Scott H. Medina

N-acetylgalactosamine-functionalized dendrimers as hepatic cancer cell-targeted carriers

There is an urgent need for novel polymeric carriers that can selectively deliver a large dose of chemotherapeutic agents into hepatic cancer cells to achieve high therapeutic activity with minimal systemic side effects. PAMAM dendrimers are characterized by a unique branching architecture and a large number of chemical surface groups suitable for coupling of chemotherapeutic agents. In this article, we report the coupling of N-acetylgalactosamine (NAcGal) to generation 5 (G5) of poly(amidoamine) (PAMAM-NH₂) dendrimers via peptide and thiourea linkages to prepare NAcGal-targeted carriers used for targeted delivery of chemotherapeutic agents into hepatic cancer cells. We describe the uptake of NAcGal-targeted and non-targeted G5 dendrimers into hepatic cancer cells (HepG2) as a function of G5 concentration and incubation time. We examine the contribution of the asialoglycoprotein receptor (ASGPR) to the internalization of NAcGal-targeted dendrimers into hepatic cancer cells through a competitive inhibition assay. Our results show that uptake of NAcGal-targeted G5 dendrimers into hepatic cancer cells occurs via ASGPR-mediated endocytosis. Internalization of these targeted carriers increased with the increase in G5 concentration and incubation time following Michaelis-Menten kinetics characteristic of receptor-mediated endocytosis. These results collectively indicate that G5-NAcGal conjugates function as targeted carriers for selective delivery of chemotherapeutic agents into hepatic cancer cells.

Injectable bioadhesive hydrogels with innate antibacterial properties

Surgical site infections cause significant postoperative morbidity and increased healthcare costs. Bioadhesives used to fill surgical voids and support wound healing are typically devoid of antibacterial activity. Here, we report novel syringe-injectable bioadhesive hydrogels with inherent antibacterial properties prepared from mixing polydextran aldehyde (PDA) and branched polyethylenimine (PEI). These adhesives kill both Gram-negative and Gram–positive bacteria, while sparing human erythrocytes. An optimal composition of 2.5 wt % oxidized dextran and 6.9 wt % PEI sets within seconds forming a mechanically rigid (~1700 Pa) gel offering a maximum adhesive stress of ~ 2.8 kPa. A murine infection model showed that the adhesive is capable of killing S. pyogenes introduced subcutaneously at the bioadhesive’s surface, with minimal inflammatory response. The adhesive was also effective in a cecal ligation and puncture model, preventing sepsis and significantly improving survival. These bioadhesives represent novel, inherently antibacterial materials for wound filling applications.

A multiphase transitioning peptide hydrogel for suturing ultrasmall vessels

Many surgeries are complicated by the need to anastomose, or reconnect, micron-scale vessels. Although suturing remains the gold standard for anastomosing vessels, it is difficult to place sutures correctly through collapsed lumen, making the procedure prone to failure. Here, we report a multi-phase transitioning peptide hydrogel that can be injected into the lumen of vessels to facilitate suturing. The peptide, which contains a photocaged glutamic acid, forms a solid-like gel in a syringe and can be shear-thin delivered to the lumen of collapsed vessels (where it distends the vessel), and the space between two vessels (where it is used to approximate the vessel ends). Suturing is performed directly through the gel. Light is used to initiate the final gel-sol phase transition that disrupts the hydrogel network, allowing the gel to be removed and blood flow to resume. This gel adds a new tool to the armamentarium for micro- and supermicrosurgical procedures.

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 conjugates 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.

Targeting Hepatic Cancer Cells with PEGylated Dendrimers Displaying N -Acetylgalactosamine and SP94 Peptide Ligands

Poly(amidoamine) (PAMAM) dendrimers are branched water-soluble polymers defined by consecutive generation numbers (Gn) indicating a parallel increase in size, molecular weight, and number of surface groups available for conjugation of bioactive agents. In this article, we compare the biodistribution of N-acetylgalactosamine (NAcGal)-targeted [(14) C]1 -G5-(NH2 )5 -(Ac)108 -(NAcGal)14 particles to non-targeted [(14) C]1 -G5-(NH2 )127 and PEGylated [(14) C]1 -G5-(NH2 )44 -(Ac)73 -(PEG)10 particles in a mouse hepatic cancer model. Results show that both NAcGal-targeted and non-targeted particles are rapidly cleared from the systemic circulation with high distribution to the liver. However, NAcGal-targeted particles exhibited 2.5-fold higher accumulation in tumor tissue compared to non-targeted ones. In comparison, PEGylated particles showed a 16-fold increase in plasma residence time and a 5-fold reduction in liver accumulation. These results motivated us to engineer new PEGylated G5 particles with PEG chains anchored to the G5 surface via acid-labile cis-aconityl linkages where the free PEG tips are functionalized with NAcGal or SP94 peptide to investigate their potential as targeting ligands for hepatic cancer cells as a function of sugar conformation (α versus β), ligand concentration (100-4000 nM), and incubation time (2 and 24 hours) compared to fluorescently (Fl)-labeled and non-targeted G5-(Fl)6 -(NH2 )122 and G5-(Fl)6 -(Ac)107 -(cPEG)15 particles. Results show G5-(Fl)6 -(Ac)107 -(cPEG[NAcGalβ ])14 particles achieve faster uptake and higher intracellular concentrations in HepG2 cancer cells compared to other G5 particles while escaping the non-specific adsorption of serum protein and phagocytosis by Kupffer cells, which make these particles the ideal carrier for selective drug delivery into hepatic cancer cells.

An Intrinsically Disordered Peptide Facilitates Non-Endosomal Cell Entry.

