Through the comprehensive study design, we integrated the biomaterial, in vitro, and in vivo investigations to propose a novel combination of carboplatin with hydrogel and surveyed its optimized treatment combining with RT for glioma. According to the analyses of drug release profile, biocompatibility, tumor control, survival, and side effects in this study, we demonstrated hydrogel carboplatin as an effective, simplified, and safe modality for local drug delivery to combine with RT for treating mice glioma, which provides a further clinical application for brain tumor treatment.
4.2.1 Effectiveness of hydrogel carboplatin combined with RT for tumor control
We demonstrated that the hydrogel dye diffused 7 to 9 mm from the injection site after intratumoral injection, which provides adequate distribution extent of hydrogel carboplatin in brain tumors after excision clinically. Regarding to the carboplatin dose, we used different dosages of carboplatin combined with RT to investigate the synergistic treatment effect. In the first-stage in vivo experiment using low-dose carboplatin (3 µg/g), the tumor control effect of ACR and HCR treatment groups was not different from R treatment group, which suggests low-dose carboplatin is not sufficient for tumor control. In the second-stage in vivo
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experiment using high-dose carboplatin (15 µg/g), the tumor control effect and survival of both HCR and ACR treatment groups were significantly better than R, HC and AC treatment groups. We designed the timing of RT on the second and third days after hydrogel carboplatin injection according to the drug release profile of hydrogel carboplatin and the tumor growth course of mice to maximize the CCRT synergistic effect. The tumor control effect of high-dose CCRT resulted in the higher complete response rate and longer survival time than carboplatin or RT alone. These results demonstrated that the synergistic effect of combining high-dose carboplatin with RT is more robust than RT or carboplatin alone. Clinically, most malignant gliomas progress or recur at the surgical cavity after tumor excision and RT [16-18]. Considering the clinical demand and our study results, the local delivery of hydrogel carboplatin at surgical cavity combined with RT is an effective treatment for malignant gliomas.
4.2.2 Convenience of hydrogel carboplatin administration to combine with RT
The administration of drug by thermogelling hydrogel carrier provides a different drug delivery option compared with wafer implant and aqueous drug via CED implant during surgery. The hydrogel form presents better conformal ability compared with wafer, and better attachment compared with aqueous form [42]. Our results indicates that once oxi-HA/ADH carboplatin was injected into in vivo condition at 37 °C, oxi-HA/ADH carboplatin formed gel in 17 seconds [67]. This gelling time of oxi-HA/ADH carboplatin is adequate for intratumoral injection and hydrogel stabilization for mice subcutaneous tumor model. For human brain tumor application, longer drug gelling time is required, which can be achieved
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by increasing the concentration of ADH in the hydrogel, ranging from 2 to 3 min at 37 °C [54]. The thermogelation (thermal-dependent gelling) characteristic provides adequate time for drug injection and stabilization [68], which makes hydrogel carboplatin more adaptable to the shape of surgical cavity, easier adhesion to the tissue, and full coverage of surgical cavity compared with wafer-form and aqueous drugs [42].
Our results of low-dose and high-dose carboplatin experiments indicated that the synergistic effect with RT requires sufficient carboplatin dose. To maintain the adequate carboplatin dose takes drug via CED with daily infusion [49, 51] or slow release via drug carrier, including wafer [41] or hydrogel [55-58]. The clinical use of CED or wafer is limited due to its inconvenience and potential side effects [42, 52, 53]. Using the features of slow and steady release of hydrogel and synergistic effect of carboplatin with RT [46, 47], we compounded and applied the hydrogel carboplatin to treat the mice glioma. By comparing the effects of single injection of hydrogel carboplatin with multiple injection of aqueous carboplatin to investigate the feasibility and convenience of hydrogel carboplatin. Comparing the HC verse AC and HCR versus ACR groups respectively of the second-stage in vivo experiment, the tumor control effects and survivals of single dose hydrogel carboplatin (300 µg) were comparable with the aqueous carboplatin with daily dose (100 µg) for 3 days. The results indicate that the single injection of hydrogel carboplatin facilitates the drug administration without compromising the anti-tumor effect. When combining with RT, the single injection of hydrogel carboplatin simplified the concurrent RT course without compromising the synergistic effects.
One published study using covalent linking of platinum and hydrogel to compose hydrogel polymer–platinum, which made the platinum release lasted more than 60 days [68].
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Clinically, the RT course duration for malignant glioma ranges from 3 to 6 weeks and hydrogel carboplatin is feasible for single injection during surgery rather than daily local infusion via CED. The degradation time of hydrogel ranges from 4 to 6 weeks after injection [67], which avoids interfering the neuroimaging follow-up in 8 to 12 weeks after treatment competed. Our results and published data indicates that using local and single injection of hydrogel carboplatin to combine with RT is a feasible treatment modality for malignant glioma [68, 70].
4.2.3 Safety of hydrogel carboplatin with RT
According to our in vitro study, the WST-1, LDH test, and LIVE/DEAD staining validated the biocompatibility of oxi-HA/ADH hydrogel. In the first-stage in vivo experiment, the tumor growth curves of R and HRT groups showed no significant difference, which suggests no additional toxicity of hydrogel when combining RT. In the second-stage in vivo experiment, through the white blood count, and renal and liver function tests, no systemic side effect was detected under the high-dose (15 µg/g) of either hydrogel carboplatin single dose or aqueous carboplatin daily injection for 3 days. In all treatment groups, no drug injection, radiation or CCRT-related skin ulcers were observed and the transitory weight loss was acceptable.
Due to the size scale of mice, the total drug dose for single injection was limited.
Comparing with human brain, the drug dose loaded by hydrogel is able to be higher.
According to the mice tumor growth schedule, we design the total drug release in 4 days and RT on the second and third days after first drug dose injection. The drug release time can be
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prolonged up to 5 weeks technically [54] to fit the clinical CCRT course for malignant gliomas. In order to measure the tumor volume serially, we adopted the subcutaneous implant model rather than brain implant model. Our results demonstrated the intratumoral drug delivery, but the effect to normal brain tissue or its tolerance dose requires further investigation. The influences on tumor growth and treatment effect resulting from the environment differences between subcutaneous tissue and brain needs more survey.