Intracellular activation of anticancer therapeutics using polymeric bioorthogonal nanocatalysts

Abstract

Bioorthogonal catalysis provides a promising strategy for imaging and therapeutic applications, providing controlled in situ activation of pro-dyes and prodrugs. In this work, we present the use of a polymeric scaffold to encapsulate transition metal catalysts (TMCs), generating bioorthogonal ‘polyzymes’. These polyzymes enhanced the stability of TMCs, protecting the catalytic centers from deactivation in biological media. The therapeutic potential of these polyzymes was demonstrated by the transformation of a nontoxic prodrug to an anticancer drug (mitoxantrone), leading to the cancer cell death in vitro.

TOC

Ruthenium catalysts are encapuslated into the hydrophobic pocket of polymers to provide bioorthogonal ‘polyzymes’ with enhanced catalyst stability. These polyzymes uncage maging and anticancer therapeutics in cells.

Graphical Abstract

Introduction

Bioorthogonal chemistry is a versatile tool for the generation of imaging and therapeutic agents inside living systems, using reactions cannot be performed by bioprocesses.^{[1–2,3,4,5,6,7,8]} Traditionally, bioorthogonal chemistry has been treated as the “ligation” reaction for two biologically inert components.^{[9,-10,11,12]} Recently, transition metal catalyst (TMCs) mediated bond-cleavage bioorthogonal catalysis has shown potential for biomedical applications, combining efficient catalysis with a broad range of possible transformations.^{[13–14,15,16,17,18]} ‘Naked’ TMCs have challenges arising from stability, concerns that have been addressed by loading TMCs onto/in inorganic nanomaterials,^{[19–20,21,22,23]} including gold nanoparticles,^{[24–25,26,27,28,29,30]} metal-organic frameworks,^[31] and mesoporous silica nanoparticles,^[32,33] providing ‘nanozymes’ for imaging and therapeutic applications.^[34] Engineering the surface functionalization of these nano-scaffolds can impart stimuli responsiveness,^[16,23] biostability,^[21] and localize the nanozyme at desired target sites such as biofilms,^[17] cells,^[35] inflamed areas^[36] and the extracellular matrix.^[18]

Inorganic nanomaterials present issues of clearance when employed biomedically.^[37,38] Polymers provide a versatile alternative, featuring ready functionalization,^[39,40] high biocompatibility,^[41] and scalability.^[42] Polymer scaffolds fabricated using living ring-opening metathesis polymerization (ROMP) are particularly attractive. ROMP tolerates a wide range of functional groups on monomer,^[43] and can provide biomimetic semi-rigid backbones through appropriate choice of monomer,^[44] making ROMP polymers useful scaffolds for TMC encapsulation.^[45–46,47]

We report here the fabrication of a ruthenium-based ‘polyzyme’ employing a poly(oxanorbornene imide) scaffold ^[48] for in vitro anti-cancer demonstration. The polymeric scaffolds employed features cationic hydrophobic imide functionalization, imparting both water solubility and inducing polymer self-assembly around the ruthenium TMC. These polyzymes maintained high catalytic activity in aqueous solution as well as in biological conditions, in contrast to free TMCs. Polyzymes were readily internalized into cells via endocytosis, and were able to efficiently activate pro-fluorophores inside cells. Therapeutic potential was established through intracellular activation of a non-toxic mitoxantrone prodrug in vitro, leading to the efficient killing of the cancer cells. Taken together, polyzymes provide a favorable platform for in situ imaging and therapeutic applications.

Result and Discussion

Poly(oxanorborneneimide) (PONI) polymers (Figure 1) were synthesized by ROMP (detail in supporting information Figure S1–5), providing a well-controlled and stable platform. The polymer features a hydrophobic sidechain with a permanently cationic trimethylammonium headgroup. The cationic headgroup imparts water solubility and facilitates uptake of the polyzyme through endocytosis. The hydrophobic side chain dictates self-assembly of the polymers into nanoparticles, ^[49] providing a hydrophobic environment suitable for encapsulation of hydrophobic TMCs.

