The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in hep G2 cells

Abstract

The development of macromolecules as drugs and drug carriers requires knowledge of their fate in cells. To this end, we studied the internalization and subcellular fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in Hep G2 (human hepatocellular carcinoma) cells. Semiquantitative fluorometry confirmed that galactose was an effective ligand for receptormediated endocytosis for Hep G2 cells. The rate of internalization of a galactose-targeted copolymer was almost 2 orders of magnitude larger than that of the nontargeted copolymer. Confocal fluorescent microscopy of both fixed and live cells revealed that the polymer entered the cells by endocytosis. After longer incubation times (typically >8 hours), polymer escaped from small vesicles and distributed throughout the cytoplasm and nuclei of the cells. Polymer that entered the cytoplasm tended to accumulate in the nucleus. Microinjection of the HPMA copolymers into cells' cytoplasm and nuclei indicated that the polymers partitioned to the nucleus. The data from fixed cells was confirmed by microscopy of live, viable cells. To examine the effect of the fluorescent dye on the intracellular fate, polymers with fluorescein, Oregon Green 488, Lissamine rhodamine B, and doxorubicin were tested; no significant differences were observed.

Synthesis and Functions of Well-defined Polymer-drug Conjugates as Efficient Nanocarriers for Intravesical Chemotherapy of Bladder Cancera

“…Such a result is not without precedence. Jensen et al have reported the cytoplasmic localization and nucleus entry of HPMA copolymers in Hep G2 cells before. After 24 h of incubation, the green fluorescence remains unchanged, but the red fluorescence of DOX exhibited a similar distribution as free DOX, indicating that the released DOX acts the same function as free DOX.…”

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“…Because drug-polymer conjugates are not easily recognized by their receptors, their therapeutic activity depends mainly on the amount of free drug released from the conjugate. It usually takes longer for non-targeted polymer-drug conjugates, such as P-Dex, to be endocytosed and incorporated into the lysosomes where the acidic environment would act to release the Dex [37]. The free Dex must then escape from the lysosomal compartment to deliver its anti-inflammatory effect.…”

Cathepsin B-cleavable doxorubicin prodrugs for targeted cancer therapy

“…4B), contains N-acylated galactosamine (GalN), which was designed to be recognized by ASGPR in HepG2 human hepatocellular carcinoma cells (59,82) and individual members of the galectin family (e.g., galectin-3) in human colon adenocarcinoma (83,84). Galectin-3 is expressed in normal tissues and highly expressed in neoplastic tissues (85–87); although the exact opposite has been shown to occur (88,89).…”