Controlled Release Of Proteins Research Articles

Bioinspired conformational changes: an adaptable mechanism for bio-responsive protein delivery

Hydrogels that respond to their environment have been extensively used as controlled drug delivery devices. An emerging trend is to form these “dynamic” hydrogels from polymers that undergo conformational changes. Indeed, nanometer scale polymer conformational changes have translated to macroscopic changes in hydrogel properties and controlled the release of encapsulated drugs. This review will focus on the mechanisms that control protein release from dynamic hydrogels. Specifically, we will highlight emerging mechanisms to form dynamic hydrogels, whose functional nature is derived from nature-inspired polymer conformational changes. Pertinent results from the literature will be examined to illustrate how these nanometer scale polymer conformational changes influence therapeutic protein release from dynamic hydrogels.

An aptamer-functionalized hydrogel for controlled protein release: A modeling study

An analytical model is developed for predicting the controlled release of platelet-derived growth factor-BB (PDGF-BB) from an aptamer-functionalized hydrogel. The release kinetics are determined by the equilibrium constant of aptamer–protein interactions, which significantly increases the characteristic release time scale. The reduction of the cumulative protein release can also be modulated by tuning the binding affinity of the aptamer. The analysis describes the underlying transport and reaction mechanisms for the controlled protein release well. Particularly, the non-Fickian release kinetics are represented by a self-similar release profile. The model is validated by experiments and direct numerical calculation. It can be applied to release kinetics involving multiple aptamers and proteins.

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.

Identifying Factors Controlling Protein Release from Combinatorial Biomaterial Libraries via Hybrid Data Mining Methods

Polyanhydrides are a class of degradable biomaterials that have shown much promise for applications in drug and vaccine delivery. Their properties can be tailored for controlled drug release, drug/protein stability, and immune regulation (adjuvant effect). Identifying the relationship between the molecular structures of the polymers and the drug release kinetics profiles would help understand the release mechanism and aid in the accurate prediction of drug release and the rational design of polymer-based drug carrier systems. The molecular structure descriptors that had the most impact on the release kinetics were identified using a prediction/optimization data mining approach. Using this new approach for modeling nonlinear release kinetics behavior, we determined that the descriptors which had the greatest effect on the release kinetics were the number of backbone -COO- nonconjugated bonds, the number of aromatic rings, and the number of -CH₂- bonds.

Electrically Polarized Biphasic Calcium Phosphates: Adsorption and Release of Bovine Serum Albumin

In this study, we applied electrical polarization technique to increase adsorption and control protein release from biphasic calcium phosphate (BCP). Three different biphasic calcium phosphate (BCP) composites, with hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), were processed and electrically polarized. Our study showed that stored charge was increased in the composites with the increase in HAp percentage. Adsorption of bovine serum albumin (BSA), as a model protein, on the poled as well as unpoled surfaces of the composites was studied. The highest amount of BSA adsorption was obtained on positively poled surfaces of each composite. Adsorption isotherm study suggested a multilayer adsorption of BSA on the BCP composites. The effect of electrical polarization on BSA release kinetics from positively charged BCP surfaces was studied. A gradual increase in percent BSA release from positively charged BCP surfaces with decreasing stored charge was observed. Our study showed that the BCP based composites have the potential to be used as a drug or growth factor delivery vehicle.

Photopolymerized Thermosensitive Poly(HPMAlactate)-PEG-Based Hydrogels: Effect of Network Design on Mechanical Properties, Degradation, and Release Behavior

Photopolymerized thermosensitive A-B-A triblock copolymer hydrogels composed of poly(N-(2-hydroxypropyl)methacrylamide lactate) A-blocks, partly derivatized with methacrylate groups to different extents (10, 20, and 30%) and hydrophilic poly(ethylene glycol) B-blocks of different molecular weights (4, 10, and 20 kDa) were synthesized. The aim of the present study was to correlate the polymer architecture with the hydrogel properties, particularly rheological, swelling, degradation properties and release behavior. It was found that an increasing methacrylation extent and a decreasing PEG molecular weight resulted in increasing gel strength and cross-link density, which tailored the degradation profiles from 25 to more than 300 days. Polymers having small PEG blocks showed a remarkable phase separation into polymer- and water-rich domains, as demonstrated by confocal microscopy. Depending on the hydrophobic domain density, the loaded protein resides in the hydrophilic pores or is partitioned into hydrophilic and hydrophobic domains, and its release from these compartments is tailored by the extent of methacrylation and by PEG length, respectively. As the mechanical properties, degradation, and release profiles can be fully controlled by polymer design and concentration, these hydrogels are suitable for controlled protein release.