Figure 1.

a. The activation of pro-Rhodamine (pro-Rho) by ruthenium catalysts. b. Free catalysts are rapidly deactivated in biological environments, while polyzymes prevented deactivation, maintaining high catalytic reactivity in living cells.

Polyzymes were generated by encapsulating catalysts [Cp*Ru(cod)Cl] (Cp* = pentamethylcyclopentadienyl, cod = 1,5-cyclooctadiene) ^[50] into polymeric self-assemblies through nanoprecipitation. The successful encapsulation of Ru catalysts into polymeric scaffold was demonstrated by the UV absorption of catalysts at 350 nm (Figure S6). Transmission electron microscopy (TEM, Figure S7) indicated a diameter of ~30 nm (dry). Dynamic light scattering (DLS, Figure S7) showed that the size of the self-assemblies was ~80 nm in solution, indicating some degree of swelling of self-assembled NP in aqueous media. TMC encapsulation was quantified using inductively coupled plasma mass spectrometry (ICP-MS), indicating ~5 ruthenium catalysts per polymer in the polyzyme. (Table S1).

The catalytic efficiency of polyzymes was quantified through uncaging of non-fluorescent pro-Rhodamine 110 (pro-Rho) to generate fluorescent Rhodamine (Figure 1, Figure S8 for the calibration curve of Rhodamine). As shown in the Figure 2, a rapid increase of fluorescence was observed after adding polyzymes into the solution of pro-Rho. Similar catalytic efficiency was observed in 10% serum. In contrast, free Ru catalysts at the same concentration (calculated based on ICP data) suffered a ~50% loss of activity in water and a >80% decrease in activity in serum. The reason for the deactivation could be the non-specific binding to serum proteins or oxidation by enzymes.^[51,52] We further tested the stability of polyzymes in PBS and 10% serum condition. As shown in Figure S9, polyzymes were stable in both condition for at least three days. These results indicated that the hydrophobic interior of polyzymes effectively shielded TMCs from both water and serum components.

Figure 2.

The kinetic study of polyzymes (200 nM, containing 1 μM catalysts) and free catalysts (1 μM) converting pro-Rho to fluorescent dye at 37°C in a. buffer saline and b. 10% serum. The rate was calculated by the calibration curve of rhodamine and showed in c.

We next investigated the intracellular efficacy of the polyzymes. Polyzymes showed little effect on cell viability over a wide range of concentrations, as demonstrated through Alamar Blue assay after 24 h incubation (Figure S7, minimal effects observed ≤ 800 nM) The cellular uptake of polyzymes was determined by tracking Ru using ICP-MS. As shown in Figure S10, maximum cellular uptake of polyzymes (~48% uptake efficiency) was reached after 12 h incubation.

Intracellular activity and localization were obtained by tagging the polyzyme with a red fluorophore (cyanine-3, Figure S11–14), allowing tracking of the polyzyme. Cells were treated with polyzymes at 500 nM for 12 h and washed three times to remove any non-uptaken polyzyme. Fresh media containing pro-Rho (100 μM) was then added and incubated overnight. In these co-localization studies, cells featured bright punctuate green fluorescence that co-localized with red fluorescence from polymer scaffold (Figure 3), indicative of pro-dye activation in endo/lysosomal compartments. As a negative control, pro-Rho alone showed no fluorescence in either green or red channels. To test the catalytic stability of polyzymes, HeLa cells were treated with polyzymes (500 nM) followed by adding pro-Rho (100 μM) after further 0 day, 1 day, 2 day and 3 day incubation. Figure S15 indicated that polyzymes maintained promising catalytic activity inside cells for more than three days.

Figure 3.

