Reswelling Rate Research Articles

Evaluation of porous bacterial cellulose produced from foam templating with different additives and its application in 3D cell culture

Bacterial cellulose (BC) is used in biomedical applications due to its unique material properties such as mechanical strength with a high water-absorbing capacity and biocompatibility. Nevertheless, native BC lacks porosity control which is crucial for regenerative medicine. Hence, developing a simple technique to change the pore sizes of BC has become an important issue. This study combined current foaming BC (FBC) production with incorporation of different additives (avicel, carboxymethylcellulose, and chitosan) to form novel porous additive-altered FBC. Results demonstrated that the FBC samples provided greater reswelling rates (91.57 % ~ 93.67 %) compared to BC samples (44.52 % ~ 67.5 %). Moreover, the FBC samples also showed excellent cell adhesion and proliferation abilities for NIH-3T3 cells. Lastly, FBC allowed cells to penetrate to deep layers for cell adhesion due to its porous structure, providing a competitive scaffold for 3D cell culture in tissue engineering.

A Novel Method for Preparing Poly(vinyl alcohol) Hydrogels: Preparation, Characterization, and Application

This work provides a new method to prepare poly(vinyl alcohol) (PVA) hydrogel. Compared with the traditional repeated freeze–thaw method, the physical cross-linking method was adopted to prepare a high-strength hydrogel in one step. The morphology, melting, and crystallization behavior and mechanical properties of the hydrogel were investigated. The hydrogel has a high water content and reswelling rate, as well as a high melting temperature and mechanical strength. It also has a stable cross-linked structure in the temperature range of 25–65 °C. The hydrogel fracture surface shows ductile and brittle fracture morphology. The protrusions in the hydrogel three-dimensional topography are more numerous and homogeneous when the solvent preparation ratio is 4–6. On the basis of the good plasticity of the hydrogels, tubular hydrogels with inner diameters of 1–6 mm are also prepared to widen their applications.

Synthesis and characterization of stereoregular poly(N-ethylacrylamide) hydrogel by using Y(OTf)3 Lewis acid

Macroporous poly(N-ethylacrylamide) (PNEAM) hydrogels have been prepared in a methanol-water (1:1, v/v) mixture in the presence of 0, 0.05, 0.1, 0.15, and 0.2 M yttrium trifluoromethanesulfonate [Y(OTf)3] Lewis acid (LA) concentrations. SEM micrographs show that the resulted hydrogels are highly porous. Swelling ratios of all the hydrogels in water decrease with the increase in the temperature, and the hydrogels show lower critical solution temperature (LCST) volume phase transition at around 75 °C. LCST of PNEAM homopolymers gradually increases with the increase in the isotacticity of the polymers. Moreover, the hydrogels show cononsolvency in different methanol-water mixtures at 50 °C. Deswelling rate of the hydrogels prepared in the presence of LA is higher than the gel prepared in the absence of LA and this rate increases with increase in the LA concentration. The reswelling rate of the hydrogels also follows the same order, that is reswelling rate also increases with the increase in the LA loading in the gels. All these results are explained on the basis of the formation of the highly porous hydrogels with a higher isotactic PNEAM chain segment in a methanol-water mixture in the presence of LA.

Role of N-vinyl-2-pyrrolidinone on the thermoresponsive behavior of PNIPAm hydrogel and its release kinetics using dye and vitamin-B12 as model drug

Temperature-sensitive hydrogels hold great promise in biological applications as they can respond to changes in physiological temperature to produce a desired effect like controlled drug delivery. In this study, a series of poly(N-isopropylacrylamide-co-N-vinyl-2-pyrrolidinone) thermosensitive hydrogels were synthesized by radical copolymerization of NIPAm with 1-vinyl-2-pyrrolidinone (NVP). By altering the initial NIPAm/NVP mole ratios, copolymers were synthesized to have their own distinctive lower critical solution temperature which was established using differential scanning calorimetry. The swelling behavior of the hydrogel was analyzed gravimetrically and it was observed that reswelling rate increases with increasing NVP mole ratio. Further characterizations of the hydrogels were performed using Fourier transform infrared spectroscopy and scanning electron microscopy. Release kinetics with respect to temperature was studied using methylene blue dye solution and vitamin B12. Kinetic modeling of the release profile revealed that the release mechanism is a non-Fickian diffusion mechanism. These results suggested that this material has potential application as intelligent drug carriers. The quantities of residual monomers in the PIV4 hydrogel were determined by HPLC method, and the results show almost complete conversion.

