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Drug Delivery

Drug Delivery - Poster Session E

Poster X19 - Tumor targeted self-synergistic anticancer therapy based on dual stimuli-responsive redox regulating polymeric prodrug micelles

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Cancer cells are more vulnerable to oxidative stress compared to normal cells because of massive reactive oxygen species (ROS) generation and altered redox balance. Based on the biological difference between cancer cells and normal cells, the co-delivery of ROS generating agents and glutathione (GSH) depleting agents has gained increasing attention as a promising anticancer therapeutic strategy. Cinnamaldehyde (CA) is known to exhibit anticancer activity by generating a large amount of ROS. Quinone methide (QM) is an intermediate generated from the hydrolysis of phenyl boronate, rapidly alkylating GSH to elicit anticancer actions. We developed amphiphilic polymer (pCB) as a polymeric prodrug of CA and QM to realize concurrent delivery of CA and QM, fully maximizing their anticancer effects. pCB incorporates covalently CA and QM precursor via acid-cleavable acetal linkage and hydrogen peroxide (H2O2)-cleavable boronate linkage, respectively. Their hydrophobic core was loaded with boronated retinoic acid prodrug (BRDP), a dimeric prodrug of all-trans retinoic acid (atRA), which release therapeutic atRA and QM. Their surface was decorated with tripeptide (Arg-Gly-Asp, RGD) for binding integrin-overexpressing cells. RGD-decorated and BRDP-loaded pCB micelles (t-pCBRD) were activated by acidic pH and H2O2 to release CA and QM, leading to oxidative stress amplification. BRDP as a payload was also released to synergize pCB micelles. Therapeutic potential of t-pCBRD was examined using a mouse xenograft model. We anticipate that t-pCBRD presents great translational potential for oxidative cancer therapy based on tumor stimuli-responsiveness, self-synergistic oxidative stress amplification and minimal toxicity to normal cells.

Materials and

Methods:  pCB polymer was synthesized from the Michael addition reaction of CA-containing diamine, boronate containing diacrylate and PEG-containing acrylate at room temperature for 3 days. After solvent evaporation, pCB was obtained as a solid resin. RGD-decorated and BRDP-loaded pCB micelles (t-pCBRD) were prepared by self-assembly of pCB, BRDP and DSPE-PEG-RGD at a weight ratio of 1.0:0.2:0.1. The hydrodynamic diameter of t-pCBRD was determined by dynamic light scattering and the morphology was examined using transmission emission microscopy. The acid and H2O2-responsiveness of t-pCBRD was investigated by measuring their size and morphology. The ability of t-pCBRD to elevate oxidative stress was evaluated by measuring the intracellular level of ROS in A549 cells using DCFH-DA as a ROS probe. To verify the apoptotic cell death by t-pCBRD, cells were stained with Annexin-V-APC and observed under the microscope. The effects of t-pCBRD on mitochondrial membrain potential was examined using JC-1 dye. In the animal studies, A549 cells (2×106 cells) were subcutaneously injected into the nude BALB/c mice. When tumor volumes reached 30~50 mm3, t-pCB (20mg/kg) - BRDP (4mg/kg) mix, t-CBRD micelles (10, 20 mg/kg) were injected through retro-orbital injection. Tumor volume and body weight were monitored for 27 days. Tumors and major organ organs were collected for histological examination.

Results, Conclusions, and Discussions: pCB was synthesized via the Michael addition polymerization reaction of CA-containing diamine, QM precursor-containing diacrylate and PEG-methyl ether acrylate at a molar ratio of 1:0.9:0.1. The chemical structure of pCB was verified by 1H NMR analysis and its number average molecular weight was determined to be 8.6 kDa by diffusion-ordered NMR spectroscopy. NMR analysis also revealed that pCB releases CA and QM in response to acidic pH and H2O2. Under the aqueous condition, pCB could self-assemble to form micelles that have a mean hydrodynamic diameter of 190 nm. pCB micelles were stable during the incubation with 75% serum protein, but rapidly dissociated with the reduction of pH to 5.0. t-pCBRD was prepared from the self-assembly of pCB, BRDP and DSPE-PEG-RGD, which have a mean diameter of 200 nm. The surface modification with RGD was confirmed by the significant changes of zeta potential from pCB micelles. After incubation with pCB micelles, the level of intracellular ROS was markedly increased. pCB micelles generated ROS more than the equivalent CA. We also found that pCB micelles significantly decreased the level of intracellular GSH. These observations demonstrate that the superior oxidative stress amplifying activity of pCB micelles over the equivalent CA could be explained by the combined effects of ROS-generating CA and GSH-depleting QM. In the animal study, t-pCBRD accumulated in tumor sites preferentially and significantly suppressed the tumor growth. Histological analysis revealed that t-pCBRD induces apoptotic cell death without noticeable harmful effects in major organs.

Acknowledgements (Optional):  This study was supported by the grant of National Research Foundation of Korea (RS-2024-00335085).