Enhancement of sustained and controlled protein release using polyelectrolyte complex-loaded injectable and thermosensitive hydrogel

In this study, we aimed at developing controlled and sustained protein release formulations using a combination system of polyelectrolyte complexes (PECs) and thermosensitive poly(organophosphazene) hydrogels as an injectable gel-depot system. In the protein-loaded hydrogel system, the loaded proteins were released rapidly through diffusion regardless of viscosities and mass loss of the gels because of the small hydrodynamic size of the proteins. To suppress protein diffusion and increase protein size, we induced PECs between negatively charged proteins (BSA, gelatin-type B 75 bloom, α-amylase, and hGH) and polycations (protamine, polyethylenimine, poly- l-lysine, and poly- l-arginine (PLA)) via an electrostatic interaction and loaded the PECs into the hydrogels. The formations of PECs were affected by molecular weight, p I (or p K a), and types of amine group of the used polycations. Unlike other polycations, PLA formed a large uniform complex with BSA, and the PLA/protein complex-loaded hydrogel showed the slowest protein release behavior. In the PEC-loaded hydrogel system, the protein release could also be controlled by viscosities of the gel and weight ratios of polycations and proteins, although the activities of the proteins were decreased in proportion to the PLA amounts. These results suggest that the PEC-loaded injectable and thermosensitive poly(organophosphazene) hydrogel has considerable potential for creating a sustained protein delivery system by using the PEC via electrostatic interaction.

Hyaluronic acid hydrogels with controlled degradation properties for oriented bone regeneration

Non-healing fractures can result from trauma, disease, or age-related bone loss. While many treatments focus on restoring bone volume, few try to recapitulate bone organization. However, the native architecture of bone is optimized to provide its necessary mechanical properties. Hyaluronic acid (HA) hydrogel scaffold systems with tunable degradation properties were developed for the controlled delivery of osteoinductive and angiogenic growth factors, thus affecting the quantity and quality of regenerated tissue. HA hydrogels were designed to degrade at fast, intermediate, and slow rates due to hydrolysis and further provided controlled release of cationic proteins due to electrostatic interactions. Scaffolds delivering bone morphogenetic protein-2 (BMP-2) were evaluated in a rat calvarial bone critical size defect model. BMP-2 delivery from the HA hydrogels had a clear osteoinductive effect in vivo and, for all hydrogel types, BMP-2 delivery resulted in significant mineralization compared to control hydrogels. The temporal progression of this effect could be modulated by altering the degradation rate of the scaffold. All three degradation rates tested resulted in similar amounts of mineral formation at the latest (six week) time point examined. Interestingly, however, the fastest and slowest degrading scaffolds seemed to result in more organized bone than the intermediate degrading scaffold, which was designed to degrade in 6-8 weeks to match the healing time. Additionally, healing could be enhanced by co-delivery of vascular endothelial growth factor along with BMP-2.

One-Step Immobilization of Protein-Encapsulated Core/Shell Particles onto Nanofibers

A novel method is described to functionalize nanofibers to form a nanocomposite with core/shell particles in order to control protein release. The nanocomposite is produced by electrically neutralizing negatively charged poly(lactic acid) nanofibers with positively charged poly[(lactic acid)-co-(glycolic acid)] particles via a one-step electrohydrodynamic jetting process. The protein-encapsulated core/shell particles exhibited no significant initial burst release or denaturation. The protein release profile was controlled by porosity and protein/polymer interactions. The method may be promising to engineer intelligent scaffolds that can fulfill the needs of biomimetic materials.

Encapsulation of BSA in polylactic acid–hyperbranched polyglycerol conjugate nanoparticles: preparation, characterization, and release kinetics

Nanoparticles based on an amphiphilic copolymer with polylactic acid (PLA) grafted onto hyperbranched polyglycerol (HPG) were prepared by the use of BSA as a model protein. The characteristics of the nanoparticles were evaluated using particle size analyzer, transmission electron microscopy, and X-ray photoelectron spectroscopy. The secondary structure of BSA released from nanoparticles were analysed by circular dichroism experiments. Cell viability of nanoparticles was also evaluated by using NIH 3T3 cells. The mechanism of BSA release was studied by fitting experimental data to three model equations. Results indicated that BSA diffusion and the polymeric relaxation jointly governed the overall release process. The detailed analysis of BSA release was performed using the first-order kinetic model equation, which gave a good fit to the experimental release data. The influence of different copolymer structures and BSA loading capacities on release profiles were also evaluated for the potential of using nanoparticles as controlled release protein delivery systems.

