A thermoreversible poly(choline phosphate) based universal biomembrane adhesive.

A new monomer, 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl methacrylate (AEO4 MA), and its direct atom transfer radical polymerization (ATRP) into poly(AEO4 MA), then "clicked" with prop-2-ynyle choline phosphate (CP) to produce a poly(choline phosphate) are described. This polymer exhibits a lower critical solution temperature (LCST) at ≈ 32 °C, and provides a universal thermally reversible biomembrane adhesive, which can rapidly both bind to any mammalian cell membrane and internalize into the cytoplasm of nucleated cells below the LCST. Moving above the LCST reverses cell surface binding. The use of ATRP implies that such polymers can be applied to modify the surfaces of a wide range of biomaterials. The capacity to bind and immobilize cells at room temperature and release them above the LCST should be particularly useful for in vitro cell manipulation and tissue engineering applications.

A pH and thermosensitive choline phosphate-based delivery platform targeted to the acidic tumor microenvironment.

Solid tumors generally exhibit an acidic microenvironment which has been recognized as a potential route to distinguishing tumor from normal tissue for purposes of drug delivery or imaging. To this end we describe a pH and temperature sensitive polymeric adhesive that can be derivatized to carry drugs or other agents and can be tuned synthetically to bind to tumor cells at pH 6.8 but not at pH 7.4 at 37 °C. The adhesive is based on the universal reaction between membrane phosphatidyl choline (PC) molecules and polymers derivatized with multiple copies of the inverse motif, choline phosphate (CP). The polymer family we use is a linear copolymer of a CP terminated tetraethoxymethacrylate and dimethylaminoethyl (DMAE) methacrylate, the latter providing pH sensitivity. The copolymer exhibits a lower critical solution temperature (LCST) just below 37 °C when the DMAE is uncharged at pH 7.4 but the LCST does not occur when the group is charged at pH 6.8 due to the ionization hydrophilicity. At 37 °C the polymer binds strongly to mammalian cells at pH 6.8 but does not bind at pH 7.4, potentially targeting tumor cells existing in an acidic microenvironment. We show the binding is strong, reversible if the pH is raised and is followed rapidly by cellular uptake of the fluorescently labeled material. Drug delivery utilizing this dually responsive family of polymers should provide a basis for targeting tumor cells with minimal side reactions against untransformed counterparts.

Design of long circulating nontoxic dendritic polymers for the removal of iron in vivo.

Patients requiring chronic red blood cell (RBC) transfusions for inherited or acquired anemias are at risk of developing transfusional iron overload, which may impact negatively on organ function and survival. Current iron chelators are suboptimal due to the inconvenient mode of administration and/or side effects. Herein, we report a strategy to engineer low molecular weight iron chelators with long circulation lifetime for the removal of excess iron in vivo using a multifunctional dendritic nanopolymer scaffold. Desferoxamine (DFO) was conjugated to hyperbranched polyglycerol (HPG) and the plasma half-life (t1/2) in mice is defined by the structural features of the scaffold. There was a 484 fold increase in t1/2 between the DFO (5 min) versus the HPG-DFO (44 h). In an iron overloaded mouse model, efficient iron excretion by HPG-DFO in the urine and feces was demonstrated (p = 0.0002 and 0.003, respectively) as was a reduction in liver, heart, kidney, and pancreas iron content, and plasma ferritin level (p = 0.003, 0.001, 0.001, 0.001, and 0.003, respectively) compared to DFO. Conjugates showed no apparent toxicity in several analyses including body weight, serum lactate dehydrogenase level, necropsy analysis, and by histopathological examination of organs. These findings were supported by in vitro biocompatibility analyses, including blood coagulation, platelet activation, complement activation, red blood cell aggregation, hemolysis, and cell viability. This nanopolymer-based chelating system would potentially benefit patients suffering from transfusional iron overload.

Thermal reversal of polyvalent choline phosphate, a multivalent universal biomembrane adhesive.

Multivalent macromolecular associations are widely observed in biological systems and are increasingly being utilized in bioengineering, nanomedicine, and biomaterial applications. Control over such associations usually demands an ability to reverse the multivalent binding. While in principle this can be done with binding site competitive inhibitors, dissociation is difficult in practice due to limited site accessibility when the macromolecule is bound. We demonstrate here efficient binding reversal of multivalent linear copolymers that adhere to any mammalian cell via the universal mechanism based on choline phosphate (CP) groups binding to phosphatidyl choline (PC)-containing biomembranes. Using a smart linear polymer exhibiting a lower critical solution temperature (LCST), we take advantage of the thermal contraction of the polymer above the LCST, which reduces accessibility of the CP groups to cell membrane PC lipids. The polymer construct can then desorb from the cell surface, reversing all effects of multivalent polymer adhesion on the cell.

ATRP synthesis of poly(2-(methacryloyloxy)ethyl choline phosphate): a multivalent universal biomembrane adhesive.

A new monomer, 2-(methacryloyloxy)ethyl choline phosphate, and its direct polymerization into a polyvalent choline phosphate are described, providing a universal biomembrane adhesive exhibiting rapid, strong attachment to any mammalian cell membrane and fast internalization, properties of great value in applications such as tissue engineering and drug delivery.

Branched multifunctional polyether polyketals: variation of ketal group structure enables unprecedented control over polymer degradation in solution and within cells.

