PNIPAAm Hydrogels Research Articles

From poly(N‐isopropylacrylamide)‐block‐poly(ethylene oxide)‐block‐poly(N‐isopropylacrylamide) triblock copolymer to poly(N‐isopropylacrylamide)‐block‐poly(ethylene oxide) hydrogels: Synthesis and rapid deswelling and reswelling behavior of hydrogels

AbstractPoly(N‐isopropylacrylamide)‐block‐poly(ethylene oxide)‐block‐poly(N‐isopropylacrylamide) (PNIPAAm‐b‐PEO‐b‐PNIPAAm) triblock copolymer was synthesized via the reversible addition‐fragmentation chain transfer/macromolecular design via the interchange of xanthate (RAFT/MADIX) process with xanthate‐terminated poly(ethylene oxide) (PEO) as the macromolecular chain transfer agent. The successful synthesis of the ABA triblock copolymer inspired the preparation of poly(N‐isopropylacrylamide)‐block‐poly(ethylene oxide) (PNIPAAm‐b‐PEO) copolymer networks with N,N′‐methylenebisacrylamide as the crosslinking agent with the similar approach. With the RAFT/MADIX process, PEO chains were successfully blocked into poly(N‐isopropylacrylamide) (PNIPAAm) networks. The unique architecture of PNIPAAm‐b‐PEO networks allows investigating the effect of the blocked PEO chains on the deswelling and reswelling behavior of PNIPAAm hydrogels. It was found that with the inclusion of PEO chains into the PNIPAAm networks as midblocks, the swelling ratios of the hydrogels were significantly enhanced. Furthermore, the PNIPAAm‐b‐PEO hydrogels displayed faster response to the external temperature changes than the control PNIPAAm hydrogel. The accelerated deswelling and reswelling behaviors have been interpreted based on the formation of PEO microdomains in the PNIPAAm networks, which could act as the hydrophilic tunnels to facilitate the diffusion of water molecules in the PNIPAAm networks. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012

Preparation, characterisation and controlled drug release from thermosensitive hybrid hydrogels

Temperature sensitive hydrogels are probably the most commonly studied class of environmentally sensitive polymer systems in drug delivery research. In this study, polyvinyl alcohol (PVA) matrix with nanosized pores was obtained by treatment with silica and glutaraldehyde. Then, the internal pores of the dry PVA matrix were filled with poly(N-isopropylacrylamide) (PNIPAAm) hydrogels to form a thermoresponsive hybrid PVA/PNIPAAm hydrogel for controlled drug release. The morphology and functional groups of the hybrid hydrogel were characterised by SEM and Fourier transform infrared spectroscopy. The swelling kinetics and the temperature dependence of equilibrium swelling ratio in distilled water were also investigated. Rhodamine B was loaded to the hybrid hydrogels for release study, and the release rate was fast when the temperature was above the lower critical solution temperature of PNIPAAm. The release data suggest that an excellent controlled release can be achieved by the hybrid hydrogel without losing the intelligent properties of the PNIPAAm hydrogel.

In vitro study of the interactions of galactosylated thermo-responsive hydrogels with cells

Abstract The aim of this work is to explore the ability of poly(N-isopropylacrylamide) (NIPAAm) with galactosylated acrylate (GAC) hydrogels as cell scaffolds for HL-7702 cells to proliferate, keep function of albumin and urea synthesis and detach. The results indicate poly(NIPAAm-co-GAC) hydrogels with weight ratio of 0.2 could support cells to adhere, proliferate and maintain their liver functions for 7 days. An intact cells sheet could spontaneously detach from poly(NIPAAm-co-GAC) (r = 0.2) hydrogels without treating with any enzymes. And detached cells show more integrate morphology (H&E staining) and higher proliferation index (cell cycle analysis) than cells detached by trypsin treatment. The results also reveal excessive GAC introduced to PNIPAAm hydrogels could induce apoptotic cell death of adhered cells.

Thermoresponsive nanocomposite double network hydrogels.

The utility and efficacy of thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) hydrogels as smart materials is limited by their physical properties. In this study, we sought to design PNIPAAm nanocomposite hydrogels which displayed enhanced mechanical properties as well as deswelling-reswelling kinetics but without reducing equilibrium swelling or altering the convenient volume phase transition temperature (VPTT) of PNIPAAm. PNIPAAm hydrogels were formed as double networks (DN) comprised of a tightly crosslinked 1st network and a loosely crosslinked 2nd network. In addition, polysiloxane nanoparticles of two different average diameters (~50 nm and ~200 nm) were incorporated during formation of the 1st or 2nd network. The influence of the hydrogel composition on VPTT, morphology, equilibrium swelling, deswelling-reswelling kinetics and mechanical properties was evaluated. We observed that DN hydrogels formed with ~200 nm polysiloxane nanoparticles introduced during formation of the 1st network achieved the best combination of the desired properties.

