Oncotically Driven Control over Glycocalyx Dimension for Cell Surface Engineering and Protein Binding in the Longitudinal Direction.

Here we present a simple technique for re-directing reactions on the cell surface to the outermost region of the glycocalyx. Macromolecular crowding with inert polymers was utilized to reversibly alter the accessibility of glycocalyx proteoglycans toward cell-surface reactive probes allowing for reactivity control in the longitudinal direction ('z'-direction) on the glycocalyx. Studies in HUVECs demonstrated an oncotically driven collapse of the glycocalyx brush structure in the presence of crowders as the mechanism responsible for re-directing reactivity. This phenomenon is consistent across a variety of macromolecular agents including polymers, protein markers and antibodies which all displayed enhanced binding to the outermost surface of multiple cell types. We then demonstrated the biological significance of the technique by increasing the camouflage of red blood cell surface antigens via a crowding-enhanced attachment of voluminous polymers to the exterior of the glycocalyx. The accessibility to Rhesus D (RhD) and CD47 proteins on the cell surface was significantly decreased in crowding-assisted polymer grafting in comparison to non-crowded conditions. This strategy is expected to generate new tools for controlled glycocalyx engineering, probing the glycocalyx structure and function, and improving the development of cell based therapies.

Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution.

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.

Intravenously injected human apolipoprotein A-I rapidly enters the central nervous system via the choroid plexus.

BACKGROUND: Brain lipoprotein metabolism is dependent on lipoprotein particles that resemble plasma high-density lipoproteins but that contain apolipoprotein (apo) E rather than apoA-I as their primary protein component. Astrocytes and microglia secrete apoE but not apoA-I; however, apoA-I is detectable in both cerebrospinal fluid and brain tissue lysates. The route by which plasma apoA-I enters the central nervous system is unknown. METHODS AND RESULTS: Steady-state levels of murine apoA-I in cerebrospinal fluid and interstitial fluid are 0.664 and 0.120 µg/mL, respectively, whereas brain tissue apoA-I is ≈10% to 15% of its levels in liver. Recombinant, fluorescently tagged human apoA-I injected intravenously into mice localizes to the choroid plexus within 30 minutes and accumulates in a saturable, dose-dependent manner in the brain. Recombinant, fluorescently tagged human apoA-I accumulates in the brain for 2 hours, after which it is eliminated with a half-life of 10.3 hours. In vitro, human apoA-I is specifically bound, internalized, and transported across confluent monolayers of primary human choroid plexus epithelial cells and brain microvascular endothelial cells. CONCLUSIONS: Following intravenous injection, recombinant human apoA-I rapidly localizes predominantly to the choroid plexus. Because apoA-I mRNA is undetectable in murine brain, our results suggest that plasma apoA-I, which is secreted from the liver and intestine, gains access to the central nervous system primarily by crossing the blood-cerebrospinal fluid barrier via specific cellular mediated transport, although transport across the blood-brain barrier may also contribute to a lesser extent.

Enhancement of biological reactions on cell surfaces via macromolecular crowding.

The reaction of macromolecules such as enzymes and antibodies with cell surfaces is often an inefficient process, requiring large amounts of expensive reagent. Here we report a general method based on macromolecular crowding with a range of neutral polymers to enhance such reactions, using red blood cells (RBCs) as a model system. Rates of conversion of type A and B red blood cells to universal O type by removal of antigenic carbohydrates with selective glycosidases are increased up to 400-fold in the presence of crowders. Similar enhancements are seen for antibody binding. We further explore the factors underlying these enhancements using confocal microscopy and fluorescent recovery after bleaching (FRAP) techniques with various fluorescent protein fusion partners. Increased cell-surface concentration due to volume exclusion, along with two-dimensionally confined diffusion of enzymes close to the cell surface, appear to be the major contributing factors.

The size-dependent efficacy and biocompatibility of hyperbranched polyglycerol in peritoneal dialysis.

