Construction of Enzyme-Responsive Micelles Based on Theranostic Zwitterionic Conjugated Bottlebrush Copolymers with Brush-on-Brush Architecture for Cell Imaging and Anticancer Drug Delivery

Abstract

Bottlebrush copolymers with different chemical structures and compositions as well as diverse architectures represent an important kind of material for various applications, such as biomedical devices. To our knowledge, zwitterionic conjugated bottlebrush copolymers integrating fluorescence imaging and tumor microenvironment-specific responsiveness for efficient intracellular drug release have been rarely reported, likely because of the lack of an efficient synthetic approach. For this purpose, in this study, we reported the successful preparation of well-defined theranostic zwitterionic bottlebrush copolymers with unique brush-on-brush architecture. Specifically, the bottlebrush copolymers were composed of a fluorescent backbone of polyfluorene derivate (PFONPN) possessing the fluorescence resonance energy transfer with doxorubicin (DOX), primary brushes of poly(2-hydroxyethyl methacrylate) (PHEMA), and secondary graft brushes of an enzyme-degradable polytyrosine (PTyr) block as well as a zwitterionic poly(oligo (ethylene glycol) monomethyl ether methacrylate-co-sulfobetaine methacrylate) (P(OEGMA-co-SBMA)) chain with super hydrophilicity and highly antifouling ability via elegant integration of Suzuki coupling, NCA ROP and ATRP techniques. Notably, the resulting bottlebrush copolymer, PFONPN₉-g-(PHEMA₁₅-g-(PTyr₁₆-b-P(OEGMA₆-co-SBMA₆)₂)) (P₂) with a lower MW ratio of the hydrophobic side chains of PTyr and hydrophilic side chains of P(OEGMA-co-SBMA) could self-assemble into stabilized unimolecular micelles in an aqueous phase. The resulting unimolecular micelles showed a fluorescence quantum yield of 3.9% that is mainly affected by the pendant phenol groups of PTyr side chains and a drug-loading content (DLC) of approximately 15.4% and entrapment efficiency (EE) of 90.6% for DOX, higher than the other micelle analogs, because of the efficient supramolecular interactions of π-π stacking between the PTyr blocks and drug molecules, as well as the moderate hydrophilic chain length. The fluorescence of the PFONPN backbone enables fluorescence resonance energy transfer (FRET) with DOX and visualization of intracellular trafficking of the theranostic micelles. Most importantly, the drug-loaded micelles showed accelerated drug release in the presence of proteinase K because of the enzyme-triggered degradation of PTyr blocks and subsequent deshielding of P(OEGMA-co-SBMA) corona for micelle destruction. Taken together, we developed an efficient approach for the synthesis of enzyme-responsive theranostic zwitterionic conjugated bottlebrush copolymers with a brush-on-brush architecture, and the resulting theranostic micelles with high DLC and tumor microenvironment-specific responsiveness represent a novel nanoplatform for simultaneous cell image and drug delivery.

Keywords: FRET; brush-on-brush; enzyme-responsive; theranostic; zwitterionic.

Scheme 1

Schematic representation of the synthesis, drug loading, cellular uptake, and rapid enzyme-responsive drug release of the zwitterionic conjugated bottlebrush copolymers.

Scheme 2

Synthesis of monomers (1) and (2), the backbone of azido-functionalized Polyfluorene (PFONPN-g-N₃), alkyne-PHEMA, l-tyrosine N-carboxyanhydride (Tyr-NCA), and CHO-Br₂.

Scheme 3

Synthesis of hydrophilic bottlebrush copolymers PFONPN-g-(PHEMA-g-(PTyr-b-P(OEGMA-co-SBMA)₂)) and PFONPN-g-(PHEMA-g-(PTyr-b-POEGMA₂)) via the approach of “graft from”.

Figure 1

SEC elution traces (dRI signals) of macroinitiator I₁-I₆ (a) and amphiphilic bottlebrush copolymers P₁, P₃, and P₅ (b), as well as SEC elution traces (UV signals) of macroinitiator I₁, I₃-I₆ (c).

Figure 2

¹H-NMR spectra of (a) PFONPN-g-(PHEMA-g-(PTyr-NH₂)), (b) PFONPN-g-(PHEMA-g-(PTyr-Br₂)), and (c) PFONPN-g-(PHEMA-g-(PTyr-b-P(OEGMA-co-SBMA)₂)) in DMSO-d₆.

Figure 3

Size distributions of P₁ (a), and P₂ (b) micelles with a polymer concentration of 0.5 mg/mL in DMF; P₁ (c), and P₂ (d) with different polymer concentrations in water.

Figure 4

Size distributions of P₁ (a), and P₂ (b) micelles in water; TEM images of P₁ (c), and P₂ (d) micelles in water at a polymer concentration of 0.5 mg/mL (scale bar: 100 nm).

Figure 5

Size distributions of P₁ (a), and P₂ (b) micelles in HEPES (10 mM, pH 7.4) and P₁ (c), and P₂ (d) micelles in response to proteinase K (6 U/mL) in HEPES (pH 7.4, 10 mM) at 37 °C for 12 h at a polymer concentration of 0.5 mg/mL.

Figure 6

Size distributions of DOX@P₂-1 (a), and DOX@P₂-2 (b) micelles in water; TEM images of DOX@P₂-1 (c), and DOX@P₂-2 (d) micelles in water at a polymer concentration of 0.5 mg/mL (scale bar: 100 nm).

Figure 7

In vitro release of DOX in the presence or absence of proteinase K (6 U/mL) in DOX@P₂-1 micelles (0.5 mg/mL) at different pH values of 7.4 and 5.0 at 37 °C.

Figure 8

Fluorescence excitation and emission spectra of the conjugated backbone of PFONPN and P₂ in DMSO (a), as well as P₂ and DOX in water (b), and fluorescence emission spectra of P₂ added with a different feed ratio of DOX from 1–6% (c) and 7–10% (d) with excitation at 390 nm in water.

Figure 9

Fluorescence microscopy images of DOX, P, and DOX@P₂-1 (green for PFONPN moiety) micelles uptake in Bel-7402 cells (nuclei stained blue with DAPI) (scale bar: 200 μm).

Figure 10

Quantitative measurements of the average fluorescence intensities of free DOX and DOX@P₂-1 by flow cytometry in Bel-7402 cells (n = 3, *** p < 0.001) (a); In vitro cytotoxicity of P₂ micelles in L02 cells and Bel-7402 (b) as well as DOX, DOX@P₂-1 in L02 cells (c), and Bel-7402 cells (d).