Charged Lipids Modulate the Phase Separation in Multicomponent Membranes.

Phase separation in lipid membranes controls the organization of membrane components and thus regulates membrane-mediated processes. Membrane phase behavior is influenced by the molecular properties of its components and their relative concentrations. Charged lipid species are among the most essential components of lipid membranes, and their impact on the membrane phase behavior is yet to be fully understood. Aiming to provide insight into this impact, this paper investigates how the presence and amount of anionic and cationic lipids affect the phase behavior of multicomponent membranes. Membranes of ternary composition DOPC:DPPC:Chol with two distinct molar ratios were used to test the hypothesis that inclusion of charged lipids with saturated tails, beyond a certain concentration, would impede phase separation in an otherwise phase-separating membrane. Fluorescence microscopy examination of electroformed giant liposomes revealed that when more than half of DOPC in the examined mixtures was replaced with DOPA or DOTAP, phase separation in liposomes was somewhat suppressed, and this effect increased with increasing charged lipid content. This effect depended on the membrane surface charge density as the half-maximal effect was observed at around 0.0072 C Å-2 in all examined cases. The phase-separation suppressing effect of DOPA was neutralized when oppositely charged lipid DOTAP was included in the mixture. Likewise, presence of divalent cation Ca2+ in the solution neutralized the impact of negatively charged DOPA. These results underline the detrimental influence of surface charge density on membrane phase behavior. More importantly, these findings suggest that the charged lipid content in membranes may be a regulator of their phase behavior and open new opportunities for the design of synthetic lipid membranes.

Microtubule binding kinetics of membrane-bound kinesin-1 predicts high motor copy numbers on intracellular cargo.

Bidirectional vesicle transport along microtubules is necessary for cell viability and function, particularly in neurons. When multiple motors are attached to a vesicle, the distance a vesicle travels before dissociating is determined by the race between detachment of the bound motors and attachment of the unbound motors. Motor detachment rate constants (koff) can be measured via single-molecule experiments, but motor reattachment rate constants (kon) are generally unknown, as they involve diffusion through the bilayer, geometrical considerations of the motor tether length, and the intrinsic microtubule binding rate of the motor. To understand the attachment dynamics of motors bound to fluid lipid bilayers, we quantified the microtubule accumulation rate of fluorescently labeled kinesin-1 motors in a 2-dimensional (2D) system where motors were linked to a supported lipid bilayer. From the first-order accumulation rate at varying motor densities, we extrapolated a koff that matched single-molecule measurements and measured a 2D kon for membrane-bound kinesin-1 motors binding to the microtubule. This kon is consistent with kinesin-1 being able to reach roughly 20 tubulin subunits when attaching to a microtubule. By incorporating cholesterol to reduce membrane diffusivity, we demonstrate that this kon is not limited by the motor diffusion rate, but instead is determined by the intrinsic motor binding rate. For intracellular vesicle trafficking, this 2D kon predicts that long-range transport of 100-nm-diameter vesicles requires 35 kinesin-1 motors, suggesting that teamwork between different motor classes and motor clustering may play significant roles in long-range vesicle transport.

Organic Semiconductor Nanotubes for Electrochemical Devices.

Electrochemical devices that transform electrical energy to mechanical energy through an electrochemical process have numerous applications ranging from soft robotics and micropumps to autofocus microlenses and bioelectronics. To date, achievement of large deformation strains and fast response times remains a challenge for electrochemical actuator devices operating in liquid wherein drag forces restrict the actuator motion and electrode materials/structures limit the ion transportation and accumulation. We report results for electrochemical actuators, electrochemical mass transfers, and electrochemical dynamics made from organic semiconductors (OSNTs). Our OSNTs electrochemical device exhibits high actuation performance with fast ion transport and accumulation and tunable dynamics in liquid and gel-polymer electrolytes. This device demonstrates an excellent performance, including low power consumption/strain, a large deformation, fast response, and excellent actuation stability. This outstanding performance stems from enormous effective surface area of nanotubular structure that facilitates ion transport and accumulation resulting in high electroactivity and durability. We utilize experimental studies of motion and mass transport along with the theoretical analysis for a variable-mass system to establish the dynamics of the electrochemical device and to introduce a modified form of Euler-Bernoulli's deflection equation for the OSNTs. Ultimately, we demonstrate a state-of-the-art miniaturized device composed of multiple microactuators for potential biomedical application. This work provides new opportunities for next generation electrochemical devices that can be utilized in artificial muscles and biomedical devices.

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays.

Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar[5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(± 0.3) × 10(-14) cm(3)/s or 3.5(± 1.0) × 10(8) water molecules per s, which is in the range of AQPs (3.4 ∼ 40.3 × 10(8) water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10(8) water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼ 10(7) water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼ 2.6 × 10(5) pores per µm(2)) is two orders of magnitude higher than that of CNT membranes (0.1 ∼ 2.5 × 10(3) pores per µm(2)). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.

A model for the interfacial kinetics of phospholipase D activity on long-chain lipids.

The membrane-active enzyme phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond in phospholipids and plays a critical role in cell signaling. This catalytic reaction proceeds on lipid-water interfaces and is an example of heterogeneous catalysis in biology. Recently we showed that planar lipid bilayers, a previously unexplored model membrane for these kinetic studies, can be used for monitoring interfacial catalytic reactions under well-defined experimental conditions with chemical and electrical access to both sides of the lipid membrane. Employing an assay that relies on the conductance of the pore-forming peptide gramicidin A to monitor PLD activity, the work presented here reveals the kinetics of hydrolysis of long-chain phosphatidylcholine lipids in situ. We have developed an extension of a basic kinetic model for interfacial catalysis that includes product activation and substrate depletion. This model describes the kinetic behavior very well and reveals two kinetic parameters, the specificity constant and the interfacial quality constant. This approach results in a simple and general model to account for product accumulation in interfacial enzyme kinetics.

On Fusogenicity of Positively Charged Phased-Separated Lipid Vesicles: Experiments and Computational Simulations.

This paper studies the fusogenicity of cationic liposomes in relation to their surface distribution of cationic lipids and utilizes membrane phase separation to control this surface distribution. It is found that concentrating the cationic lipids into small surface patches on liposomes, through phase-separation, can enhance liposome's fusogenicity. Further concentrating these lipids into smaller patches on the surface of liposomes led to an increased level of fusogenicity. These experimental findings are supported by numerical simulations using a mathematical model for phase-separated charged liposomes. Findings of this study may be used for design and development of highly fusogenic liposomes with minimal level of toxicity.

Direct Laser 3D Printing of Organic Semiconductor Microdevices for Bioelectronics and Biosensors.

Fabrication of conductive and bioactive microdevices has garnered tremendous attention in the emerging biomedical fields, particularly organic bioelectronics and biosensing. Direct laser 3D printing based on two-photon polymerization (TPP) has shown great promise in construction of well-defined and multi-functional microdevices. Herein, we present a novel photosensitive resin for fabrication of highly conductive and bioactive microstructures via TPP. This resin is based on poly(ethylene glycol) diacrylate that is doped with poly (3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (organic semicoductor), and laminin (extracellular matrix protein) or glucose oxidase (biorecognition enzyme). We demonstrate the fabrication of hybrid microelectrodes, bioactive microstructures for cellular adhesion / spreading, and high-performance glucose biosensors. Clinical Relevance- Conductive and bioactive microelectronic devices based on the formulated resin can be utilized for neural recording / stimulation, tissue engineering, and biosensing applications.

Nanoparticle Rigidity for Brain Tumor Cell Uptake.

Nanoparticles (NPs) have emerged as versatile and widely used platforms for a variety of biomedical applications. For delivery purposes, while some of NPs' physiochemical aspects such as size and shape have been extensively studied, their mechanical properties remain understudied. Recent studies have reported NPs' rigidity as a significant factor for their cell interactions and uptake. Here, we aim to study how NPs' rigidity affects their interactions with brain glioma tumor cells. To produce NPs with different rigidities, we encapsulate poly(ethylene glycol) diacrylate (PEGDA) of different volume ratios (0, 10, 30 v/v%) within the lumen of nanoliposomes and study the uptake of these NPs in a glioblastoma cell line U87. PEGDA with volume ratios of 10 and 30% were selected to provide a significant increase of the elastic modulus of the hydrogel (0.1 to 4 MPa) as determined by compression testing. Dynamic light scattering (DLS) and zeta potential measurements indicated that despite differences in their core formulation, all examined NPs had a similar size range (106 to 132 nm) and surface charge (-2.0 to -3.0 mV). Confocal microscopy revealed that all NP groups accumulated inside U87 cells, and flow cytometry data showed that liposomes with a gel core (10 and 30 v/v% PEGDA) had significantly higher cellular uptake (up to 9-fold), compared to liposomes with an aqueous core. Notably, we did not find any substantial difference between the uptake of liposomes with PEGDA core of 10 and 30% volume ratios. Clinical Relevance- By providing an insight into how NP rigidity influences glioma tumor cellular uptake, this work would enable development of more effective therapeutics for brain cancer.

