Intracellular Trafficking and Subcellular Distribution of a Large Array of HPMA Copolymers

Abstract

The basic physicochemical properties that determine the distribution and fate of synthetic macromolecules in living cells were characterized using fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers. Twelve different classes of water-soluble copolymers were created by incorporating eight different functionalized comonomers. These comonomers possessed functional groups with positive or negative charges or contained short hydrophobic peptides. The copolymers were fractionated to create parallel “ladders” consisting of 10 fractions of narrow polydispersity with molecular weights ranging from 10 to 200 kDa. The intracellular distributions were characterized for copolymer solutions microinjected into the cytoplasm of cultured ovarian carcinoma cells. Even the highest molecular weight HPMA copolymers were shown to quickly and evenly diffuse throughout the cytoplasm and remain excluded from membrane-bound organelles, regardless of composition. The exceptions were the strongly cationic copolymers, which demonstrated a pronounced localization to microtubules. For all copolymers, nuclear entry was consistent with passive transport through the nuclear pore complex (NPC). Nuclear uptake was shown to be largely dictated by the molecular weight of the copolymers, however, detailed kinetic analyses showed that nuclear import rates were moderately, but significantly, affected by differences in comonomer composition. HPMA copolymers containing amide-terminated phenylalanine-glycine (FG) sequences, analogous to those found in the NPC channel protein, demonstrated a potential to regulate import to the nuclear compartment. Kinetic analyses showed that 15 kDa copolymers containing GGFG, but not those containing GGLFG, peptide pendant groups altered the size-exclusion characteristics of NPC-mediated nuclear import.

Endocytic uptake of a large array of HPMA copolymers: Elucidation into the dependence on the physicochemical characteristics

“…The HPMA copolymers were prepared as described in [24]. The general copolymerization scheme, structures of comonomers used, and example of molecular weight profiles are shown in Scheme 1.…”

PEG-poly(amino acid) Block Copolymer Micelles for Tunable Drug Release

“…On the other hand, intracellular pH is more acidic, reaching 5.0 in endosomes and lysosomes (32). Such differences in pH throughout the body would permit these micelles to control the conditions of drug release in various locations differentially (33,34). DOX, a widely used anticancer drug, is used in this study as a model drug because it has a ketone group at its C13 position and also possesses the spectrometric advantages over detection of color and fluorescence.…”

Design of smart HPMA copolymer-based nanomedicines

“…As a conduit for non - biological macromolecules, the NPCs have been shown to transmit PEG-coated gold colloid particles 4–7 nm in diameter [55]. The basic physico-chemical properties that determine the distribution and fate of synthetic macromolecules in living cells were characterized using fluorescently-labeled HPMA copolymers [56]. Twelve different classes of water-soluble copolymers were created by incorporating eight different functionalized comonomers.…”

“…HPMA copolymers containing amide-terminated phenylalanine-glycine (FG) sequences, analogous to those found in the NPC channel protein, demonstrated a potential to regulate import to the nuclear compartment. Kinetic analyses showed that 15 kDa copolymers containing GG FG , but not those containing GGL FG , peptide pendant groups altered the size-exclusion characteristics of NPC-mediated nuclear import [56]. One possible explanation is that the GGFG moieties were able to weakly bind to FG-domain crosslinks in a way that altered the dynamics of a putative nucleoporin hydrogel structure, whereas GGLFG peptides would be expected to bind more strongly and not allow a rapid transfer of crosslinks in the hydrogel-like structure of the nucleopore proteins [57].…”

“…A) Structure of monomers and polymers [37,56]; B) Impact of Mw and structural factors on transport of macromolecules from the cytoplasm into the nucleus [56]; C) Internalization of positively and negatively charged macromolecules into prostate cancer cells [37]. …”