Towards chamber specific heart-on-a-chip for drug testing applications.

Modeling of human organs has long been a task for scientists in order to lower the costs of therapeutic development and understand the pathological onset of human disease. For decades, despite marked differences in genetics and etiology, animal models remained the norm for drug discovery and disease modeling. Innovative biofabrication techniques have facilitated the development of organ-on-a-chip technology that has great potential to complement conventional animal models. However, human organ as a whole, more specifically the human heart, is difficult to regenerate in vitro, in terms of its chamber specific orientation and its electrical functional complexity. Recent progress with the development of induced pluripotent stem cell differentiation protocols, made recapitulating the complexity of the human heart possible through the generation of cells representative of atrial & ventricular tissue, the sinoatrial node, atrioventricular node and Purkinje fibers. Current heart-on-a-chip approaches incorporate biological, electrical, mechanical, and topographical cues to facilitate tissue maturation, therefore improving the predictive power for the chamber-specific therapeutic effects targeting adult human. In this review, we will give a summary of current advances in heart-on-a-chip technology and provide a comprehensive outlook on the challenges involved in the development of human physiologically relevant heart-on-a-chip.

Recapitulating pancreatic tumor microenvironment through synergistic use of patient organoids and organ-on-a-chip vasculature.

Tumor progression relies heavily on the interaction between the neoplastic epithelial cells and their surrounding stromal partners. This cell cross-talk affects stromal development, and ultimately the heterogeneity impacts drug efflux and efficacy. To mimic this evolving paradigm, we have micro-engineered a three-dimensional (3D) vascularized pancreatic adenocarcinoma tissue in a tri-culture system composed of patient derived pancreatic organoids, primary human fibroblasts and endothelial cells on a perfusable InVADE platform situated in a 96-well plate. Uniquely, through synergistic engineering we combined the benefits of cellular fidelity of patient tumor derived organoids with the addressability of a plastic organ-on-a-chip platform. Validation of this platform included demonstrating the growth of pancreatic tumor organoids by monitoring the change in metabolic activity of the tissue. Investigation of tumor microenvironmental behavior highlighted the role of fibroblasts in symbiosis with patient organoid cells, resulting in a six-fold increase of collagen deposition and a corresponding increase in tissue stiffness in comparison to fibroblast free controls. The value of a perfusable vascular network was evident in drug screening, as perfusion of gemcitabine into a stiffened matrix did not show the dose-dependent effects on tumor viability as those under static conditions. These findings demonstrate the importance of studying the dynamic synergistic relationship between patient cells with stromal fibroblasts, in a 3D perfused vascular network, to accurately understand and recapitulate the tumor microenvironment.

Engineering microenvironment for human cardiac tissue assembly in heart-on-a-chip platform.

Organ-on-a-chip systems have the potential to revolutionize drug screening and disease modeling through the use of human stem cell-derived cardiomyocytes. The predictive power of these tissue models critically depends on the functional assembly and maturation of human cells that are used as building blocks for organ-on-a-chip systems. To resemble a more adult-like phenotype on these heart-on-a-chip systems, the surrounding micro-environment of individual cardiomyocyte needs to be controlled. Herein, we investigated the impact of four microenvironmental cues: cell seeding density, types and percentages of non-myocyte populations, the types of hydrogels used for tissue inoculation and the electrical conditioning regimes on the structural and functional assembly of human pluripotent stem cell-derived cardiac tissues. Utilizing a novel, plastic and open-access heart-on-a-chip system that is capable of continuous non-invasive monitoring of tissue contractions, we were able to study how different micro-environmental cues affect the assembly of the cardiomyocytes into a functional cardiac tissue. We have defined conditions that resulted in tissues exhibiting hallmarks of the mature human myocardium, such as positive force-frequency relationship and post-rest potentiation.

Hemocompatibility of Degrading Polymeric Biomaterials: Degradable Polar Hydrophobic Ionic Polyurethane versus Poly(lactic-co-glycolic) Acid.

The use of degradable polymers in vascular tissue regeneration has sparked the need to characterize polymer biocompatibility during degradation. While tissue compatibility has been frequently addressed, studies on polymer hemocompatibility during degradation are limited. The current study evaluated the differences in hemocompatibility (platelet response, complement activation, and coagulation cascade initiation) between as-made and hydrolyzed poly(lactic-co-glycolic) acid (PLGA) and degradable polar hydrophobic ionic polyurethane (D-PHI). Platelet activation decreased (in whole blood) and platelet adhesion decreased (in blood without leukocytes) for degraded versus as-made PLGA. D-PHI showed minimal hemocompatibility changes over degradation. Leukocytes played a major role in mediating platelet activation for samples and controls, as well as influencing platelet-polymer adhesion on the degraded materials. This study demonstrates the importance of assessing the blood compatibility of biomaterials over the course of degradation since the associated changes in surface chemistry and physical state could significantly change biomaterial hemocompatibility.

Alteration of blood clotting and lung damage by protamine are avoided using the heparin and polyphosphate inhibitor UHRA.

Anticoagulant therapy-associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis.

