N‐Acetylgalactosamine‐Targeted Delivery of Dendrimer‐Doxorubicin Conjugates Influences Doxorubicin Cytotoxicity and Metabolic Profile in Hepatic Cancer Cells

Abstract

This study describes the development of targeted, doxorubicin (DOX)-loaded generation 5 (G5) polyamidoamine dendrimers able to achieve cell-specific DOX delivery and release into the cytoplasm of hepatic cancer cells. G5 is functionalized with poly(ethylene glycol) (PEG) brushes displaying N-acetylgalactosamine (NAcGal) ligands to target hepatic cancer cells. DOX is attached to G5 through one of two aromatic azo-linkages, L3 or L4, achieving either P1 ((NAcGal -PEGc) -G5-(L3-DOX) ) or P2 ((NAcGal -PEGc) -G5-(L4-DOX) ) conjugates. After confirming the conjugates' biocompatibility, flow cytometry studies show P1/P2 achieve 100% uptake into hepatic cancer cells at 30-60 × 10 m particle concentration. This internalization correlates with cytotoxicity against HepG2 cells with 50% inhibitory concentration (IC ) values of 24.8, 1414.0, and 237.8 × 10 m for free DOX, P1, and P2, respectively. Differences in cytotoxicity prompted metabolomics analysis to identify the intracellular release behavior of DOX. Results show that P1/P2 release alternative DOX metabolites than free DOX. Stable isotope tracer studies show that the different metabolites induce different effects on metabolic cycles. Namely, free DOX reduces glycolysis and increases fatty acid oxidation, while P1/P2 increase glycolysis, likely as a response to high oxidative stress. Overall, P1/P2 conjugates offer a platform drug delivery technology for improving hepatic cancer therapy.

Dendrimer-doxorubicin conjugates exhibit improved anticancer activity and reduce doxorubicin-induced cardiotoxicity in a murine hepatocellular carcinoma model

“…We[ 24 – 27 , 29 ] and others[ 30 – 33 ] have previously discussed the utility of using G5 PAMAM dendrimers as targeted drug delivery vehicles. G5 PAMAM dendrimers exhibit a unique combination of properties that give them advantages over other nanocarrier options, such as their high water solubility, biocompatibility, controllable synthesis, and size/weight properties that extend circulation time and allow penetration into tumor tissue.…”

“…We previously reported the synthetic techniques used to create P1 and P2 particles. [ 27 ] We used similar techniques to fabricate the particles presented here, and the complete synthesis along with nuclear magnetic resonance (NMR) and time-of-flight matrix-assisted laser desorption/ionization (MALDI) spectra can be found in the Supporting Information . We relied on NMR and MALDI data to establish the molecular weights of P1 and P2 particles, as well as the density of loaded NAcGal β -PEG c and L(x)-DOX linkages.…”

“…We reported the development of enzyme-activated polymer-DOX nano-conjugates as a potential new therapy for HCC ( Fig 1 ). [ 24 – 27 ] Specifically, we conjugated DOX molecules to water-soluble generation 5 (G5) of poly(amidoamine) (PAMAM) dendrimers via aromatic azo-linkages forming G5-DOX conjugates, which are selectively recognized and cleaved by azoreductase enzymes expressed by hepatic cancer cells ( Fig 2 ). [ 26 ] Targeting hepatic cancer cells was achieved by conjugating N -acetylgalactosamine (NAcGal β ) sugar molecules to the free tip of a PEG brush anchored to the G5 surface via acid-labile cis-aconityl ( c ) linkages ( Fig 2 ), which proved to achieve rapid internalization into hepatic cancer cells while avoiding recognition and uptake by healthy hepatocytes.…”

“…[ 26 ] Targeting hepatic cancer cells was achieved by conjugating N -acetylgalactosamine (NAcGal β ) sugar molecules to the free tip of a PEG brush anchored to the G5 surface via acid-labile cis-aconityl ( c ) linkages ( Fig 2 ), which proved to achieve rapid internalization into hepatic cancer cells while avoiding recognition and uptake by healthy hepatocytes. [ 25 , 27 ] In these reports, we describe the synthesis of two NAcGal β -targeted nano-conjugates where DOX molecules are attached to the G5 surface via two different enzyme-sensitive azo-linkages (i.e. L3 -DOX or L4 -DOX) yielding NAcGal β -PEG c -G5- L3 -DOX or NAcGal β -PEG-G5- L4 -DOX, named P1 and P2, respectively ( Fig 2 ).…”

Dendrimers: Amazing Platforms for Bioactive Molecule Delivery Systems

“…The above can be considered as an advantage because key parts of the glycoproteins can be obtained and grafted on the surface of nanoparticles (NP) to promote high-efficiency binding to hepatic tumor cells. N-acetylgalactosamine (NAcGal) ligands on a NP surface achieves selective intake into hepatic cancer cells [101][102][103]. Moreover, Kurivilla et al synthesized G5-dendrimers containing NAcGal ligands tri-valent (NAcGal3) attached to the surface through a PEG linker and measured their ability to achieve hepatic cancer cells in comparison to mono-valent ligands [104] (Scheme 6).…”