3.2.1 Biomaterial investigation
3.2.1.1 Characterization of oxi-HA/ADH hydrogel and hydrogel carboplatin by FTIR analysis
The FTIR spectrum (Figure 12A1) demonstrated the aldehyde functional group of oxi-HA at frequency 1730 cm-1 [59]. The spectrum peak at 1730 cm-1 of oxi-HA disappeared after mixing oxi-HA with ADH, which correlated to the consumption of aldehyde to form the imine bond between oxi-HA and ADH and suggested the hydrogel formation [54, 67].
The appearance of a new forming peak at 1528 cm-1 of oxi-HA/ADH was associated with the N—H function group of ADH [54, 67]. The appearance of a new forming peak at 545 cm-1 of oxi-HA/ADH hydrogel carboplatin (Figure 12A2) was associated with the Pt—N function group of hydrogel carboplatin [59, 83]. Our results are comparable with published study that the oxi-HA, ADH, and caboplatin compound into hydrogel carboplatin [54, 83].
3.2.1.2 Gelling time of oxi-HA/ADH hydrogel by rheometer
The rheological properties demonstrated that the gelation of oxi-HA/ADH started at temperature higher than 27.6 °C (Figure 13, A1) and the gelling time of oxi-HA/ADH from liquid state to gel state were 17 seconds at 37 °C (body temperature) (Figure 13, A2) [59].
27
3.2.1.3 Degradation property of oxi-HA/ADH hydrogel
At 72 and 120 h, degradation percentages for oxi-HA/ADH (Figure 14) were 5.2% and 18.2%, respectively, which means less than 10% hydrogel was degraded within the first 3 days and more than 20% hydrogel started to degrade after 5 days [59]. The slow degradation property of oxi-HA/ADH hydrogel provides a stable carrier to load drug.
3.2.1.4 Drug release of hydrogel carboplatin by ICP-MS
Figure 15 demonstrates the cumulative release profile of carboplatin from oxi-HA/ADH hydrogel [59]. The ICP-MS result demonstrates two phases of the carboplatin release from hydrogel, including a burst release of 63.7% during the first 24 h, followed by a steady release of 16.6% over the 24 to 96 h. The drug release profile of carboplatin hydrogel illustrated that carboplatin was released up to 80.3% in 96 h, which provide 4 days to combine with RT after drug injection.
3.2.2 In vitro investigation
3.2.2.1 Biocompatibility of hydrogel
The WST-1 analysis (Figure 16A) demonstrated the cell viability of 3T3 cells cultured in oxi-HA/ADH hydrogel extraction medium was not significantly different compared with those in the control and negative control groups (p = 0.644) [59]. The LDH assay (Figure 16B) indicated the cytotoxicity oxi-HA/ADH is not significantly different from the negative control group (p = 0.173) [59].
28
The Live/Death staining (Figure 17) illustrated that nearly all the 3T3 cells were viable in the oxi-HA/ADH hydrogel after 3 days’ cultivation [59]. Our results, including WST-1 analysis, LDH assay, and Live/Death staining, demonstrated no evidence of toxicity of oxi-HA/ADH hydrogel, which is comparable with the published data [54].
3.2.2.2 IC
50of carboplatin to ALCS1C1 cells
The IC50 (Figure 18) test demonstrated that the in vitro concentrations of carboplatin to inhibit 50% ALCS1C1 cells to proliferation was 44.4 and 18.5 µg/mL after 1-day and 3-day treatment, respectively [59]. Considering the IC50 of carboplatin to AL1SC1 cells, drug carrying ability of hydrogel, and published data of Intratumoral carboplatin injection the adequate carboplatin concentration for in vivo experiment, we adopted the carboplatin concentration of 2 mg/mL for our in vivo investigation [56, 78].
3.2.3 In vivo investigation
3.3.1 First-stage in vivo experiment (low-dose carboplatin)
Five treatment groups, including S (N = 5), R (N = 5), HRT (N = 5), ACR (N = 5), and HCR (N = 5), were analyzed in the first-stage in vivo experiment. One mouse in HRT group and and 2 mice in HCR group died early due to the anesthesia procedure.
