The effect of glutathione as chain transfer agent in PNIPAAm-based thermo-responsive hydrogels for controlled release of proteins.

PURPOSE: To control degradation and protein release using thermo-responsive hydrogels for localized delivery of anti-angiogenic proteins. METHODS: Thermo-responsive hydrogels derived from N-isopropylacrylamide (NIPAAm) and crosslinked with poly(ethylene glycol)-co-(L-lactic acid) diacrylate (Acry-PLLA-b-PEG-b-PLLA-Acry) were synthesized via free radical polymerization in the presence of glutathione, a chain transfer agent (CTA) added to modulate their degradation and release properties. Immunoglobulin G (IgG) and the recombinant proteins Avastin® and Lucentis® were encapsulated in these hydrogels and their release was studied. RESULTS: The encapsulation efficiency of IgG was high (75-87%) and decreased with CTA concentration. The transition temperature of these hydrogels was below physiological temperature, which is important for minimally invasive therapies involving these materials. The toxicity from unreacted monomers and free radical initiators was eliminated with a minimum of three buffer extractions. Addition of CTA accelerated degradation and resulted in complete protein release. Glutathione caused the degradation products to become solubilized even at 37°C. Hydrogels prepared without glutathione did not disintegrate nor released protein completely after 3 weeks at 37°C. PEGylation of IgG postponed the burst release effect. Avastin® and Lucentis® released from degraded hydrogels retained their biological activity. CONCLUSIONS: These systems offer a promising platform for the localized delivery of proteins.

A study of the intrinsic autofluorescence of poly (ethylene glycol)-co-(L-lactic acid) diacrylate.

Poly (ethylene glycol)-co-(L-Lactic acid) diacrylate (PEG-PLLA-DA) copolymers have been extensively investigated for a number of applications in medicine. PEG-PLLA-DA is biodegradable and the human body can process its degradation products. In this study, we describe the autofluorescence of PEG-PLLA-DA copolymers and compared it to the fluorescence of poly(ethylene glycol) diacrylate (PEG-DA) and the precursor molecules used for their synthesis. In addition, we examined the influence of pH on the fluorescence spectra. We found that PEG-PLLA-DA exhibits higher fluorescence than PEG-DA and all reagents involved in the synthesis of PEG-PLLA-DA. The fluorescence of PEG-PLLA-DA was affected by pH with fluorescence decreasing at high pH values. At high pH, PEG-PLLA-DA could not polymerize into hydrogels and exhibited a dramatic decrease in autofluorescence, suggesting that hydrolysis of the ester bond affected its autofluorescence. At low pH, PEG-PLLA-DA exhibited higher fluorescence and it was able to form crosslinked hydrogels. The autofluorescence of PEG-PLLA-DA could be exploited to monitor polymer degradation and material structure without the need to introduce exogenous fluorescent probes. The origin of fluorescence is not clear at this point in time but it appears to result from a synergetic effect of both lactate units and diacrylate groups in the PEG-PLLA-DA backbone. The observed autofluorescence of PEG-PLLA-DA persists after reaction of the acrylate groups in the polymerization reaction. This autofluorescence is advantageous because it could assist in the study of polymers used for drug delivery and tissue engineering applications.

Generation of Photopolymerized Microparticles Based on PEGDA Using Microfluidic Devices. Part 1. Initial Gelation Time and Mechanical Properties of the Material.

Photopolymerized microparticles are made of biocompatible hydrogels like Polyethylene Glycol Diacrylate (PEGDA) by using microfluidic devices are a good option for encapsulation, transport and retention of biological or toxic agents. Due to the different applications of these microparticles, it is important to investigate the formulation and the mechanical properties of the material of which they are made of. Therefore, in the present study, mechanical tests were carried out to determine the swelling, drying, soluble fraction, compression, cross-linking density (Mc) and mesh size (ξ) properties of different hydrogel formulations. Tests provided sufficient data to select the best formulation for the future generation of microparticles using microfluidic devices. The initial gelation times of the hydrogels formulations were estimated for their use in the photopolymerization process inside a microfluidic device. Obtained results showed a close relationship between the amount of PEGDA used in the hydrogel and its mechanical properties as well as its initial gelation time. Consequently, it is of considerable importance to know the mechanical properties of the hydrogels made in this research for their proper manipulation and application. On the other hand, the initial gelation time is crucial in photopolymerizable hydrogels and their use in continuous systems such as microfluidic devices.