Poly(ethylene oxide)-grafted poly(N-isopropylacrylamide) networks: Preparation, characterization and rapid deswelling and reswelling behavior of hydrogels

Poly(ethylene oxide)-grafted poly(N-isopropylacrylamide) networks (PNIPAAm-g-PEO) were prepared via the reversible addition–fragmentation chain transfer polymerization (RAFT) of N-isopropylacrylamide with trithiocarbonate-terminated poly(ethylene oxide) and N,N′-methylenebisacrylamide as the chain transfer agent and the crosslinking agent, respectively. It was found that the PNIPAAm-g-PEO copolymer networks were microphase-separated and that PEO microdomains were dispersed in the PNIPAAm matrix. The hydrogel behavior of the PNIPAAm-g-PEO networks was investigated using swelling, deswelling and reswelling tests. The PNIPAAm-g-PEO hydrogels displayed faster responses to external temperature changes than did the control PNIPAAm hydrogel.

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.

Synthesis and properties of thermo- and pH-sensitive poly(N-isopropylacrylamide)/polyaspartic acid IPN hydrogels

Abstract Various interpenetrating polymer network (IPN) hydrogels with sensitivity to temperature and pH were prepared by introducing the pH-sensitive polymer polyaspartic acid (PASP) hydrogel, into the poly(N-isopropylacrylamide) (PNIPAAm) hydrogel system for the purpose of improving its response rate to temperature. The morphologies and thermal behavior of the prepared IPN hydrogels were studied by both scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The IPN hydrogels showed a large and uneven porous network structure, without showing the common PNIPAAm hydrogel structure. The paper moreover studied their swelling properties, such as temperature dependence of equilibrium swelling ratio, shrinking kinetics, re-swelling kinetics and oscillatory swelling behavior in water. The swelling experiment results revealed that IPN hydrogels exhibited much faster shrinking and re-swelling in function of the composition ratio of the two network components. These fast responsive hydrogels foster potential applications in biomedical and biotechnology fields.

Development and characterization of thermosensitive hydrogels based on poly(N‐isopropylacrylamide) and calcium alginate

AbstractThis work refers to the synthesis and characterization of thermosensitive hydrogels based on interpenetrating polymer networks (IPNs) of poly(N‐isopropylacrylamide) (PNIPAAm) and calcium alginate in the form of films. The influence of the crosslinking degree of PNIPAAm and alginate content on thermal, swelling, mechanical, and morphological properties of hydrogels is investigated in detail. Characterization of pure PNIPAAm hydrogels and IPN hydrogels was performed by FTIR, DSC, DMA, and SEM. In addition, the studies of equilibrium swelling behavior as well as swelling, deswelling, and reswelling kinetics are performed. The results obtained imply the benefits of synthesizing IPNs based on PNIPAAm and calcium alginate over pure PNIPAAm hydrogels. The presence of calcium alginate contributes to the improvement of mechanical properties, the deswelling rate of hydrogels, and the network porosity, without altering the thermosensitivity of PNIPAAm significantly. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

In situ entrapment of urease in cryogels of poly(N‐isopropylacrylamide): An effective strategy for noncovalent immobilization of enzymes

AbstractSupermacroporous poly(N‐isopropylacrylamide) (PNIPAAm) cryogels containing urease were prepared via UV irradiation technique and hydrogen peroxide as initiator. Specifically, due to the cryostructuration phenomenon urease molecules were embedded into the dense cryogel walls. Thus, although the enzyme is physically entrapped, the system exhibited remarkable resistance against leaking due to the dense polymer network formed in the cryogel walls. The immobilized urease can catalyze the hydrolysis of urea in a broad temperature range in both batch and flow regime. The interconnected macropores assist for unhindered diffusion of the substrate and reaction products through the gel, thus, paving the way for consecutive reuse at a constant activity, in contrast to the conventional PNIPAAm hydrogel. Due to the spongy‐like morphology PNIPAAm cryogels containing urease can be exploited as highly permeable membrane for direct removal of traces of urea from continuously flowing feed solutions. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Thermoresponsive Nanocomposite Hydrogels: Transparency, Rapid Deswelling and Cell Release