In vivo efficacy, toxicity and biodistribution of ultra-long circulating desferrioxamine based polymeric iron chelator.

Desferrioxamine (DFO) is currently in clinical use to remove iron from transfusion-dependent patients with ß-thalassemia major, sickle-cell anemia and the myelodysplastic syndromes. However, its short half-life, burdensome, subcutaneous mode of administration and propensity to cause neurotoxicity at high doses greatly hinder its use. Thus, developing an optimized version of DFO with extended half-life, and reduced toxicity is a major goal. Using high molecular weight (MW), non-toxic, hyperbranched polyglycerol with high functionality, we demonstrate that the efficacy of DFO can be tuned with considerable reduction in toxicity. Using zebrafish embryos and mice, we tested toxicity, iron removal efficacy with low dosing and the biodistribution of ultra-long circulating DFO (ULC-DFO) conjugates. There was no significant difference in the mortality and development of zebrafish embryos upon exposure to ULC-DFO. Similarly, body weights and serum lactate dehydrogenase levels in mice treated with ULC-DFO remained within the normal range throughout the tolerance study. Moreover, ULC-DFO is significantly more effective than low MW DFO in promoting iron removal both from organs and via urine in iron overloaded mice despite using a moderate, once-weekly dosing schedule. This is probably due to the extended circulation half-life of ULC-DFO. The MW of ULC-DFO influences the accumulation and biodistribution, with highest MW (637 KDa) associated with up to 12% accumulation in the liver. In contrast, ULC-DFO with MWs of 75 KDa and lower were associated with relatively low organ accumulation, indicating that biodistribution of ULC-DFO can be tuned. Since ULC-DFO has improved iron removal properties, longer plasma retention time and possesses excellent biocompatibility, it represents a polymer conjugate with high clinical utility in comparison to DFO for the treatment of transfusion dependent iron overload. More importantly, ULC-DFO is anticipated to reduce the requirement for prolonged subcutaneous infusion of DFO.

Blood Components Interactions to Ionic and Nonionic Glyconanogels.

Nanogels are prominent examples of "smart" nanomaterials, which are designed to incorporate biologically relevant (macro)molecules for systemic delivery. Although these systems are carefully engineered, only a handful of studies discuss the blood compatibility of nanogels, and no systematic studies are available on how the presence of net or surface charges impacts the hemocompatibility of these nanomaterials. Therefore, in this study, temperature responsive, galactose based nanogels bearing net positive, negative, or neutral charges, either in the core or shell of nanogels, are prepared and are subsequently evaluated for their blood compatibility profiles. The nanogels containing neutral core and shell, cationic core with neutral shell, anionic core with neutral shell, neutral core with cationic shell, and neutral core with anionic shell are prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization approach. The evaluation of complement activation, blood clot formation, platelet activation, red blood cells aggregation, and hemolysis provides a detailed analysis of structure activity relationship of blood compatibility profile of these nanogels. The data reveal that the physical and biological (blood compatibility) properties can be carefully tuned by embedding the charges in the core of temperature-responsive nanomaterials, protected by neutral carbohydrate based shells.

Interaction of blood components with cathelicidins and their modified versions.

Cationic antimicrobial peptides (cAMPs) serve as effective components of the innate host defense against microbial infections. cAMPs often show broad-spectrum antimicrobial activity, but narrow-band activity is also observed. Despite their great potential, the polycationic nature of cAMPs could cause serious side effects once in the bloodstream which may limit their applications. However, there is very limited knowledge available on AMPs interaction with blood components in spite of the fact that the most likely route of administration to treat systemic microbial infections for these peptides is intravenous, where they immediately come in contact with all blood components. In order to evaluate the therapeutic potential of cAMPs as new alternative to antibiotics, we investigated the impact of cathelicidin related cAMPs on red blood cell lysis, aggregation, platelet activation, blood coagulation, and complement activation. The influence of cAMPs on blood depends on hydrophobicity and number of charges in the peptides. The hemolytic activity of cathelicidin (bactenecin) variants was much less than that of indolicidin due to their lower hydrophobicity. Except indolicidin, none of the peptides induce platelet activation. Some of bactenecin variants (R3, Sub3 and W3) with higher charge inhibited the blood coagulation. The cAMPs did not activate or inhibit complement at the concentrations studied, expect for the peptide (Sub3). Our data shows that it is important to investigate cAMP-based drug candidates regarding their interaction with blood components early on in the development process. We anticipate that this new knowledge on blood interaction of antimicrobial peptides will help to design peptides with a better therapeutic window and with less side effects.

Conjugation of aurein 2.2 to HPG yields an antimicrobial with better properties.

