Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

Harvard Medical School

A variety of important biological events are mediated by the multivalent interaction between relevant oligosaccharides and multiple saccharide receptors on lectins, toxins, and cell surfaces; a variety of glycopolymeric materials have therefore been investigated in studies aimed at manipulating these events. The synthesis of protein- and polypeptide-based glycopolymers via protein engineering and other methods offers opportunities to control both the number and the spacing of saccharides on a scaffold, as well as the conformation of the polymer backbone, and will therefore facilitate the structure-based design of polymers for inhibition of multivalent binding events. In initial studies, we have synthesized a family of galactose-functionalized glycopolymers with a poly(L-glutamic acid) backbone, in which the density and linker length of the pendant carbohydrate moiety were varied. The composition of the glycopolymers was determined via (1)H NMR spectroscopy, and the impact of saccharide density and linker length, as well as the potential for these polypeptide-based glycopolymers to act as high-affinity inhibitors of the cholera toxin, has been indicated via competitive enzyme-linked immunosorbent assay and fluorescence titration experiments. The results of these studies suggest strategies for optimizing the binding of linear glycopolymers to bacterial toxins and will aid in the design of additional protein-based materials for studying the impact of multivalency, spacing, and backbone rigidity in a variety of biologically relevant binding events.

Effects of polymer structure on the inhibition of cholera toxin by linear polypeptide-based glycopolymers.

Hydrogels formed from (meth)acrylated poly(ethylene glycol) precursors are commonly used in a variety of biomedical applications ranging from tissue engineering to biosensors. While this approach has proven quite diverse, a major limitation to this approach is the heterogeneities and nonidealities that arise in the gels from the chain polymerization process, which increases the difficulty in relating the network structure to the final physical properties of the gel. Here we have exploited the specificity and fidelity of the [3+2] cycloaddition reaction to synthesize hydrogels with controlled architectures and improved mechanical properties. Moreover, we demonstrate a general approach toward the integration of multifunctional photoreactive polypeptide sequences into the network structure that provides a facile way to independently tune the 3D chemical and physical properties of the gel. Standard photolithographic techniques were used to generate a variety of two- and three-dimensional patterns as well as controlled biochemical gradients within existing preformed hydrogels.

Three-dimensional biochemical patterning of click-based composite hydrogels via thiolene photopolymerization.

Many recognition events important in biology are mediated via multivalent interactions between relevant oligosaccharides and multiple saccharide receptors present on lectins, viruses, toxins, and cell surfaces. Because of the important role played by protein-carbohydrate interactions in these pathogenic recognition events and in other human diseases, considerable effort has been devoted toward the development of multivalent polymeric ligands for carbohydrate-binding proteins. In this work, we report the synthesis of new polypeptide-based glycopolymers produced via a combination of protein engineering and chemical methods. These methodologies permit control over the number and the spacing of saccharides on the scaffold, as well as the conformation of the polymer backbone, and allow a more purposeful design of polymers for manipulation of multivalent binding events. Two families of galactose-bearing glycopolypeptides with random coil conformations, [(AG)(3)PEG](y) (y = 10 and 16) and {[(AG)(2)PSG](2)[(AG)(2)PEG][(AG)(2)PSG](2)}(y) (y = 6), have been synthesized. The carboxylic acid functionality of the glutamic acid residues allowed subsequent modification with amino-saccharides to yield the desired glycopolypeptides; selective placement of the glutamic acid group permitted investigation of the effects of multivalency and saccharide spacing on toxin inhibition. In addition, a family of galactose-functionalized PGA-based glycopolymers of varying molecular weights was also synthesized to compare the effects of backbone flexibility and hydrodynamic volume, relative to the recombinant glycopolypeptides, on toxin inhibition. Glycopolypeptides were characterized via (1)H NMR, MALDI-TOF mass spectrometry, SDS-PAGE analysis, and spectrophotometric assays. They were tested as inhibitors of the binding of the cholera toxin B subunit via direct enzyme-linked assays. The data from these experiments confirm the relevance of appropriate saccharide spacing on controlling the binding event and also indicate the influence of chain extension in improving inhibition.

Effects of Saccharide Spacing and Chain Extension on Toxin Inhibition by Glycopolypeptides of Well-Defined Architecture.

Click chemistry provides extremely selective and orthogonal reactions that proceed with high efficiency and under a variety of mild conditions, the most common example being the copper(I)-catalysed reaction of azides with alkynes. While the versatility of click reactions has been broadly exploited, a major limitation is the intrinsic toxicity of the synthetic schemes and the inability to translate these approaches into biological applications. This manuscript introduces a robust synthetic strategy where macromolecular precursors react through a copper-free click chemistry, allowing for the direct encapsulation of cells within click hydrogels for the first time. Subsequently, an orthogonal thiol-ene photocoupling chemistry is introduced that enables patterning of biological functionalities within the gel in real time and with micrometre-scale resolution. This material system enables us to tailor independently the biophysical and biochemical properties of the cell culture microenvironments in situ. This synthetic approach uniquely allows for the direct fabrication of biologically functionalized gels with ideal structures that can be photopatterned, and all in the presence of cells.

Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments.

Administration of a recombinant peptide of Periostin (rPN) has recently been shown to stimulate cardiomyocyte proliferation and angiogenesis after myocardial infarction (MI) [1]. However, strategies for targeting the delivery of rPN to the heart are lacking. Intrapericardial administration of drug-eluting hydrogels may provide a clinically viable strategy for increasing myocardial retention, therapeutic efficacy, and bioactivity of rPN and to decrease systemic re-circulation.

Intrapericardial delivery of gelfoam enables the targeted delivery of Periostin peptide after myocardial infarction by inducing fibrin clot formation.

Self-assembling, concentrated, lipid-based oxygen microparticles (LOMs) have been developed to administer oxygen gas when injected intravenously, preventing organ injury and death from systemic hypoxemia in animal models. Distinct from blood substitutes, LOMs are a one-way oxygen carrier designed to rescue patients who experience life-threatening hypoxemia, as caused by airway obstruction or severe lung injury. Here, we describe methods to manufacture large quantities of LOMs using an in-line, recycling, high-shear homogenizer, which can create up to 4 liters of microparticle emulsion in 10 minutes, with particles containing a median diameter of 0.93 microns and 60 volume% of gas phase. Using this process, we screen 30 combinations of commonly used excipients for their ability to form stable LOMs. LOMs composed of DSPC and cholesterol in a 1:1 molar ratio are stable for a 100 day observation period, and the number of particles exceeding 10 microns in diameter does not increase over time. When mixed with blood in vitro, LOMs fully oxygenate blood within 3.95 seconds of contact, and do not cause hemolysis or complement activation. LOMs can be manufactured in bulk by high shear homogenization, and appear to have a stability and size profile which merit further testing.

Bulk Manufacture of Concentrated Oxygen Gas-Filled Microparticles for Intravenous Oxygen Delivery.

Auto-assemblage, concentr&eacute;e, lipides &agrave; base d&#39;oxyg&egrave;ne microparticules (LOM) ont &eacute;t&eacute; d&eacute;velopp&eacute;s pour administrer l&#39;oxyg&egrave;ne gazeux lorsqu&#39;elle est inject&eacute;e par voie intraveineuse, emp&ecirc;chant d&#39;orgue blessures et de d&eacute;c&egrave;s d&#39;hypox&eacute;mie syst&eacute;mique chez les mod&egrave;les animaux. Distincte de substituts sanguins, LOM est un vecteur d&#39;oxyg&egrave;ne sens unique con&ccedil;u pour sauver les patients qui &eacute;prouvent une hypox&eacute;mie mortelles, comme caus&eacute;e par l&#39;obstruction des voies respiratoires ou de l&eacute;sions pulmonaires graves. Nous d&eacute;crivons ici les m&eacute;thodes pour fabriquer de grandes quantit&eacute;s de LOM en utilisant un en ligne, le recyclage, l&#39;homog&eacute;n&eacute;isateur de cisaillement &eacute;lev&eacute;, ce qui peut cr&eacute;er jusqu&#39;&agrave; 4 litres d&#39;&eacute;mulsion de microparticules en 10 minutes, avec des particules contenant un diam&egrave;tre moyen de 0,93 microns et 60 % de volume de la phase gazeuse. Gr&acirc;ce &agrave; ce processus, nous l&#39;&eacute;cran 30 combinaisons des excipients couramment utilis&eacute;s pour leur capacit&eacute; &agrave; former des LOM stable. LOM compos&eacute; du CSCB et cholest&eacute;rol dans un rapport molaire de 1:1 sont stables pendant une p&eacute;riode de 100 jours d&#39;observation, et le nombre de particules de plus de 10 microns de diam&egrave;tre n&#39;augmente pas avec le temps. Lorsqu&#39;il est m&eacute;lang&eacute; avec du sang in vitro, LOM enti&egrave;rement oxyg&eacute;ner le sang dans les 3,95 secondes de contact et faire pas cause h&eacute;molyse ou compl&eacute;ment activation. LOM peut &ecirc;tre fabriqu&eacute;s en vrac par cisaillement &eacute;lev&eacute; homog&eacute;n&eacute;isation et semble avoir une stabilit&eacute; et une taille de profil qui m&eacute;ritent des essais suppl&eacute;mentaires.

Fabrication en vrac de microparticules de remplissage de gaz oxyg&egrave;ne concentr&eacute; pour l&#39;apport d&#39;oxyg&egrave;ne par voie intraveineuse.