Thermal modulation reversibly switches poly(N-isopropylacrylamide) (PNIPAAm) hydrogels between a water-swollen and a deswollen state which is useful for a variety of biomedical applications. The utility and efficiency of PNIPAAm hydrogels requires tailoring their rate of deswelling/reswelling, mechanical properties and/or optical clarity. In the current work, we prepared novel thermoresponsive nanocomposite hydrogels comprised of a PNIPAAm hydrogel matrix and polysiloxane colloidal nanoparticles (~54 nm ave. diameter) via in situ photopolymerization of aqueous solutions of NIPAAm monomer, N,N'-methylenebisacrylamide (BIS, crosslinker), photoinitiator and 0.5-4.0 wt% polysiloxane nanoparticles (wt% solids of nanoparticles with respect to NIPAAm weight) at ~7 °C. At these nanoparticle concentrations, the nanocomposite hydrogels were more optically transparent versus those prepared with analogous larger nanoparticles (~219 nm ave. diameter). The volume phase transition temperature (VPTT) of the nanocomposite hydrogels was conveniently unaltered versus that of the pure PNIPAAm hydrogel. Incorporation of nanoparticles caused enhancement in modulus as well as the extent and rate of deswelling. When cooled from 37 °C to 25 °C, mouse smooth muscle precursor cells (10T1/2) were effectively detached from nanocomposite hydrogel surfaces due to hydrogel swelling.

Thermodynamic investigation of thermoresponsive xanthan‐poly (N‐isopropylacrylamide) hydrogels

AbstractPolysaccharide‐based hydrogels, such as xanthan maleate/poly(N‐isopropylacrylamide) (PNIPAAm) interpenetrated polymer networks, are thermostimulable materials of interest for the controlled release of biologically active components due to conformation changes at the low critical‐solution temperature (LCST) PNIPAAm phase transition. The phase transition of these interpenetrated polymer network hydrogels, where PNIPAAm is in a ‘confined’ environment, was examined by high resolution magic angle spinning nuclear magnetic resonance and differential scanning calorimetry. High resolution magic angle spinning nuclear magnetic resonance spectroscopy allows the accurate determination of LCST and an evaluation of the corresponding thermodynamic data. More particularly, the evolution of these data as a function of the composition of the hydrogel, and of the external parameters such as pH and ionic strength, was considered. LCST shows a minimal value with increasing xanthan content. Moreover, it was possible to calculate, as a function of temperature, the fraction of NIPAAm which remains uncollapsed. The data obtained for pure PNIPAAm hydrogels are in good agreement with recently published results. The phase transition of PNIPAAm in a diphasic hydrogel is broader when PNIPAAm is ‘confined’ within an interpenetrated polymer network than in a pure PNIPAAm crosslinked network. The widening of the transition with increasing xanthan content indicates a reduction of the PNIPAAm interchain aggregation in a network structure. Copyright © 2011 Society of Chemical Industry

Dual‐responsive semi‐interpenetrating network beads based on calcium alginate/poly(N‐isopropylacrylamide)/poly(sodium acrylate) for sustained drug release

AbstractTo improve the mechanical strength of natural hydrogels and to obtain a sustained drug‐delivery device, temperature‐/pH‐sensitive hydrogel beads composed of calcium alginate (Ca‐alginate) and poly(N‐isopropylacrylamide) (PNIPAAm) were prepared in the presence of poly(sodium acrylate) (PAANa) with ultrahigh molecular weight (Mη ≥ 1.0 × 107) as a strengthening agent. The influence of PAANa content on the properties, including the beads stability, swelling, and drug‐release behaviors, of the hydrogels was evaluated. Scanning electron microscopy and oscillation experiments were used to analyze the structure and mechanical stability of the hydrogel beads, respectively. The results show that stability of the obtained Ca‐alginate/PNIPAAm hydrogel beads strengthened by PAANa the alginate/poly(N‐isopropyl acrylamide) hydrogel bead (SANBs) was significantly improved compared to that of the beads without PAANa (NANBs) at pH 7.4. The swelling behavior and drug‐release capability of the SANBs were markedly dependent on the PAANa content and on the environmental temperature and pH. The bead sample with a higher percentage of PAANa exhibited a lower swelling rate and slower drug release. The drug release profiles from SANBs were further studied in simulated intestinal fluid, and the results demonstrated here suggest that SANBs could serve as a potential candidate for controlled drug delivery in vivo. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Poly(N-vinylpyrrolidone)-grafted poly(N-isopropylacrylamide) copolymers: Synthesis, characterization and rapid deswelling and reswelling behavior of hydrogels

