Benjamin F.L. Lai

Multilayered coating on titanium for controlled release of antimicrobial peptides for the prevention of implant-associated infections

Prevention of bacterial colonization and formation of a bacterial biofilm on implant surfaces has been a challenge in orthopaedic surgery. The treatment of implant-associated infections with conventional antibiotics has become more complicated by the emergence of multi-drug resistant bacteria. Antimicrobial eluting coatings on implants is one of the most promising strategies that have been attempted. This study reports a controlled release of an antimicrobial peptide (AMP) from titanium surface through a non-cytotoxic multilayered coating. Three layers of vertically oriented TiO2 nanotubes, a thin layer of calcium phosphate coating and a phospholipid (POPC) film were impregnated with a potent broad-spectrum AMP (HHC-36). The coating with controlled and sustained release of AMP was highly effective against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. No cytotoxicity to osteoblast-like cells (MG-63) was observed. Moderate platelet activation and adhesion on the implant surface with no observable activation in solution, and very low red blood cell lysis was observed on the implant. This multi-layer assembly can be a potential approach to locally deliver AMPs to prevent peri-implant infection in orthopaedics without being toxic to host cells.

Influence of architecture of high molecular weight linear and branched polyglycerols on their biocompatibility and biodistribution

The availability of long circulating, multifunctional polymers is critical to the development of drug delivery systems and bioconjugates. The ease of synthesis and functionalization make linear polymers attractive but their rapid clearance from circulation compared to their branched or cyclic counterparts, and their high solution viscosities restrict their applications in certain settings. Herein, we report the unusual compact nature of high molecular weight (HMW) linear polyglycerols (LPGs) (LPG - 100; M(n) - 104 kg mol(-1), M(w)/M(n) - 1.15) in aqueous solutions and its impact on its solution properties, blood compatibility, cell compatibility, in vivo circulation, biodistribution and renal clearance. The properties of LPG have been compared with hyperbranched polyglycerol (HPG) (HPG-100), linear polyethylene glycol (PEG) with similar MWs. The hydrodynamic size and the intrinsic viscosity of LPG-100 in water were considerably lower compared to PEG. The Mark-Houwink parameter of LPG was almost 10-fold lower than that of PEG. LPG and HPG demonstrated excellent blood and cell compatibilities. Unlike LPG and HPG, HMW PEG showed dose dependent activation of blood coagulation, platelets and complement system, severe red blood cell aggregation and hemolysis, and cell toxicity. The long blood circulation of LPG-100 (t(1/2β,) 31.8 ± 4 h) was demonstrated in mice; however, it was shorter compared to HPG-100 (t(1/2β,) 39.2 ± 8 h). The shorter circulation half life of LPG-100 was correlated with its higher renal clearance and deformability. Relatively lower organ accumulation was observed for LPG-100 and HPG-100 with some influence of on the architecture of the polymers. Since LPG showed better biocompatibility profiles, longer in vivo circulation time compared to PEG and other linear drug carrier polymers, and has multiple functionalities for conjugation, makes it a potential candidate for developing long circulating multifunctional drug delivery systems similar to HPG.

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.

Hyperbranched Glycopolymers for Blood Biocompatibility

Carbohydrate-based drug and gene delivery carriers are becoming extremely popular for in vitro and in vivo applications. These carriers are found to be nontoxic and can play a significant role in targeted delivery. However, the interactions of these carriers with blood cells and plasma components are not well explored. To the best of our knowledge, there are currently no reports that explore the role of carbohydrate based carriers for blood biocompatibility. Hyperbranched glycopolymers of varying molecular weights are synthesized by reversible addition-fragmentation chain transfer polymerization (RAFT) and are studied in detail for their biocompatibility, including hemocompatibility and cytotoxicity against different cell lines in vitro. The hemocompatibility studies (such as hemolysis and platelet activation) indicate that hyperbranched glycopolymers of varying molecular weights produced are highly hemocompatible and do not induce clot formation, red blood cell aggregation, and immune response. Hence, it can be concluded that glycopolymers functionalized carriers can serve as an excellent candidate for various biomedical applications. In addition, cytotoxicity of these hyperbranched polymers is studied in primary and malignant cell lines at varying concentrations using cell viability assay.