Time Controlled Protein Release from Layer‐by‐Layer Assembled Multilayer Functionalized Agarose Hydrogels

DOI: 10.1002/adfm.200901172 In the final print version of this manuscript, Figure 2 was produced incorrectly, resulting in a loss of fidelity in the microscopy images. The corrected image can be found below. The editorial office apologises for any inconvenience caused by this error.

Time Controlled Protein Release from Layer-by-Layer Assembled Multilayer Functionalized Agarose Hydrogels.

Axons of the adult central nervous system exhibit an extremely limited ability to regenerate after spinal cord injury. Experimentally generated patterns of axon growth are typically disorganized and randomly oriented. Support of linear axonal growth into spinal cord lesion sites has been demonstrated using arrays of uniaxial channels, templated with agarose hydrogel, and containing genetically engineered cells that secrete brain-derived neurotrophic factor (BDNF). However, immobilizing neurotrophic factors secreting cells within a scaffold is relatively cumbersome, and alternative strategies are needed to provide sustained release of BDNF from templated agarose scaffolds. Existing methods of loading the drug or protein into hydrogels cannot provide sustained release from templated agarose hydrogels. Alternatively, here it is shown that pH-responsive H-bonded poly(ethylene glycol)(PEG)/poly(acrylic acid)(PAA)/protein hybrid layer-by-layer (LbL) thin films, when prepared over agarose, provided sustained release of protein under physiological conditions for more than four weeks. Lysozyme, a protein similar in size and isoelectric point to BDNF, is released from the multilayers on the agarose and is biologically active during the earlier time points, with decreasing activity at later time points. This is the first demonstration of month-long sustained protein release from an agarose hydrogel, whereby the drug/protein is loaded separately from the agarose hydrogel fabrication process.

Light controlled protein release from a supramolecular hydrogel

Using the inclusion complex of trans azobenzene and cyclodextrin as a photo-switchable crosslinker, a dextran based photo-responsive hydrogel system has been constructed and employed for a light controlled protein release system.

Injectable Microsphere/Hydrogel Combination Systems for Localized Protein Delivery

Injectable delivery systems for therapeutic proteins (e.g., hydrogels and microspheres) have attracted wide attention. Hydrogels, however, may release their hydrophilic contents too rapidly in a large initial burst, and phagocytes may clear microspheres within a relatively short time period after administration. We hypothesized that microsphere/hydrogel combination systems could achieve a controlled and sustained release of proteins as an injectable delivery system. To test this hypothesis, we prepared PLGA microspheres containing a model protein and mixed these with alginate gels. The mixing ratio of the components was the primary controlling parameter of the protein release. This approach could be useful for development of injectable and localized drug delivery systems.

Polyelectrolyte nanoparticles based on water-soluble chitosan–poly( l-aspartic acid)–polyethylene glycol for controlled protein release

Water-soluble chitosan (WSC)–poly( l-aspartic acid) (PASP)–polyethylene glycol (PEG) nanoparticles (CPP nanoparticles) were prepared spontaneously under quite mild conditions by polyelectrolyte complexation. These nanoparticles were well dispersed and stable in aqueous solution, and their physicochemical properties were characterized by turbidity, FTIR spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), and zeta potential. PEG was chosen to modify WSC–PASP nanoparticles to make a protein-protective agent. Investigation on the encapsulation efficiency and loading capacity of the bovine serum albumin (BSA)-loaded CPP nanoparticles was also conducted. Encapsulation efficiency was obviously decreased with the increase of initial BSA concentration. Furthermore, its in vitro release characteristics were evaluated at pH 1.2, 2.5, and 7.4. In vitro release showed that these nanoparticles provided an initial burst release, followed by a slowly sustained release for more than 24 h. The BSA released from CPP nanoparticles showed no significant conformational change compared with native BSA, which is superior to the BSA released from nanoparticles without PEG. A cell viability study suggested that the nanoparticles had good biocompatibility. This nanoparticle system was considered promising as an advanced drug delivery system for the peptide and protein drug delivery.

