Profiling target engagement and cellular uptake of cRGD-decorated clinical-stage core-crosslinked polymeric micelles

Abstract

Polymeric micelles are increasingly explored for tumor-targeted drug delivery. CriPec® technology enables the generation of core‐crosslinked polymeric micelles (CCPMs) based on thermosensitive (mPEG-b-pHPMAmLacn) block copolymers, with high drug loading capacity, tailorable size, and controlled drug release kinetics. In this study, we decorated clinical-stage CCPM with the αvβ3 integrin-targeted cyclic arginine-glycine-aspartic acid (cRGD) peptide, which is one of the most well-known active targeting ligands evaluated preclinically and clinically. Using a panel of cell lines with different expression levels of the αvβ3 integrin receptor and exploring both static and dynamic incubation conditions, we studied the benefit of decorating CCPM with different densities of cRGD. We show that incubation time and temperature, as well as the expression levels of αvβ3 integrin by target cells, positively influence cRGD-CCPM uptake, as demonstated by immunofluorescence staining and fluorescence microscopy. We demonstrate that even very low decoration densities (i.e., 1 mol % cRGD) result in increased engagement and uptake by target cells as compared to peptide-free control CCPM, and that high cRGD decoration densities do not result in a proportional increase in internalization. In this context, it should be kept in mind that a more extensive presence of targeting ligands on the surface of nanomedicines may affect their pharmacokinetic and biodistribution profile. Thus, we suggest a relatively low cRGD decoration density as most suitable for in vivo application. Graphical Abstract

Photodynamically Tumor Vessel Destruction Amplified Tumor Targeting of Nanoparticles for Efficient Chemotherapy

“…RGD-modified micelles (PPRMs) can target the integrin alphavbeta3 (αvβ3) which is highly expressed in the tumor neovascular endothelial cells and multiple tumors cells, such as breast cancer, lung cancer, and malignant melanoma cells. − Immortalized human umbilical vein endothelial cells (HUVEC) have high expression of αvβ3 integrin, which commonly used as an in vitro model of tumor neovascularization study . The quantitative cellular uptake of PPM and PPRM in the HUVEC at different time points was analyzed by flow cytometry.…”

Polymeric Micellar Systems—A Special Emphasis on “Smart” Drug Delivery

“…Typically, ligands are conjugated to the outer ends of the hydrophilic segment to modify polymeric micelles for active targeting [ 65 ]. Targeting ligands used to create polymeric micellar systems are typically categorized as (a) small molecular weight molecules (e.g., folic acid [ 66 ], sialic acid [ 67 ], biotin [ 68 ]); (b) antibodies and their fragments [ 69 ]; (c) peptides and proteins such as trans-activator of transcription (TAT) peptide [ 70 ] and arginine–glycine–aspartic acid (RGD) peptide [ 71 ]; and (d) aptamers [ 72 ]. Due to their high binding affinity and specificity for tumor cells, antibodies and peptides are proven to be superior ligands.…”

Design, development and clinical translation of CriPec®-based core-crosslinked polymeric micelles

“…Concerning the functionalization of CCPM surface with ligands for active targeting strategies (e.g., RGD [71], folate) and/or imaging markers (e.g., Cy7 [72]; 89 Zr [73]) for theranostics applications, parts of the PEG terminus of block copolymer can be modified with an azide moiety (mainly 1 -5 %), thus enabling ligands functionalized with click probes to be irreversibly conjugated to the surface of CCPM via copper-free click chemistry [74] (Fig. 3B).…”

“…By modifying (a fraction) of the PEG-termini of the block copolymers with an azide moiety (1-5 %), ligands derived with click probes can be irreversibly conjugated to the CCPM surface by copper-free click chemistry [74]. This versatile surface functionality allows the use of API-loaded CCPM also for active targeting strategies, imaging applications, as well as vaccine development [54,62,[71][72][73].…”

“…To this end, rhodamine-labeled CCPM were decorated with increasing densities (0-5 mol%) of cyclic RGD peptide (cRGD), and a panel of both amb3 integrin positive (TNFa-activated HUVEC, and 4 T1 breast cancer cells) and amb3 integrin negative cells (A431 epidermoid carcinoma cells) for assessing cRGD CCPM functionality was used (Fig. 4A) [71]. The obtained results showed the uptake of cRGD CCPM to be time-and temperature-dependent, with the highest uptake at 24 h post incubation at 37 °C (Fig.…”