(T1430-07-38) Active Targeting of Colorectal Cancer Using Chemotherapy-Loaded Nanoparticles Functionalized with Folate Receptor-α (FRα) Ligand, Pemetrexed

Purpose: Colorectal cancer (CRC) is the third most frequent cause of cancer deaths in the United States and more than 50,000 patients die from CRC each year. These alarming statistics highlight the inadequacies of current treatment modalities (e.g., chemotherapy and radiotherapy) particularly for advanced CRC. Chemotherapeutics that comprise the first and second-line treatments for advanced CRC have substantial systemic side effects that limit treatment effectiveness. This underscores the pressing need for innovative therapeutic strategies that selectively target neoplastic cells while sparing healthy tissues. Tumor-targeting nanoparticles (TTNPs) present a viable solution to these challenges. TTNPs are capable of delivering therapeutic agents directly to neoplastic cells, thereby augmenting treatment efficacy and mitigating systemic toxicity. To achieve this objective, biocompatible polymers were fabricated de novo, linked to a novel targeting moiety, pemetrexed, a folic acid receptor-α (FRα) ligand. FRα is known to be overexpressed by 34% of human CRCs while not being expressed by most healthy tissues. The overexpression FRα in aggressive CRC makes it an appealing target for tumor-specific drug delivery. The current research landscape is primarily focused on the utilization of folic acid as a targeting moiety, however, pemetrexed has a higher affinity for FRα and has never been used before as a targeting ligand. This project aims to evaluate the ability of pemetrexed conjugated polymers to generate TTNPs loaded with chemotherapeutic agents and test them in a murine FRα⁺ CRC model.

Methods: Pemetrexed was employed to synthesize tumor-targeting PLGA-PEG-pemetrexed polymers via a ring opening polymerization (ROP) reaction of lactide and glycolide. The resultant tumor-targeting polymer was used to produce TTNPs. The targeting efficiency of TTNPs loaded with coumarin 6 (C6) was evaluated using CT26 cells, a FRα-expressing CRC cell line, through cellular uptake studies, employing flow cytometry and confocal microscopy. The ability of TTNPs to accumulate in FRα-expressing tumors was assessed in CT26 tumor-bearing BALB/C mice using In Vivo Imaging System (IVIS) imaging. The anticancer activity and safety profile of paclitaxel (PTX)-loaded TTNPs were evaluated by in vivo antitumor efficacy testing and body weight measurements, respectively. CT26 tumor-bearing BALB/C mice were dosed with PTX-loaded TTNPs, Taxol (positive control), empty TTNPs (negative control) and non-targeting formulations (NTNPs) via IV tail injection equivalent to 10 mg/kg of PTX, two doses one week apart.

Results: Pemetrexed terminated PLGA-PEG copolymer was successfully synthesized by ROP of lactide and glycolide using PEG-pemetrexed as an initiator in the presence of the catalyst, Sn(Oct)2. The polymer structure was validated by NMR spectroscopy. The calculated molecular weight of the polymer was ~20 kDa and lactide:glycolide ratio was 50:50. The tumor targeting polymer was used to synthesize PTX-loaded TTNPs using a single emulsion method. TTNPs had a hydrodynamic diameter of 160 nm and a Zeta potential of -21 mV. Flow cytometry experiments showed that the uptake of TTNPs by CT26 cells was significantly higher compared to NTNPs made with commercially available PEG-PLGA. These results were reproducible in two other CRC cell lines, an aggressive BRCA1 knockdown HT29 variant and SW620 cell lines. Pretreatment of CT26 cells with folic acid (1 mM for 2 hr) prior to incubation with TTNPs led to a decrease in the internalization of TTNPs, rendering the uptake levels similar to NTNPs. An in vivo syngeneic CRC model (CT26) was used to assess the tumor targeting and the anti-tumor capabilities of TTNPs. IVIS generated in vivo and ex vivo images showed that TTNPs had accumulated in the tumor more significantly compared to NTNPs and had a significantly higher tumor/liver signal ratio. Treatment with TTNPs was superior to that of NTNPs and Taxol, which indicated that the delivery of PTX in TTN improved antitumor efficacy. There was no significant change in mice weights which suggested that PTX-loaded TTNPs were well tolerated by the mice.

Conclusion: In this work, a single-step, solvent-free ROP procedure was used to create PLGA-PEG-pemetrexed copolymers, where pemetrexed acts as a tumor-targeting moiety by targeting FRα. The tumor-targeting polymer successfully self-assembled into TTNPs using a single emulsion nanoformulation technique. The TTNPs showed higher uptake by CRC cells expressing FRα, and higher tumor accumulation in in vivo tumor models compared to NTNPs. In vivo antitumor efficacy of PTX-loaded TTNPs was significantly enhanced compared to NTNPs or Taxol. Thus, pemetrexed represents a promising targeting ligand directed towards tumors expressing FRα such as aggressive CRCs.

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Acknowledgements: This work was funded by the GastroIntestinal Research Foundation.

Figure 1: In vitro cellular uptake of TTNPs and NTNPs loaded with C6. A) Flow cytometric overlays of CT26 cells treated with C6-TTNPs (Blue), C6-NTNPs (Orange), and blank-TTNPs (Pink) at a concentration 100 µg/mL for 1 hour in addition to untreated control (Green). B) Flow cytometric-generated relative mean fluorescence intensities of CT26 exposed to TTNPs with pretreatment with 1 mM folic acid TTNPs+FA, NTNPs with pretreatment with 1 mM folic acid NTNPs+FA, TTNPs and NTNPs respectively. n=4, one-way ANOVA, * p-value < 0.05 and ** p-value < 0.01, *** p-value < 0.001.

Figure 2: (A) Real-time whole-body imaging of CT26 tumor-bearing female BALB/C mice at different time points after tail vein injection of DiR-loaded TTNPs and DiR-loaded NTNPs at a dose of 4 mg per mouse, dorsal view. (B) Quantitative image analysis by relative fluorescent intensity per unit area as Tumor/Liver ratio, n=3, t-test, * p-value < 0.05. (C) Ex vivo images of tumor and major organs (heart, lungs, liver, kidneys and spleen) 96 h post-intravenous injection.

Figure 3: Tumor growth profiles for CT26 tumor-bearing BALB/C mice, I.V. injected with PTX-loaded TTNPs, NTNPs, Taxol, or blank TTNPs. n=3-4. Dose of PTX/Taxol was 10 mg/kg. Statistical analysis was performed using a one-way ANOVA. Data are presented as mean ± SD. * p-value < 0.05, *** p-value <0.001.