Disulfide-crosslinked chitosan hydrogel for cell viability and controlled protein release

Synthetic hydrogel mimics of the extracellular matrix (ECM) were prepared by cross-linking a thiol-modified chitosan (CS). CS was chemically modified using N-acetyl- l-cysteine (NAC). To minimize interference with biological function, the degree of substitution of thiol groups was kept below 50%. Solution of thiolated CS was prepared in pH 7.4 phosphate buffered saline (PBS) and crosslinked by disulfide bond formation in air. The gelation mainly depended on the content of thiol groups on thiolated CS, concentration of thiolated CS and the molecular weight of CS. Thermogravimetric analysis showed the thermal stabilities of CSS-S hydrogels. Results from SEM observation showed a porous 3D hydrogel structure with pores ranging from 5 to 30 μm. In vitro release showed that insulin and BSA release could be controlled by choosing the composition, loading and disulfide bond contents. In vitro cell compatibility of the hydrogels on NIH 3T3 cells was evaluated, indicating that the hydrogels were biocompatible and the cells could migrate into the hydrogels. Moreover, cells were viable and preserved 3D cell morphology inside the hydrogels. These results demonstrate that disulfide-crosslinked CS hydrogels, a new type of macroporous, biocompatible, synthetic polymers, are promising applications in tissue engineering, drug delivery, and cell culture.

Chitosan bearing pendant cyclodextrin as a carrier for controlled protein release

The objective of this work was to investigate the possibility of chitosan bearing β-cyclodextrin (CDen- g-CS) nanocomplexes for controlled protein release. CDen- g-CS was synthesized by a one-step procedure with N-succinylated chitosan and mono(6-(2-aminoethyl)amino-6-deoxy)-β-cyclodextrin in the presence of the water-soluble carbodiimide. The amount of β-CD grafted was up to 62.1 wt%. In vitro cytotoxicity against NIH 3T3 cells showed that CDen- g-CS was not cytotoxic and no significant difference of cytotoxicity was found between CDen- g-CS groups. Self-assembled nanocomplexes between CDen- g-CS and insulin were in the size range of 190–328 nm, with positive electrical charge (+3.7 to +25.5 mV) and high loading efficiency (37.7%). Insulin release in vitro was affected by the medium pH and the composition of copolymer. These results demonstrated that CDen- g-CS copolymer was a new promising vehicle for controlled protein release.

Recent U.S. Patents on Protein Drug Formulation: 2000-2007

This review summarizes U.S. patents on protein drug formulation issued during the period 2000-2007. The patents fall into four major areas: (i) protein stabilization in solution and solid formulations, (ii) polymer-conjugated and chemically modified proteins, (iii) controlled release protein formulations, and (iv) protein formulation for novel routes of administration. Patents issued in each of the four areas are described and possible areas for additional research are noted.

Effects of photochemical crosslinking on the microstructure of collagen and a feasibility study on controlled protein release

Photochemical crosslinking is an emerging technique able to modify the physicochemical properties of collagen. However, whether this technique can be used to modify collagen-based structures for drug delivery has not been studied. This study demonstrated that the microporous structure of photochemically crosslinked collagen was affected by rose Bengal and laser energy level. Using the optimized process parameters, the authors fabricated photochemically crosslinked collagen structures encapsulated with sample proteins and demonstrated that photochemical crosslinking reduced the initial burst effect and protein release without compromising the protein bioactivity. The fiber meshwork in collagen structures was also characterized, and it was found that photochemical crosslinking did not significantly alter the mesh size. This study reports the effects of photochemical crosslinking on the microstructure of collagen structures and suggests the feasibility of using photochemically crosslinked collagen structures for controlled protein release.

Engineering of multifunctional gels integrating highly efficient growth factor delivery with endothelial cell transplantation

Transplantation of Bcl-2-transduced human umbilical vein endothelial cells (ECs) in protein gels into the gastrocnemius muscle improves local reperfusion in immunodeficient mouse hosts with induced hind limb ischemia. We tested the hypothesis that incorporation of local, sustained growth factor delivery could enhance and accelerate this effect. Tissue engineering scaffolds often use synthetic polymers to enable controlled release of proteins, but most synthetic delivery systems have major limitations, most notably hydrophobicity and inefficient protein loading. Here, we report the development of a novel alginate-based delivery system for vascular endothelial growth factor-A(165) (VEGF) that exhibits superior loading efficiency and physical properties to previous systems in vitro. In vivo, VEGF released from alginate microparticles within protein gels was biologically active and, when combined with EC transplantation, led to increased survival of transplanted cells at 28 days. The composite graft described also improved early (14 days) tissue perfusion and late (28 days) muscle myoglobin expression, a sign of recovery from ischemia, compared with EC transplantation and VEGF delivery separately. We conclude that our improved approach to sustained VEGF delivery in tissue engineering is useful in vivo and that the integration of high efficiency protein delivery enhances the therapeutic effect of protein gel-based EC transplantation.