<i>In Vivo</i> Biological Evaluation of High Molecular Weight Multifunctional Acid-Degradable Polymeric Drug Carriers with Structurally Different Ketals

Understanding the influence of degradable chemical moieties on <i>in vivo</i> degradation, tissue distribution, and excretion is critical for the design of novel biodegradable drug carriers. Polyketals have recently emerged as a promising therapeutic delivery platform due to their ability to degrade under mild acidic intracellular compartments and generation of nontoxic degradation products. However, the effect of chemical structure of the ketal groups on the <i>in vivo</i> degradation, biodistribution, and pharmacokinetics of water-soluble ketal-containing polymers has not been explored. In the present work, we synthesized high molecular weight, water-soluble biodegradable hyperbranched polyglycerols (BHPGs) through the incorporation of structurally different ketal groups into the main chain of highly biocompatible polyglycerols. BHPGs showed pH and ketal group structure dependent degradation in buffer solutions. When the polymers were intravenously administered in mice, a strong dependence of <i>in vivo</i> degradation, biodistribution, and clearance on the ketal group structure was observed. All the BHPGs demonstrated degradation and clearance <i>in vivo</i>, with minimal tissue accumulation. Interestingly, an unanticipated degradation behavior of BHPGs with structurally different ketal groups was observed <i>in vivo</i> in comparison to their degradation in buffer solutions. BHPGs with cyclohexyl ketal (CHK) and cyclopentyl ketal (CPK) groups degraded much faster and were cleared from circulation much rapidly, while BHPG with glycerol hydroxy butanone ketal (GHBK) group degraded at a much slower rate and exhibited similar plasma half-life as that of nondegradable HPG. BHPG-GHBK also showed significantly lower tissue accumulation than nondegradable HPG after 30 days of administration. The difference in <i>in vivo</i> degradation may be attributed to the difference in hydrophobic characteristics of different ketal containing polymers, which may change their interaction with proteins and cells <i>in vivo</i>. This is the first study that demonstrates the influence of chemical structure of ketal groups on <i>in vivo</i> degradation and circulation profile of polymers, and through proper surface modifications, these polymers would be useful as multifunctional drug carriers

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

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

Lectin Interactions on Surface-Grafted Glycostructures: Influence of the Spatial Distribution of Carbohydrates on the Binding Kinetics and Rupture Forces

We performed quantitative analysis of the binding kinetics and affinity of carbohydrate–lectin binding and correlated them directly with the molecular and structural features of ligands presented at the nanoscale within the glycocalyx mimicking layers on surfaces. The surface plasmon resonance analysis identified that the mode of binding changed from multivalent to monovalent, which resulted in a near 1000-fold change in the equilibrium association constant, by varying the spatial distribution of carbohydrate ligands within the surface-grafted polymer layer. We identified, for the first time, that the manner in which the ligands presented on the surface has great influence on the binding at the first stage of bivalent chelating, not on the binding at the second stage. The rupture forces measured by atomic force microscope force spectroscopy also indicated that the mode of binding between lectin and ligands changed from multiple to single with variation in the ligand presentation. The dependence of lectin binding on the glycopolymer composition and grafting density is directly correlated with the nanoscale presentation of ligands on a surface, which is a determining factor in controlling the clustering and statistical effects contributing to the enhanced bindin

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

Chain Length and Grafting Density Dependent Enhancement in the Hydrolysis of Ester-Linked Polymer Brushes

Poly­(<i><i>N</i>,<i>N</i></i>-dimethylacrylamide) (PDMA) brushes with different grafting density and chain length were grown from an ester group-containing initiator using surface-initiated polymerization. Hydrolysis of the PDMA chains from the surface was monitored by measuring thickness of the polymer layer by ellipsometry and extension length by atomic force microscopy. It was found that the initial rate of cleavage of one end-tethered PDMA chains was dependent on the grafting density and chain length; the hydrolysis rate was faster for high grafting density brushes and brushes with higher molecular weights. Additionally, the rate of cleavage of polymer chains during a given experiment changed by up to 1 order of magnitude as the reaction progressed, with a distinct transition to a lower rate as the grafting density decreased. Also, polymer chains undergo selective cleavage, with longer chains in a polydisperse brush being preferentially cleaved at one stage of the hydrolysis reaction. We suggest that the enhanced initial hydrolysis rates seen at high grafting densities and high chain lengths are due to mechanical activation of the ester bond connecting the polymer chains to the surface in association with high lateral pressure within the brush. These results have implications for the preparation of polymers brushes, their stability under harsh conditions, and the analysis of polymer brushes from partial hydrolysates

