P. Oommen

Microencapsulation of cells, including islets, within stable ultra-thin membranes of maleimide-conjugated PEG-lipid with multifunctional crosslinkers

The encapsulation of islets of Langerhans (islets) and insulin-secreting cells within a semi-permeable membrane has been suggested as a safe and simple technique for islet transplantation to attenuate early graft loss and avoid immunosuppressive therapy. The total volume of these implants tends, however, to increase upon encapsulation of the islets and cells within the polymer membrane, limiting transport between encapsulated cells and the surrounding tissue. Ultra-thin membranes could potentially overcome these diffusion limitations to provide for clinically applicable implants. Here we propose a method to encapsulate islets and cells within a stable ultra-thin polymer membrane using poly(ethylene glycol)-conjugated phospholipid bearing a maleimide group (Mal-PEG-lipids) and multiple interactive polymers (e.g., 4-arm PEG-Mal and 8-arm PEG-SH). When Mal-PEG-lipids were added to islet and cell suspensions, spontaneous incorporation into a cell surface occurred from the micelles at an equilibrium state. The addition of 4-arm PEG-Mal and 8-arm PEG-SH to the mixture induced a substantial increase in the membrane thickness because a number of Mal-PEG-lipid micelles were involved in the membrane formation at the micrometer level. No appreciable increase in islet volume was observed after microencapsulation by this method. Microencapsulation of islets with the polymer membranes, which showed semi-permeability, did not impair insulin release in response to glucose stimulation, even after 7 days. The polymer membrane structure surrounding the islets and cells was well maintained for at least 30 days. In addition, the membrane formed showed much lower thrombogenicity and inhibited complement activation upon exposure to human whole blood and serum.

Critical assessment of rhBMP-2 mediated bone induction: An in vitro and in vivo evaluation

Understanding the influence of formulation and storage conditions on rhBMP-2 bioactivity is extremely important for its clinical application. Reports in the literature show that different research groups employ different parameters such as formulation conditions, storage, doses for in vivo applications etc. that makes it difficult to correlate results from different experiments. We therefore decided to rationalize these anomalies by performing a basic study on such parameters using two commercially available BMPs. Our in vitro experiments suggest that BMPs from different sources have significant differences in their bioactivity. The clinically approved rhBMP-2 (InductOs®; BMP-P) showed superior stability, compared to rhBMP-2 from R&D Systems (BMP-R) at physiological pH (determined by ALP assay). This BMP-P also showed lower binding to polypropylene Eppendorf tube. The BMP-R almost lost its bioactivity within 30 min at physiological pH and also shows more adhesion to plastic surfaces. This aggregation behavior was unequivocally ascertained by performing light scattering studies of the two BMPs, which revealed linear aggregation with time for BMP-R unlike BMP-P. The in vitro results were also reflected in the in vivo experiments, in a rat ectopic model with injectable hyaluronic acid (HA) hydrogel as BMP carrier. After 7 weeks post-implantation we observed larger bone volume with oriented collagen in the BMP-P group but a smaller bone with disoriented collagen in the BMP-R case. Our results highlight the large difference in activity between seemingly identical substances and also the importance of proper handling of such sensitive proteins.

Tailored doxorubicin-hyaluronan conjugate as a potent anticancer glyco-drug: an alternative to prodrug approach.

Releasibility of doxorubicin from drug-conjugates is believed to be a prerequisite for its anti-cancer activity. Here, a new glyco-drug approach that circumvents the releasibility restriction is reported, opening a new possibility to design efficient, target specific drug delivery system. It is discovered that stable amide coupling of doxorubicin (DOX) tohyaluronan (HA) shows dose dependent cytotoxicity to CD44 positive human coloncancer cells (HCT116) as compared to human breast cancer cells(MCF-7) and mouse fibroblast cells (NIH-3T3), which express less CD44 receptor. This direct conjugation approach is an easy scalable strategy that could be adopted to design innocuous anti-tumor nanoparticle formulations.

