Diana Velluto

Device design and materials optimization of conformal coating for islets of Langerhans

Significance Cell encapsulation with biocompatible and permeable hydrogels may allow transplantation without immunosuppression. As an alternative to standard microencapsulation approaches that create single-sized capsules around cell clusters of different sizes, we have designed and optimized a novel approach for conformal coating of islets of Langerhans, resulting in thin, complete, and uniform coatings of similar thickness on differently sized islets. Coated islets exhibited no delay in glucose-stimulated insulin release or loss of function during culture, which is often observed with naked islets. The conformal coating reduces transplant volume relative to traditional encapsulation approaches. When transplanted in syngeneic diabetic mice, conformally coated islets restored and maintained euglycemia for more than 100 d with no foreign body reaction and normal revascularization. Encapsulation of islets of Langerhans may represent a way to transplant islets in the absence of immunosuppression. Traditional methods for encapsulation lead to diffusional limitations imposed by the size of the capsules (600–1,000 μm in diameter), which results in core hypoxia and delayed insulin secretion in response to glucose. Moreover, the large volume of encapsulated cells does not allow implantation in sites that might be more favorable to islet cell engraftment. To address these issues, we have developed an encapsulation method that allows conformal coating of islets through microfluidics and minimizes capsule size and graft volume. In this method, capsule thickness, rather than capsule diameter, is constant and tightly defined by the microdevice geometry and the rheological properties of the immiscible fluids used for encapsulation within the microfluidic system. We have optimized the method both computationally and experimentally, and found that conformal coating allows for complete encapsulation of islets with a thin (a few tens of micrometers) continuous layer of hydrogel. Both in vitro and in vivo in syngeneic murine models of islet transplantation, the function of conformally coated islets was not compromised by encapsulation and was comparable to that of unencapsulated islets. We have further demonstrated that the structural support conferred by the coating materials protected islets from the loss of function experienced by uncoated islets during ex vivo culture.

PEG-b-PPS Diblock Copolymer Aggregates for Hydrophobic Drug Solubilization and Release: Cyclosporin A as an Example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block.

Nano-sized drug-loaded micelles deliver payload to lymph node immune cells and prolong allograft survival

By delivering immunomodulatory drugs in vivo directly to lymph nodes draining an injection site, an opportunity exists to increase drug bioavailability to local immune cells. Importantly, particles smaller than 100 nm are efficiently transported through lymphatic vessels to draining lymph nodes. To investigate whether this approach could be used for local delivery of immunomodulatory drugs, amphiphilic poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) block copolymers forming 50 nm micelles were used to encapsulate hydrophobic drugs. Micelle drainage was determined using fluorescent micelles and showed effective targeting of multiple immune cell subsets in lymph nodes. For functional studies of our formulations, two approaches were considered. To evaluate the efficacy of anti-inflammatory drug delivery, dendritic cell activation was shown to be prevented when mice were pretreated with micelles loaded with the glucocorticoid mometasone and then challenged with the TLR9 ligand, CpG. To evaluate whether immunosuppressive drug-loaded micelles were effective in prolonging MHC-mismatched allograft survival, BALB/c mice were treated for 14 consecutive days with drug-loaded micelles following transplantation of allogenic C57BL/6 tail skin. Micelles loaded with a mixture of rapamycin and tacrolimus prolonged allograft survival by 2-fold. Our results indicate that the drug-loaded micelle approach effectively targets the draining lymph nodes and exhibits proper immune regulation.

Controlled release nanoparticle-embedded coatings reduce the tissue reaction to neuroprostheses

Controlled release coatings were developed for neuroprostheses with the aim of combating the tissue reaction following implantation in the brain. The coatings consist of poly(propylene sulfide) drug-eluting nanoparticles embedded in a poly(ethylene oxide) matrix. The nanoparticles are loaded with dexamethasone, an anti-inflammatory drug known to have an effect on the cells activated during the damage caused by implantation. The nanoparticles are not affected by the coating process and the drug remains bioactive after it is released. The coating was applied to microfabricated cortical neuroprostheses consisting of platinum and polyimide. Coated drug-eluting devices were implanted in the cortex of rats. After implantation the matrix dissolves, exposing the electrode surfaces, while the nanoparticles remain in the vicinity of the tissue-implant interface. Using electrical impedance spectroscopy and comparative histology, a long-term decrease in the tissue response in comparison to control devices was observed. These coatings can therefore be used to increase the reliability and long-term efficacy of neuroprostheses.

Extracellular matrix binding mixed micelles for drug delivery applications

We present the formation of collagen-binding mixed micelles and their potential suitability to deliver therapeutic drugs to the vessel wall. We modified poly(ethylene oxide)-bl-poly(propylene oxide)-bl-poly(ethylene oxide) (Pluronic F-127) to display sulfate groups on the terminus of the PEO block to act as a heparin mimics and bind to collagen in the extracellular matrix. This functionalized macroamphiphile was incorporated into a mixed micelle with poly(propylene sulfide)-bl-poly(ethylene oxide), a macroamphiphile that demonstrates improved micellar stability relative to Pluronic F-127 micelles. The mixed micelles were examined using analytical ultracentrifugation, dynamic light scattering, transmission electron microscopy, and measures of the critical micellar concentration using surface tensiometry. Encapsulation and in vitro release of Sirolimus, an immunosuppressant drug of interest in coronary artery treatment, was considered as an example. Mixed micelles with the sulfate functionality demonstrated enhanced binding to collagen I coated surfaces, suggestive of the potential for binding to the extracellular milieu.