Confocal images of HeLa cells incubated with or without Cy3-polyzymes (12 h, 500 nM), followed by incubation with pro-Rho(100 μM). Scale bar = 50 μm. Cells incubated with prodye only (a. to d.) showed no fluorescence inside cells, as expected. Cells treated with Cy3-polyzymes and prodye (e) showed bright green fluorescence from the activation of pro-Rho (f). The green fluorescence colocalized with red fluorescence (g) from Cy3-polyzymes (h), confirming the activation was due to the catalytic effect of polyzymes. I. The activation of pro-Mitoxantrone (pro-Mit) by polyzymes. J. Cell viability of HeLa cells after the pro-Mit activation by polyzymes.

The therapeutic potential of the polyzyme was probed via activation of prodrugs. The pharmacophore of anticancer drug mitoxantrone (Mit) was blocked by allyloxycarbonyl to generate prodrug (pro-Mit) (Figure S16–S18 for synthesis detail). Pro-Mit then deprotected by polyzymes inside cells to generate highly toxic anticancer drugs (Figure 3I). For the in vitro therapeutic study, HeLa cells were pre-incubated with polyzymes (500 nM) or free catalysts (2.5 μM) for 12 h, washed, and then incubated with concentrations of pro-Mit up to 5 μM for 24 h. Pro-Mit alone and Mit alone were used as negative control and positive control, respectively. As shown in Figure 3J, cells incubated with polyzymes and pro-Mit showed dose-dependent elevated toxicity, while the incubation with the controls free catalysts and pro-Mit exhibited minimum toxicity to cancer cells. Cancer cells were stained by apoptotic probe YO-PRO-1 to further demonstrate the therapeutic efficacy. As shown in Figure S19, green fluorescence from cells treated with polyzymes and pro-Mit indicated effective induction of apoptosis from the polyzyme-based therapy.

Conclusions

In summary, polymer nanoparticles offer effective protection for transition metal catalysts, providing polyzyme systems for imaging and therapeutic applications. The hydrophobic interior of these polymers isolated TMCs from deactivating biological environments, without clearance issues raised using inorganic nanoparticle analogs. These polyzymes provided efficient intracellular activation of prodyes and prodrugs in vitro. This study reveals that polyzymes provide an alternative direction for disease diagnosis and treatment through in situ generation of imaging and therapeutic agents.

Experimental Section

Kinetic study: 10 μL of pro-Rho in dimethyl sulfoxide (DMSO) stock solution was added into 96 well black plate, followed by adding 100 μL of polyzymes or free catalysts solution in PBS buffer or PBS buffer with 10% serum, obtaining the final concentration of 10 μM of pro-Rho and 200 nM of polyzymes or 1 μM free catalysts. The kinetic study was based on tracking the increase of fluorescence intensity of Rhodamine (λ_ex = 488 nm, λ_em = 521 nm) by Molecular Devices SpectraMax M2 microplate reader at 37 °C. Each experiment comprised three replicates.

Confocal imaging of catalysis in HeLa cells:

HeLa cells were seeded at 100k in confocal dish one day prior the experiment. Cells were washed with PBS and added with fresh cell culture media containing 500 nM Cy-3 polyzymes for 12 hours. Afterwards, cells were washed with PBS for three time to fully removed unabsorbed polyzymes. Fresh media containing 100 μM of pro-Rho was added to HeLa cells and incubated overnight. On the next day, HeLa cells were washed with PBS and applied to Nikon spinning disk confocal microscopy (60x).

Prodrug activation:

HeLa cells were plated in 96 well plate at the density of 7k per well 24 hours before the experiment. During the experiment, 500 nM polyzymes were incubated with cells for 12 hours followed by multiple washing with PBS. Cell culture media containing pro-Mit (0, 0.5, 1, 2, and 5 μM) was then added to HeLa cells and incubated for 24 hours. After that, cells were fully washed with PBS and supplemented with 10% Alamar Blue in cell culture media. After 3 hours of incubation, the supernatant was transferred to a black plate, and the cell viability was determined by measuring the fluorescence at 570 nm (excitation) and 590 nm (emission).