Convenient and Controllable Synthesis of Poly(2-oxazoline)-Conjugated Doxorubicin for Regulating Anti-Tumor Selectivity

Abstract

Polyethylene glycol (PEG)-doxorubicin (DOX) conjugation is an important strategy to improve toxicity and enhance clinically therapeutic efficacy. However, with the frequent use of PEG-modified drugs, the accumulation of anti-PEG antibodies has become a tough issue, which limits the application of PEG-drug conjugation. As an alternative solution, poly(2-oxazoline) (POX)-DOX conjugation has shown great potential in the anti-tumor field, but the reported conjugation process of POX with DOX has drawbacks such as complex synthetic steps and purification. Herein, we propose a convenient and controllable strategy for the synthesis of POX-DOX conjugation with different chain lengths and narrow dispersity by N-boc-2-bromoacetohydrazide-initiated 2-ethyl-oxazoline polymerization and the subsequent deprotection of the N-Boc group and direct reaction with DOX. The DOX-PEtOx conjugates were firstly purified, and the successful conjugations were confirmed through various characterization methods. The synthetic DOX-PEtOx_n conjugates reduce the toxicity of DOX and increase the selectivity to tumor cells, reflecting the promising application of this POX-DOX conjugation strategy in drug modification and development.

Keywords: anti-tumor selectivity; facile synthesis; poly(2-oxazoline); poly(2-oxazoline)-doxorubicin conjugation.

Scheme 1

(A) Synthesis of N-boc-2-bromoacetohydrazide; (B) Synthetic route of hydrazide poly(2-ethyl-oxazoline)s; (C) Conjugation of doxorubicin and PEtOx.

Figure 1

Schematic illustration of the design and drug modification property of DOX−P(EtOx)n. Host cells were killed when treated with free DOX, whereas the cells remained alive after treatment with DOX−PEtOx conjugates.

Figure 2

(A) ¹H NMR characterization of protected polymers (D₂O as solvent, 600 MHz), a, b, c and d represented the characteristics of H at different positions in the chemical structure of the polymers, the ¹H NMR spectra was cut to 0.5–3.7 ppm because this range includes the characteristic peaks, and the complete spectra can be found in the Supporting Information; (B) GPC traces of PEtOx of three chain lengths; (C) GPC characterization of PEtOx of three chain lengths. M_n and Đ were determined by GPC using DMF as the mobile phase at a flow rate of 1 mL/min. DP was determined by ¹H NMR. M_n,theo is the theoretical number average molecular weight; M_n,GPC is the obtained number average molecular weight analyzed by GPC; DP is the obtained degree of polymerization; Đ means the dispersity.

Figure 3

HPLC spectrum of DOX, PEtOx and DOX−PEtOx conjugates: (A) Comparation among DOX, PEtOx₅ and DOX−PEtOx₅; (B) Comparation among DOX, PEtOx₁₀ and DOX-PEtOx₁₀; (C) Comparation among DOX, PEtOx₂₀ and DOX-PEtOx₂₀. The analysis was performed with a SHIMADZU LC 20AR HPLC System equipped with a Luna Omega 5 μm polar C18 column. All samples (DOX, PEtOx and DOX−PEtOx) were analyzed with the same method: 20–80% phase B for 8 min; 80% phase B for 7 min; 80–20% phase B for 8 min, with 1 mL/min flow rate, detected at the wavelength of 220 nm.

Figure 4

FTIR spectra of DOX, PEtOx and DOX−PEtOx conjugates: (A) Comparison among DOX, PEtOx₅ and DOX−PEtOx₅; (B) Comparison among DOX, PEtOx₁₀ and DOX−PEtOx₁₀; (C) Comparison among DOX, PEtOx₂₀ and DOX−PEtOx₂₀; (D) The summary of the FTIR signals of characteristic groups of DOX and polymers.

Figure 5

MALDI-TOF MS spectrum of DOX-PEtOx conjugates: (A) DOX−PEtOx₅; (B) DOX−PEtOx₁₀; (C) DOX−PEtOx₂₀. The 2,5-dihydroxybenzoic acid (DHB) was used as the matrix dissolved in acetonitrile (MeCN) at a concentration of 20 mg/mL. All conjugates were dissolved in MeCN at the concentration of 10 mg/mL. The matrix and conjugates were mixed with v/v 5:1 and used as samples for MALDI-TOF MS characterization. The data were analyzed by data explorer and origin.

Figure 6

Mammalian cytotoxicity (IC₅₀, μg/mL) of three DOX−PEtOx conjugates toward (A) HUVEC cells and (B) COS-7 cells determined by MTT assay; (C) anticancer activity (IC₅₀, μg/mL) of conjugates against B16 cells determined by MTT assay. Free DOX was used as control. Three replicates were conducted in each experiment; all the experiments were repeated for twice.

Figure 7

Selectivity index of DOX−PEtOx calculated from: (A) IC₅₀(HUVEC)/IC₅₀(B16); (B) IC₅₀(COS-7)/IC₅₀(B16).

Figure 8

Proof used to form unstable self-assembling micelles at aqueous solution: (A) Size distribution of DOX−PEtOx conjugates characterized by DLS measurement thrice; (B) Tyndall observation of DOX−PEtOx conjugates.