In Situ Targeting RGD-Modified Cyclodextrin Inclusion Complex/Hydrogel Hybrid System for Enhanced Glioblastoma Therapy

Abstract

Background/Objectives: Glioblastoma (GBM) remains the most aggressive primary brain tumor, characterized by high malignancy, recurrence rate, and dismal prognosis, thereby demanding innovative therapeutic strategies. In this study, we report a novel in situ targeting inclusion complex hydrogel hybrid system (DOX/RGD-CD@Gel) that integrates doxorubicin (DOX) with RGD-conjugated cyclodextrin (RGD-CD) and a thermosensitive hydrogel for enhanced GBM therapy. Methods: The DOX/RGD-CD@Gel system was prepared by conjugating doxorubicin (DOX) with RGD-modified cyclodextrin (RGD-CD) and embedding it into a thermosensitive hydrogel. The drug delivery and antitumor efficacy of this system were evaluated in vitro and in vivo. Results: In vitro and in vivo evaluations demonstrated that DOX/RGD-CD@Gel significantly enhanced cytotoxicity compared to free DOX or DOX/CD formulations. The targeted delivery system effectively promoted apoptosis and inhibited cell proliferation and metastasis in GBM cells. Moreover, the hydrogel-based system exhibited prolonged drug retention in the brain, as evidenced by its temperature- and pH-responsive release characteristics. In a GBM mouse model, DOX/RGD-CD@Gel significantly suppressed tumor growth and improved survival rates. Conclusions: This study presents a paradigm of integrating a targeted inclusion complex with a thermosensitive hydrogel, offering a safe and efficacious strategy for localized GBM therapy with potential translational value.

Keywords: cyclodextrin; doxorubicin; glioblastoma; hydrogel; inclusion complex.

Scheme 1

Formation and combined antiglioma mechanism of DOX/RGD-CD@Gel.

Figure 1

The effects of DOX/RGD-CD on GL261 cell activity and the underlying mechanisms. (A) The effect of DOX/RGD-CD on the cytotoxicity of GL261 cells. (B) The effect of DOX/RGD-CD on the apoptosis of GL261 cells. (C) The effect of DOX/RGD-CD on the live/death of GL261 cells. (D) The effect of DOX/RGD-CD on the expression of apoptosis-related proteins in GL261 cells. (E) The semi-quantitative statistical results of apoptosis-related protein expression (n = 3, $\overline{\mathrm{x}}$ ± SD, * p < 0.05).

Figure 2

The effects of DOX/RGD-CD on the migration and invasion of GL261 cells. (A) The effect of DOX/RGD-CD on the migration of GL261 cells. (B) The effect of DOX/RGD-CD on the invasion of GL261 cells. (C) The relative migration rate of GL261 cells after the treatment with DOX/RGD-CD. (D) The relative invasion rate of GL261 cells after the treatment with DOX/RGD-CD. (E) The effect of DOX/RGD-CD on the expression of invasion-related proteins in GL261 cells. (F) Semi-quantitative statistical results of the invasion-related proteins. (n = 3, $\overline{\mathrm{x}}$ ± SD, *^,# p < 0.05).

Figure 3

Cellular uptake and uptake mechanism of DOX/RGD-CD by GL261 cells. (A) The uptake of DOX/RGD-CD by GL261 cells observed via CLSM. (B) The effect of uptake inhibitors on the uptake of DOX/RGD-CD by GL261 cells observed via CLSM.

Figure 4

Physicochemical characterization of DOX/RGD-CD@Gel. (A) Representative SEM image of DOX/RGD-CD@Gel. (B) The temperature-responsive phase transition process of DOX/RGD-CD@Gel. (C) Rheological characterization of DOX/RGD-CD@Gel. (D) Cytotoxicity of Blank@Gel. (E) Erythrocyte hemolysis of Blank@Gel. (F) In vitro DOX release from DOX/RGD-CD@Gel in different pH release media (n = 3, $\overline{\mathrm{x}}$ ± SD, * p < 0.05).

Figure 5

The biodistribution and therapeutic effect of DOX/RGD-CD@Gel in orthotopic GBM mice. (A) In vivo distribution of different formulations over time. (B) Semi-quantitative statistical results of fluorescence intensity in A. (C) The inhibitory effect of DOX/RGD-CD@Gel on the growth of orthotopic GBM observed by in vivo bioluminescence imaging. (D) Statistical analysis of orthotopic GBM growth. (E) Survival curve of orthotopic GBM mice. (F) Effects of DOX/RGD-CD@Gel on the body weight of orthotopic GBM mice (n = 6, $\overline{\mathrm{x}}$ ± SD, *^,# p < 0.05).

Figure 6

Effect of DOX/RGD-CD@Gel on the apoptosis and invasive microenvironment in orthotopic GBM tissue. (A) The HE staining of orthotopic GBM tissue slices. (B) Expression of Ki-67 staining of orthotopic GBM tissue. (C) Expression of TUNEL staining of orthotopic GBM tissue. (D) Semi-quantitative statistical results of Ki-67 expression. (E) Semi-quantitative statistical results of TUNEL staining. (F) The effect of DOX/RGD-CD@Gel on the expression of apoptosis-related proteins in orthotopic GBM tissue. (G) Semi-quantitative statistical results of apoptosis-related proteins. (H) The effect of DOX/RGD-CD@Gel on the expression of EMT-related protein in orthotopic GBM tissue. (I) Semi-quantitative statistical results of EMT-related proteins (n = 3, $\overline{\mathrm{x}}$ ± SD, *^,# p < 0.05).

Figure 7

Preliminary safety evaluation of DOX/RGD-CD@Gel in mice. (A) HE staining of the heart and kidney tissue in mice. (B) Effects of DOX/RGD-CD@Gel on the content of LDH activity in serum of mice. (C,D) Effects of the DOX/RGD-CD@Gel on the contents of BUN and CREA in serum of mice. (E,F) Effects of DOX/RGD-CD@Gel on the content of ALT and AST activity in serum of mice. The earthy yellow area indicates the normal ranges (n = 3, $\overline{\overline{\mathrm{x}}}$ ± SD).