Cellular delivery of doxorubicin via pH-controlled hydrazone linkage using multifunctional nano vehicle based on poly(β-l-malic acid)

Abstract

Doxorubicin (DOX) is currently used in cancer chemotherapy to treat many tumors and shows improved delivery, reduced toxicity and higher treatment efficacy when being part of nanoscale delivery systems. However, a major drawback remains its toxicity to healthy tissue and the development of multi-drug resistance during prolonged treatment. This is why in our work we aimed to improve DOX delivery and reduce the toxicity by chemical conjugation with a new nanoplatform based on polymalic acid. For delivery into recipient cancer cells, DOX was conjugated via pH-sensitive hydrazone linkage along with polyethylene glycol (PEG) to a biodegradable, non-toxic and non-immunogenic nanoconjugate platform: poly(β-l-malic acid) (PMLA). DOX-nanoconjugates were found stable under physiological conditions and shown to successfully inhibit in vitro cancer cell growth of several invasive breast carcinoma cell lines such as MDA-MB-231 and MDA-MB- 468 and of primary glioma cell lines such as U87MG and U251.

Keywords: brain and breast cancer; doxorubicin; nanoconjugate; pH-controlled hydrazine linkage; polymalic acid.

Figure 1

Schematic presentation of the drug delivery system.

Figure 2

SEC-HPLC chromatograph of nanoconjugate P/PEG(5%)/GH-DOX(5%) after purification using size exclusion chromatography. Eluents were scanned at different wavelengths: (A) 220 nm (near the absorbance maximum for PMLA); (B) 480 nm (absorbance maximum for DOX); (C) 570 nm (DOX fluorescence, excitation at 475 nm) and (D) 220–600 nm wavelength range indicating degree of absorbance by a color code (red > yellow > green > blue > magenta).

Figure 3

Release kinetics of DOX from nanoconjugate P/PEG(5%)/GH-DOX(5%) at pH 5.0 (red) and pH 7.4 (green), at 37 °C.

Figure 4

Effect of nanoconjugate P/PEG(5%)/GH-DOX(5%), P/PEG(5%)/GH(5%) and free DOX on cell viability of human breast cancer cell lines MDA-MB-231, MDA-MB-468 and human glioma cell lines U87MG, U251. The concentrations refer to DOX content.

Scheme I

Synthesis of glycine Boc-hydrazide. Reagents and conditions: (i) N,N′-Dicyclohexylcarbodiimide (DCC), Ethyl acetate, 0 °C 2 h, RT 4 h, yield 78%; (ii) H₂/Pd-C, MeOH, RT 16 h, yield 80%.

Scheme II

Synthesis of P/PEG(5%)/GH-DOX(5%). Reagents and conditions: (i) Mixture of NHS and DCC, Acetone, DMF, RT 3 h, followed by addition of mPEG₅₀₀₀-NH₂, RT 45 min, and by glycine hydrazide, 2 h yield 67%; (ii) DOX.HCl, molecular sieves 4 Å, 10–18 mesh, DMF, RT 16 h, yield 72%.