Glucose is a common osmotic agent for peritoneal dialysis (PD), but has many adverse side effects for patients with end-stage renal disease. Recently, hyperbranched polyglycerol (HPG) has been tested as an alternative osmotic agent for PD. This study was designed to further examine the efficacy and biocompatibility of HPG over a range of different molecular weights. HPGs of varying molecular weights (0.5 kDa, 1 kDa, 3 kDa) were evaluated in a preclinical rodent model of PD. HPG PD solutions were standardized for osmolality and compared directly to conventional glucose-based Physioneal™ PD solution (PYS). The efficacy of HPG solutions was measured by their ultrafiltration (UF) capacity, solute removal, and free water transport; biocompatibility was determined in vivo by the histological analysis of the peritoneal membrane and the cell count of detached peritoneal mesothelial cells (PMCs) and neutrophils, and in vitro cytotoxicity to cultured human PMCs. All the different sized HPGs induced higher UF and sodium removal over a sustained period of time (up to 8 h) compared to PYS. Urea removal was significantly higher for 1-3 kDa than PYS, and was similar for 0.5 kDa. Our analyses indicated that the peritoneal membrane exhibited more tolerance to the HPG solutions compared to PYS, evidenced by less submesothelial injury and neutrophil infiltration in vivo, and less cell death in cultured human peritoneal mesothelial cells. Free water transport analysis of HPG indicated that these molecules function as colloids and induce osmosis mainly through capillary small pores. We attribute the differences in the biocompatibility and osmotic activity of different sized HPGs to the differences in the polymer bound water measured by differential scanning calorimetry. These preclinical data indicate that compared to PYS, low MW HPGs (0.5-3 kDa) produces superior fluid and waste removal with better biocompatibility profile, suggesting that they are promising osmotic agents for PD.

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.

Antigens protected functional red blood cells by the membrane grafting of compact hyperbranched polyglycerols.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen, glucose, and ions. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane and mask RBC surface antigens.

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.

Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.

Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic delivery applications. The intermediate sized polymers (PEG or HPG) which showed greater antigen camouflage at lower grafting concentrations have significant potential in transfusion as universal red blood donor cells.

In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells.

The in vivo circulation of hyperbranched polyglycerol (HPG) grafted red blood cells (RBCs) was investigated in mice. The number of HPG molecules grafted per RBC was measured using tritium labeled HPGs ((3)H-HPG) of different molecular weights; the values ranged from 1 × 10(5) to 2 × 10(6) molecules per RBC. HPG-grafted RBCs were characterized in vitro by measuring the electrophoretic mobility, complement mediated lysis, and osmotic fragility. Our results show that RBCs grafted with 1.5 × 10(5) HPG molecules per RBC having molecular weights 20 and 60 kDa have similar characteristics as that of control RBCs. The in vivo circulation of HPG-grafted RBCs was measured by a tail vain injection of (3)H-HPG60K-RBC in mice. The radioactivity of isolated RBCs, whole blood, plasma, different organs, urine and feces was evaluated at different time intervals. The portion of (3)H-HPG60K-RBC that survived the first day in mice (52%) remained in circulation for 50 days. Minimal accumulation radioactivity in organs other than liver and spleen was observed suggesting the normal clearance mechanism of modified RBCs. Animals gained normal weights and no abnormalities observed in necropsy analysis. The stability of the ester-amide linker between the RBC and HPG was evaluated by comparing the clearance rate of (3)H-HPG60K-RBC and PKH-26 lipid fluorescent membrane marker labeled HPG60K-RBCs. HPG modified RBCs combine the many advantages of a dendritic polymer and RBCs, and hold great promise in systemic drug delivery and other applications of functional RBC.

Osmotic release of bioactive VEGF from biodegradable elastomer monoliths is the same in vivo as in vitro.

The feasibility of generating an extended period of linear release of therapeutic proteins from photo-cross-linked, biodegradable elastomer monolithic devices in vitro has been previously demonstrated. The release is driven primarily by the osmotic pressure generated upon the dissolution of the encapsulated particles within the polymer. The osmotic pressure is provided by co-incorporation into the particle of trehalose as an osmotigen. Herein, we demonstrate that the release rate of a therapeutic protein, vascular endothelial growth factor (VEGF), by this osmotic pressure mechanism is the same in vivo as found in vitro. (125) I-VEGF was colyophilized with trehalose and serum albumin and distributed as particles throughout a photo-cross-linked elastomer composed of trimethylene carbonate, ε-caprolactone, and d,l-lactide. The release of VEGF from the device was monitored by measuring the decrease in radioactivity within the devices in vitro and within explanted devices that had been implanted subcutaneously in the dorsal area of Wistar rats. The released VEGF remained bioactive in vivo, inducing the formation of blood vessels that contained red blood cells. Furthermore, the released trehalose was well tolerated by the surrounding tissue.

VEGF-induced angiogenesis following localized delivery via injectable, low viscosity poly(trimethylene carbonate).

