Balancing the stability and drug activation in adaptive nanoparticles potentiates chemotherapy in multidrug-resistant cancer

Rationale: Prodrug strategies that render the drug temporarily inactive through a cleavable linkage are able to modulate the physicochemical properties of drugs for adaptive nanoparticle (NP) formulation. Here we used cabazitaxel as a model compound to test the validity of our “balancing NP stability and specific drug activation” strategy. Methods: Cabazitaxel is conjugated to hydrophobic polylactide fragments with varying chain lengths via a self-immolation linkage, yielding polymeric prodrugs that can be reactivated by reductive agents in cells. Following a nanoprecipitation protocol, cabazitaxel prodrugs can be stably entrapped in amphiphilic polyethylene-block-polylactide matrices to form core-shell nanotherapies with augmented colloidal stability. Results: Upon cellular uptake followed by intracellular reduction, the NPs spontaneously release chemically unmodified cabazitaxel and exert high cytotoxicity. Studies with near-infrared dye-labeled NPs demonstrate that the nanodelivery of the prodrugs extends their systemic circulation, accompanied with increased drug concentrations at target tumor sites. In preclinical mouse xenograft models, including two paclitaxel-resistant xenograft models, the nanotherapy shows a remarkably higher efficacy in tumor suppression and an improved safety profile than free cabazitaxel. Conclusion: Collectively, our approach enables more effective and less toxic delivery of the cabazitaxel drug, which could be a new generalizable strategy for re-engineering other toxic and water-insoluble therapeutics.


Synthesis of hydroxyl-terminated polylactide (pLA 15 )
Hydroxyl-terminated polylactide (pLA 15 ) was synthesized by ringopening polymerization. Briefly, D,L -lactide (91.57 mmol, 13.19 g) and S3 triethylene glycol monomethyl ether (11.02 mmol, 1.81 g) dissolved in 50 mL toluene were added into a round-bottom flask, followed by an addition of stannous octoate (Sn(Oct) 2 ). After stirring for 12 h at 140°C, the solvent was removed by evaporation under vacuum. Subsequently, the residue was dissolved in dichloromethane (DCM) and then precipitated in excess cold diethyl ether and filtered. The molecular weight of pLA 15 was determined by 1 H NMR.

Synthesis of hydroxyl-terminated polylactide (pLA 50 )
Hydroxyl-terminated polylactide (pLA 50 ) was also synthesized by ringopening polymerization. Briefly, D,L -lactide (120.45 mmol, 17.35 g) and triethylene glycol monomethyl ether (3.96 mmol, 0.65 g) dissolved in 50 mL toluene were added into a round-bottom flask, followed by an addition of stannous octoate (Sn(Oct) 2 ). After stirring for 12 h at 140°C, the solvent was removed by evaporation under vacuum. Subsequently, the residue was dissolved in DCM and then precipitated in excess cold diethyl ether and filtered. The molecular weight of pLA 50 was determined by 1 H NMR. and bis(2-hydroxyethyl) disulfide (7.00 mmol, 1.08 g) dissolved in 6 mL DCM were added into a round-bottom flask, followed by an addition of DIEA (1.40 mmol, 0.18 g). After stirring for 4 h at 45°C, the solvent was removed by evaporation under vacuum and then was washed by 5% citric acid, saturated NaHCO 3 , and brine. The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and evaporated under vacuum. The crude residue was purified by flash column chromatography on silica gel (hexane/ethyl acetate=2:1) to afford the conjugate 1 (light yellow solid, 1.6 g, 47.0%).   4, 152.3, 145.5, 125.4, 121.8, 66.8, 36.8.

Analysis of molecular weight distribution
The molecular weight distribution of the pLA n -SS-CTX conjugates (n = 15 or 50) was analyzed by gel permeation chromatography (GPC) measurement (Waters 1525/2414, USA) at 35°C. S10 Characterization of pLA n -SS-CTX NPs.

TEM and SEM analysis
Samples were prepared by dipping pLA n -SS-CTX NPs (at 0.3 mg/mL cabazitaxel equivalent concentration) onto a 300-mesh copper grid.
Following 2 min of deposition, the surface solvent was removed with filter papers and dried at room temperature. Subsequently, samples were positively stained with 2 wt% aqueous uranyl acetate solution for 1 min.
Finally, pLA n -SS-CTX NPs were observed on TECNAL 10 (Philips). For SEM analysis, samples were directly observed with Nova Nano 450 without positive staining.

Dynamic Light Scattering (DLS)
The hydrodynamic diameters (D H ) and distribution and zeta potentials for pLA n -SS-CTX NPs were measured by a Malvern Nano-ZS90 instrument (Malvern, UK) at 25 °C.

Determination of drug loading and encapsulation efficiency
The drug loading and encapsulation efficiency of pLA n -SS-CTX NPs were determined with analytical RP-HPLC. Briefly, pLA n -SS-CTX NP solutions were centrifuged at 12000 rpm for 10 min to remove the free agents. Subsequently, supernatant was collected and hydrolyzed with 0.1 M NaOH at 37 °C for 2 h. The final hydrolytic product (i.e., benzoic acid) was used to quantify the amounts of cabazitaxel. The encapsulation efficiency (EE) and drug loading (DL) of cabazitaxel in NPs were calculated as the following formulas:

Evaluation of the stability of pLA n -SS-CTX NPs
pLA n -SS-CTX NPs were incubated in DI water and DI water supplemented with 10% FBS (v/v) at 37 °C. The hydrodynamic diameters (D H ) and zeta potential were monitored over several days.

CTX NPs
A series of pLA n -SS-CTX NP solutions ranging from 1.0×10 -5 to 1.0 mg/mL were prepared for the measurement of scattering intensity. All samples were detected on a Malvern Nano-ZS90 instrument (Malvern, UK) at 25 °C. The critical micelle concentrations (CMCs) were determined by plotting the scattering intensity as a function of the logarithm of concentration.

Acridine orange/ethidium bromide (AO/EB) staining assay
The cell apoptosis induced by cabazitaxel formulations was assessed with acridine orange-ethidium bromide (AO/EB) staining assay. DU145 and HeLa/PTX cells were seeded into 48-well plates and cultured for 24 h at 37 °C. Then, cells were exposed to free CTX and pLA n -SS-CTX NPs for 48 h (2 nM cabazitaxel-equivalence for DU145 cells, 6 nM cabazitaxelequivalence for HeLa/PTX cells). The medium for each well was removed and replenished with AO/EB staining solutions premixed at a ratio of 1:1 (v/v). After washing with PBS, the cells were immediately imaged by fluorescence microscopy.

Hemolysis assay
We