Orthogonal Covalent Entrapment of Cargo into Biodegradable Polymeric Micelles via Native Chemical Ligation

Abstract

Polymeric micelles (PMs) are promising platforms for enhanced tissue targeting of entrapped therapeutic agents. Strategies to circumvent premature release of entrapped drugs include cross-linking of the micellar core as well as covalent attachment of the drug cargo. The chemistry employed to obtain cross-linked micelles needs to be mild to also allow entrapment of fragile molecules, such as certain peptides, proteins, oligonucleotides, and fluorescent dyes. Native chemical ligation (NCL) is a mild bio-orthogonal reaction between a N-terminal cysteine residue and a thioester that proceeds under physiological conditions. Here, we designed a trifunctional cross-linker containing two cysteine residues for the micelle core-cross-linking reaction and an azide residue for ring-strained alkyne conjugation of fluorescent dyes. We applied this approach to thermosensitive methoxypolyethylene glycol-b-N-(2-hydroxypropyl)methacrylamide-lactate (mPEG-b-HPMAmLac_n) based block copolymers of a core-cross-linked polymeric micelle (CCPM) system by attaching thioester residues (using ethyl thioglycolate-succinic anhydride, ETSA) for NCL cross-linking with the trifunctional cross-linker under physiological conditions. By use of mild copper-free click chemistry, we coupled fluorescent dyes, Sulfo.Cy5 and BODIPY, to the core via the azide residue present on the cross-linker by triazole ring formation. In addition, we employed a recently developed cycloheptyne strain promoted click reagent (TMTHSI, CliCr) in comparison to the frequently employed cyclooctyne derivative (DBCO), both achieving successful dye entrapment. The size of the resulting CCPMs could be tuned between 50 and 100 nm by varying the molecular weight of the thermosensitive block and ETSA content. In vitro cell experiments showed successful internalization of the dye entrapped CCPMs, which did not affect cell viability up to a polymer concentration of 2 mg/mL in PC3 cells. These fluorescent dye entrapped CCPMs can be applied in diagnostic imaging and the chemistry developed in this study serves as a steppingstone toward covalently entrapped fragile drug compounds with tunable release in CCPMs.

Scheme 1. Synthesis of the Azide Containing Di-cysteine Crosslinker (Compound 5)

Scheme 2. Synthesis of mPEG₅₀₀₀-b-P(HPMAmLac₁-co-HPMAmLac₂) Polymers (polymer Pn) by Free Radical Polymerization Using a (Methoxy polyethylene glycol)₂-4,4-azobis(4-cyanopentanoic acid) ((mPEG₅₀₀₀)₂-ABCPA) Macroinitiator and Subsequent ETSA Coupling to Attain Polymer Pn-En

Scheme 3. One Pot Reaction of the Trifunctional Crosslinker with BCN-OH

BCN-OH is clicked to the trifunctional cross-linker (compound 5) in DMSO and dried for analysis to confirm the azide based coupling.

Scheme 4. One Pot Reaction of the Trifunctional Cross-Linker with the DBCO Functionalized Sulfo.Cy5

Sulfo.Cy5-DBCO is clicked to the trifunctional cross-linker (compound 5), after which the disulfide bonds are reduced using TCEP for NCL cross-linking of the cross-linker-Cy5 conjugate with Pn-En to obtain CCPMs.

Scheme 5. One Pot Reaction of the Trifunctional Cross-Linker with the TMTHSI Functionalized BODIPY Dye

BODIPY-TMTHSI is clicked to the trifunctional cross-linker (compound 5), after which the disulfide bonds are reduced using TCEP for NCL cross-linking of the cross-linker-BODIPY conjugate with Pn-En to obtain CCPMs.

Figure 1

Effect of increasing concentrations of SDS on cross-linked micelles (filled shapes) and non-cross-linked micelles (hollow shapes) as measured by DLS at 25 °C.

Figure 2

Degradation characteristics of P100-E15 based CCPMs under physiological conditions (pH 7.4, 37 °C) at a polymer concentration of 7.5 mg/mL. Derived count rate (blue circles), Z-Ave (green circles), and PDI (black squares) were determined by DLS measurements at 37 °C. Lactic acid formed % (red squares) was determined by UPLC.

Figure 3

Confocal laser scanning microscopy images of PC3 cells incubated for 24 h with empty CCPMs (A) or BODIPY loaded CCPMs (B). Cells were treated with 2 μg/mL Hoechst nuclei stain (blue signal) prior to microscopy. Scale bar: 30 μm. Cell viability was determined in triplicate by MTS staining for BODIPY loaded CCPMs (C).