Aggregation Behavior of Poly(ethylene glycol-bl-propylene sulfide) Di- and Triblock Copolymers in Aqueous Solution

Block copolymers of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-PPS) have recently emerged as a new macromolecular amphiphile capable of forming a wide range of morphologies when dispersed in water. To understand better the relationship between stability and morphology in terms of the relative and absolute block compositions, we have synthesized a collection of PEG-PPS block copolymers and quantified their critical aggregation concentration and observed their morphology using cryogenic transmission electron microscopy after thin film hydration with extrusion and after solvent dispersion from tetrahydrofuran, a solvent for both blocks. By understanding the relationship between aggregate character and block copolymer architecture, we have observed that whereas the relative block lengths control morphology, the stability of the aggregates upon dilution is determined by the absolute block length of the hydrophobic PPS block. We have compared results obtained with PEG-PPS to those obtained with poly(ethylene glycol)-bl-poly(propylene oxide)-bl-poly(ethylene glycol) block copolymers (Pluronics). The results reveal that the PEG-PPS aggregates are substantially more stable than Pluronic aggregates, by more than an order of magnitude. PEG-PPS can form a wide variety of stable or metastable morphologies in dilute solution within normal time and temperature ranges, whereas Pluronics can generally form only spherical micelles under the same conditions. On the basis of these results, block copolymers of PEG with poly(propylene sulfide) may present distinct advantages over those with poly(propylene glycol) for a number of applications.

Investigating the acoustic release of doxorubicin from targeted micelles

The main problem associated with the administration of anti-cancer medication is that the drug is delivered throughout the body causing undesirable side effects. Therefore, it is important to synthesize drug carriers capable of minimizing the adverse side effects of chemotherapy by preferentially targeting tumor cells both actively (e.g. a folate receptor) and using external stimulus (e.g. ultrasound). In this paper, we report the synthesis of Pluronic P105 micelles with a folate targeting moiety (with a yield of 48%) containing doxorubicin (Dox). We applied low frequency ultrasound as an external stimulus and measured the amount of release of Dox from these folated micelles. The results showed that the percent drug release increases as the power intensity of ultrasound increases. The maximum amount of release (14%) was measured at 5.4 W/cm(2). A power density threshold at approximately 0.55 W/cm(2) exists below which no statistically significant release was observed. This lower threshold suggests that cavitation plays an important role in triggering drug release from targeted micelles.

Effects of fullerene guests on the stability of 1-palmitoyl-2-oleoylphosphatidylcholine liposomes

Membrane stability of extruded large unilamellar vesicles () formed by 1-palmitoyl-2-oleoylphosphatidylcholine () containing fullerene C or an amphiphilic fullerene derivative, 2-[2-(2-fulleropyrrolidin-1-ylethoxy)-ethoxy]-ethanol (), has been investigated by spectrofluorimetrically monitoring the spontaneous release of entrapped carboxyfluorescein (). Under controlled conditions of temperature, osmolarity and pH, these guests increase the stability of the liposomal membrane as shown by the decrease in the rate of outflux of . The stability conferred to the liposomes by C and has been compared with that conferred by the well-known stabilizing guest 1,2-dipalmitoyl--glycero-3-phosphoethanolamine--[methoxy-(polyethylene glycol)-2000] ammonium salt (). The addition of amphiphilic molecules, such as non-ionic surfactants, which intercalate into the membrane bilayer, and of sucrose or NaCl, which induce a hyposmotic stress, has been extensively investigated in order to get information on how to modulate the release of entrapped . This information could hopefully be useful in the formulation of new drug delivery systems as well as for getting a deeper understanding of the mechanisms of the formation/enclosure of channels through the membrane. The viscosity and the micropolarity of the membrane have been measured fluorimetrically by using pyrene as the probe. An interesting increase of the gel-liquid crystal phase transition temperature has been observed for POPC liposomes hosting C.

Characterization of cationic liposomes. Influence of the bilayer composition on the kinetics of the liposome breakdown

The cationic large unilamellar mixed liposomes from 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and didodecyldimethylammonium bromide (DDAB) or dioctadecyldimethylammonium bromide (DODAB) were prepared. The influence of the addition of Triton X-100 (TX-100) or octaethylene glycol mono-n-dodecylether (C(12)E(8)) on the membrane integrity was investigated turbidimetrically. The stability of the liposomal systems was estimated by monitoring fluorimetrically at 25 °C the rate of spontaneous and surfactant-induced release of entrapped 5(6)-carboxyfluorescein (CF). In order to evaluate the interaction of the cationic DODAB guest with the host POPC membrane, the main phase transition temperatures (T(m)) were determined by electron paramagnetic resonance spectroscopy (EPR). All the results obtained show that the presence of DODAB and DDAB stabilizes the POPC liposomes. The extent of stabilization depends on the concentration and nature of the cationic guest.

Sorting live stem cells based on Sox2 mRNA expression.

While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB+SSEA1+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB− cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.