Hemolytic Activity of pH-Responsive Polymer-Streptavidin Bioconjugates

Drug delivery systems that increase the rate and/or quantity of drug release to the cytoplasm are needed to enhance cytosolic delivery and to circumvent nonproductive cell trafficking routes. We have previously demonstrated that poly(2-ethylacrylic acid) (PEAAc) has pH-dependent hemolytic properties, and more recently, we have found that poly(2-propylacrylic acid) (PPAAc) displays even greater pH-responsive hemolytic activity than PEAAc at the acidic pHs of the early endosome. Thus, these polymers could potentially serve as endosomal releasing agents in immunotoxin therapies. In this paper, we have investigated whether the pH-dependent membrane disruptive activity of PPAAc is retained after binding to a protein. We did this by measuring the hemolytic activity of PPAAc−streptavidin model complexes with different protein to polymer stoichiometries. Biotin was conjugated to amine-terminated PPAAc, which was subsequently bound to streptavidin by biotin complexation. The ability of these samples to disrupt red blood cell membranes was investigated for a range of polymer concentrations, a range of pH values, and two polymer-to-streptavidin ratios of 3:1 and 1:1. The results demonstrate that (a) the PPAAc−streptavidin complex retains the ability to lyse the RBC lipid bilayers at low pHs, such as those existing in endosomes, and (b) the hemolytic ability of the PPAAc−streptavidin complex is similar to that of the free PPAAc

Internalization of novel non-viral vector TAT-streptavidin into human cells

BACKGROUND: The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT(47–57)-streptavidin (TAT-SA, 60 kD). SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes. RESULTS: By confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accumulate in perinuclear vesicles in both cancer and non-cancer cell lines. The uptake studies in living cells with various fluorescent endocytic markers and inhibiting agents suggested that TAT-SA is internalized into cells efficiently, using both clathrin-mediated endocytosis and lipid-raft-mediated macropinocytosis. When endosomal release of TAT-SA was enhanced through the incorporation of a biotinylated, pH-responsive polymer poly(propylacrylic acid) (PPAA), nuclear localization of TAT-SA and TAT-SA bound to biotin was markedly improved. Additionally, no significant cytotoxicity was detected in the TAT-SA constructs. CONCLUSION: This study demonstrates that TAT-SA-PPAA is a potential non-viral vector to be utilized in protein therapeutics to deliver biotinylated molecules both into cytoplasm and nucleus of human cells

Antibody Targeting Facilitates Effective Intratumoral SiRNA Nanoparticle Delivery to HER2-Overexpressing Cancer Cells

The therapeutic potential of RNA interference (RNAi) has been limited by inefficient delivery of short interfering RNA (siRNA). Tumor-specific recognition can be effectively achieved by antibodies directed against highly expressed cancer cell surface receptors. We investigated the utility of linking an internalizing streptavidinconjugated HER2 antibody to an endosome-disruptive biotinylated polymeric nanocarrier to improve the functional cytoplasmic delivery of siRNA in breast and ovarian cancer cells in vitro and in an intraperitoneal ovarian cancer xenograft model in vivo, yielding an 80% reduction of target mRNA and protein levels with sustained repression for at least 96 hours. RNAi-mediated site specific cleavage of target mRNA was demonstrated using the 5\u27 RLM-RACE (RNA ligase mediated-rapid amplification of cDNA ends) assay. Mice bearing intraperitoneal human ovarian tumor xenografts demonstrated increased tumor accumulation of Cy5.5 fluorescently labeled siRNA and 70% target gene suppression after treatment with HER2 antibody-directed siRNA nanocarriers. Detection of the expected mRNA cleavage product by 5\u27 RLM-RACE assay confirmed that suppression occurs via the expected RNAi pathway. Delivery of siRNA via antibody-directed endosomolytic nanoparticles may be a promising strategy for cancer therapy

Pretargeted Radioimmunotherapy Using Genetically Engineered Antibody-Streptavidin Fusion Proteins for Treatment of Non-Hodgkin Lymphoma

Pretargeted radioimmunotherapy (PRIT) using streptavidin (SAv)-biotin technology can deliver higher therapeutic doses of radioactivity to tumors than conventional RIT. However, “endogenous” biotin can interfere with the effectiveness of this approach by blocking binding of radiolabeled biotin to SAv. We engineered a series of SAv FPs that down-modulate the affinity of SAv for biotin, while retaining high avidity for divalent DOTA-bis-biotin to circumvent this problem

Biomolecular Materials That Talk and Listen

Pat Stayton of the University of Washington Department of Bioengineering presented a lecture on October 10, 2002 at 11:00 am in the Suddath Seminar Room, IBB Building, Georgia Tech Campus."Smart" or "intelligent" materials are those that reversibly change their structural and functional properties in response to environmental signals such as a change in temperature. Nature has itself perfected smart polymers in the form of proteins. A hallmark of many proteins is their ability to change their structural and functional properties in response to specific physical changes. That they can do this reversibly, in a continuously cyclical fashion, is a remarkable materials property. The molecular mechanisms that proteins use to sense and respond provide interesting paradigms for the development of new smart polymer based biotechnologies. As with nature and proteins, we have been working to develop systems where the environmentally responsive changes in polymer structure and physical properties are directly coupled to biofunctionality. These biofunctional smart polymers provide "listening" elements that reversibly modulate protein (or other biomolecules) activity in the device setting. Other biofunctional smart polymers are designed to directly enhance intracellular trafficking of biomolecular therapeutics, by destabilizing biological membranes in response to compartmental pH changes. In this talk, I will provide an overview of hybrid polymer biomolecule systems that are designed for applications in gene and protein delivery, diagnostics, microfluidics, and chip/array biotechnologies. These systems merge the impressive recognition and biofunctional properties of biomolecules, with the impressive responsiveness and chemical versatility of functional polymers

Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier

close-up view, English portion of sign about Hadrian's Gate, July 201