Affinity-based design of a synthetic universal reversal agent for heparin anticoagulants

A safe, synthetic anticoagulant-reversal agent based on a dendritic polymer is effective against all the clinical heparin anticoagulants and may be a treatment for bleeding in high-risk surgical procedures. One Drug to Rule Them All To clot or not to clot—that is the question. Prevention of blood clotting—anticoagulation—is preferred during surgical procedures or in blood vessels where clots can cause blockage. In fact, heparin-based anticoagulant drugs are used broadly for such purposes. But on the flip side, these anticoagulants are associated with bleeding risks that make close monitoring and neutralization necessary. Currently, only protamine has been clinically approved as an antidote to heparin-based anticoagulants; but the drug displays some adverse effects and is impotent against certain heparins and heparin-related medications. Now, Shenoi et al. describe a fully synthetic dendritic polymer–based universal heparin reversal agent (UHRA) that functions via multivalent presentation of branched cationic heparin binding groups (HBGs). The authors varied the agent’s scaffold, protective shell, and number and array of HBGs to develop an antidote that neutralized all clinically used heparin-related anticoagulants. The UHRA displayed safety and efficacy in animal models of heparin-induced bleeding. The new therapeutic may one day benefit patients in situations where the goal is to clot—such as in the treatment of excessive bleeding during anticoagulant therapy or high-risk surgery. Heparin-based anticoagulant drugs have been widely used for the prevention of blood clotting during surgical procedures and for the treatment of thromboembolic events. However, bleeding risks associated with these anticoagulants demand continuous monitoring and neutralization with suitable antidotes. Protamine, the only clinically approved antidote to heparin, has shown adverse effects and ineffectiveness against low–molecular weight heparins and fondaparinux, a heparin-related medication. Alternative approaches based on cationic molecules and recombinant proteins have several drawbacks including limited efficacy, toxicity, immunogenicity, and high cost. Thus, there is an unmet clinical need for safer, rapid, predictable, and cost-effective anticoagulant-reversal agents for all clinically used heparins. We report a design strategy for a fully synthetic dendritic polymer–based universal heparin reversal agent (UHRA) that makes use of multivalent presentation of branched cationic heparin binding groups (HBGs). Optimization of the UHRA design was aided by isothermal titration calorimetry studies, biocompatibility evaluation, and heparin neutralization analysis. By controlling the scaffold’s molecular weight, the nature of the protective shell, and the presentation of HBGs on the polymer scaffold, we arrived at lead UHRA molecules that completely neutralized the activity of all clinically used heparins. The optimized UHRA molecules demonstrated superior efficacy and safety profiles and mitigated heparin-induced bleeding in animal models. This new polymer therapeutic may benefit patients undergoing high-risk surgical procedures and has potential for the treatment of anticoagulant-related bleeding problems.

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.

Biodegradable polyglycerols with randomly distributed ketal groups as multi-functional drug delivery systems

Biodegradable multi-functional polymeric nanostructures that undergo controlled degradation in response to physiological cues are important in numerous biomedical applications including drug delivery, bio-conjugation and tissue engineering. In this paper, we report the development of a new class of water soluble multi-functional branched biodegradable polymer with high molecular weight and biocompatibility which demonstrates good correlation of in vivo biodegradation and in vitro hydrolysis. Main chain degradable hyperbranched polyglycerols (HPG) (20-100 kDa) were synthesized by the introduction of acid labile groups within the polymer structure by an anionic ring opening copolymerization of glycidol with ketal-containing epoxide monomers with different ketal structures. The water soluble biodegradable HPGs with randomly distributed ketal groups (RBHPGs) showed controlled degradation profiles in vitro depending on the pH of solution, temperature and the structure of incorporated ketal groups, and resulted in non-toxic degradation products. NMR studies demonstrated the branched nature of RBHPGs which is correlating with their smaller hydrodynamic radii. The RBHPGs and their degradation products exhibited excellent blood compatibility and tissue compatibility based on various analyses methods, independent of their molecular weight and ketal group structure. When administered intravenously in mice, tritium labeled RBHPG of molecular weight 100 kDa with dimethyl ketal group showed a circulation half life of 2.7 ± 0.3 h, correlating well with the in vitro polymer degradation half life (4.3 h) and changes in the molecular weight profile during the degradation (as measured by gel permeation chromatography) in buffer conditions at 37 °C. The RBHPG degraded into low molecular weight fragments that were cleared from circulation rapidly. The biodistribution and excretion studies demonstrated that RBHPG exhibited significantly lower tissue accumulation and enhanced urinary and fecal excretion when compared to non-degradable HPG of similar molecular weight. Excellent biocompatibility together with in vivo degradability and clearance of RBHPGs make them attractive for the development of multi-functional drug delivery systems.

Synthesis, characterization, and biocompatibility of biodegradable hyperbranched polyglycerols from acid-cleavable ketal group functionalized initiators.