Autoassemblanti, concentrato, ossigeno basati sui lipidi microparticelle (LOM) sono state sviluppate per amministrare gas ossigeno quando viene iniettato per via endovenosa, prevenire lesioni di organi e morte da ipossiemia sistemica in modelli animali. Lom distinti dai sostituti del sangue, sono un trasportatore di ossigeno unidirezionale progettato per salvare i pazienti che avvertono ipossiemia life-threatening, come causato da ostruzione delle vie respiratorie o polmonari gravi lesioni. Qui, descriviamo i metodi per produrre grandi quantit&agrave; di Lom utilizzando un in linea, riciclaggio, omogeneizzatore ad alta-taglio, che pu&ograve; creare fino a 4 litri di emulsione di microparticelle in 10 minuti, con particelle contenenti un diametro mediano di 0,93 micron e 60% di volume della fase gassosa. Utilizzando questo processo, abbiamo schermo 30 combinazioni di eccipienti comunemente utilizzati per la loro capacit&agrave; di formare Lom stabile. Lom &egrave; composto da Lotusflower e colesterolo in rapporto molare 1:1 sono stabili per un periodo di osservazione di 100 giorni, e il numero di particelle superiori a 10 micron di diametro non aumenta nel tempo. Quando sono mescolati con sangue in vitro, Lom completamente ossigenare il sangue in 3,95 secondi di contatto e fare non causa emolisi o complemento attivazione. Lom possono essere prodotte in massa da high shear omogeneizzazione e sembrano avere una stabilit&agrave; e dimensioni profilo che legittimano ulteriori test.

Fabbricazione di massa di microparticelle riempiti di Gas ossigeno concentrato per la fornitura di ossigeno per via endovenosa.

Numerous mouse models have utilized Cre-loxP technology to modify gene expression. Adverse effects of Cre recombinase activity have been reported, including in the heart. However, the mechanisms associated with cardiac Cre toxicity are largely unknown. Here, we show that expression of Cre in cardiomyocytes induces a DNA damage response, resulting in cardiomyocyte apoptosis, cardiac fibrosis and cardiac dysfunction. In an effort to increase the recombination efficiency of a widely used tamoxifen-sensitive Cre transgene under control of the α-myosin-heavy-chain promoter (αMHC-MerCreMer), we observed myocardial dysfunction and decreased survival, which were dependent on the dose of tamoxifen injected. After excluding a Cre-independent contribution by tamoxifen, we found that Cre induced myocardial fibrosis, activation of pro-fibrotic genes and cardiomyocyte apoptosis. Examination of the molecular mechanisms showed activation of DNA damage response signaling and p53 stabilization in the absence of loxP sites, suggesting that Cre induced illegitimate DNA breaks. Cardiomyocyte apoptosis was also induced by expressing Cre using adenoviral transduction, indicating that the effect was not dependent on genomic integration of the transgene. Cre-mediated homologous recombination at loxP sites was dose-dependent and had a ceiling effect at ∼80% of cardiomyocytes showing recombination. By titrating the amount of tamoxifen to maximize recombination while minimizing animal lethality, we determined that 30 μg tamoxifen/g body weight/day injected on three consecutive days is the optimal condition for the αMHC-MerCreMer system to induce recombination in the Rosa26-lacZ strain. Our results further highlight the importance of experimental design, including the use of appropriate genetic controls for Cre expression.

Moderate and high amounts of tamoxifen in αMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death.

Tissue hypoxia is a final common pathway that leads to cellular injury and death in a number of critical illnesses. Intravenous injections of self-assembling, lipid-based oxygen microbubbles (LOMs) can be used to deliver oxygen gas, preventing organ injury and death from systemic hypoxemia. However, current formulations exhibit high polydispersity indices (which may lead to microvascular obstruction) and poor shelf-lives, limiting the translational capacity of LOMs. In this study, we report our efforts to optimize LOM formulations using a mixture response surface methodology (mRSM). We study the effect of changing excipient proportions (the independent variables) on microbubble diameter and product loss (the dependent variables). By using mRSM analysis, the experimental data were fit using a reduced Scheffé linear mixture model. We demonstrate that formulations manufactured from 1,2-distearoyl-sn-glycero-3-phosphocholine, corn syrup, and water produce micron-sized microbubbles with low polydispersity indices, and decreased product loss (relative to previously described formulations) when stored at room temperature over a 30-day period. Optimized LOMs were subsequently tested for their oxygen-releasing ability and found to have similar release kinetics as prior formulations. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2014.

Brian D. Polizzotti

Department of Cardiology,
Boston Children's Hospital,
Department of Cardiology, Boston Children's Hospital

Harvard Medical School

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Brian D. Polizzotti

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cardiology,Boston Children's Hospital,Department of Cardiology, Boston Children's Hospital

Harvard Medical School

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Department of Cardiology,
Boston Children's Hospital,
Department of Cardiology, Boston Children's Hospital