Injectable light-assisted thermo-responsive methylcellulose-sodium humate hydrogel proposed for photothermal ablation and localized delivery of cisplatin

This study aimed to develop injectable light-assisted thermo-responsive methylcellulose hydrogels filled with sodium humate, which were proposed for photothermal ablation and localized cisplatin delivery. Sodium humate converts light energy from laser beams into thermal energy, which causes methylcellulose to gel, thereby controlling the release of chemotherapy agents. Meanwhile, light emission causes to the photothermal ablation of tumor cells. For determining the optimal production conditions, different concentrations of sodium humate and light emission times were investigated. Results show that hydrogel uniformity is highly dependent on variables. An increase in sodium humate concentration and emission time resulted in a slight reduction in swelling ratio and an increase in durability. According to the simulation conditions, the cisplatin release profile was consistent with a non-Fickian mechanism with a predominant erosion contribution. In conjugation with increasing light emission time and sodium humate content, the storage modulus and viscosity increased, demonstrating hydrogel’s sol-gel transition and long-lasting durability. The intrinsic fluorescence spectroscopy study revealed that the hydrogel-model protein complex empowered hydrogel bio-performance. Laser emission and cisplatin release synergistically reduced the number of viable osteosarcoma cell lines, suggesting the possibility of tumor ablation. This study describes the potential of simultaneous photothermal therapy and chemotherapy in osteosarcoma treatment, laying the groundwork for future preclinical and clinical trials.


Experimental:
The potential of hydrogels to interact with water molecules was evaluated by water content measurement. So, the dried hydrogels were weighted (Wi), and after incubation of samples in 30 ml of the PBS solution at 37±0.5 °C, the wet weight (Ww) was measured at a pre-determined time (6,12, and 24 hours). Finally, the water content of hydrogels was then calculated using Eq. 1 [1]: Water content (%) = [swelling / (swelling+1)]*100 (1) Results:

 Water content
As indicated in Fig. S1, MS1 hydrogels show low storage modulus compared with other experimental groups. Herein, MS2 and MS3 gels indicated about 80 times higher stability than MS1. Although MS2 hydrogels indicated higher storage modulus in low strain than MS3, a larger linear viscoelastic region in MS3 hydrogels presented higher structural integrity. The stable performance of MS3 hydrogels with high storage modulus and stiffness may arise from binding energy, as was observed in other literature [2][3][4][5]. Besides, the higher potential of MS3 hydrogels to produce thermal energy (affected by the concentration of sodium humate and exposure time) and promoting cross-linking should not be ignored.

 Release
The release data are fitted in mathematical models to indicate the release kinetics and mechanism. According to the correlation coefficient (R 2 ) value, the best release model is selected for cisplatin release from MS1, MS2, and MS3 hydrogel. The mathematical kinetics models of zero-order, first-order [6], Weibull [7], Higuchi [8], Hixson-Crowell [9], and Korsmeyer-Peppas [10]. (Eqs. 1-6) are used for the release data fitting: Weibull: Hixson-Crowell: Where M0, Mt, and M∞ represent the drug released amount at time zero, t, and infinity, respectively; t represents release time. In the Hixson-Crowell model, w0 and wt represent the drug weight at time zero and t, respectively. The k0, k1, kH, kHC, and kKP are the release kinetic constants in zero-order, first-order, the Higuchi, Hixson-Crowell, and Korsmeyer-Peppas models, respectively. In the Weibull model, a and b variables are constants. In the Korsmeyer-Peppas, the n variable is the release exponent, indicating the drug's release mechanism. The results of the fitting are summarized in Table S2. 5 The results indicate that the relevant data are well fitted to the Korsmeyer-Peppas model. So, the release exponent in Korsmeyer-Peppas model is calculated. Accordingly, the value of release exponent explore the release mechanism since n = 0.5, 0.5 < n < 1, n = 1 and n > 1 representing Fickian diffusion, anomalous (non-Fickian) diffusion (i.e. by both diffusion and erosion [11]), case II transport (zero-order (time-independent) release) and super case II transport, respectively [10].
Accordingly, due to the values of release exponent in the Korsmeyer-Peppas model, which are obtained between 0.5 and 1, the anomalous (non-Fickian) diffusion is the mechanism of cisplatin release from MS1, MS2, and MS3 hydrogels. The results are summarized in Table S3. According to the cisplatin release mechanism from MS1, MS2, and MS3 hydrogels, which is accompanied by diffusion and erosion processes; the Kopcha release model is used to explore the exact contribution of diffusion and erosion using the Eq. 7 [12]: Where t represents the release time; A and B represent the diffusion and erosion terms, respectively. The contribution of diffusion and erosion in the release mechanism is demonstrated by A to B ratio. The contribution ratio is shown in forms of A/B=1, A/B>1, and A/B<1, describing as equal contribution between diffusion and erosion, the diffusion predominates over erosion, and the erosion predominates over diffusion, respectively [9][10][11]13]. The obtained parameters of the Kopcha release model are summarized in Table S3. According to the results, the non-Fickian process occurred for the cisplatin release from MS1, MS2, and MS3 hydrogels. Also, the Kopcha

 Protein interaction
Herein, the interaction mechanism of the MS2 hydrogel with the model proteins is investigated using the intrinsic fluorescence changes. As shown in Fig S2, the intrinsic fluorescence intensities of HSA and HB are regularly decreased with each addition of MS2 hydrogel at 25°C and 37 °C.
Hence, to indicate the mechanism of quenching, the Stern-Volmer equation (Eq. 8) is used as follows: The quenching mode is a combination of static and dynamic since the Stern-Volmer plot for both proteins is non-linear at 25 and 37 °C (Fig. S3 (A, B)). The graphs have upward curvature revealing that one quenching mechanism is dominated during complex formation [14]. Besides, it means rising temperature drives the quenching mechanism toward the dynamic. Nevertheless, it shows that the complex formation between the hydrogel and the model proteins is unstable with 9 increasing temperature. The slope deviation of graphs demonstrates that hydrogel interacts with the model proteins differently, which leads to the distinct complex stability for HSA and HB. It is In this regard, fa is the fraction of accessible fluorophores [15]. The results are shown in Fig.   S3 (C, D) and the compensatory behavior of the quenching mechanism and alterations in fa and KSV, the biomolecular quenching rate constant determines the interaction mechanism. The obtained values of constant kq are more than the maximum diffusion rate of molecules (2×10 10 M -1 s -1 ) in the 11 hydrogel interaction with the model proteins at 25 and 37 °C (Table S4). It elucidates that static quenching is dominant in the quenching mechanism. Therefore, a complex is formed between the hydrogel and the model proteins, which rising temperature causes complex instability.