Mild and Efficient Strategy for Site-Selective Aldehyde Modification of Glycosaminoglycans : Tailoring Hydrogels with Tunable Release of Growth Factor

Aldehydes have been used as an important bioorthogonal chemical reporter for conjugation of large polymers and bioactive substances. However, generating aldehyde functionality on carbohydrate-based biopolymers without changing its native chemical structure has always persisted as a challenging task. The common methods employed to achieve this require harsh reaction conditions, which often compromise the structural integrity and biological function of these sensitive molecules. Here we report a mild and simple method to graft aldehydes groups on glycosaminoglycans (GAGs) in a site-selective manner without compromising the structural integrity of the biopolymer. This regio-selective modification was achieved by conjugating the amino-glycerol moiety on the carboxylate residue of the polymer, which allowed selective cleavage of pendent diol groups without interfering with the C2-C3 diol groups of the native glucopyranose residue. Kinetic evaluation of this reaction demonstrated significant differences in second-order reaction rate for periodate oxidation (by four-orders of magnitude) between the two types of vicinal diols. We employed this chemistry to develop aldehyde modifications of sulfated and nonsulfated GAGs such as hyaluronic acid (HA), heparin (HP), and chondroitin sulfate (CS). We further utilized these aldehyde grafted GAGs to tailor extracellular matrix mimetic injectable hydrogels and evaluated its rheological properties. The composition of the hydrogels was also found to modulate release of therapeutic protein such as FGF-2, demonstrating controlled release (60%) for over 14 days. In short, our result clearly demonstrates a versatile strategy to graft aldehyde groups on sensitive biopolymers under mild conditions that could be applied for various bioconjugation and biomedical applications such as drug delivery and regenerative medicine.

Multifunctional Hyaluronic Acid and Chondroitin Sulfate Nanoparticles: Impact of Glycosaminoglycan Presentation on Receptor Mediated Cellular Uptake and Immune Activation.

Hyaluronic acid (HA) and chondroitin sulfate (CS) polymers are extensively used for various biomedical applications, such as for tissue engineering, drug delivery, and gene delivery. Although both these biopolymers are known to target cell surface CD44 receptors, their relative cellular targeting properties and immune activation potential have never been evaluated. In this article, we present the synthesis and characterization of novel self-assembled supramolecular HA and CS nanoparticles (NPs). These NPs were developed using fluorescein as a hydrophobic component that induced amphiphilicity in biopolymers and also efficiently stabilized anticancer drug doxorubicin (DOX) promoting a near zero-order drug release. The cellular uptake and cytotoxicity studies of these NPs in different human cancer lines, namely, human colorectal carcinoma cell line HCT116 and human breast cancer cell line MCF-7 demonstrated dose dependent cytotoxicity. Interestingly, both NPs showed CD44 dependent cellular uptake with the CS-DOX NP displaying higher dose-dependent cytotoxicity than the HA-DOX NP in different mammalian cells tested. Immunological evaluation of these nanocarriers in an ex vivo human whole blood model revealed that unlike unmodified polymers, the HA NP and CS NP surprisingly showed platelet aggregation and thrombin-antithrombin complex formation at high concentrations (0.8 mg/mL). We also observed a clear difference in early- and late-stage complement activation (C3a and sC5b-9) with CS and CS NP triggering significant complement activation at high concentrations (0.08-0.8 mg/mL), unlike HA and HA NP. These results offer new insight into designing glycosaminoglycan-based NPs and understanding their hematological responses and targeting ability.