Multiphoton Lithography of Organic Semiconductor Devices for 3D Printing of Flexible Electronic Circuits, Biosensors, and Bioelectronics.

In recent years, 3D printing of electronics have received growing attention due to their potential applications in emerging fields such as nanoelectronics and nanophotonics. Multiphoton lithography (MPL) is considered the state-of-the-art amongst the microfabrication techniques with true 3D fabrication capability owing to its excellent level of spatial and temporal control. Here, a homogenous and transparent photosensitive resin doped with an organic semiconductor material (OS), which is compatible with MPL process, is introduced to fabricate a variety of 3D OS composite microstructures (OSCMs) and microelectronic devices. Inclusion of 0.5 wt% OS in the resin enhances the electrical conductivity of the composite polymer about 10 orders of magnitude and compared to other MPL-based methods, the resultant OSCMs offer high specific electrical conductivity. As a model protein, laminin is incorporated into these OSCMs without a significant loss of activity. The OSCMs are biocompatible and support cell adhesion and growth. Glucose-oxidase-encapsulated OSCMs offer a highly sensitive glucose sensing platform with nearly tenfold higher sensitivity compared to previous glucose biosensors. In addition, this biosensor exhibits excellent specificity and high reproducibility. Overall, these results demonstrate the great potential of these novel MPL-fabricated OSCM devices for a wide range of applications from flexible bioelectronics/biosensors, to nanoelectronics and organ-on-a-chip devices.

Tumor Targeted Delivery of an Anti-Cancer Therapeutic: An In Vitro and In Vivo Evaluation.

The limited effectiveness of current therapeutics against malignant brain gliomas has led to an urgent need for development of new formulations against these tumors. Chelator Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) presents a promising candidate to defeat gliomas due to its exceptional anti-tumor activity and its unique ability to overcome multidrug resistance. The goal of this study is to develop a targeted nano-carrier for Dp44mT delivery to glioma tumors and to assess its therapeutic efficacy in vitro and in vivo. Dp44mT is loaded into poly(ethylene glycol) (PEG)ylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) decorated with glioma-targeting ligand Interlukin 13 (IL13). IL13-conjugation enhanced the NP uptake by glioma cells and also improved their transport across an in vitro blood-brain-barrier (BBB) model. This targeted formulation showed an outstanding toxicity towards glioma cell lines and patient-derived stem cells in vitro, with IC50 values less than 125 nM, and caused no significant death in healthy brain microvascular endothelial cells. In vivo, when tested on a xenograft mouse model, IL13-conjugated Dp44mT-NPs reduced the glioma tumor growth by ≈62% while their untargeted counterparts reduced the tumor growth by only ≈16%. Notably, this formulation does not cause any significant weight loss or kidney/liver toxicity in mice, demonstrating its great therapeutic potential.

An Improved in Vitro Blood-Brain Barrier Model for Applications in Therapeutics' Delivery to Brain.

Blood-brain barrier (BBB) imposes a major obstacle for entry of therapeutics to brain. In vitro BBB models that can provide reliable prediction of therapeutics' ability to cross BBB are thus, critical for the advancement of brain therapeutics. Towards the development of an improved BBB model, here we studied the individual and combinatorial effect of few different culture conditions on the quality of the commonly used trans-well BBB model. Specifically, we investigated how the addition of (i) astrocyte co-culture, (ii) astrocyte-conditioned media (ACM), and (iii) astrocyte co-culture along with ACM, affects the characteristics of BBB. The resultant BBB models were characterized for trans-endothelial electrical resistance (TEER), permeability, and expression of a tight-junction protein ZO-1. We found that addition of ACM and astrocytes, individually, had similar impact on BBB's TEER, increasing it by ~2 fold. Interestingly, the presence of both astrocytes and ACM had a significantly greater impact on TEER and increased it by ~3 fold. Addition of ACM, with and without astrocyte co-culture, led to a reduction in permeability of this BBB model. Moreover, addition of ACM and astrocyte co-culture, both individually and in combination, led to a noticeable increase in ZO-1 expression in the BBB endothelial cells. These findings provide a new approach for further improvement of the commonly used trans-well BBB system.

Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ.

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.

Gramicidin pores report the activity of membrane-active enzymes.

