Cristina F. Stefanescu

Magnetically triggered nanocomposite membranes: a versatile platform for triggered drug release.

Drug delivery devices based on nanocomposite membranes containing thermoresponsive nanogels and superparamagnetic nanoparticles have been demonstrated to provide reversible, on-off drug release upon application (and removal) of an oscillating magnetic field. We show that the dose of drug delivered across the membrane can be tuned by engineering the phase transition temperature of the nanogel, the loading density of nanogels in the membrane, and the membrane thickness, allowing for on-state delivery of model drugs over at least 2 orders of magnitude (0.1-10 μg/h). The zero-order kinetics of drug release across the membranes permit drug doses from a specific device to be tuned according to the duration of the magnetic field. Drugs over a broad range of molecular weights (500-40000 Da) can be delivered by the same membrane device. Membrane-to-membrane and cycle-to-cycle reproducibility is demonstrated, suggesting the general utility of these membranes for drug delivery.

Near-infrared-actuated devices for remotely controlled drug delivery

Significance Devices that release a drug in response to a remote trigger would enable on-demand control of the timing and dose of drug released. They would allow the patient or physician to adjust therapy precisely to a target effect, thus improving treatment and reducing toxicity. We have developed implantable reservoirs that release a drug when irradiated with near-infrared laser light. The release rate was correlated to laser intensity, with negligible leakage between doses. Devices containing aspart, a fast-acting analog of insulin, were implanted in diabetic rats and were able to achieve glycemic control upon irradiation. Such devices can be loaded with a wide range of drugs to treat a variety of clinical indications. A reservoir that could be remotely triggered to release a drug would enable the patient or physician to achieve on-demand, reproducible, repeated, and tunable dosing. Such a device would allow precise adjustment of dosage to desired effect, with a consequent minimization of toxicity, and could obviate repeated drug administrations or device implantations, enhancing patient compliance. It should exhibit low off-state leakage to minimize basal effects, and tunable on-state release profiles that could be adjusted from pulsatile to sustained in real time. Despite the clear clinical need for a device that meets these criteria, none has been reported to date to our knowledge. To address this deficiency, we developed an implantable reservoir capped by a nanocomposite membrane whose permeability was modulated by irradiation with a near-infrared laser. Irradiated devices could exhibit sustained on-state drug release for at least 3 h, and could reproducibly deliver short pulses over at least 10 cycles, with an on/off ratio of 30. Devices containing aspart, a fast-acting insulin analog, could achieve glycemic control after s.c. implantation in diabetic rats, with reproducible dosing controlled by the intensity and timing of irradiation over a 2-wk period. These devices can be loaded with a wide range of drug types, and therefore represent a platform technology that might be used to address a wide variety of clinical indications.

In vivo performance of a drug-eluting contact lens to treat glaucoma for a month

For nearly half a century, contact lenses have been proposed as a means of ocular drug delivery, but achieving controlled drug release has been a significant challenge. We have developed a drug-eluting contact lens designed for prolonged delivery of latanoprost for the treatment of glaucoma, the leading cause of irreversible blindness worldwide. Latanoprost-eluting contact lenses were created by encapsulating latanoprost-poly(lactic-co-glycolic acid) films in methafilcon by ultraviolet light polymerization. In vitro and in vivo studies showed an early burst of drug release followed by sustained release for one month. Contact lenses containing thicker drug-polymer films demonstrated released a greater amount of drug after the initial burst. In vivo, single contact lenses were able to achieve, for at least one month, latanoprost concentrations in the aqueous humor that were comparable to those achieved with topical latanoprost solution, the current first-line treatment for glaucoma. The lenses appeared safe in cell culture and animal studies. This contact lens design can potentially be used as a treatment for glaucoma and as a platform for other ocular drug delivery applications.

Elasticity and safety of alkoxyethyl cyanoacrylate tissue adhesives

Cyanoacrylate glues are easily applied to wounds with good cosmetic results. However, they tend to be brittle and can induce local tissue toxicity. A series of cyanoacrylate monomers with a flexible ether linkage and varying side-chain lengths was synthesized and characterized for potential use as tissue adhesives. The effect of side-chain length on synthesis yield, physical and mechanical properties, formaldehyde generation, cytotoxicity in vitro and biocompatibility in vivo were examined. The incorporation of etheric oxygen allowed the production of flexible monomers with good adhesive strength. Monomers with longer side-chains were found to have less toxicity both in vitro and in vivo. Polymerized hexoxyethyl cyanoacrylate was more elastic than its commercially available and widely used alkyl analog 2-octyl cyanoacrylate, without compromising biocompatibility.