Fast deswelling and highly extensible poly(N-isopropylacrylamide)-hectorite clay nanocomposite cryogels prepared by freezing polymerization

Poly(N-isopropylacrylamide) (PNIPAm)-hectorite clay nanocomposite cryogels (NC-C gels) were prepared by freezing polymerization at −18 °C of NIPAm monomer in the aqueous hectorite clay suspensions. The clay concentration CxLS was found to be a key factor determining the characters of the NC-C gels, because the average interparticle distance became smaller than the clay diameter when CxLS > 6 w/v%. The NC-C gels of CxLS ≤ 6.0 w/v% were heterogeneous at the nascent state and became homogeneous after 24 h thawing, which endowed the cryogels self-improved mechanical strength and elongation up to 900%. On the contrary, for the NC-C gels of CxLS > 6.0 w/v%, the extensibility was about half of the corresponding normal NC gels, suggesting that the heterogeneity cannot be homogenized after 24 h thawing. The deswelling and reswelling rate of the NC-C gels of CxLS > 6 w/v% was significantly accelerated due to the micropore structure formed by the molten ice crystals. The micro-pores were observed with SEM and CLSM from the cryogels with the clay content higher than 6 w/v%. The extensibility of the NC-C gels was much higher than other cryogels with high deswelling rate, promising some possible applications.

Synthesis and Study of the Properties of Stereocontrolled Poly(N-isopropylacrylamide) Gel and Its Linear Homopolymer Prepared in the Presence of a Y(OTf)3 Lewis Acid: Effect of the Composition of Methanol–Water Mixtures as Synthesis Media

Poly(N-isopropylacrylamide) (PNIPAM) hydrogels and the corresponding linear homopolymers were synthesized in different methanol-water mixtures (x(m) = 0, 0.13, 0.21, 0.31, 0.43, 0.57, and 0.76, where x(m) is the mole fraction of methanol) in the presence of 0.1 M Y(OTf)(3) Lewis acid. The isotacticity (meso dyad (m), %) and cloud-point temperature of these homopolymers were gradually increased and decreased, respectively, with the increase in the x(m) values of the synthesis solvent mixtures. Moreover, the corresponding linear PNIPAM homopolymers prepared in the absence of Y(OTf)(3) showed an almost constant isotacticity of m = 45% and a cloud-point temperature of 33.0 °C. A SEM study revealed that the resulting hydrogels were highly porous except for the gels prepared at x(m) = 0 and 0.76. The swelling ratios of these hydrogels in water at different temperatures and in different methanol-water mixtures at 20 °C and the deswelling rate and the reswelling rate of these hydrogels were studied. All of these swelling results were compared with that of the corresponding gels prepared in the absence of a Lewis acid (Biswas, C. S.; Patel, V. K.; Vishwakarma, N. K.; Mishra, A. K.; Bhimireddi, R.; Rai, R.; Ray, B. J. Appl. Polym. Sci.2012, DOI: 10.1002/app.36318) and explained on the basis of the porosity of the gel, the state of aggregation and isotacticity of the PNIPAM chain segment, and the cononsolvency of the methanol-water mixture toward the PNIPAM chain segment.

Rapid Responsive Behaviors of the Dual Stimuli-Sensitive Poly(DEA-co-DMAEMA) Hydrogel via Comb-Type Grafted Polymeriziation

The dual-sensitive, comb-type grafted hydrogels were synthesized by grafting polymeric chains with freely mobile ends, which were composed of both N,N-diethylacrylamide (DEA) and (2-dimethylamino) ethyl methacrylate (DMAEMA), onto the backbone of crosslinked poly(DEA-co-DMAEMA) networks. The structure of the poly(DEA-co-DMAEMA) macromonomer was characterized by Nuclear Magnetic Resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR). The effects of the feed components, temperature, and pH on the swelling behavior of the hydrogels were studied. With the increase of the poly(DEA-co-DMAEMA) macromonomer content, the thermo- and pH-sensitivity of the hydrogels improved. The G-PDD hydrogels exhibited improved thermo- and pH-sensitive characteristics, such as faster deswelling and reswelling rates and great oscillating deswelling/swelling behavior, and the level of improvement depended on the poly(DEA-co-DMAEMA) macromonomer content. The proposed dual thermo- and pH-sensitive, comb-type grafted poly(DEA-co-DMAEMA) hydrogel has various potential applications.

