Harnessing photochemical internalization with dual degradable nanoparticles for combinatorial photo-chemotherapy

Abstract

Light-controlled drug delivery systems constitute an appealing means to direct and confine drug release spatiotemporally at the site of interest with high specificity. However, the utilization of light-activatable systems is hampered by the lack of suitable drug carriers that respond sharply to visible light stimuli at clinically relevant wavelengths. Here, a new class of self-assembling, photo- and pH-degradable polymers of the polyacetal family is reported, which is combined with photochemical internalization to control the intracellular trafficking and release of anticancer compounds. The polymers are synthesized by simple and scalable chemistries and exhibit remarkably low photolysis rates at tunable wavelengths over a large range of the spectrum up to the visible and near infrared regime. The combinational pH and light mediated degradation facilitates increased therapeutic potency and specificity against model cancer cell lines in vitro. Increased cell death is achieved by the synergistic activity of nanoparticle-loaded anticancer compounds and reactive oxygen species accumulation in the cytosol by simultaneous activation of porphyrin molecules and particle photolysis.

Figure 1. Synthesis, degradation and self-assembly properties of the block copolymer.

(a) Synthetic route of the polycondensation reaction. For the precursor polymer, 2-nitroresorcinol and cyclohexyl divinyl ether were added in equimolar amounts with 1% PPTS (THF, N₂, 12 h, 55% yield). For the block copolymer, 2 kDa PEG monohydroxy terminated was used with 1% PPTS (see experimental section for the detailed description), (b) the dual stimulus-mediated degradation of the polymers occurs either by ultraviolet/visible photolysis by single (365 nm), double (532 nm) or multi (1,064 nm, Supplementary Fig. 8) photon excitation, or acidolysis at mildly acidic pH (ca. 5.2) and (c) our proposed generalized strategy involves the self-assembly of the block copolymers in aqueous solutions, which allows for efficient loading of drug cocktails (that is, CPT and HP) that can be released by application of mild light irradiation and/or owing to the intracellular pH drop in the late endosomes upon cellular uptake.

Figure 2. Polymer thin film photolysis studies.

(a) Characteristic ultraviolet/visible and (b) FT-IR spectra of the non-exposed and exposed polymer films at 365 nm radiation demonstrating typical absorbance and transmittance peaks of the photoproducts formed, (c) optical microscopy image showing the pattern formation after laser ablation of the polymer film with a single laser pulse (532 nm, 20 mJ cm⁻²) by application of a TEM grid as photomask; simultaneous Fresnel patterns in the squares of the photomask shown as a close-up image. Scale bar, 200 μm (expansion scale bar, 50 μm). (d) Digital photograph of the ablated areas that can be seen with the naked eye on a piece of a polymer coated silicon wafer. Scale bar, 10 mm.

Figure 3. Polymer and NP photolysis in solution and light/pH-controlled CPT release.

(a) ¹H NMR spectra of the initial polymer solution in d-chloroform (non-exposed) (in black) with the corresponding spectrum (in red) of the laser-irradiated sample showing characteristic diminution of the acetal proton (peak e) and the appearance of acetaldehyde (peaks i and f) and 2-nitroresorcinol (peak k) protons, (b) TEM microphotograph of polyacetal nanoparticles and (c) % CPT release from NPs at different pH and laser irradiation conditions (the green stripe represents the irradiation interval), and digital photograph of laser-exposed and non-exposed polymer solution. Scale bar, 500 nm.

Figure 4. NPs cell uptake and cytotoxicity studies.

(a) Increased cellular uptake of the NPs compared with the non-encapsulated drugs, (b) characteristic fluorescence microscopy image of HeLa cells incubated with drug-loaded NPs, the red filter has been used to track the distinct red emission profile of HP in the cytoplasm and (c) cell death rates in respect to irradiation using different laser sources and control samples (namely, free HP or CPT, free CPT/HP and lasers alone). Scale bar, 100 μm.

Figure 5. Photochemical internalization augmented by NPs photo–chemo-lysis.

The proposed mechanism of action involves the cellular uptake of the drug-loaded NPs followed by synergistic photo–chemo-degradation, which leads to enhanced drug liberation and endosome photo-disruption via HP activation and hydrophobization; cell death is augmented by the combination of the CPT-topoisomerase I interaction and the phototoxic pathway via ROS accumulation in the cytoplasm.