Local toxicity from local anesthetic polymeric microparticles.

BACKGROUND:Local tissue injury from sustained-release formulations for local anesthetics can be severe. There is considerable variability in reporting of that injury. We investigated the influence of the intrinsic myotoxicity of the encapsulated local anesthetic (lidocaine, low; bupivacaine, high) on tissue reaction in rats. METHODS:Cytotoxicity from a range of lidocaine and bupivacaine concentrations was measured in C2C12 myotubes over 6 days. Rats were given sciatic nerve blocks with 4 microparticulate formulations of lidocaine and bupivacaine: 10% (w/w) lidocaine poly(lactic-co-glycolic) acid (PLGA), 10% (w/w) bupivacaine PLGA, 50% (w/w) lidocaine PLGA, and 50% (w/w) bupivacaine PLGA. Effectiveness of nerve blockade was assessed by a modified hotplate test and weightbearing measurements. Myotoxicity was scored in histologic sections of injection sites. Bupivacaine and lidocaine release kinetics from the particles were measured. RESULTS:Median sensory blockade duration for 50% (w/w) lidocaine was 255 (90–540) minutes versus 840 (277–1215) minutes for 50% (w/w) bupivacaine (P = 0.056). All microparticulate formulations resulted in myotoxicity. The choice of local anesthetic did not influence the severity of myotoxicity. Median myotoxicity scores for 50% (w/w) lidocaine compared with 50% (w/w) bupivacaine at 4 days were 3.4 (2.1–4.2) vs 3.3 (2.9–3.5) (P = 0.44) and at 14 days 1.9 (1.8–2.4) vs 1.7 (1.3–1.9) (P = 0.23), respectively. CONCLUSIONS:Lidocaine and bupivacaine PLGA microspheres resulted in similar degrees of myotoxicity, irrespective of drug loading. Intrinsic myotoxicity did not predict tissue injury from sustained release of these anesthetics. Caution is warranted in the use of such devices near muscle and nerve.

An in situ cross-linking hybrid hydrogel for controlled release of proteins

There is a clear need for methods to provide a safe controlled release of therapeutic proteins, either to achieve and maintain high local protein concentrations, or for sustained systemic delivery. We have developed a protein delivery system that combines in situ cross-linkable polysaccharide hydrogels with gelatin. This formulation is injectable, easy to apply, and obviates the need for organic solvents or potentially toxic cross-linking agents in the formulation process. The cross-linked polysaccharides themselves (comprising hyaluronic acid, dextran and/or carboxymethylcellulose) provided prolonged release of fluorescently labeled albumin (FITC-albumin). The duration of release was markedly extended by the incorporation of gelatin into the formulation: FITC-albumin and interleukin-2 (IL-2) were released over the course of more than 3 weeks. The IL-2 maintained >70% activity throughout that time. Gelatin also accelerated the gelation time of the hydrogels, and reduced their swelling in phosphate-buffered saline. The composite hydrogel (dextran-carboxymethylcellulose-gelatin) showed minimal cytotoxicity in vitro, and benign tissue reaction after subcutaneous injection in rats.

Polydopamine coatings enhance biointegration of a model polymeric implant

The biointegration of implants affects their function, stability and safety. Although most research on this topic has focused on bone and other hard tissues, biointegration with soft tissues is important in numerous applications, such as in prosthetic corneas. Here, we have adapted polydopamine-based adhesive surface chemistry to enhance the biointegration with soft tissue of a model polymer—poly(methyl methacrylate) (PMMA), commonly used in prosthetic corneas. Polydopamine coating (PDA) and subsequent modification with the cell-adhesive peptide RGD (PDA-PEG-RGD) significantly enhanced cellular proliferation of corneal epithelial cells and keratocytes without causing excessive secretion of pro-inflammatory cytokines (e.g.IL-6) by either cell type. PDA adhered tightly to collagen gels, while PDA-PEG-RGD and uncoated PMMA did not. PDAs adhesion to collagen was greatly reduced by preincubation in serum. Tissue reaction to both polydopamine-coated surfaces was benign after 45 days of subcutaneous implantation. However, in contrast to the findings with collagen gels, PDA-PEG-RGD bound much more tightly to tissue than did PDA—although both bound better than unmodified PMMA. Polydopamine-based surface chemistries are potentially useful in enhancing tissue integration of implants with soft tissues.