Synthesis, characterization, and drug release properties of poly(N‐isopropylacrylamide) gels prepared in methanol–water cononsolvent medium

AbstractPoly(N‐isopropylacrylamide) (PNIPAM) hydrogels were simply prepared by free radical polymerization in different methanol–water mixture. A scanning electron microscopy study revealed that the freeze‐dried hydrogels were macroporous. The swelling ratios in water at 20°C of the resulting hydrogels followed the order: X0.43>X0.21>X0.76 ≈ X0.57>X0.31>X0.13>X0.06>X0, where Xm denotes a gel prepared in a methanol–water mixture with m mole fraction of methanol (xm). Below the lower critical solution temperature, the swelling ratio values of all of the hydrogels gradually decreased with the increase in the temperature. The complete collapse of the PNIPAM chain of all the gels occurred at about 38°C, whereas the same was observed at about 35°C for the conventional gel prepared in water. The swelling ratio values of all the PNIPAM gels in the methanol–water mixtures with different xm values at 20°C passed through a minimum in the cononsolvency zone. The deswelling rates of the hydrogels decreased in the following order: X0.43> X0.31> X0.21> X0.57> X0.76 ≈ X0.13> X0.06> X0. The reswelling rates of these hydrogels decreased in the following order: X0> X0.31> X0.06 ≈ X0.13 > X0.76> X0.57> X0.21> X0.43. The release rates of the Tramadol Hydrochloride drug at 37°C from the drug‐loaded hydrogels were almost same for all of the hydrogels. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Synthesis and characterization of porous poly(N‐isopropylacrylamide) hydrogels prepared in ethanol–water mixtures

AbstractPoly(N‐isopropylacrylamide) (PNIPAM) hydrogels were prepared by free‐radical polymerization in different ethanol–water mixtures. A scanning electron microscopy study revealed that the resulting hydrogels were macroporous. The swelling ratios of the resultant hydrogels in water at 20°C followed this order: X0.34 ≈ X0.68 > X0.48 > X0.09 > X0.04 > X0, where Xa denotes a gel prepared in an ethanol–water solvent mixture with an ethanol molar fraction of a. Below the lower critical solution temperature, the swelling ratio values of all of the hydrogels gradually decreased with increasing temperature. The complete collapse of the PNIPAM chain of all of these gels occurred at about 38°C, whereas the same was observed at about 35°C for the conventional gel prepared in water. The swelling ratio values of all of the PNIPAM gels with different molar fractions of ethanol at 20°C passed through a minimum in the cononsolvency zone. The deswelling rates of the hydrogels decreased in the following order: X0.34 > X0.48 > X0.68 > X 0.09 > X0.04 > X0. The reswelling rates of these hydrogels decreased in the following order: X0 > X0.04 ≈ X0.48 > X0.09 ≈ X0.68 > X 0.34. The freeze‐drying process decreased the swelling ratios but increased the deswelling and reswelling properties of the PNIPAM gels. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Fast-reversible light-driven hydrogels consisting of spirobenzopyran-functionalized poly(N-isopropylacrylamide)

We introduced different spirobenzopyran derivatives into poly(N-isopropylacrylamide) gels and evaluated the effects of spontaneous ring-opening rates of the spirobenzopyrans on light-responsive volume change behaviors of the gels. Rates of reswelling from the light-induced shrunken state of the spirobenzopyran-functionalized poly(N-isopropylacrylamide) (p(Sp-NIPAAm)) gels increased with increasing ring-opening rates of spirobenzopyrans in the gels. Bending motion of a rod-like p(Sp-NIPAAm) gel was invoked by local light-irradiation because of asymmetric shrinkage of the gel. Two types of bending motions were observed depending on the reswelling rates of the gels; a gel with a fast reswelling rate was steadily bent only during light-irradiation, whereas another gel with a slow reswelling rate showed a continuous bending motion even after the irradiation. In addition, a sheet-like gel with a fast reswelling rate was operated as a micro-conveyor whose conveying direction was arbitrarily controlled with a light-irradiation.