Encapsulation of Biological Agents in Hydrogels for Therapeutic Applications.

Hydrogels are materials specially suited for encapsulation of biological elements. Their large water content provides an environment compatible with most biological molecules. Their crosslinked nature also provides an ideal material for the protection of encapsulated biological elements against degradation and/or immune recognition. This makes them attractive not only for controlled drug delivery of proteins, but they can also be used to encapsulate cells that can have therapeutic applications. Thus, hydrogels can be used to create systems that will deliver required therapies in a controlled manner by either encapsulation of proteins or even cells that produce molecules that will be released from these systems. Here, an overview of hydrogel encapsulation strategies of biological elements ranging from molecules to cells is discussed, with special emphasis on therapeutic applications.

Detection of proteins and bacteria using an array of feedback capacitance sensors.

An integrated array of micron-dimension capacitors, originally developed for biometric applications (fingerprint identification), was engineered for detection of biological agents such as proteins and bacteria. This device consists of an array of 93,184 (256 x 364) individual capacitor-based sensing elements located underneath a thin (0.8 microm) layer of glass. This glass layer can be functionalized with organosilane-based monolayers to provide groups amenable for the immobilization of bioreceptors such as antibodies, enzymes, peptides, aptamers, and nucleotides. Upon functionalization with antibodies and in conjunction with signal amplification schemes that result in perturbation of the dielectric constant around the captured antigens, this system can be used as a detector of biological agents. Two signal amplification schemes were tested in this work: one consisted of 4 microm diameter latex immunobeads and a second one was based on colloidal gold catalyzed reduction of silver. These signal amplification approaches were demonstrated and show that this system is capable of specific detection of bacteria (Escherichia coli) and proteins (ovalbumin). The present work shows proof-of-principle demonstration that a simple fingerprint detector based on feedback capacitance measurements can be implemented as a biosensor. The approach presented could be easily expanded to simultaneously test for a large number of analytes and multiple samples given that this device has a large number of detectors. The device and required instrumentation is highly portable and does not require expensive and bulky instrumentation because it relies purely on electronic detection.

Sequential formation of covalently bonded hydrogel multilayers through surface initiated photopolymerization.

A novel method for the sequential formation of hydrogel multilayers is described. Formation of the first layer is based on surface initiated photopolymerization of hydrogel precursors on eosin derivatized surfaces. In order to attach subsequent layers it is necessary to be able to functionalize intermediate hydrogel layers with eosin. In the present work, this is accomplished by introducing poly(ethylene glycol) amino acrylate (NH2-PEG-Acr) along with other hydrogel precursors such as poly(ethylene glycol) diacrylate (PEG-DA) on the intermediate layers. The pendant amine groups allow functionalization of the intermediate layers with eosin for subsequent photopolymerization of new hydrogel layers. The process can be repeated sequentially to construct multilayered hydrogel membranes. The NH2-PEG-Acr monomer can be formed by coupling cysteamine to PEG-DA by a conjugate addition reaction. The approach to multilayer formation could allow the incorporation of specific functionalities or compositions within each hydrogel layer so that multifunctional membranes can be formed. It could also be implemented, through proper photopatterning procedures, for the formation of 3-D hydrogel structures. The mild photopolymerization conditions employed using visible (514 nm), rather than ultraviolet light would make this technique especially attractive for tissue engineering, drug delivery, biomaterials, biosensor development and other situations where the elements incorporated are sensitive to UV light exposure.

Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface.

A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained.

Investigation of lysine acrylate containing poly(N-isopropylacrylamide) hydrogels as wound dressings in normal and infected wounds.

The design of materials for cutaneous wound dressings has advanced from passive wound covers to bioactive materials that promote skin regeneration and prevent infection. Crosslinked poly(N-isopropylacrylamide) (PNIPAAm)-based hydrogels have been investigated for a number of biomedical applications. While these materials can be used for drug delivery, limited cell interactions restrict their biological activity. In this article, acryoyl-lysine (A-Lys) was incorporated into poly(ethylene glycol) crosslinked PNIPAAm to enhance biological activity. A-Lys could be incorporated into the hydrogels to improve cellular interaction in vitro, while maintaining swelling properties and thermoresponsive behavior. Polyhexamethylene biguanide, an antimicrobial agent, could be encapsulated and released from the hydrogels and resulted in decreased bacteria counts within 2 hours. Two in vivo animal wound models were used to evaluate the hydrogel wound dressing. First, application of the hydrogels to a rodent cutaneous wound healing model resulted in significant increase in healing rate when compared with controls. Moreover, the hydrogels were also able to decrease bacteria levels in an infected wound model. These results suggest that PNIPAAm hydrogels containing A-Lys are promising wound dressings due to their ability to promote healing and deliver active antimicrobial drugs to inhibit infection.