Duration and local toxicity of sciatic nerve blockade with coinjected site 1 sodium-channel blockers and quaternary lidocaine derivatives.

Background and Objectives Quaternary lidocaine derivatives (QLDs) have recently received much attention because of their potential application in prolonged or sensory-selective local anesthesia. However, associated tissue toxicity is an impeding factor that makes QLDs unfavorable for clinical use. Based on the proposed intracellular site of action, we hypothesized that nerve blocks obtained from lower concentrations of QLDs would be enhanced by the coapplication of extracellularly acting site 1 sodium-channel blocker, resulting in prolonged block duration but with minimal tissue toxicity. Methods Quaternary lidocaine derivatives (QX-314 or QX-222), site 1 sodium-channel blockers (tetrodotoxin [30 &mgr;M] or saxitoxin [12.5 &mgr;M]), or both were injected in the vicinity of the sciatic nerve. Thermal nociceptive block was assessed using a modified hot plate test; motor block by a weight-bearing test. Tissue from the site of injection was harvested for histological assessment. Results Coapplication of 25 mM QX-314 or 100 mM QX-222 with site 1 sodium-channel blockers produced an 8- to 10- fold increase in the duration of nerve blocks (P < 0.05), compared with QLDs or site 1 blockers alone. Quaternary lidocaine derivatives elicited severe myotoxicity; this was not exacerbated by coinjection of the site 1 sodium-channel blockers. Conclusions Coadministration of site 1 sodium-channel blockers and QLDs greatly prolongs the duration of peripheral nerve block without enhancing local tissue injury, but minimal myotoxicity still persists. It is not clear that the risks of QLDs are outweighed by the benefits in providing prolonged nerve blockade.

Nanogel scavengers for drugs: Local anesthetic uptake by thermoresponsive nanogels

The use of functional nanogels based on poly(N-isopropylacrylamide) for effectively scavenging compounds (here, the model drug bupivacaine) is demonstrated using an in vitro cell-based assay. Nanogels containing higher loadings of acidic functional groups or more core-localized functional group distributions bound more bupivacaine, while nanogel size had no significant effect on drug binding. Increasing the dose of nanogel applied also facilitated more bupivacaine binding for all nanogel compositions tested. Binding was driven predominantly by acid-base interactions between the nanogels (anionic) and bupivacaine (cationic) at physiological pH, although both non-specific absorption and hydrophobic partitioning also contributed to drug scavenging. Nanogels exhibited minimal cytotoxicity to multiple cell types and were well tolerated in vivo via peritoneal injections, although larger nanogels caused limited splenic toxicity at higher concentrations. The cell-based assay described herein is found to facilitate more robust drug uptake measurements for nanogels than conventional centrifugation-based assays, in which nanogels can be compressed (and thus drug released) during the measurement.

Microgels for Efficient Protein Purification

Immobilized metal affinity chromatography (IMAC) is the most frequently used method for the separation and purification of histidine-tagged (His-tagged) proteins.[1] In this technique, the high affinity of metal ions, such as nickel or copper, to a tag sequence of the protein of interest creates strong, yet reversible binding.[2] A major limitation of current systems is their inefficiency in purifying many recombinant proteins, particularly when present in their native state or in low concentrations in the cell lysate.[3,4] Performance deficiencies are caused in part by the clogging or destruction of matrix micropores by undissolved salts and other compounds during particle synthesis,[5] limiting the surface area accessible for binding[6,7] (Figure S1 in the Supporting Information). Low surface metal density can further impair efficiency.[8,9] To overcome these limitations, some alternative chelator complexes have been suggested, including magnetic anoparticles,[10] cobalt-based affinity resins (Talon),[11] and gels.[12] However, the efficiency of these systems have not been clearly proven to be superior to others, they are relatively expensive, and they may require longer operation times.[13]