Synthesis and Characterization of Stereocontrolled Poly(N-isopropylacrylamide) Hydrogel Prepared in the Presence of Y(OTf)3 Lewis Acid

Macroporous poly(N-isopropylacrylamide) (PNIPAM) hydrogels have been prepared in methanol-water (1:1, v/v) mixture in the presence of 0, 0.05, 0.1, 0.15, and 0.2 M Y(OTf)(3) Lewis acid concentrations. Synthesis of the corresponding linear PNIPAM homopolymers in the absence of a cross-linker keeping all other conditions the same shows that the isotacticity (meso dyad, %) and the cloud point temperature of the resulted in polymers increases and decreases, respectively, with the increase in the concentration of the Lewis acid. SEM micrographs reveal that the resulted hydrogels are highly porous. Swelling ratios of all the hydrogels in water decrease with the increase in the temperature. Moreover, swelling ratios of all the hydrogels in different methanol-water mixtures pass through a minimum in the co-nonsolvency zone, and the co-nonsolvency zone shifts toward the lower methanol-content solvent mixture with gradual increase in the Lewis acid concentration. Deswelling rate of the hydrogel prepared in methanol-water (1:1, v/v) mixture is much faster than that of the conventional hydrogel prepared in water. Moreover, the deswelling rate slightly increases with the hydrogels prepared with the increasing concentrations of Lewis acid. But, the reswelling rate of the hydrogels follows almost the reverse order. All these results have been explained on the basis of the formation of highly porous hydrogels with higher isotactic PNIPAM chain segment owing to the faster polymerization rate in the methanol-water mixture in the presence of Lewis acid and the co-nonsolvency behavior of the methanol-water (1:1, v/v) mixture toward PNIPAM chain segment in the PNIPAM hydrogel.

Preparation of pH Sensitive Polyacrylic Acid(PAAc) Macroporous Hydrogels in Aqueous NaCl Solutions and their Characterization

In this study, macroporous, pH sensitive polyacrylic acid hydrogels were prepared by using different concentration of aqueous NaCl solutions as the polymerization medium. The optical photos showed that the pore morphologies of the hydrogels were dependent on the concentration of NaCl. Swelling studies showed an increase in equilibrium swelling ratio as the NaCl concentration in the polymerization medium increased from 0 to 0.7mol/L. The hydrogels exhibited stronger sensitivity to pH changes, and showed much faster reswelling and deswelling response rates with the NaCl concentration increasing in the polymerization medium.

Temperature-sensitive PVA/PNIPAAm semi-IPN hydrogels with enhanced responsive properties

A series of temperature-sensitive hydrogels of semi-interpenetrating polymeric networks (semi-IPN) composed of poly( N-isopropylacrylamide) (PNIPAAm) and poly(vinyl alcohol) (PVA) were prepared by radical polymerization. The PNIPAAm networks were cross-linked by N, N′-methylenebisacrylamide in the presence of linear PVA. The reaction processes were investigated by rheometry using oscillatory deformation tests. It was found that gelations were very fast and the modulus reached equilibrium within about 12.5 min. The prepared semi-IPN hydrogels were characterized for their morphologies and thermal behaviors by scanning electron microscopy and differential scanning calorimetry, respectively. The interior network structures of the semi-IPN matrix became more porous with increasing PVA. In comparison to the conventional PNIPAAm gel, the newly reported semi-IPN hydrogels exhibited the same lower critical solution temperature. Their swelling properties, such as temperature dependence of equilibrium swelling ratio, shrinking kinetics and reswelling kinetics in water, were also studied. Experimental data indicated that the shrinking and reswelling rates of the semi-IPN hydrogels were much faster than those of the conventional PNIPAAm hydrogels. With this novel approach, water absorption and response properties could be adjusted by tuning the feed ratio of NIPAAm and PVA. These fast responsive hydrogels exhibited improved temperature sensitivity and swelling properties compared to the conventional PNIPAAm hydrogel, which would be critical and desirable for a gel to find potential applications in biomedical fields, such as drug delivery systems and sensors.

