High efficiency loading of micellar nanoparticles with a light switch for enzyme-induced rapid release of cargo

Abstract

Polymeric nanoscale materials able to target and accumulate in the tumor microenvironment (TME) offer promising routes for a safer delivery of anticancer drugs. By reaching their targets before significant amounts of drug are released, such materials can reduce off-target side effects and maximize drug concentration in the TME. However, poor drug loading capacity and inefficient nanomaterial penetration into the tumor can limit their therapeutic efficacy. Herein, we provide a novel approach to achieve high loading profiles while ensuring fast and efficient drug penetration in the tumor. This is achieved by co-polymerizing light-sensitive paclitaxel with monomers responsive to tumor-associated enzymes, and assembling the resulting di-block copolymers into spherical micelles. While light exposure enables paclitaxel to decouple from the polymeric backbone into light-activated micelles, enzymatic digestion in the TME initiates its burst release. Through a series of in vitro cytotoxicity assays, we demonstrate that these light-switch micelles hold greater potency than covalently linked, non-triggered micelles, and enable therapeutic profiles comparable to that of the free drug.

Figure 1.. Light-activable micelles (LAMs) for the delivery of paclitaxel (PTX) to the tumor microenvironment (TME).

Schematic representation of the enzyme-responsive LAMs. UV irradiation of inactive micelles (IMs) causes PTX cleavage and release into the hydrophobic pocket switching IMs to active micelles (AMs). Cleavage of the outer peptide shell by proteases results in a morphological switch and burst PTX release.

Figure 2.. Synthesis of light-activable block copolymers (LAPs).

Ring-opening metathesis polymerization (ROMP) of functionalized monomers lead to the incorporation of (i) (PTX) containing the UV-cleavable linker (red), (ii) MMP-responsive peptide (blue) and (iii) NIR Cy5.5 fluorescent dye (green). Polymerizations were terminated via the addition of (iv) ethyl vinyl ether

Figure 3.. Enzyme-triggered size and morphology switch of LAMs.

A) Size of inactive micelles (IMs), B) size of active micelles (AMs) before and C) after 18 h incubation with the model enzyme thermolysin as determined by dynamic light scattering (DLS) measurements. Changes in micelles morphology was investigated via both (D-F) dry state transmission electron microscopy (TEM) imaging (scale bars 200 nm), as well as confocal fluorescence microscopy of Cy5.5 labeled micelles in PBS G) before and H) after enzymatic cleavage. All settings were kept constant between images. Scale bars 50 μm and 10 μm

Figure 4.. UV irradiation triggers PTX cleavage from the polymer backbone and results in micelles activation.

(A) RP-HPLC traces of IMs before and (B) after 30 min UV irradiation at 365 nm (AMs). (C) RP-HPLC traces of the filtrate deriving from AM centrifugal filtration. RP-HPLC gradient: 10 to 80 % acetonitrile in 45 min. (D) ESI-MS spectra of the peak eluted at R_t 24.5 min in the chromatogram reported in (C): PTX [M+H]⁺ = 854.35.

Figure 5.. LAMs cytotoxicity in HT1080 cell line is comparable to that of free PTX.

(A) Structure of the control ester di-block copolymer (EP) and B) DLS before (blue) and after (red) enzymatic cleavage. (C) Live fluorescence imaging of the resulting Cy 5.5-labeled EMs in cell medium after 24 h incubation with HT-1080 cells. Micelle aggregates are indicated in red (Cy5.5) and the cell nuclei are stained in blue (Hoechst). Scale bars 10 μm. Transmitted light images as well as more examples are reported in the Supporting Information. (D) HT1080 cell viability upon treatment with either EMs or free PTX. Cell viability was determined after 72 h via Cell Titer Blue (CTB) assay and the reported curves are the result of independent triplicate experiments with each condition performed in triplicate. The same results for LAMs before and after UV or both UV and enzymatic degradation are reported in the bottom panel (E-H).