Role of Thermo-responsiveness and Poly(ethylene glycol) Diacrylate Cross-link Density on Protein Release from Poly(N-isopropylacrylamide) Hydrogels.

Thermo-responsive hydrogels have shown promise as injectable materials for local drug delivery. However, the phase-induced changes in polymer properties of N-isopropylacrylamide (NIPAAm) can pose additional challenges for achieving controlled protein release. In this work, thermo-responsive hydrogels derived from NIPAAm and cross-linked with poly(ethylene glycol) diacrylate (PEG-DA) were synthesized via free radical polymerization. The volume phase transition temperature (VPTT) of the hydrogels ranged from 32.9°C to 35.9°C. Below the VPTT, swelling ratios of the hydrogels decreased with cross-linker concentration, and showed a sharp drop (at least 4-fold) upon phase change. Protein encapsulation efficiency was high (84-90%) and decreased with cross-linker concentration. Release of bovine serum albumin, a model protein, at body temperature was significantly higher than at room temperature (67% at 37°C compared to 44% at 23°C after 48 h). The release kinetics of proteins from the hydrogels were initially expected to be a function of cross-link density. However, at the hydrogel compositions explored in this work, protein release did not change significantly with cross-linker mol fraction. The thermo-responsive hydrogels offer a promising platform for the localized delivery of proteins.

The effects of cross-linked thermo-responsive PNIPAAm-based hydrogel injection on retinal function.

There is significant interest in biomaterials that provide sustained release of therapeutic molecules to the retina. Poly(N-isopropylacrylamide) (PNIPAAm)-based materials have received significant attention as injectable drug delivery platforms due to PNIPAAm's thermo-responsive properties at approximately 32 °C. While the drug delivery properties of PNIPAAm materials have been studied extensively, there is a need to evaluate the safety effects of hydrogel injection on retinal function. The purpose of this study was to examine the effect of poly(ethylene glycol) diacrylate (PEG-DA) crosslinked PNIPAAm hydrogel injection on retinal function. Utilizing scanning laser ophthalmoscopy (SLO), optical coherent tomography (OCT), and electroretinography (ERG), retinal function was assessed following hydrogel injection. In region near the hydrogel, there was a significant decrease in arterial and venous diameters (∼4%) and an increase in venous blood velocity (∼8%) 1 week post-injection. Retinal thickness decreased (∼6%) at 1 week and the maximum a- and b-wave amplitudes of ERG decreased (∼15%). All data returned to baseline values after week 1. These data suggest that the injection of PEG-DA crosslinked PNIPAAm hydrogel results in a small transient effect on retinal function without any long-term effects. These results further support the potential of PNIPAAm-based materials as an ocular drug delivery platform.

Surface-grafted hybrid material consisting of gold nanoparticles and dextran exhibits mobility and reversible aggregation on a surface.

Gold nanoparticles linked to linear carboxylated dextran chains were attached to 3-aminopropyltriethoxysilane-functionalized glass surfaces. This method provides novel hybrid nanostructures on a surface with the unique optical properties of gold nanoparticles. The particles attached to the surface retain the capability to aggregate and disaggregate in response to their environment. This procedure presents an alternative method to the immobilization of gold nanoparticles onto planar substrates. Compared to gold nanoparticle monolayers, larger particle surface densities were obtained. Exposure to hydrophobic environments changes the conformation of the hydrophilic dextran chains, causing the gold nanoparticles to aggregate and inducing changes in the absorption spectrum such as red-shifting and broadening of the plasmon absorption peaks. These changes, characteristic of particle aggregation, are reversible. When the substrates are dried and then immersed in an aqueous environment, these changes can be visually observed in a reversible fashion and the sample changes color from the red color of colloidal gold to a bluish-purple color of aggregated nanoparticles. Surface-bound nanoparticles that retain their mobility when attached to a surface by means of a flexible polymer chain could expand the use of aggregation-based assays to solid substrates.