Thermosensitive Poly( N-isopropylacrylamide-co-acrylonitrile) Hydrogels with Rapid Response

Abstract Acrylonitrile (AN) was copolymerized with N-isopropylacrylamide (NIPA) to synthesize thermosensitive hydrogels, and the on-off switch behavior of poly(NIPA-co-AN) hydrogels with different fraction of hydrophobic component (AN) was investigated. It is found that the lower critical solution temperature (LCST), the swelling ratio at certain temperature and the reswelling rate of poly(NIPA-co-AN) hydrogels decreased as AN unit fraction in copolymers increased. In order to improve the responsive rate of poly(NIPA-co-AN) hydrogels, they were further treated by surface crosslinking using N, N′-methylene bisacrylamide (BIS) as a crosslinking agent. The swelling and deswelling behaviors of these copolymers were compared with those of the untreated hydrogels. The results indicated that the responsive rate of poly(NIPA-co-AN) hydrogel was improved by surface crosslinking. The resulting hydrogels bearing cyano groups with fast response have potential applications in the field of drug-controlled release and immobilization of biomolecules.

Preparation and characterization of novel physically cross-linked hydrogels composed of poly(vinyl alcohol) and amine-terminated polyamidoamine dendrimer.

Poly(vinyl alcohol) (PVA) and polyamidoamine (PAMAM) dendrimers are water-soluble, biocompatible and biodegradable polymers, which have been widely applied in biomedical fields. In this paper, novel physically cross-linked hydrogels composed of PVA and amine-terminated PAMAM dendrimer G6-NH(2) were prepared by cyclic freezing/thawing treatment of aqueous solutions containing PVA and G6-NH(2). The FT-IR analysis and elemental analysis indicated that PAMAM dendrimer G6-NH(2) was successfully introduced into the formed hydrogels, possibly via hydrogen bonds among hydroxyl groups, amide groups and amino groups in PVA and PAMAM dendrimer in the process of freezing-thawing cycle. Compared with physically cross-linked PVA hydrogel, PVA/G6-NH(2) hydrogels show higher swelling ratios and faster re-swelling rate due to the higher hydrophilicity of PAMAM dendrimer G6-NH(2). Higher contents of G6-NH(2) in PVA/G6-NH(2) hydrogels resulted in higher swelling ratios and faster re-swelling rates. With increasing freezing/thawing cyclic times, the swelling ratios and re-swelling rates of PVA/G6-NH(2) hydrogels decreased, which is similar to that of physically cross-linked PVA hydrogel. Combining the special host property of polyamidoamine dendrimer, these novel physically cross-linked hydrogels are expected to have potential use in drug delivery, including improving drug-loading amounts in hydrogels and prolonging drug release time. Swelling ratios of physically cross-linked PVA/G6-NH(2)-50 hydrogels prepared by three, six, nine freezing/thawing cycles. The swelling equilibrium experiments were carried out in distilled water at 25 degrees C.

The Influence of Cold Treatment on Properties of Temperature-Sensitive Poly(N-isopropylacrylamide) Hydrogels

Poly(N-isopropylacrylamide) (PNIPAAm) hydrogel exhibits a response to external temperature variation and shrinks in volume abruptly as the temperature is increased above its lower critical solution temperature. It has great potential applications in biomedical fields. A rapid response rate is essential, especially when this material is designed as an on–off switch for targeted drug delivery. However, due to the appearance of a thick, dense skin layer on the hydrogel surface during the shrinking process, the deswelling rate of conventional PNIPAAm gels is low. In this article, a novel method is proposed to modify the surface morphology of PNIPAAm gel, in which the swollen gels are frozen at low temperature (−20°C). The scanning electron micrographs revealed that a fishnet-like skin layer appeared on the surfaces of the cold-treated gels. Dramatically rapid deswelling was achieved with the cold-treated gels since the fishnet-like structure with numerous small pores prevented the formation of a dense, thick skin layer during the deswelling process, which commonly occurs in normal PNIPAAm hydrogels. Prolonging the cold treatment from 1 day to 10 days resulted in a slightly higher deswelling rate. Rearrangement of the hydrogel matrix structure during the freezing process might contribute to the formation of the fishnet-like skin layer. The water uptake of the hydrogels increased nearly in proportion to the square root of time, indicating that the reswelling rate of hydrogels was controlled predominantly by water diffusion into the network. However, there were no significant differences in the equilibrated swelling ratio and reswelling kinetics at room temperature (22°C) between normal gels and cold-treated gels, which implied that cold treatment did not change bulk porosity and gel tortuosity much.

