EGFR-Targeted Nanobody Functionalized Polymeric Micelles Loaded with mTHPC for Selective Photodynamic Therapy

Abstract

meta-Tetra(hydroxyphenyl)chlorin (mTHPC) is one of the most potent second-generation photosensitizers, clinically used for photodynamic therapy (PDT) of head and neck squamous cell carcinomas. However, improvements are still required concerning its present formulation (i.e., Foscan, a solution of mTHPC in ethanol/propylene glycol (40:60 w/w)), as mTHPC has the tendency to aggregate in aqueous media, e.g., biological fluids, and it has limited tumor specificity. In the present study, polymeric micelles with three different diameters (17, 24, and 45 nm) based on benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) (PCL n -PEG; n = 9, 15, or 23) were prepared with mTHPC loadings ranging from 0.5 to 10 wt % using a film-hydration method as advanced nanoformulations for this photosensitizer. To favor the uptake of the micelles by cancer cells that overexpress the epidermal growth factor receptor (EGFR), the micelles were decorated with an EGFR-targeted nanobody (named EGa1) through maleimide-thiol chemistry. The enhanced binding of the EGFR-targeted micelles at 4 °C to EGFR-overexpressing A431 cells, compared to low-EGFR-expressing HeLa cells, confirmed the specificity of the micelles. In addition, an enhanced uptake of mTHPC-loaded micelles by A431 cells was observed when these were decorated with the EGa1 nanobody, compared to nontargeted micelles. Both binding and uptake of targeted micelles were blocked by an excess of free EGa1 nanobody, demonstrating that these processes occur through EGFR. In line with this, mTHPC loaded in EGa1-conjugated PCL 23 -PEG (EGa1-P 23 ) micelles demonstrated 4 times higher photocytotoxicity on A431 cells, compared to micelles lacking the nanobody. Importantly, EGa1-P 23 micelles also showed selective PDT against A431 cells compared to the low-EGFR-expressing HeLa cells. Finally, an in vivo pharmacokinetic study shows that after intravenous injection, mTHPC incorporated in the P 23 micelles displayed prolonged blood circulation kinetics, compared to free mTHPC, independently of the presence of EGa1. Thus, these results make these micelles a promising nanomedicine formulation for selective therapy.

π-π-Stacked Poly(ε-caprolactone)-b-poly(ethylene glycol) Micelles Loaded with a Photosensitizer for Photodynamic Therapy

“…On the other hand, micelles from the corresponding benzyl-terminated polymer (entry 8, Table 1) showed substantially decreased size and size distribution (23 nm with PDI of 0.3, Figure 2A). This result is in accordance with our previous studies, in which it was demonstrated that micelles based on PCL 9 -PEG with a benzyl end group showed smaller size (17 nm), lower PDI (<0.1), and better stability than the non-benzylated polymer micelles [24,38]. Figure 2A shows that the different micelles based on P(CL/TMC-Bz)-PEG or P(TMC-Bz)-PEG block copolymers with different amounts of pendant benzyl groups (Entries 2-4, 6 and 7, Table 1) had small hydrodynamic diameters that slightly increased with the increasing chain length of the hydrophobic blocks (from 17-23 nm) with PDIs < 0.1 (Figure 2A).…”

“…After the reaction (4 h at room temperature), the unreacted free thiols were blocked by reaction with maleimide (4 h at room temperature), and subsequently, the unreacted maleimide and unreacted Cy7 were removed by dialysis against THF/water (50:50 volume ratio) for 3 days. The successful coupling of Cy7 with the polymer and complete removal of free Cy7 was demonstrated by GPC analysis, with which the amount of Cy7 in the copolymer was quantified using UV-Vis detection at 755.5 nm, showing 17% coupling efficiency, as reported previously [38]. On average, one polymer chain carried 0.17 Cy7 label.…”

“…For in vivo studies, Cy7-labeled polymers were obtained by first synthesizing a block copolymer containing pendant thiol groups (i.e., poly(ε-caprolactone-co-2,2-bis-thiomethyl-trimethylene carbonate)-b-poly(ethylene glycol) (P(CL 18 -TTC 7.5 )-PEG)), and allowing this polymer to react with maleimide-functionalized near-infrared (NIR) fluorophore Cyanine7 (Cy7) (Lumiprobe Corporation, Hannover, Germany), as described previously (Scheme S1, Supplementary Materials) [38]. In short, CL was copolymerized with TTC using mPEG-OH as an initiator and MSA as a catalyst (molar ratio of CL/TTC/mPEG-OH was 18/8/1), following the same procedure as described in Section 2.2.2.1 for the synthesis of P(CL/TMC-Bz)-PEG.…”

“…The collected blood samples were centrifuged at 1000× g for 15 min at 4°C. The plasma supernatant was collected, extracted using acetonitrile/DMSO (4:1, v/v), and analyzed by high-performance liquid chromatography (HPLC) and a LI-COR Odyssey imaging system to quantify the amount of mTHPC and Cy7-labeled micelles, respectively, as previously described [38]. Plasma concentration curves were analyzed by non-compartmental analysis with the PKSolver add-in for Microsoft Excel [40].…”

“…The mixture was then centrifuged at 15,000× g for 10 min. Next, 50 µL of the obtained supernatant was injected into the HPLC system consisting of a Waters X Select Charged Surface Hybrid (CSH) C18 3.5 µm 4.6 × 150 mm column coupled with a fluorescence detector set at λ ex 420 nm and λ em 650 nm to analyze mTHPC concentration [38]. The mobile phase was 0.1 % trifluoroacetic acid in acetonitrile/water (60:40, v/v) at a flow rate of 1 mL/min.…”