignments and the respective chemica for DPC and phenol.  The removal of by-product phenol and the cross-linker diphenyl carbonate was analyzed using 1 H-NMR. Figure A3 shows the 1 H-NMR spectra of NSs, DPC, and phenol. None of the characteristic peaks in DPC (H1, 7.40 ppm; H2, 7.29 ppm; H3, 7.25 ppm) nor phenol (−OH, 5.35 ppm; H1, 7.20 ppm; H2, 6.93 ppm; H3, 6.83 ppm) were observed in the 1 H-NMR spectra of NSs, thus confirming the elimination of by-products and residual cross-linker. Table A2 shows the proton assignments and the respective chemical shifts for DPC and phenol.

Appendix C.2. FT-IR of NSs and Phenol
The removal of the phenol was also analyzed using FT-IR. Figure A4 shows the FT-IR spectra of NSs and phenol. The NSs bands were observed at 3388 cm −1 (−OH, stretching), 2929 cm −1 (C-H stretching), 1030, and 1079 cm −1 (C-O stretching), 1368, 1235, and 1155 cm −1 (−OH bending), and 1760 cm −1 (C=O stretching). The fact that the most characteristic bands ascribed to phenol, namely 3230 cm −1 (−OH stretching), 1410, and 1320 cm 1-(C-O stretching), were not detected in the NSs spectra suggests that the by-product was removed successfully.

Appendix C.2. FT-IR of NSs and Phenol
The removal of the phenol was also analyzed using FT-IR. Figure A4 shows the FT-IR spectra of NSs and phenol. The NSs bands were observed at 3388 cm −1 (−OH, stretching), 2929 cm −1 (C-H stretching), 1030, and 1079 cm −1 (C-O stretching), 1368, 1235, and 1155 cm −1 (−OH bending), and 1760 cm −1 (C=O stretching). The fact that the most characteristic bands ascribed to phenol, namely 3230 cm −1 (−OH stretching), 1410, and 1320 cm −1 (C-O stretching), were not detected in the NSs spectra suggests that the byproduct was removed successfully.

Appendix C.2. FT-IR of NSs and Phenol
The removal of the phenol was also analyzed using FT-IR. Figure A4 shows the FT-IR spectra of NSs and phenol. The NSs bands were observed at 3388 cm −1 (−OH, stretching), 2929 cm −1 (C-H stretching), 1030, and 1079 cm −1 (C-O stretching), 1368, 1235, and 1155 cm −1 (−OH bending), and 1760 cm −1 (C=O stretching). The fact that the most characteristic bands ascribed to phenol, namely 3230 cm −1 (−OH stretching), 1410, and 1320 cm 1-(C-O stretching), were not detected in the NSs spectra suggests that the by-product was removed successfully.

Photothermal Efficiency of AuNRs
The photothermal efficiency of AuNRs and the ICs-AuNRs systems are shown in this section, as reported by previous studies involving AuNRs (for further details on the methodology, see references [42,67,81]). Photothermal efficiency indicates the system's ability to convert incident light into thermal energy. The increase in temperature and the calculated photothermal efficiencies for AuNRs and the ICs-AuNRs are summarized in Figure A6 and Table A4, respectively. The decrease in the photothermal efficiencies in the ICs-AuNRs systems can be ascribed to the effect of the organic matrix in heat dissipation.

Photothermal Efficiency of AuNRs
The photothermal efficiency of AuNRs and the ICs-AuNRs systems are shown in this section, as reported by previous studies involving AuNRs (for further details on the methodology, see references [42,67,81]). Photothermal efficiency indicates the system's ability to convert incident light into thermal energy. The increase in temperature and the calculated photothermal efficiencies for AuNRs and the ICs-AuNRs are summarized in Figure A6 and Table A4, respectively. The decrease in the photothermal efficiencies in the ICs-AuNRs systems can be ascribed to the effect of the organic matrix in heat dissipation.

Photothermal Efficiency of AuNRs
The photothermal efficiency of AuNRs and the ICs-AuNRs systems are shown in this section, as reported by previous studies involving AuNRs (for further details on the methodology, see references [42,67,81]). Photothermal efficiency indicates the system's ability to convert incident light into thermal energy. The increase in temperature and the calculated photothermal efficiencies for AuNRs and the ICs-AuNRs are summarized in Figure A6 and Table A4, respectively. The decrease in the photothermal efficiencies in the ICs-AuNRs systems can be ascribed to the effect of the organic matrix in heat dissipation.