Preparation of fast responsive, temperature-sensitive poly(N-isopropylacrylamide) hydrogel

A new poly(N-isopropylacrylamide) (PNIPA) hydrogel was prepared by decreasing the temperature of polymerization/crosslinking reaction below the freezing point (-18 °C). This PNIPA hydrogel shows a large swelling ratio at room temperature and exhibits a fast deswelling and reswelling rate in response to external temperature changes. These properties are attributed to the macroporous and regularly arranged network of this PNIPA hydrogel.

Synthesis and characterization of thermally reversible macroporous poly(N‐isopropylacrylamide) hydrogels

AbstractMacroporous hydrogels are characterized by large pore sizes, high pore volumes, and high specific surface area. Besides these characteristics, macroporous hydrogels based on thermally reversible polymers respond to temperature changes much faster than hydrogels prepared by a conventional method. Crosslinked poly(N‐isopropylacrylamide) (polyNIPAAm) forms a thermally reversible hydrogel which shows a lower critical solution temperature (LCST) ca. 33°C in aqueous solutions. We have synthesized thermally reversible polyNIPAAm hydrogels having macroporous structures by a new method. These macroporous hydrogels have large pore volumes, large average pore sizes, and faster macromolecule permeation rates in comparison to conventional polyNIPAAm hydrogels synthesized by a conventional method. Compared with conventional polyNIPAAm hydrogels, the macroporous polyNIPAAm hydrogels have higher swelling ratios at temperatures below the LCST and exhibit faster deswelling and reswelling rates. The deswelling rates are especially rapid. These thermally reversible macroporous hydrogels may be very useful in controlled active agent delivery and toxin removal, as well as dewatering of solutions. Peptides or proteins may behave as if they were in bulk solution within the large aqueous pores, and this may reduce their inactivation when such gels are used for their storage and later release. The gels may also be useful in microrobotic devices due to their fast response to temperature. © 1992 John Wiley & Sons, Inc.

Modulation of salt permeabilities of intestinal brush-border membrane vesicles by micromolar levels of internal calcium

A possible modulation of ion permeabilities of rat intestinal brush-border membrane vesicles by Ca 2+, a putative second messenger of salt secretion, was explored by three independent methods: (1) measurements of [ 3H]glucose accumulation driven by a Na + gradient; (2) stopped-flow spectrophotometry of salt-induced osmotic swelling; (3) 86Rb +, 22Na + and 36Cl − flux measurements. Cytoskeleton-deprived membrane vesicles were prepared from isolated brushborders by thiocyanate treatment. Intravescicular Ca 2+ levels were varied by preincubating vesicles in Ca-EGTA buffers in the presence of the Ca 2+-ionophore A23187. At Ca free 2+ > 10 −5 M , initial Na +-dependent glucose uptake in the presence of a 0.1 M NaSCN gradient (but not in its absence) was inhibited by about 50 per cent as compared to EGTA alone ( ED 50≅10 −6 M Ca 2+ ). By contrast, initial rates of 22Na + uptake and reswelling rates of vesicles exposed to a NaSCN gradient were increased at least 2-fold by 10 −5 M Ca free 2+. Both observations are compatible with a Ca 2+-induced increase of the Na +-permeability of the vesicle membrane. The modulation of ion transport was fully reversible and critically dependent on internal Ca 2+, suggesting a localization of Ca 2+-sensor sites at the inner surface of the microvillous membrane. As shown by radiotracer and osmotic swelling measurements, micromolar Ca 2+ additionally increased the flux rate of K +, Rb +, Cl − and NO 3 − but did not change the membrane permeability for small uncharged molecules, including glucose and mannitol. The effect of Ca 2+ on ion permeabilities could be blocked by Ba 2+ (10 −3 M) or Mg 2+ (10 −2 M), but not by amiloride (10 −3 M), apamin (2 · 10 −7 M), trifluoperazine (10 −4 M) or quinine (5 · 10 −4 M). At present it is unclear whether Ca 2+ activates a nonselective cation and anion channel or multiple highly selective channels in the vesicle membrane.