Poly(N-isopropylacrylamide)- block-poly(N-vinylpyrrolidone) diblock copolymer (PNIPAAm- b-PVPy) was successfully synthesized via sequential reversible addition-fragmentation chain transfer/macromolecular design via the interchange of xanthate (RAFT/MADIX) process, in which the chain transfer agent of xanthate was in situ afforded via the reaction of isopropylxanthic disulfide (DIP) with 2,2-azobisisobutylnitrile (AIBN). The RAFT/MADIX technique was employed to prepare the poly(N-vinylpyrrolidone)-grafted poly(N-isopropylacrylamide) copolymers (PNIPAAm- g-PVPy) with N,N-methylenebisacrylamide as the crosslinking agent. The comb-like PNIPAAm- g-PVPy copolymer networks with PVPy as the pendent chains were characterized by means of Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS). The hydrogel behavior of PNIPAAm- g-PVPy networks was investigated in terms of swelling, deswelling and reswelling tests. With the inclusion of PVPy chains, the swelling ratios of the hydrogels were significantly enhanced compared to the control PNIPAAm hydrogel. It is found that the PVPy-modified PNIPAAm hydrogels displayed faster response to the external temperature changes than the control PNIPAAm hydrogel. The improved thermoresponsive properties of hydrogels are ascribed to the formation of the comb-like architectures in the copolymer networks.

Hepta(3,3,3-trifluoropropyl) Polyhedral Oligomeric Silsesquioxane-capped Poly(N-isopropylacrylamide) Telechelics: Synthesis and Behavior of Physical Hydrogels

Hepta(3,3,3-trifluoropropyl) polyhedral oligomeric silsesquioxane (POSS)-capped poly(N-isopropylacrylamide) (PNIPAAm) telechelics with variable lengths of PNIPAAm midblocks were synthesized by the combination of reversible addition-fragmentation chain transfer polymerization (RAFT) and the copper-catalyzed Huisgen 1,3-cycloaddition (i.e., click chemistry). The POSS-capped trithiocarbonate was synthesized and used as the chain transfer agent for the RAFT polymerization of N-isopropylacrylamide. The organic-inorganic amphiphilic telechelics were characterized by means of nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). Atomic force microscopy (AFM) shows that all the POSS-capped PNIPAAm telechelics exhibited microphase-separated morphologies, in which the POSS terminal groups were self-assembled into the microdomains and dispersed into the continuous PNIPAAm matrices. The POSS nanodomains could behave as the physical cross-linking sites and as a result the physical hydrogels were formed while these POSS-capped PNIPAAm telechelics were subjected to the solubility tests with water. These physical hydrogels possessed well-defined volume phase transition phenomena and displayed rapid reswelling and deswelling thermoresponsive behavior compared to control PNIPAAm hydrogel.

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.

Probing mobility and structural inhomogeneities in grafted hydrogel films by fluorescence correlation spectroscopy

We employed fluorescence correlation spectroscopy to investigate the effect of crosslinking density, annealing in the dry state, temperature, and solvent quality on the one-dimensional swelling, permeability, and mobility of tracer molecules in thermoresponsive hydrogel films. These consist of a carboxylated poly(N-isopropylacrylamide) derivative (PNIPAAm) covalently anchored to glass substrates. Upon increasing the temperature beyond the collapse transition at about 32 °C, the gels shrunk from the swollen to a collapsed state. A molecular dye (Alexa 647) and green fluorescent protein were chosen as tracers as they display only weak interaction with the carboxylated PNIPAAm. At large swelling ratios (low temperatures) the hydrogel layers are spatially homogeneous and both tracers show single Fickian diffusion. Diffusion coefficients scale with the PNIPAAm volume fraction. Upon temperature increase a qualitatively different behavior is observed already in the pretransition region (25–32 °C) concurrently with moderate swelling ratios (<4). This is manifested by an additional, faster Fickian diffusion and structural inhomogeneities, which are also found by optical waveguide mode spectroscopy. Above the collapse transition all diffusants are expelled from the hydrogels at a limiting swelling ratio ∼1.5. Subtle differences in the solvent quality influence the diffusion of tracers in the PNIPAAm hydrogel films. In the transition temperature range structural inhomogeneities at the nanoscale appeared.

