Polyarginine segments in block copolypeptides drive both vesicular assembly and intracellular delivery

Abstract

Polymeric vesicles are a relatively new class of nanoscale self-assembled materials that show great promise as robust encapsulants. Compared with liposomes, use of polymeric building blocks for membrane formation allows increased stability, stimuli responsiveness and chemical diversity, which may prove advantageous for drug-delivery applications . A major drawback of most polymeric vesicles is the lack of biofunctionality, which restricts their ability to interact with cells and tissues. We have prepared vesicles composed of polyarginine and polyleucine segments that are stable in media, can entrap water soluble species, and can be processed to different sizes and prepared in large quantities. The remarkable feature of these materials is that the polyarginine segments both direct structure for vesicle formation and provide functionality for efficient intracellular delivery of the vesicles. This unique synergy between nanoscale self-assembly and inherent peptide functionality provides a new approach for design of multifunctional materials for drug delivery.

Arginine‐rich cell‐penetrating peptides

“…The diffuse staining was not due to the release of peptide from endosomes as it appeared first and the punctate staining later, and it continued to occur at 4°C when endocytosis is inhibited. Several other studies have also reported cellular uptake when the cells have been incubated at 4°C [26,[51][52][53]. It has also been observed that blocking specific endocytotic pathways does not affect the ability of TAT peptide to enter cells.…”

Use of Methionine Alkylation to Prepare Cationic and Zwitterionic Block Copolypeptide Vesicles

“…In addition, due to its use in animal feeds, more methionine is produced chemically than any other amino acid, [18] which lowers its cost and makes this route a very economical process for synthesis of ionic amphiphilic copolypeptides. followed by dialysis gave polydisperse, unilamellar vesicles with average diameters of a few microns, [5,12] as determined by optical microscopy and laser scanning confocal microscopy (LSCM) (Figure 1a-d). These structures are consistent with results obtained for many other block copolypeptide amphiphiles of similar block composition, [5,12] validating the chosen sequence design.…”

“…followed by dialysis gave polydisperse, unilamellar vesicles with average diameters of a few microns, [5,12] as determined by optical microscopy and laser scanning confocal microscopy (LSCM) (Figure 1a-d). These structures are consistent with results obtained for many other block copolypeptide amphiphiles of similar block composition, [5,12] validating the chosen sequence design. Similar to previous work, [5,12] these new vesicles were found to be capable of encapsulating polar cargos, such as Texas Red labeled dextran (M n = 3000 Da) (Figure 1e).…”

“…peptides, functional side chains) either be protected during synthesis or conjugated to a material postpolymerization, [5] alkylated M containing amphiphilic copolypeptides were readily prepared by Table S1). [7] The segment lengths were based on those previously found to promote vesicle formation using other amino acids, i.e .an approximately 3:1 ratio of hydrophilic to hydrophobic segment lengths, [5,12,13] and phenylalanine was incorporated within the rod-like, permanently hydrophobic leucine segment to enhance membrane flexibility. [17] Subsequent alkylation of this hydrophobic precursor in water, using either iodomethane or iodoacetic acid as model alkylating agents, directly gave the desired amphiphilic sulfonium derivatives, M M 65 (L 0.5 /F 0.5 ) 20 or M C 65 (L 0.5 /F 0.5 ) 20 , in high yield and purity, respectively (Scheme 1, see SI).…”

“…L-Phenylalanine and L-leucine NCAs were synthesized by phosgenation and purified by recrystallization. [12,13] L-Methionine NCA was prepared by phosgenation and purified by anhydrous column chromatography. [6] α-Methoxy-ω-isocyanoethyl-poly(ethylene glycol) 45 (mPEG 45 -NCO) was prepared by reacting α-methoxy-ω-aminoethyl-poly(ethylene glycol) 45 (mPEG 45 -NH 2 , M n = 2000 g mol -1 , Nanocs) with phosgene in THF for 16 h. [6] All block copolypeptides were synthesized using (PMe 3 ) 4 Co initiator, [14] and the synthesis of M 65 (L 0.5 /F 0.5 ) 20 has been previously described.…”