Aurein 2.2 is an antimicrobial peptide (AMP) whose mechanism of action is quite well-understood and that has good activity against Gram-positive bacteria. It is, however, highly cytotoxic. Poly(ethylene glycol) (PEG) conjugation (PEGylation) of protein and peptide drugs has been used for decades to improve their in vivo efficacy and blood circulation by enhancing the biocompatibility of the protein or peptide in question. However, the relatively large hydrodynamic size, high intrinsic viscosity, the limited number of functional groups available for conjugation, and immunogenicity of high molecular weight PEG limits its use in bioconjugation applications. Recently, hyperbranched polyglycerol (HPG) has been gaining attention as an alternative to PEG due to its excellent biocompatibility. Here, for the first time, we report the synthesis of HPG conjugates of antimicrobial peptides. Aurein 2.2 peptide was conjugated to high molecular weight HPG with a varying number of peptides per polymer, and the biocompatibility and antimicrobial activity of the conjugates were investigated. The antimicrobial activity of the peptide and its conjugates were determined by measuring the minimal inhibitory concentration (MIC) against Staphylococcus aureus and Staphylococcus epidermidis. The interaction of aurein 2.2 peptide and the conjugates with a model bacterial biomembrane was investigated using CD spectroscopy to understand the mode of action of the conjugates. The biocompatibility of the AMP-polymer conjugates was investigated by measuring red cell lysis, platelet activation and aggregation, complement activation, blood coagulation, and cell toxicity. Our results show that the size of the conjugates and the peptide density influence the biocompatibility of the antimicrobial conjugates. These results will help to further define the properties of HPG-AMP conjugates and set the stage for development of better therapeutic agents.

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

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.

Modulation of complement activation and amplification on nanoparticle surfaces by glycopolymer conformation and chemistry.

The complement system plays an integral part of a host's innate immunity, and its activation is highly dependent on the chemistry and structure of a "foreign" target surface. We determined that the conformational state of glycopolymer chains, defined by the grafting density (chains/nm(2)), on the nanoparticle (NP) surface acts as a "molecular switch" for complement activation and amplification, and the protein corona on the NP surface dictates this process. A grafting density threshold was determined, below which minimal complement activation was observed and above which substantial complement activation was detected. The glycopolymer-grafted NPs activated complement via the alternative pathway. The chemical structure of pendent sugar units on the grafted polymer was also an important determinant for complement activation. NPs grafted with glucose-containing polymer activated complement at a lower grafting density compared to NPs grafted with galactose-containing polymer. Analysis of complement activation products C3a and SC5b-9 followed a similar pattern. Complement activation on the NP surface was independent of particle size or concentration for a given conformational state of grafted polymer. To gain insight into a putative surface-dependent mechanism of complement activation, we determined the nature of adsorbed protein corona on various NPs through quantitative mass spectrometry. Elevated levels of two pro-complement proteins, factors B and C3, present on the NP surface grafted with glycopolymer chains at high grafting density compared to low grafting density surface, may be responsible for its complement activity. Galactose polymer modified NPs adsorbed more of the negative regulator of complement, factor H, than the glucose surface, providing an explanation for its lower level of complement activation.

Hybrid polyglycerols with long blood circulation: synthesis, biocompatibility, and biodistribution.

Multifunctional polymers with defined structure and biocompatibility are critical to the development of drug delivery systems and bioconjugates. In this article, the synthesis, in vitro blood compatibility, cell viability, in vivo circulation, biodistribution, and clearance of hybrid copolymers based on linear and branched polyglycerol are reported. Hybrid polyglycerols (M(n) ≈ 100 kDa) are synthesized with different compositions (15-80 mol% linear polyglycerol). Relatively small hydrodynamic size and radius of gyration of the hybrid polyglycerols suggest that they are highly compact functional nanostructures. The hybrid polyglycerols show excellent blood compatibility as determined by measuring their effects on blood coagulation, red blood cell aggregation, hemolysis, platelet, and complement activation. The cell viability in presence of hybrid polyglycerols is excellent up to 10 mg mL(-1) concentration and is similar to both dextran and polyvinyl alcohol. Furthermore, tritium labeled hybrid polyglycerol shows long blood circulation (t(1/2ß)= 34 h) with minimal organ accumulation in mice. Multifunctionality, compact nature, biocompatibility, and the long blood circulation make these polymers attractive for the development of bioconjugates and drug delivery systems.

Abnormal blood clot formation induced by temperature responsive polymers by altered fibrin polymerization and platelet binding.

Thermoresponsive polymers (TRPs) have been extensively investigated as smart devices, drug delivery systems and protein conjugates due to their unique phase transition properties. Here, we report the unusual influence of TRPs in blood clotting and the mechanism by which TRPs change the three dimensional organization of blood clot structure. Ten different TRPs with lower critical solution temperatures ranged from 26 to 80 °C are studied. TRPs altered the fibrin polymerization by increasing the rate of protofibril aggregation, decreased the fibrin fiber diameter and changed the platelet integration within the clot. The mechanical properties of the clot decreased considerably in presence of TRPs due to the poor platelet binding. The poor integration of platelets within the clot is not due to the inhibition of platelet activation by TRPs but may due to the unusual organization of fibrin structure. The plasma phase of the blood coagulation is not affected in presence of TRPs. We anticipate that our results will have significant implications on the use of TRPs in applications where blood contact is essential. These observations may also open up new avenues, for example, in the design of new generation antithrombotics.

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.

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.

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.

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.

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(1/2ß,) 31.8 ± 4 h) was demonstrated in mice; however, it was shorter compared to HPG-100 (t(1/2ß,) 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.

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.

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.