An infrared-light responsive graphene-oxide incorporated poly(N-isopropylacrylamide) hydrogel nanocomposite

We demonstrate a new light-responsive poly(N-isopropylacrylamide) (PNIPAAm) hydrogel nanocomposite incorporating glycidyl methacrylate functionalized graphene oxide (GO–GMA) instead of metallic nanoparticle fillers. GO–GMA is synthesized by esterification with glycidyl methacrylate. GO–GMA incorporated hydrogels are synthesized by photopolymerization of NIPAAm including 4-dimethylaminopyridine (DMAP) and N,N′-methylenebisacrylamide (NMBA) in GO–GMA dispersed dimethyl sulfoxide (DMSO) solution. Results show that such a nanocomposite hydrogel can undergo a large volumetric change in response to infrared (IR) light illumination, due to the highly efficient photothermal conversion of GO–GMA. It also exhibits significantly larger water uptake compared to conventional PNIPAAm hydrogel by 3 times. Based on the developed IR-responsive nanocomposite hydrogel, we have also realised a microvalve that controls the fluidic flow within a microfluidic channel through remote IR light actuation.

Poly(acrylic acid)-grafted Poly(N-isopropyl acrylamide) Networks: Preparation, Characterization and Hydrogel Behavior

Poly(acrylic acid)-grafted poly(N-isopropylacrylamide) co-polymer networks (PNIPAAm-g-PAA) were prepared via the reversible addition-fragmentation transfer (RAFT) polymerization of N-isopropyl- acrylamide (NIPAAm) with trithiocarbonate-terminated PAA as a macromolecular chain-transfer agent in the presence of N,N-methylenebisacrylamide. The PNIPAAm-g-PAA co-polymer networks were characterized by means of Fourier transform infrared spectroscopy, differential scanning calorimetry and small-angle X-ray scattering. It is found that the PNIPAAm-g-PAA co-polymer networks were microphase-separated, in which the microdomains of PNIPAAm-PAA interpolymer complexes were dispersed into the PNIPAAm matrix. The PNIPAAm-g-PAA hydrogels displayed a dual response to temperature and pH values. The thermoresponsive properties of PNIPAAm-g-PAA networks were investigated. Below the volume phase transition temperatures, the PNIPAAm-g-PAA hydrogels possessed much higher swelling ratios than control PNIPAAm hydrogel. In terms of swelling, deswelling and reswelling tests, it is judged that the PNIPAAm-g-PAA hydrogels displayed faster response to the external temperature changes than control PNIPAAm hydrogel. The improved thermoresponsive properties of hydrogels are ascribed to the formation of PAA-grafted PNIPAAm networks, in which the water-soluble PAA chains behave as the hydrophiphilic tunnels and allow water molecules to go through and, thus, to accelerate the diffusion of water molecules.

Thermoswitchable Electrokinetic Ion-Enrichment/Elution Based on a Poly(N-isopropylacrylamide) Hydrogel Plug in a Microchannel

This paper reports a novel protocol consisting of the thermomodulated electrokinetic enrichment, elution, and separation of charged species based upon a thermoswitchable swelling-shrinking property of a poly(N-isopropylacrylamide), PNIPAAm, hydrogel. A 0.2-1 mm long PNIPAAm hydrogel plug was photopolymerized inside a glass microfluidic channel to produce a composite device consisting of the PNIPAAm hydrogel plug and the glass microchannel (abbreviated as plug-in-channel). After voltage was applied to the composite device, anions, such as FITC, could be enriched at the cathodic end of the PNIPAAm plug when the temperature of the plug was kept below its lower critical solution temperature (LCST, ∼32 °C). The concentrated analytes could then be eluted by electroosmotic flow when the temperature of the plug was heated above the LCST. The mechanism of the thermoswitchable ion enrichment/elution process was studied with the results presented. The analytical potential of the composite device was demonstrated for the temperature-modulated preconcentration, elution, and separation of FITC-labeled amino acids.

Fabrication of Degradable Microsphere/PNIPAAm Hydrogel Combination Systems for Protein Delivery

A novel protein delivery system with incorporating PLGA microspheres into porous PNIPAAm hydrogel for sustained delivery of proteins was successfully developed. Distribution of PLGA microspheres in the hydrogel was determined by SEM, and the in vitro protein release characteristics of combination system were investigated by UV-vis spectroscopy. The SEM results showed that PLGA microspheres were encased into the interconnected porous structure of PNIPAAm hydrogel. In vitro release of BSA from combination system exhibited a less burst release followed by a slower sustained release for 20 days. The slow release after the initial burst release was due to the low permeability of BSA in PLGA microspheres. The controlled release of BSA encapsulated within microspheres embedded in scaffolds was better controlled when compared to delivery from PNIPAAm hydrogels alone. The combination systems showed great promise for application in protein delivery.