Herein we report the synthesis of biodegradable hyperbranched polyglycerols (BHPGs) having acid-cleavable core structure by anionic ring-opening multibranching polymerization (ROMBP) of glycidol using initiators bearing dimethyl and cyclohexyl ketal groups. Five different multifunctional initiators carrying one to four ketal groups and two to four hydroxyl groups per molecule were synthesized. The hydroxyl carrying initiators containing one ketal group per molecule were synthesized from ethylene glycol. An alkyne-azide click reaction was used for synthesizing initiators containing multiple cyclohexyl ketal linkages and hydroxyl groups. The synthesized BHPGs exhibited monomodal molecular weight distributions and polydispersity in the range of 1.2 to 1.6, indicating the controlled nature of the polymerizations. The polymers were relatively stable at physiological pH but degraded at acidic pH values. The polymer degradation was dependent on the type of ketal structure present in the BHPG; polymers with cyclohexyl ketal groups degraded at much slower rates than those with dimethyl ketal groups at a given pH. Good control of polymer degradation was achieved under mild acidic conditions by changing the structure of ketal linkages. A precise control of the molecular weight of the degraded HPG was achieved by controlling the number of ketal groups within the core, as revealed from the gel permeation chromatography (GPC) analyses. The decrease in the polymer molecular weights upon degradation was correlated well with the number of ketal groups in their core structure. Our data support the suggestion that glycidol was polymerized uniformly from all hydroxyl groups of the initiators. BHPGs and their degradation products were highly biocompatible, as measured by blood coagulation, complement activation, platelet activation, and cell viability assays. The controlled degradation profiles of these polymers together with their excellent biocompatibility make them suitable for drug delivery and bioconjugation applications.

Carbohydrate Structure Dependent Hemocompatibility of Biomimetic Functional Polymer Brushes on Surfaces

Glycocalyx mimicking glycopolymer brushes presenting mannose, galactose and glucose residues in the pyranose form, similar to those present on cell surfaces, were synthesized on planar substrates (Si wafer, gold chip) and monodispersed polystyrene (PS) particles, and the interaction of blood to these surfaces were studied using various methods with the goal of producing a hemocompatible surface. Surface plasmon resonance (SPR) spectroscopy and gel analyses showed that the total protein adsorption from plasma was greatly reduced, as low as 24.3 ng/cm(2) from undiluted plasma on the glucose carrying brush. The protein adsorption decreased with increasing grafting density of the brushes. It was also found that the protein adsorption varied with the anticoagulant used for blood collection; much higher amount of protein was adsorbed from heparinzied plasma than citrated plasma. Proteomics protein identification analysis revealed that protein adsorption from plasma depended on the type of sugar residue present on the surface as well as the type of anticoagulant. All the three types of glycopolymer brushes showed similar level of platelet activation as that of buffer control irrespective of the nature of carbohydrate residue. However, the number of adhered platelet and their morphology depended on the type of carbohydrate residue present on the brush. On glucose brush, the extent of platelet adhesion and spreading was significantly lowered compared to other brushes. All the glycopolymer brushes were neutral to blood coagulation as indicated by thromboelastography analysis. The glucose brush gave a slightly longer initial coagulation time suggesting that this surface may be more biocompatible. Our data demonstrate that the structure of carbohydrate residue is an important factor in the design of synthetic blood contacting surface based on glycopolymer.

Inhibitory Effect of Hydrophilic Polymer Brushes on Surface-Induced Platelet Activation and Adhesion

Poly(N,N-dimethylacrylamide) (PDMA) brushes are successfully grown from unplasticized poly(vinyl chloride) (uPVC) by well-controlled surface-initiated atom transfer radical polymerization (SI-ATRP). Molecular weights of the grafted PDMA brushes vary from ≈ 35,000 to 2,170000 Da, while the graft density ranges from 0.08 to 1.13 chains · nm(-2). The polydispersity of the grafted PDMA brushes is controlled within 1.20 to 1.80. Platelet activation (expression of CD62) and adhesion studies reveal that the graft densities of the PDMA brushes play an important role in controlling interfacial properties. PDMA brushes with graft densities between 0.35 and 0.50 chains · nm(-2) induce a significantly reduced platelet activation compared to unmodified uPVC. Moreover, the surface adhesion of platelets on uPVC is significantly reduced by the densely grafted PDMA brushes. PDMA brushes that have high molecular weights lead to a relatively lower platelet activation compared to low-molecular-weight brushes. However, the graft density of the brush is more important than molecular weight in controlling platelet interactions with PVC. PDMA brushes do not produce any significant platelet consumption in platelet rich plasma. Up to a seven-fold decrease in the number of platelets adhered on high graft density brushes is observed compared to the bare PVC surface. Unlike the bare PVC, platelets do not form pseudopodes or change morphology on PDMA brush-coated surfaces.