Effect of N-acetylgalactosamine ligand valency on targeting dendrimers to hepatic cancer cells.

The display of N-acetylgalactosamine (NAcGal) ligands has shown great potential in improving the targeting of various therapeutic molecules to hepatocellular carcinoma (HCC), a severe disease whose clinical treatment is severely hindered by limitations in delivery of therapeutic cargo. We previously used the display of NAcGal on generation 5 (G5) polyamidoamine (PAMAM) dendrimers connected through a poly(ethylene glycol) (PEG) brush (i.e. G5-cPEG-NAcGal; monoGal) to effectively target hepatic cancer cells and deliver a loaded therapeutic cargo. In this study, we were interested to see if tri-valent NAcGal ligands (i.e. NAcGal3) displayed on G5 dendrimers (i.e. G5-cPEG-NAcGal3; triGal) could improve their ability to target hepatic cancer cells compared to their monoGal counterparts. We therefore synthesized a library of triGal particles, with either 2, 4, 6, 8, 11, or 14 targeting branches (i.e. cPEG-NAcGal3) attached. Conventional flow cytometry studies showed that all particle formulations can label hepatic cancer cells in a concentration-dependent manner, reaching 90-100% of cells labeled at either 285 or 570 nM G5, but interestingly, monoGal labeled more cells at lower concentrations. To elucidate the difference in internalization of monoGal versus triGal conjugates, we turned to multi-spectral imaging flow cytometry and quantified the amount of internalized (I) versus surface-bound (I0) conjugates to determine the ratio of internalization (I/I0) in all treatment groups. Results show that regardless of NAcGal valency, or the density of targeting branches, all particles achieve full internalization and diffuse localization throughout the cell (I/I0 ∼ 3.0 for all particle compositions). This indicates that while tri-valent NAcGal is a promising technique for targeting nanoparticles to hepatic cancer cells, mono-valent NAcGal is more efficient, contrary to what is observed with small molecules.

Gas Embolization in a Rodent Model of Hepatocellular Carcinoma Using Acoustic Droplet Vaporization.

Trans-arterial embolization is a commonly used therapy in unresectable hepatocellular carcinoma. Current methods involve the careful placement of an intraarterial catheter and the deposition of embolizing particles. Gas embolotherapy has been proposed as an embolization method with the potential for high spatial resolution without the need for a catheter. This method involves vaporizing intravenouslyadministered droplets into gas bubbles using focused ultrasound - a process termed acoustic droplet vaporization. The bubbles can become lodged in the vasculature, thereby creating an embolus. Here, we initially demonstrate the feasibility of achieving significant targeted embolization with this method in the rat cremaster using intravital microscopy. The therapy was then tested in an ectopic xenograft mouse model of hepatocellular carcinoma. Gas embolotherapy was shown to maintain the tumor volume at baseline over a twoweek treatment course while control groups showed significant tumor growth. These preliminary results demonstrate thatgas embolotherapy could serve as an effective noninvasive method for the management of unresectable hepatocellular carcinoma.

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

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 ((NAcGalß -PEGc)16.6 -G5-(L3-DOX)11.6 ) or P2 ((NAcGalß -PEGc)16.6 -G5-(L4-DOX)13.4 ) conjugates. After confirming the conjugates' biocompatibility, flow cytometry studies show P1/P2 achieve 100% uptake into hepatic cancer cells at 30-60 × 10-9 m particle concentration. This internalization correlates with cytotoxicity against HepG2 cells with 50% inhibitory concentration (IC50 ) values of 24.8, 1414.0, and 237.8 × 10-9 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.