Phospholipases constitute a ubiquitous class of membrane-active enzymes that play a key role in cellular signaling, proliferation, and membrane trafficking. Aberrant phospholipase activity is implicated in a range of diseases including cancer, inflammation, and myocardial disease. Characterization of these enzymes is therefore important, both for improving the understanding of phospholipase catalysis and for accelerating pharmaceutical and biotechnological applications. This paper describes a novel approach to monitor, in situ and in real-time, the activity of phospholipase D (PLD) and phospholipase C (PLC) on planar lipid bilayers. This method is based on lipase-induced changes in the electrical charge of lipid bilayers and on the concomitant change in ion concentration near lipid membranes. The approach reports these changes in local ion concentration by a measurable change in the single channel ion conductance through pores of the ion channel-forming peptide gramicidin A. This enzyme assay takes advantage of the amplification characteristics of gramicidin pores to sense the activity of picomolar to nanomolar concentrations of membrane-active enzymes without requiring labeled substrates or products. The resulting method proceeds on lipid bilayers without the need for detergents, quantifies enzyme activity on native lipid substrates within minutes, and provides unique access to both leaflets of well-defined lipid bilayers; this method also makes it possible to generate planar lipid bilayers with transverse lipid asymmetry.

Optimization of the Single Emulsion Method for Encapsulation of a Cancer Drug in Nanoparticles.

The goal of this study is to apply and optimize the single emulsion technique for encapsulation of an anti-tumor drug, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), in nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA), as a step towards targeted delivery of this drug. We previously showed that the nanoprecipitation technique can effectively produce PLGA NPs carrying this drug. Here, we aim to examine the single emulsion technique as an alternative for the preparation of these NPs and to compare the resultant NPs to those from nanoprecipitation. We fabricated NPs with variations in (i) injection rate, (ii) the amount of surfactant poly (vinyl alcohol) (PVA) in aqueous phase, and (iii) concentration of PLGA in the organic phase. These NPs were characterized for size, surface potential, and encapsulation efficiency. The results revealed that increasing the injection rate (from manual addition to 90 mL/hr via syringe pump) greatly reduced the size of NPs (by 48%) and decreasing the PVA concentration in the aqueous phase (from 5 to 1% w/v) further reduced the NP size (by 32%) to 329 nm. All tested NP formulations had negative surface potential, suggesting good colloidal stability for these NPs. Focusing on the optimal injection rate and PVA percentage, we found that reducing the concentration of PLGA, from 100 to 1 mg/mL, significantly reduced the NP size to 136 nm, which is close to the optimal range for cancer therapeutic delivery. NPs produced by this method had a high encapsulation efficiency of 77% for Dp44mT and reducing the PLGA concentration slightly lowered this value to 74%. Overall, these NPs were comparable to those produced by nanoprecipitation and can thus, serve as an effective alternative for delivery of Dp44mT to cancer cells.

Preparation and characterization of size-controlled glioma spheroids using agarose hydrogel microwells.

Treatment of glioblastoma, the most common and aggressive type of primary brain tumors, is a major medical challenge and the development of new alternatives requires simple yet realistic models for these tumors. In vitro spheroid models offer attractive platforms to mimic the tumor behavior in vivo and have thus, been increasingly applied for assessment of drug efficacy in various tumors. The aim of this study was to produce and characterize size-controlled U251 glioma spheroids towards application in glioma drug evaluation studies. To this end, we fabricated agarose hydrogel microwells with cylindrical shape and diameters of 70-700 µm and applied these wells without any surface modification for glioma spheroid formation. The resultant spheroids were homogeneous in size and shape, exhibited high cell viability (> 90%), and had a similar growth rate to that of natural brain tumors. The final size of spheroids depended on cell seeding density and microwell size. The spheroids' volume increased linearly with the cell seeding density and the rate of this change increased with the well size. Lastly, we tested the therapeutic effect of an anti-cancer drug, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) on the resultant glioma spheroids and demonstrated the applicability of this spheroid model for drug efficacy studies.

Conducting Polymer Microtubes for Bioactuators.

Conducting polymer (CP) actuators are promising devices for biomedical applications such as artificial muscles and drug delivery systems. Here, we report a tri-layer actuator based on poly(pyrrole) (PPy) microtubes (PPy MTs) doped with poly(sodium-p-styrenesulfonate) (PSS) and constructed on a passive layer of gold-coated poly-propylene (PP) film. The PPy MTs were fabricated using electrochemical deposition of PPy around poly(lactic-co-glycolic acid) (PLGA) fiber templates, followed by template removal. The PPy MTs were subjected to a redox process using cyclic voltammetry in 0.1 M NaPSS electrolyte solution as the potential was swept between -0.8 V and +0.4 V for 5 cycles at the scan rates of 10, 50, 100, and 200 mV/s. The bending behavior of the PPy MTs actuator was investigated by measuring the deflection of actuator tip resulting from the expansion/contraction strain of PPy MTs. The PPy MTs actuator showed a reversible bending movement during each potential cycle. The maximum deflection of actuator decreased by increasing the scan rate that was confirmed by calculating the actuation strain generated during each cycle at various scan rates.