Many cell-penetrating peptides (CPPs) fold at cell surfaces, adopting α- or β-structure that enable their intracellular transport. However, the same structural folds that facilitate cellular entry can also elicit potent membrane-lytic activity, limiting their use in delivery applications. Further, a distinct CPP can enter cells through many mechanisms, often leading to endosomal entrapment. Herein, we describe an intrinsically disordered peptide (CLIP6) that exclusively employs non-endosomal mechanisms to cross cellular membranes, while being remarkably biocompatible and serum-stable. We show that a single anionic glutamate residue is responsible for maintaining the disordered bioactive state of the peptide, defines its mechanism of cellular entry, and is central to its biocompatibility. CLIP6 can deliver membrane-impermeable cargo directly to the cytoplasm of cells, suggesting its broad utility for delivery of drug candidates limited by poor cell permeability and endosomal degradation.

Synthesis and cell-selective antitumor properties of amino acid conjugated tumor-associated carbohydrate antigen-coated gold nanoparticles

The Thomsen Friedenreich antigen (TFag) disaccharide is a tumor-associated carbohydrate antigen (TACA) found primarily on carcinoma cells and rarely expressed in normal tissue. The TFag has been shown to interact with Galectin-3 (Gal-3), one in a family of β-galactoside binding proteins. Galectins have a variety of cellular functions, and Gal-3 has been shown to be the sole galectin with anti-apoptotic activity. We have previously prepared gold nanoparticles (AuNP) coated with the TFag in various presentations as potential anti-adhesive therapeutic tools or antitumor vaccine platforms. Here we describe the synthesis of TFag-glycoamino acid conjugates attached to gold nanoparticles through a combined alkane/PEG linker, where the TFag was attached to either a serine or threonine amino acid. Particles were fully characterized by a host of biophysical techniques, and along with a control particle carrying hydroxyl-terminated linker units, were evaluated in both Gal-3 positive and negative cell lines. We show that the particles bearing the saccharides selectively inhibited tumor cell growth of the Gal-3 positive cells significantly more than the Gal-3 negative cells. In addition, the threonine-attached TF particles were more potent than the serine-attached constructs. These results support the use of AuNP as antitumor therapeutic platforms, targeted against cell lines that express specific lectins that interact with TFag.

Cancer cell surface induced peptide folding allows intracellular translocation of drug

Many lead molecules identified in drug discovery campaigns are eliminated from consideration due to poor solubility and low cell permeability. These orphaned molecules could have clinical value if solubilized and delivered properly. SVS-1 is a de novo designed peptide that preferentially folds at the surface of tumor cells, adopting a β-hairpin conformation that rapidly translocates into the cytoplasm, and ultimately nucleus, of cells. SVS-1 is stable in serum and small molecules attached to the peptide are effectively delivered to cancer cells via mechanisms involving physical translocation and, to a lesser extent, clathrin-dependent endocytosis. For example, ligating the model hydrophobic drug Paclitaxel (PTX) to SVS-1 improved its aqueous solubility by ~1000-fold and successfully delivered and released PTX to cancer cells in vitro and tumors in vivo without toxic adjuvants. These results suggest that SVS-1 can serve as a simple, effective delivery platform for molecules with poor solubility and permeability.

Glycan Alteration Imparts Cellular Resistance to a Membrane-Lytic Anticancer Peptide.

Although resistance toward small-molecule chemotherapeutics has been well studied, the potential of tumor cells to avoid destruction by membrane-lytic compounds remains unexplored. Anticancer peptides (ACPs) are a class of such agents that disrupt tumor cell membranes through rapid and non-stereospecific mechanisms, encouraging the perception that cellular resistance toward ACPs is unlikely to occur. We demonstrate that eukaryotic cells can, indeed, develop resistance to the model oncolytic peptide SVS-1, which preferentially disrupts the membranes of cancer cells. Utilizing fission yeast as a model organism, we show that ACP resistance is largely controlled through the loss of cell-surface anionic saccharides. A similar mechanism was discovered in mammalian cancer cells where removal of negatively charged sialic acid residues directly transformed SVS-1-sensitive cell lines into resistant phenotypes. These results demonstrate that changes in cell-surface glycosylation play a major role in tumor cell resistance toward oncolytic peptides.

Anti-microbial peptide facilitated cytosolic delivery of metallic gold nanomaterials

The unique photophysical properties of gold nanomaterials combined with progress in developing effective surfacefunctionalization strategies has motivated researchers to employ them as tools for use in biomedical imaging, biosensing, diagnostics, photothermal therapy, and as drug and gene delivery vehicles. However, a major challenge limiting these advancements has been the unavailability of effective strategies to deliver these and other nanocrystals into the cytoplasm of live cells. In this study, we demonstrate that the use of a chemically-synthesized anti-microbial peptide, SVS-1, can promote non-endocytic uptake of both small size gold nanoparticles (AuNPs) and larger size gold nanorods (AuNRs) into mammalian cells. For this, colloidally stable AuNP and AuNRs, surface ligated with an amine-functionalized polymer, His-PIMA-PEG-OCH3/NH2 were prepared. The amine groups allow dual, covalent attachment of cysteine terminated SVS-1 (via a thioether linkage) and NHS-ester-Texas-Red dye onto the nanocrystal surfaces. We use fluorescence microscopy to demonstrate nanocrystal staining throughout the cytoplasmic volume of the cells incubated with these conjugates. More importantly, we have conducted additional endocytosis inhibition experiments where cells were incubated with the conjugates at 4°C. Here too, the imaging data have shown significant levels of nanocrystal uptake, further verifying that physical translocation of these conjugates takes place through the cell membrane independent of endocytosis. These findings are promising and can provide critical support for the widespread applications of nanomaterials in the field of biology.