Insights into the Mechanism and Catalysis of Oxime Coupling Chemistry at Physiological pH

The dynamic covalent-coupling reaction involving α-effect nucleophiles has revolutionized bioconjugation approaches, due to its ease and high efficiency. Key to its success is the discovery of aniline as a nucleophilic catalyst, which made this reaction feasible under physiological conditions. Aniline however, is not so effective for keto substrates. Here, we investigate the mechanism of aniline activation in the oxime reaction with aldehyde and keto substrates. We also present carboxylates as activating agents that can promote the oxime reaction with both aldehyde and keto substrates at physiological pH. This rate enhancement circumvents the influence of α-effect by forming H-bonds with the rate-limiting intermediate, which drives the reaction to completion. The combination of aniline and carboxylates had a synergistic effect, resulting in a ∼14-31-fold increase in reaction rate at pD 7.4 with keto substrates. The biocompatibility and efficiency of carboxylate as an activating agent is demonstrated by performing cell-surface oxime labeling at physiological pH using acetate, which showed promising results that were comparable with aniline.

Synthesis and anticancer properties of fucoidan-mimetic glycopolymer coated gold nanoparticles.

Gold nanoparticles coated with fucoidan-mimetic glycopolymers were synthesized that displayed good colloidal stability and promising anti-cancer properties. Fucoidan mimetic glycopolymers on their own were nontoxic, while glycopolymer coated gold nanoparticles displayed selective cytotoxicity to human colon cancer cell lines (HCT116) while it was non-toxic to mouse fibroblast cells (NIH3T3).

Synthesis and energetic properties of high-nitrogen substituted bishomocubanes

Synthesis, thermodynamic characterization, and energetic properties of three novel high-nitrogen bishomocubane-based compounds DADMBHC, DTetzBHC and DPTrizDMBHC are reported here. These compounds have higher heats of formation (HoFs) and higher energy densities as compared to traditional hydrocarbon fuels. Densities, gas phase HoF and their optimized molecular structure geometries were calculated with various levels of theory. In general, the calculated HoFs of these compounds turn out to be extremely high. Ballistic properties such as vacuum specific impulse and density vacuum specific impulse were calculated using the NASA Chemical Equilibrium and Applications utility. Propulsive properties were compared with liquid bipropellants (RP1) and solid propellants (AP) and explosive properties were compared with RDX. The density specific impulse demonstrated an improvement of 35 s for DADMBHC and DTetzBHC over standard liquid hydrocarbon HTPB, thus showing promise as possible monomers to replace HTPB as a fuel-binder. The density specific impulses of these compounds were also found to be significantly higher than that of RP1, e.g. that of DADMBHC was found to be higher by 84 s, making them potentially good candidates as propellants for use under volume-limited conditions. The detonation properties showed that these compounds have low potential as explosives. TGA, coupled with IR spectroscopy, revealed that DADMBHC and DPTrizDMBHC evaporate readily while DTetzBHC decomposes partially.

Injectable and thermoresponsive pericardial matrix derived conductive scaffold for cardiac tissue engineering

Scaffolds derived from decellularized cardiac tissue offer an enormous advantage for cardiac applications as they recapitulate biophysical and cardiac specific cues. However, poor electrical conductivity and mechanical properties severely compromise the therapeutic potential of these matrices. Dispersion of multiwall carbon nanotubes (MWCTs) in these scaffolds could improve their mechanical and electrical properties. However, the inherent hydrophobicity and poor dispersibility of these materials under aqueous conditions limit their outcome. We have developed a modified MWCNT functionalized with carbodihydrazide (CDH) residues that significantly improved their dispersibility and suppressed cytotoxicity in HL-1 cardiomyocytes. We found that the doping of CDH functionalized MWCNT (CDH-MWCNT) as low as 0.5 wt% to the pericardial matrix hydrogel (PMNT) induced the necessary electrical conductivity and significantly improved the mechanical properties of the hydrogel. Cardiomyocytes cultured on a PMNT scaffold triggered proliferation and significantly increased the expression of cardiac-specific gap junction protein, namely connexin 43. Such a phenomenon was not observed when cardiomyocytes were cultured on the pericardial matrix derived gels without MWCNT or on gelatin-fibronectin coated 2D cultures. The PMNT gels displayed excellent biophysical characteristics resulting in the clustering of cardiomyocytes with synchronous contraction, which is crucial for the successful integration to the host tissue.

Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH

We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration-dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most biofriendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.