Development of a Novel Fluorescent and Injectable Thermosensitive Hydrogel as Multifunctional Modalities for Imaging and Glioblastoma Therapy

Abstract

Glioblastoma multiforme (GBM) is a highly aggressive intracranial tumor characterized by uncontrolled proliferation, neurodegeneration, and tissue infiltration. Surgical debulking, the mainstay of treatment for GBM, frequently suffers from incomplete excision owing to the tumor’s invasive nature. This incomplete removal ultimately paves the way for inevitable tumor recurrence within 6-12 months post-resection. An innovative approach to enhance treatment efficacy involves localized drug delivery systems, which administers high concentration of drugs directly into the resection cavity; thereby enhancing the therapeutic efficacy and significantly mitigating systemic exposure. In addition, the diagnosis and treatment of GBM are extremely challenging due to its intratumoral heterogeneity. Fortunately, the identification of specific molecular signatures of GBM has emerged as a better-suited technique for its effective diagnosis. The discovery of approved drugs targeting these biomarkers can effectively conquer GBM. In parallel, fluorescence imaging (FL) techniques offer substantial advantages in clinical diagnosis, including high contrast and sensitivity, non-invasiveness, and low cost. Thus, there is a growing demand for the development of multifunctional injectable systems that integrate diagnostic and therapeutic capabilities for effective detection, imaging, and targeting of GBM lesions. In this study, we systematically discovered 2734 overlapping genes that were differentially expressed between GBM and noncancerous brain tissues through meta-analysis of transcriptomic data. The relevant functions and signaling pathways of differentially expressed genes (DEGs) were detected through enrichment analysis. Subsequently, AURKA, AURKB, CDK1, CDK2, CCNB1, CCNB2, CDC20, BUB1, PLK1, and BIRC5 were screened as hub genes via maximum neighborhood component (MNC) algorithm. Furthermore, a drug-gene interaction network predicted paclitaxel (PTX) as a potential therapeutic candidate to neutralize the dysregulated effects of oncogenes. Molecular docking studies showed a stronger binding affinity of PTX with AURKA [ΔG = -9.06 kcal/mol], CDK2 [ΔG = -8.93 kcal/mol], and BIRC5 [ΔG = -7.55 kcal/mol]. We further encapsulated PTX within the mesopores of hematite (α-Fe2O3) nanoparticles (HPTX). The encapsulation efficiency (EE) and loading capacity (LC) of PTX were estimated to be 99.2 ± 0.2 % and 10.3 ± 0.08 %, respectively. The MTT assay reflected superior toxicity of HPTX (IC50 = 39.2 ng/mL) against LN229 cells compared to free PTX (IC50 = 100 ng/mL) post-24-hour treatment. Moreover, we developed ultra-small (~ 3 nm) nitrogen-doped carbon quantum dots (NCQDs) with high quantum yield (QY). NCQDs demonstrated remarkable antijamming performance and high photostability, ideal for bioimaging. Moreover, the water-soluble luminous pearls served as a biocompatible trident in cancer biology, achieving a three-pronged action of in vitro cell photostability, multi-color imaging, and migration without hampering the biological system. The histological and biochemical analysis demonstrated no overt toxicity of NCQDs in mice, even under multi-dosing situations. Further, nanofibrous polyelectrolyte (PEC) complex of chitosan (CH) and sodium alginate (SAlg) was synthesized at different volumetric ratios (CHAlg50 and CHAlg70). These nanofibers were incorporated into CH-based injectable thermoresponsible hydrogel to enhance mechanical properties, achieve sustained drug release, and ensure long-term therapeutic efficacy. Subsequently, the optimal concentrations of NCQDs and HPTX were then embedded within the thermogel matrix, enabling simultaneous diagnosis and therapy of GBM. Hydrogels formulated with CHAlg50 demonstrated favorable swelling ratio (12.9), minimal degradability, and sustained PTX release (39.14%) compared to the CHAlg70 counterparts. Mechanical characterization revealed a Young’s modulus (YM) of 12 kPa, closely mimicking the softness of human tissues (1-100 kPa). The HPTX-loaded hydrogel variant, by virtue of its slow and sustained release of PTX, exerted pronounced cytotoxicity against LN229 cells, as evidenced by MTT assay and live/dead staining. Moreover, in vitro studies highlighted the multifaceted properties of the optimized hydrogel variant in monitoring cellular uptake and inducing apoptosis in LN229 cells. The green FL of NCQDs facilitated the detection of PTX-induced apoptosis, obviating the need for multiplexed dyes. Additionally, the optimized hydrogel variant significantly downregulated the expression of the AURKA, BIRC5, and CDK2 oncogenes. In conclusion, the amalgamation of diagnostic and therapeutic moieties within a single system provides a new dimension for the potential application of injectable thermogels in cancer theranostics.