Photo- and Acid-Degradable Polyacylhydrazone-Doxorubicin Conjugates

Abstract

Light-mediated polymer degradation has attracted considerable attention in various applications, including photo-patterning, tissue engineering and photo-triggered drug delivery. In this study, we report the synthesis and characterization of a new, linear, main-chain photo- and acid-degradable copolymer based on acylhydrazone linkages. The polymer was synthesized via a step-growth copolymerization of adipic acid dihydrazide with a bifunctional poly(ethylene glycol) bearing benzaldehyde end-groups, under mild acidic conditions, to afford a hydrophilic PEG-alt-adipic acid (PEG-alt-AA) alternating copolymer. The synthesized polymer was characterized by size exclusion chromatography, proton nuclear magnetic resonance and attenuated total reflection-Fourier transform infrared spectroscopies. The main-chain photo- and acid-induced degradation of the copolymer in dimethylsulfoxide and water, respectively, was verified by UV-vis spectroscopy at light intensities as low as 0.1 mW cm^-2 at λ = 254 nm. Next, a model anticancer drug, doxorubicin (DOX), was chemically linked to the polymer chain end(s) via acylhydrazone bond(s), resulting in amphiphilic PEG-alt-adipic acid-DOX (PEG-alt-AA-DOX) polymer-drug conjugates. The conjugates were self-assembled in water to form spherical nanoparticles, as evidenced by scanning and transmission electron microscopies. The irradiation of the self-assembled PEG-alt-AA-DOX conjugates with UV light and the decrease of the solution pH resulted in the disruption of the assemblies due to the photolysis and acidolysis of the acylhydrazone bonds, and the release of the therapeutic cargo.

Keywords: acid-degradable polymers; doxorubicin; main-chain cleavage; photo-degradable polymers; polyacylhydrazones; prodrugs.

Scheme 1

Synthetic routes for the preparation of the (a) PEG_ald macromonomer, (b) PEG-alt-AA alternating copolymer and (c) PEG-alt-AA-DOX drug conjugate. (d) Schematic representation of the self-assembly process of the PEG-alt-AA-DOX conjugates and the pH- and/or light-induced disruption of the assemblies.

Figure 1

¹H NMR spectrum of PEG_ald in CDCl₃.

Figure 2

ATR–FTIR spectra (a) and SEC traces (b) of PEG before (black solid line) and after (red dashed line) esterification with 4-carboxybenzaldehyde.

Figure 3

SEC traces of the PEG-alt-AA alternating copolymer (black dashed line), the PEG_ald precursor macromonomer (wine solid line) and the PEG-alt-AA copolymer after 1 h irradiation at 254 nm (red dotted line).

Figure 4

¹H NMR spectrum of the PEG-alt-AA alternating copolymer in DMSO-d6.

Figure 5

ATR-FTIR spectra of adipic acid dihydrazide (black dashed dotted line), PEG_ald (red dashed line) and the PEG-alt-AA alternating copolymer (wine solid line).

Figure 6

(a) UV-vis absorption spectra of a 0.02 mg mL⁻¹ solution of the small diacylhydrazone molecule in DMSO upon irradiation at 254 nm, and (b) the photodegradation percentage of the diacylhydrazone as a function of the irradiation time.

Figure 7

UV-vis absorption spectra of (a) a 0.2 mg mL⁻¹ copolymer solution in DMSO, and (b) a 0.02 mg mL⁻¹ copolymer solution in H₂O at pH 7.4, upon irradiation at 254 nm. The photodegradation percentage of the PEG-alt-AA copolymer as a function of irradiation time in (c) DMSO and (d) H₂O at pH 7.4.

Figure 8

UV-vis absorption spectra of a 0.02 mg mL⁻¹ PEG-alt-AA alternating copolymer solution in H₂O before (black solid line) and after (red dashed line) irradiation for 2 h at 254 nm, and subsequently, after stirring for 24 h in the dark (blue dotted line).

Scheme 2

Proposed photodissociation pathways of PEG_ald upon irradiation with UV light.

Figure 9

¹H NMR spectra of PEG_ald (A) before, (B) after 1 h and (C) after 2 h irradiation at 254 nm in CDCl₃.

Figure 10

(a) UV-vis absorption spectrum of the PEG-alt-AA-DOX conjugate in DMSO (inset: magnified spectrum in the 340–700 nm range) and (b) fluorescence spectra of the PEG-alt-AA copolymer (black solid line), DOX (blue dashed line) and the PEG-alt-AA-DOX conjugate (red dashed dotted line).

Figure 11

SEM (a,c) and TEM (b,d) images of the self-assembled PEG-alt-AA-DOX drug conjugates before (a,b) and after (c,d) irradiation for 2 h at 254 nm.

Figure 12

Hydrodynamic diameter for the PEG-alt-AA-DOX assemblies in water before (black solid line) and after (red dashed line) irradiation for 2 h at 254 nm.

Figure 13

UV-vis absorption spectra of an aqueous PEG-alt-AA-DOX solution at (a) pH 7.4 and (b) pH 5.2 for different irradiation intervals at 254 nm. (c) Photodegradation percentage of the PEG-alt-AA-DOX nanoparticles as a function of irradiation time at pH 7.4 (red dots) and pH 5.2 (black squares).

Figure 14

Time-dependant release profile of DOX from the PEG-alt-AA-DOX assemblies upon irradiation (254 nm, 0.1 mW cm⁻²) at pH 7.4 (blue triangles), pH 5.2 (green triangles) and pH 2 (red spuares), and the control sample (without irradiation) at pH 2 (black circles).