The purpose of this study was to examine the potential of low molecular weight poly(trimethylene carbonate) for localized vascular endothelial growth factor (VEGF) delivery. Poly(trimethylene carbonate) of various molecular weights was prepared by ring-opening polymerization initiated by 1-octanol. The resultant polymers were liquid at room temperature with low glass transition temperatures and viscosities at 37 degrees C that permitted their injection through an 18 (1/2) G 1.5'' needle. Particles consisting of VEGF co-lyophilized with trehalose were mixed into the polymers and the rate of release of VEGF was assessed in vitro. With a 1% particle loading, VEGF was released from the polymer at a rate of 20 ng/day over a period of 3 weeks. This release behavior was independent of the molecular weight of polymer used. Increasing the VEGF content in the lyophilized particles did not increase the VEGF release rate, an effect attributed to the solubility limit of VEGF in the solution formed upon dissolution of the particles. The VEGF released retained its bioactivity at greater than 95% of that of as-lyophilized VEGF, as assessed using a human aortic endothelial cell proliferation assay. This high bioactivity was supported by in vivo release experiments, wherein VEGF containing polymer implants induced the generation of significantly greater numbers of blood vessels towards the polymer implant than controls. The blood vessels did not remain stable and were reduced in number by three weeks, due to the unsustained and low concentration of VEGF released. This formulation approach, of using a low viscosity polymer delivery vehicle, is potentially useful for localized delivery of acid-sensitive proteins, such as VEGF.

Long term in vivo degradation and tissue response to photo-cross-linked elastomers prepared from star-shaped prepolymers of poly(epsilon-caprolactone-co-D,L-lactide).

Long term in vivo degradation, and tissue response to, cylindrical elastomers made of photo-cross-linked star-poly(epsilon-caprolactone-co-D,L-lactide) triacrylate were investigated through subcutaneous implantation in rats. The elastomers were prepared via UV initiated crosslinking of prepolymers of equimolar amounts of monomers; a high crosslink density elastomer (ELAST 1250) was prepared from a prepolymer of 1250 Da and a low crosslink density elastomer (ELAST 7800) was prepared from a prepolymer of 7800 Da. The elastomers were characterized using cross-polarization magic angle spinning solid state (13)C NMR and attenuated total reflectance fourier transform infrared spectroscopy. The progress of the in vivo degradation process was followed by employing SEM, uniaxial tensile, mass loss, water uptake, and sol content measurements. The rate of in vivo degradation was faster than the rate of in vitro degradation for both ELAST 1250 and ELAST 7800. Long term in vivo degradation studies indicated that both elastomers undergo bulk hydrolysis along with surface erosion occurring due to the physiological environment. In the case of low cross-link density elastomers, the onset of mass loss was accompanied with an increase in both water uptake and sol content, whereas, in the case of high crosslink density elastomers, only the water uptake increased. This degradation pattern was due to crazing of the high crosslink density elastomers. ELAST 7800 cylinders were totally degraded, and ELAST 1250 cylinders had lost 80% of their mass, within 30 weeks. Aminor host reaction with minimal vascularity and inflammation was invoked, with a milder tissue response observed with more highly crosslinked cylinders.

The role of oxidation and enzymatic hydrolysis on the in vivo degradation of trimethylene carbonate based photocrosslinkable elastomers.

The in vivo degradation of trimethylene carbonate (TMC) containing elastomers was investigated, and the mechanism of degradation explored through in vitro degradation under enzymatic and oxidative conditions. The elastomers were prepared via UV initiated crosslinking of prepolymers of TMC and equimolar amounts of TMC and epsilon-caprolactone (CL). The degradation process was followed by investigating the changes in the mechanical properties, mass loss, water uptake, sol content, differential scanning calorimetry, and surface chemistry through attenuated total reflectance infrared (ATR-FTIR) spectroscopy. During in vivo degradation, TMC and TMCCL elastomers exhibited surface erosion. The tissue response was of greater intensity in the case of the TMC elastomer. Both elastomers exhibited degradation in cholesterol esterase containing solutions in vitro, but no parallels were found between the rate of in vivo degradation and the rate of in vitro degradation. Only the TMCCL elastomer degraded in lipase. Degradation in a stable superoxide anion in vitro medium was consistent with the observed in vivo degradation results, indicating a dominant role of oxidation through the secretion of this reactive oxygen species by adherent phagocytic cells in the degradation of these elastomers.