Preparation of Gel-Liposome Nanoparticles for Drug Delivery Applications.

Liposomes are amongst the most effective delivery vehicles developed to date. Despite many advantages including biocompatibility, biodegradability, and the ability to carry both hydrophilic and lipophilic compounds, liposomes suffer from low physical stability. This limitation can be effectively addressed by inclusion of a polymeric scaffold within the core of liposomes. Given the versatility of poly (ethylene glycol) (PEG) hydrogels, these polymers have a great potential for the use in liposomal core. As a step towards the development of a robust liposomal delivery platform, here we aim to develop a simple and reliable technique for the fabrication of liposomes with PEG gel cores. We assess the resultant nanoparticles using scanning electron microscopy and dynamic light scattering and demonstrate that the presented approach can successfully produce gel-liposome nanoparticles with spherical shape and 150-200 nm size. These nanoparticles are further evaluated for colloidal stability in physiological solution. Moreover, we demonstrate the versatility of this method by studying the effect of changing (A) the membrane composition in liposomes, and (B) the hydrogel concentration in liposomal core, on the formation of gel-liposome particles.

A computational study of lateral phase separation in biological membranes.

Conservative and non-conservative phase-field models are considered for the numerical simulation of lateral phase separation and coarsening in biological membranes. An unfitted finite element method is proposed to allow for a flexible treatment of complex shapes in the absence of an explicit surface parametrization. For a set of biologically relevant shapes and parameter values, the paper compares the dynamic coarsening produced by conservative and non-conservative numerical models, its dependence on certain geometric characteristics and convergence to the final equilibrium.

Development and in vitro assessment of an anti-tumor nano-formulation.

This study aims to develop a new anti-cancer formulation based on the chelator Dp44mT (Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone). Dp44mT has outstanding anti-tumor activity and the unique ability to overcome multidrug-resistance in cancer cells. This highly toxic compound has thus far only been applied in free form, limiting its therapeutic effectiveness. To reach its full therapeutic potential, however, Dp44mT should be encapsulated in a nano-carrier that would enable its selective and controlled delivery to malignant cells. As the first step towards this goal, here we encapsulate Dp44mT in nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA), characterize this nano-formulation, and evaluate its therapeutic potential against cancer cells in vitro. Our results showed that the Dp44mT-loaded NPs were homogenous in shape and size, and had good colloidal stability. These PLGA NPs also showed high encapsulation efficiency and loading capacity for Dp44mT and enabled the sustained and tunable release of this chelator. Dp44mT-NPs were uptaken by cancer cells, showed a strong and dose-dependent cytotoxicity towards these cells, and significantly increased apoptotic cell death, in both monolayer and spheroid tumor models. This formulation had a low-level of toxicity towards healthy control cells, indicating an inherent selectivity towards malignant cells. These results demonstrate the great potential of this novel Dp44mT-based nano-formulation for the use in cancer therapy.

Electrospinning of Highly Aligned Fibers for Drug Delivery Applications.

Electrospinning is a straightforward, cost-effective, and versatile technique for fabrication of polymeric micro/nanofibers with tunable structural properties. Controlling the size, shape, and spatial orientation of the electrospun fibers is crucial for utilization in drug delivery and tissue engineering applications. In this study, for the first time, we systematically investigate the effect of processing parameters, including voltage, syringe needle gauge, angular velocity of rotating wheel, syringe-collector distance, and flow rate on the size and alignment of electrospun PLGA fibers. Optimizing these parameters enabled us to produce highly aligned and monodisperse PLGA fibers (spatial orientation> 99% and coefficient of variation< 0.5). To assess the effect of fiber alignment on the release of encapsulated drugs from these fibers, we incorporated dexamethasone, an anti-inflammatory drug, within highly-aligned and randomly-oriented fibers with comparable diameters (~0.87 µm) and compared their release profiles. In-vitro release studies revealed that the aligned fibers had less burst release (~10.8% in 24 hr) and more sustained release (~8.8% average rate of change for 24 days) compared to the random fibers. Finally, the degradation modes of the aligned and random fibers after 25 days incubation were characterized and compared. The findings of this study can be applied for the development of 3D degradable aligned fibers for controlled drug release and tissue engineering applications.