Multifunctional biocompatible and biodegradable nanomaterials incorporating specific degradable linkages that respond to various stimuli and with defined degradation profiles are critical to the advancement of targeted nanomedicine. Herein we report, for the first time, a new class of multifunctional dendritic polyether polyketals containing different ketal linkages in their backbone that exhibit unprecedented control over degradation in solution and within the cells. High-molecular-weight and highly compact poly(ketal hydroxyethers) (PKHEs) were synthesized from newly designed α-epoxy-ω-hydroxyl-functionalized AB(2)-type ketal monomers carrying structurally different ketal groups (both cyclic and acyclic) with good control over polymer properties by anionic ring-opening multibranching polymerization. Polymer functionalization with multiple azide and amine groups was achieved without degradation of the ketal group. The polymer degradation was controlled primarily by the differences in the structure and torsional strain of the substituted ketal groups in the main chain, while for polymers with linear (acyclic) ketal groups, the hydrophobicity of the polymer may play an additional role. This was supported by the log P values of the monomers and the hydrophobicity of the polymers determined by fluorescence spectroscopy using pyrene as the probe. A range of hydrolysis half-lives of the polymers at mild acidic pH values was achieved, from a few minutes to a few hundred days, directly correlating with the differences in ketal group structures. Confocal microscopy analyses demonstrated similar degradation profiles for PKHEs within live cells, as seen in solution and the delivery of fluorescent marker to the cytosol. The cell viability measured by MTS assay and blood compatibility determined by complement activation, platelet activation, and coagulation assays demonstrate that PKHEs and their degradation products are highly biocompatible. Taken together, these data demonstrate the utility this new class of biodegradable polymer as a highly promising candidate in the development of multifunctional nanomedicine.

Polyvalent choline phosphate as a universal biomembrane adhesive.

Phospholipids in the cell membranes of all eukaryotic cells contain phosphatidyl choline (PC) as the headgroup. Here we show that hyperbranched polyglycerols (HPGs) decorated with the 'PC-inverse' choline phosphate (CP) in a polyvalent fashion can electrostatically bind to a variety of cell membranes and to PC-containing liposomes, the binding strength depending on the number density of CP groups per macromolecule. We also show that HPG-CPs can cause cells to adhere with varying affinity to other cells, and that binding can be reversed by subsequent exposure to low molecular weight HPGs carrying small numbers of PCs. Moreover, PC-rich membranes adsorb and rapidly internalize fluorescent HPG-CP but not HPG-PC molecules, which suggests that HPG-CPs could be used as drug-delivery agents. CP-decorated polymers should find broad use, for instance as tissue sealants and in the self-assembly of lipid nanostructures.

In vitro chelating, cytotoxicity, and blood compatibility of degradable poly(ethylene glycol)-based macromolecular iron chelators.

Desferrioxamine (DFO) is used to treat an excess accumulation of iron in the body and is currently the most commonly used iron chelator for the treatment of 'iron overload' disorder. However, the disadvantages of DFO surround its high toxicity and very short plasma half-life. Here, the detailed in vitro evaluation of a novel class of high molecular weight iron chelators based on DFO and polyethylene glycol methacrylate is reported. Reversible addition fragment chain transfer (RAFT) copolymerization afforded polymer conjugates (P-DFO) with well-controlled molecular weight (27-127 kDa) and substitution of DFO (5-26 units per chain) along the copolymer. Human umbilical vein endothelial cell (HUVEC) based cell viability assays showed that the cytotoxicity of P-DFO decreased more than 100-fold at identical concentrations of DFO. The hemocompatibilities of various P-DFO samples were determined by measuring prothrombin time (PT), activated partial thromboplastin time (APTT), thrombelastograph parameters (TEG), complement activation, platelet activation, and red blood cell aggregation. Furthermore, the iron binding properties and chelating efficiency of P-DFO were compared to DFO by measuring the spectral properties upon binding to iron(III), while the prevention of iron(III) mediated oxidation of hemoglobin was also determined. Degradation of the P-DFO conjugates via cleavable ester linkages between the polymer backbone and the PEG side chains was evaluated using gel permeation chromatography (GPC) and NMR. Since the chelating ability of DFO remains intact after conjugation to the copolymer backbone, these macromolecular, blood compatible and degradable conjugates are promising candidates as long circulating, non-toxic iron chelators.

Slx4 regulates DNA damage checkpoint-dependent phosphorylation of the BRCT domain protein Rtt107/Esc4.

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal-domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint protein kinase Mec1, although the mechanism by which Rtt107 is targeted by Mec1 after checkpoint activation is currently unclear. Slx4, a component of the Slx1-Slx4 structure-specific nuclease, formed a complex with Rtt107. Deletion of SLX4 conferred many of the same DNA-repair defects observed in rtt107delta, including DNA damage sensitivity, prolonged DNA damage checkpoint activation, and increased spontaneous DNA damage. These phenotypes were not shared by the Slx4 binding partner Slx1, suggesting that the functions of the Slx4 and Slx1 proteins in the DNA damage response were not identical. Of particular interest, Slx4, but not Slx1, was required for phosphorylation of Rtt107 by Mec1 in vivo, indicating that Slx4 was a mediator of DNA damage-dependent phosphorylation of the checkpoint effector Rtt107. We propose that Slx4 has roles in the DNA damage response that are distinct from the function of Slx1-Slx4 in maintaining rDNA structure and that Slx4-dependent phosphorylation of Rtt107 by Mec1 is critical for replication restart after alkylation damage.