Dextran-gold nanoparticle hybrid material for biomolecule immobilization and detection.

The formation of a hybrid metal-biopolymer material is described. The synthesis of this material consists of functionalizing the surface of gold nanoparticles through a series of steps that lead to epoxy-functionalized nanoparticles. These are subsequently reacted with hydroxyl moieties of the alpha-D-glucopyranosyl groups of dextran. Subsequently, the dextran chains are carboxylated through treatment with bromoacetic acid. The resultant material combines the unique optical properties of gold nanoparticles with the versatility that carboxylated dextran offers for further functionalization with biomolecules. The interaction of this material with three proteins was then investigated through changes in the plasmon resonance properties of the gold nanoparticles. Concanavalin A, a lectin that binds glucose and mannose by means of specific molecular recognition, interacts readily with this material and such interaction is easily detected using optical absorption spectroscopy. Through reaction of the carboxyl groups with (+)-biotinyl-3,6,9,-trioxaundecanediamine, a material bearing biotin groups was obtained. This could interact with streptavidin or antibiotin by means of specific molecular recognition. Further confirmation of biospecific interactions was obtained with control experiments in which the binding sites were blocked through preincubation of the proteins with the corresponding ligand in solution. Binding of these proteins was concentration-dependent over a wide concentration range. This material provides a simple and convenient colorimetric method for biospecific interaction analysis.

Quenched emission of fluorescence by ligand functionalized gold nanoparticles.

A fluorescence-based detection scheme that uses ligand functionalized gold nanoparticles is proposed. The transduction scheme is based on the strong quenching of the fluorescence emission exerted by metallic surfaces on fluorophores positioned in their immediate vicinity (< 5 nm). Binding of fluorophore-labeled anti-biotin to biotinylated gold nanoparticles resulted in decreased fluorescence emission intensity. Subsequent competitive dissociation of labeled anti-biotin with D-biotin resulted in increased fluorescence emission intensity. These interactions occurred by means of specific molecular recognition because when the binding sites of anti-biotin were saturated with D-biotin prior to interaction with the gold nanoparticles; changes in the fluorescence emission intensity were not observed.

Photopolymerization of poly(ethylene glycol) diacrylate on eosin-functionalized surfaces.

We describe a new method that allows photopolymerization of hydrogels to occur on surfaces functionalized with eosin. In this work, glass and silicon surfaces were derivatized with eosin and photopolymerization was carried out using visible light (514 nm). This mild condition may have advantages over methods that use ultraviolet (UV) light (e.g., for encapsulation of cells and proteins, in drug screening, or in biosensing applications). The hydrogel formed on the modified surface is remarkably stable for an extended period of time. The resultant hydrogel was hydrated for more than 18 months without suffering delamination from the substrate surface. This strongly suggests covalent attachment of the hydrogel to the surface. Contact angle titration measurements and X-ray photoelectron spectroscopy analysis of eosin surfaces before and after irradiation in the presence of triethanolamine suggest that the eosin radical is responsible for the covalent attachment of the gel onto the substrate surface. This method allows for 2-D patterning of hydrogels, which is demonstrated here using the microcontact printing technique. However, noncontact photolithography could be used to form similar patterns by directing light through a mask. This method can be easily implemented to form arrays of fluorophores and proteins in situ.

Biomolecular recognition on well-characterized beads packed in microfluidic channels.

We describe a new approach for the analysis of biomolecular recognition in microfluidic channels. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer (FRET). We establish a model system that detects the FLAG epitope. The assay can potentially detect subfemtomole quantities of antibody with a high signal-to-noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter-to-submicroliter volumes of analyte fluid. Kinetic and equilibrium constants for the reaction of this receptor-ligand pair are obtained through modeling of kinetic responses of the affinity microcolumn and are consistent with those obtained by flow cytometry. Because of the correlation between kinetic and equilibrium data obtained for the microcolumns, quantitative analysis can be done prior to the steady-state end point of the recognition reaction. This method has the promise of combining the utility of affinity chromatography with the advantage of direct, quantitative, and real-time analysis and the cost-effectiveness of microanalytical devices. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes and molecular assembly and microsystem-based multianalyte determinations.