Muhammad Imran ul-haq

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.

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.

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

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, Danio rerio 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.

Phase Behavior of Ketoprofen-Poly(lactic acid) Drug Particles Formed by Rapid Expansion of Supercritical Solutions

The present contribution investigates whether it is possible to form stable amorphous particles of ketoprofen-poly(lactic acid), naproxen-poly(lactic acid), and indomethacin-poly(lactic acid). Amorphization and micronization of these poorly water-soluble drugs offer a combined way to improve the solubility and enhance the dissolution rate. The particles were formed by pulsed rapid expansion of supercritical CO(2) solutions and characterized in the aerosol phase with rapid-scan infrared spectroscopy and after collection with scanning electron microscopy and X-ray diffraction. None of the three drug-poly(lactic acid) mixtures showed long-term stability on the order of weeks against the reversion from the amorphous to the crystalline state. Ketoprofen was the only drug that formed mixed amorphous particles with at least short-term stability. The long-term products turned out to be submicrometer- to micrometer-sized particles with a crystalline drug core and an amorphous poly(lactic acid) shell. Moreover, we found that the poly(lactic acid) coating stabilizes the particles against agglomeration.

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(1/2β)= 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.

Iron Binding and Iron Removal Efficiency of Desferrioxamine Based Polymeric Iron Chelators: Influence of Molecular Size and Chelator Density

Desferrioxamine (DFO) is a clinically approved, high affinity iron chelator used for the treatment of iron overload. Due to its short half-life and toxicity, DFO is administered for 8-12 h per day, 5-7 d per week. In this manuscript, the influence of molecular properties of hyperbranched polyglycerol (HPG)-DFO conjugates on their iron binding by isothermal titration calorimetry, iron removal efficiency from ferritin in presence and absence of a low molecular weight (MW) iron chelator, and protection against iron mediated oxidation of proteins is reported. The iron binding properties of HPG-DFO are slightly altered with size and DFO density of conjugates. The lower MW conjugate shows greater iron removal efficiency at room temperature, however, the efficacy of high MW conjugates increases at physiological temperature. The iron removal from ferritin by HPG-DFO conjugates increases significantly in presence of a low MW chelator, suggesting the potential of combination therapy. The molecular properties of the polymer scaffold also have influence on the prevention of iron mediated oxidation of proteins by the conjugates. The results therefore help to define the iron binding thermodynamics of HPG-DFO and their dependence on MW, and can be extended to improve the general understanding of polymeric chelator-iron interactions in situ.