Comparing with the sham group, the BLI (Figure 19) demonstrated the relatively delayed tumor progression in all treatment groups [59]. Correspondingly, the tumor volume curves (Figure 20) of the treatment groups demonstrated tumor progression after temporary
29
tumor control effects [59]. On day 24, the tumor volume analysis (excepting sham group) by one-way ANOVA showed no difference of tumor progression for RT with and without low-dose carboplatin (p = 0.787). The survival data (Table 7) revealed that all mice treated with RT alone or combined with low-dose carboplatin died within 39 days after tumor implant [59].
3.2.3.2 Second-stage in vivo experiment (high-dose carboplatin)
Seven treatment groups, including S (N = 7), H (N = 9), AC (N = 9), HC (N = 9), R (N
= 6), ACR (N = 6), and HCR (N = 6), were analyzed in the second-stage in vivo experiment.
The BLI (Figure 21) demonstrated the tumor nearly complete response in HCR and ACR groups, while tumor progression in other treatment groups [59]. The bioluminescence signal represented in radiance showed corresponding findings (Figure 22) [59].
Figure 23 [59] illustrates the tumor volume curves of different treatment modalities combination in the second-stage in vivo experiment with high-dose carboplatin (15 µg/g), including RT, aqueous carboplatin (100 µg once daily for 3 days) or hydrogel carboplatin (300 µg in a single dose on the first treatment day) alone, and combining RT with aqueous or hydrogel carboplatin, respectively. In ACR and HCR groups, the tumor volume curves demonstrate good tumor control without difference (p = 0.904), while R group demonstrates tumor progression after temporal tumor volume control and AC and HC groups demonstrate persistent tumor progression. Tumor injected with hydrogel alone showed the same tumor growth as those without any treatment.
30
The subgroup tumor volume analysis on day 24 by t-test demonstrated no significant difference of tumor control effects by comparing ACR with HCR group (p = 0.904) and AC with HC groups (p = 0.747), respectively. HCR group showed better tumor control than either RT group (p = 0.007) or the HC group (p = 0.006) [59].
Table 8 lists the survival time according to different treatment modality combinations in the second-stage [59]. Three mice in HCR group and 4 mice in ACR group had no tumor recurrence after treatment on day 104 after tumor implant. The survival curves (Figure 24) revealed that the HCR and ACR groups had 104-day survival rates of 50% and 66.7% without significant difference (p = 0.648). Other mice treated with radiation or high-dose carboplatin alone died within 40 days after tumor implant.
Figure 25A demonstrated the tumor cross section after intratumoral injection with oxi-HA/ADH hydrogel dye [59]. The blue area illustrated the diameters of dye distribution 1 day and 3 days after hydrogel dye injection were 7 and 9 mm, respectively. Our results suggested the intratumoral drug injection provides adequate drug distribution to cover the tumor area.
The response of different treatment combinations evaluated by slices of H&E stain were illustrated in Figure 25, B1 to B3 and C1 to C4, respectively [59]. The H&E stain of tumor receiving RT (Figure 25, B1) showed both cell death (yellow rectangular area at 100× and yellow arrow at 400×) and tumor cell proliferation (red rectangular area at 100× and red arrow at 400×). The H&E stain of tumor receiving either aqueous carboplatin with RT (Figure 25, B2) or hydrogel carboplatin with RT (Figure 25, B3) showed prominent cell death.
Contrastingly, H&E stain of tumor cell growth under no treatment (Figure 25, C1) and injection of hydrogel (Figure 25, C2), aqueous carboplatin (Figure 25, C3), and hydrogel carboplatin (Figure 25, C4) all showed tumor cell proliferation only.
31
Table 9 illustrated the blood analysis, and no neutropenia and renal or hepatic function impairments were detected [59]. Figure 26A demonstrated transitory weight loss in HCR and ACR of high-dose carboplatin treatment groups then recovered without death [59]. Figure 26, B1 and B2, demonstrated that no drug injection or RT-related skin ulcer was observed in 3 weeks in HCR and ACR treatment groups, respectively [59].