Injectable Dendrimer Hydrogel Delivers Melphalan in Both Conjugated and Free Forms for Retinoblastoma

Abstract

We report the successful synthesis of an injectable dendrimer hydrogel (DH) carrying melphalan, a clinical drug for retinoblastoma treatment, in both conjugated and free forms. Polyamidoamine (PAMAM) dendrimer generation 5 (G5) is surface-modified with an acid-sensitive acetal-dibenzocyclooctyne linker and then undergoes azide-alkyne cycloaddition with melphalan-PEG-N₃ conjugate to form G5-acetal-melphalan. During the DH gelation between G5-acetal-melphalan and PEG-diacrylate, free melphalan is added, resulting in a hydrogel (G5-acetal-melphalan-DH/melphalan) that carries the drug in both conjugated and free forms. Melphalan is slowly released from G5-acetal-melphalan-DH/melphalan, with the conjugated melphalan released more quickly at pH 5.3 due to acid-triggered acetal bond cleavage. The formulation's in vitro safety and efficacy were established on human corneal epithelia (HCE-2) and retinoblastoma cells (Y79). In an in vivo Y79 tumor xenograft model of retinoblastoma, intratumorally injected G5-melphalan-DH formulation prolonged tumor suppression. This injectable, multimodal, pH-responsive formulation shows promise for intravitreal injection to treat retinoblastoma.

Figure 1.

¹H NMR spectrum of G5-acetal-ene precursor in MeOD.

Figure 2.

¹H NMR spectrum of modified melphalan-PEG-N₃ in D₂O.

Figure 3.

¹H NMR of G5-acetal-melphalan spectrum in D₂O. The unreacted -ene groups remain and the peaks are circled in red.

Figure 4.

Oscillatory amplitude sweeps (A, C) and oscillatory frequency sweeps (B, D) of G5-DH^2% (A, B) and G5-melphalan-DH^2% (C, D). ● and ● represent G’,○ and ○ represent G”.

Figure 5.

In vitro drug release profiles of melphalan formulations at 37 °C.

Figure 6.

Relative cell viability based on WST-1 assay. (A) HCE-2 cells incubated with melphalan (DMSO). (B) HCE-2 cells incubated with G5-DH (curve fitting was not conducted due to its broad non-toxic dose range). (C) Y79 cells incubated with melphalan (PBS). (D) Y79 cells incubated with melphalan-PEG-N₃. (E) Comparison of cytotoxicity to Y79 cells among free melphalan, G5-DH/melphalan, and G5-melphalan-DH. n ≥ 4, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 7.

Antitumor effects of melphalan and DH formulations. (A) Relative tumor growth. (B) Kaplan-Meier survival curve. (C) Bodyweight measurements. PBS (n = 3), free melphalan (n = 5), G5-DH (n = 3), G5-DH/melphalan (n = 5), G5-melphalan-DH (n = 5), and G5-melphalan-DH/melphalan (n = 5). Arrow bars signify i.t. treatment injections. **p <0.01, ***p<0.001.

Figure 8.

H&E staining images of the tumor sections. Scale bar: 100 μm.

Scheme 1.

Preparation of dendrimer hydrogel carrying melphalan in both conjugated and free forms, i.e., G5-melphalan-DH/melphalan. (1) Synthesis of G5-acetal-DBCO. (2) Synthesis of melphalan-PEG_5K-N₃ conjugate. (3) Click reaction between G5-acetal-DBCO and melphalan-PEG-N₃ followed by crosslinking reaction with PEG-DA in the presence of free melphalan to form G5-melphalan-DH/melphalan.