Hepatocellular carcinoma (HCC) is the 2nd leading cause of cancer-related deaths every year globally. The most common form of treatment, hepatic arterial infusion (HAI), involves the direct injection of doxorubicin (DOX) into the hepatic artery. It is plagued with limited therapeutic efficacy and the occurrence of severe toxicities (e.g. cardiotoxicity). We aim to improve the therapeutic index of DOX delivered via HAI by loading the drug onto generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers targeted to hepatic cancer cells via N-acetylgalactosamine (NAcGal) ligands. DOX is attached to the surface of G5 molecules via two different enzyme-sensitive linkages, L3 or L4, to achieve controllable drug release inside hepatic cancer cells. We previously reported on P1 and P2 particles that resulted from the combination of NAcGal-targeting with L3- or L4-DOX linkages, respectively, and showed controllable DOX release and toxicity towards hepatic cancer cells comparable to free DOX. In this study, we demonstrate that while the intratumoral delivery of free DOX (1 mg/kg) into HCC-bearing nod scid gamma (NSG) mice achieves a 2.5-fold inhibition of tumor growth compared to the saline group over 30 days, P1 and P2 particles delivered at the same DOX dosage achieve a 5.1- and 4.4-fold inhibition, respectively. Incubation of the particles with human induced pluripotent stem cell derived cardiomyocytes (hiPSC CMs) showed no effect on monolayer viability, apoptosis induction, or CM electrophysiology, contrary to the effect of free DOX. Moreover, magnetic resonance imaging revealed that P1- and P2-treated mice maintained cardiac function after intraperitoneal administration of DOX at 1 mg/kg for 21 days, unlike the free DOX group at an equivalent dosage, confirming that P1/P2 can avoid DOX-induced cardiotoxicity. Taken together, these results highlight the ability of P1/P2 particles to improve the therapeutic index of DOX and offer a replacement therapy for clinical HCC treatment.

Enzyme-activated nanoconjugates for tunable release of doxorubicin in hepatic cancer cells.

We report the synthesis of a series of aromatic azo-linkers (L1-L4), which are selectively recognized and cleaved by azoreductase enzymes present in the cytoplasm of hepatic cancer cells via a NADPH-dependent mechanism. We utilized L1-L4 azo-linkers to conjugate doxorubicin to generation 5 (G5) of poly(amidoamine) dendrimers to prepare G5-L(x)-DOX nanoconjugates. We incorporated electron-donating oxygen (O) or nitrogen (N) groups in the para and ortho positions of L1-L4 azo-linkers to control the electronegativity of G5-L(x)-DOX conjugates and investigated their cleavage by azoreductase enzymes and the associated release of loaded DOX molecules. Hammett σ values of G5-L(x)-DOX conjugates ranged from -0.44 to -1.27, which is below the reported σ threshold (-0.37) required for binding to azoreductase enzymes. Results show that incubation of G5-L1-DOX (σ = -0.44), G5-L2-DOX (σ = -0.71), G5-L3-DOX (σ = -1.00), and G5-L4-DOX (σ = -1.27) conjugates with human liver microsomal (HLM) enzymes and the S9 fraction isolated from HepG2 hepatic cancer cells results in release of 4%-8%, 17%, 60%, and 100% of the conjugated DOX molecules, respectively. These results show that increasing the electronegativity (i.e. lower σ value) of L1-L4 azo-linkers increases their susceptibility to cleavage by azoreductase enzymes. Intracellular cleavage of G5-L(x)-DOX nanoconjugates, release of conjugated DOX molecules, and cytotoxicity correlated with conjugate's electronegativity (σ value) was investigated, with G5-L4-DOX conjugate exhibiting the highest toxicity towards hepatic cancer cells with an IC50 of 13 nm ± 5 nm in HepG2 cells. Cleavage of G5-L(x)-DOX conjugates was specific to hepatic cancer cells as shown by low non-specific DOX release upon incubation with non-enzymatic insect proteins and the S9 fraction isolated from rat cardiomyocytes. These enzyme-activated G5-L(x)-DOX conjugates represent a drug delivery platform that can achieve tunable and cell-specific release of the loaded cargo in hepatic cancer cells.