Therapeutic Cells via Functional Modification: Influence of Molecular Properties of Polymer Grafts on In Vivo Circulation, Clearance, Immunogenicity, and Antigen Protection

Modulation of cell surface properties via functional modification is of great interest in cell-based therapies, drug delivery, and in transfusion. We study the in vivo circulation, immuogenicity, and mechanism of clearance of hyperbranched polyglycerol (HPG)-modified red blood cells (RBCs) as a function of molecular properties of HPGs. The circulation half-life of modified cells can be modulated by controlling the polymer graft concentration on RBCs; low graft concentrations (0.25 and 0.5 mM) showed normal circulation as that of control RBCs. Molecular weight of HPG did not affect the circulation of modified RBCs. HPG grafting on RBCs reduced CD47 self-protein accessibility in a graft concentration-dependent fashion. HPG-grafted RBCs are not immunogenic, as is evident from their similar circulation profile upon repeated administration in mice and monitoring over 100 days. Histological examination of the spleen, liver, and kidneys of the mice injected with modified RBCs revealed distinct differences, such as elevated iron deposits and an increase in the number of CD45 expressing cells at high graft concentration of HPGs (1.5 mM); no changes were seen at low graft concentration. The absence of iron deposits in the white pulp region of the spleen and its presence in the red pulp region indicates that the clearance of functional RBCs occurs in the venous sinuses mechanical filtering system, similar to the clearance of unmodified senescent RBCs. HPG modification at grafting concentrations that yield long circulation in mice produced camouflage of a large number of minor blood group antigens on human RBCs, demonstrating its utility in chronic transfusion. The normal circulation, nonimmunogenic nature, and the potential to modulate the circulation time of modified cells without toxicity make this HPG-based cell surface modification approach attractive for drug delivery and other cell-based therapies

Hatching rate and morphological alterations of zebrafish embryos.

<p>DFO and L1-treated embryos hatched successfully, while ICL-670-treated embryos hatched less successfully than control due to high mortality (A). Bars missing at 1 mM are due to the death of embryos prior to hatching. <b>§</b> denoted the concentration of L1 that was excluded due to high DMSO content. There were no significant defects observed in DFO treated embryos. However, the percentage of embryos demonstrating behavioral and morphological defects increased in a time and concentration dependent manner in L1 and ICL-670-treated embryos (B). The time of onset of morphological and behavioral alterations is outlined in the figure as a separate panel. Swimming behavior was affected and bent bodies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109880#pone-0109880-g005" target="_blank"><b>Figure 5</b></a>) were observed at concentrations above 0.25 mM for ICL 670-treated embryos and at 0.5 mM for L1 treated embryos.</p

Clinically Approved Iron Chelators Influence Zebrafish Mortality, Hatching Morphology and Cardiac Function

<div><p>Iron chelation therapy using iron (III) specific chelators such as desferrioxamine (DFO, Desferal), deferasirox (Exjade or ICL-670), and deferiprone (Ferriprox or L1) are the current standard of care for the treatment of iron overload. Although each chelator is capable of promoting some degree of iron excretion, these chelators are also associated with a wide range of well documented toxicities. However, there is currently very limited data available on their effects in developing embryos. In this study, we took advantage of the rapid development and transparency of the zebrafish embryo, <i>Danio rerio</i> to assess and compare the toxicity of iron chelators. All three iron chelators described above were delivered to zebrafish embryos by direct soaking and their effects on mortality, hatching and developmental morphology were monitored for 96 hpf. To determine whether toxicity was specific to embryos, we examined the effects of chelator exposure via intra peritoneal injection on the cardiac function and gene expression in adult zebrafish. Chelators varied significantly in their effects on embryo mortality, hatching and morphology. While none of the embryos or adults exposed to DFO were negatively affected, ICL -treated embryos and adults differed significantly from controls, and L1 exerted toxic effects in embryos alone. ICL-670 significantly increased the mortality of embryos treated with doses of 0.25 mM or higher and also affected embryo morphology, causing curvature of larvae treated with concentrations above 0.5 mM. ICL-670 exposure (10 µL of 0.1 mM injection) also significantly increased the heart rate and cardiac output of adult zebrafish. While L1 exposure did not cause toxicity in adults, it did cause morphological defects in embryos at 0.5 mM. This study provides first evidence on iron chelator toxicity in early development and will help to guide our approach on better understanding the mechanism of iron chelator toxicity.</p></div

The chemical structures of clinically approved iron chelators.

<p>The chemical structures of clinically approved iron chelators.</p