Controlled Drug Loading and Release of a Stimuli-Responsive Lipogel Consisting of Poly( N -isopropylacrylamide) Particles and Lipids

Controlled Drug Loading and Release of a Stimuli-ResponsiveLipogel Consisting of Poly(N‑isopropylacrylamide) Particles andLipidsNaiyan Lu,† Kai Yang,† Jingliang Li,§ Yuyan Weng,† Bing Yuan,*,† and Yuqiang Ma*,†,‡

†Center for Soft Condensed Matter Physics and Interdisciplinary Research, Soochow University, Suzhou 215006, P. R. China‡National Laboratory of Solid State Microstructures and Department of Physics, Nanjing University, Nanjing 210093, P. R. China§Institute for Frontier Materials, Deakin University, Waurn Ponds, Vic 3216, Australia

*S Supporting Information

ABSTRACT: Environmentally responsive materials are attractive for advancebiomedicine applications such as controlled drug delivery and gene therapies.Recently, we have introduced the fabrication of a novel type of stimuli-sensitive lipogelcomposite consisting of poly(N-isopropylacrylamide) (pNIPAM) microgel particlesand lipids. In this study, we demonstrated the temperature-triggered drug releasebehavior and the tunable drug loading and release capacities of the lipogel. At roomtemperature (22 °C), no calcein was released from the lipogel over time. At bodytemperature (37 °C), the release process was significantly promoted; lipids in thelipogel acted as drug holders on the pNIPAM scaffold carrier and prolonged thecalcein release process from 10 min to 2 h. Furthermore, the loading and release ofcalcein could be effectively controlled by modulating the relative amount of lipidsincorporated in the lipogel, which can be realized by the salt-induced lipid release ofthe lipogel.

1. INTRODUCTION

There has been enormous interest in the exciting area ofmanufactured microcarriers for drug and gene delivery, amongwhich the systems based on mesoporous inorganic nano-particles (such as gold, silica, and Fe3O4) are regarded to be themost attractive.1−4 However, the in vivo applications of thesematerials are very limited because of their insufficient drugloading and holding capacities. Linear polymers and supportedlipid membranes were exploited to encapsulate these conven-tional mesoporous particles to achieve higher drug loadingcapacity, prolonged cargo retention, and controlled drug releasewithin cells.5−8 Multicomponent composites, including thosethat introduce new components to conventional materials, havebecome more attractive in recent years because of their diversestructures and combined, even improved, properties over theoriginal components. These materials are of high importance inmaterial and biomedical science.9−14

On the other hand, environmentally sensitive materials arepromising candidates in “smart” bioengineering systems.15

Among different kinds of stimuli, temperature is the mostwidely used because it is easy to control and has practicaladvantages both in vitro and in vivo.8,16 As a classic example ofsuch a “smart” polymer, poly-N-isopropylacrylamide (pNI-PAM) has attracted considerable attention and has beenstudied for diverse biomedical applications because of itsbiocompatibility.8,17,18 When dissolved in water, pNIPAMshows temperature-, pH-, or ionic strength-responsive volumephase transition at a so-called lower critical solution temper-

ature (LCST) of around 32 °C.17,18 Below the LCST, pNIPAMchains are fully soluble and the polymer is in a swollen state inwater. Above the LCST, water is excluded from the vicinity ofthe pNIPAM chains, leading to significant shrinkage of thehydrogel.19 Although pNIPAM as an environmentally sensitivepolymeric matrix, such as a hydrogel for drug carrying, has beeninvestigated intensively, pNIPAM microgel particles asspherical mesoporous containers for the fabrication of newtypes of stimuli-responsive composites have received lessattention.20,21

Recently, we have successfully developed a novel type ofstimuli-sensitive lipogel by the incorporation of lipids withpNIPAM microgel particles. The lipogel displays environ-mentally responsive transformation behaviors because of thevolume phase transition of the pNIPAM scaffold.20 In thiswork, we will show that the lipid incorporation endows thelipogel with not only improved and prolonged cargo retentionability but also tunable loading capacity and stimuli-responsiverelease of the payloads. Furthermore, because of the multi-components of the lipogel, the presence of hydrophilic andlipophilic cavities created by lipids offers the possibility for theencapsulation and delivery of various hydrophilic and lipophilicsubstances.22

Received: March 22, 2013Revised: June 10, 2013

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2. MATERIALS AND METHODS

2.1. Materials. 1,2-Dioleoyl-sn-glycero-3-phosphocholine(DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine-N-(lissaminerhodamine B sulfonyl) (Rh-PE)were purchased from Avanti Polar Lipids and used as received.Chloroform (99.7%), ethanol (99.0%), Na2CO3, PDMS, andcalcein (analytical reagent) were purchased from ShanghaiChemical Reagents Company and used without furtherpurification. The pNIPAM particle was synthesized as reported(φ ∼ 0.5).23 It consisted of 90% NIPAM monomer, 10%positively charged AEME monomer, and a small amount ofcross-linking agent BIS-acrylamide.2.2. Lipogel Preparation. Lipogels were fabricated via a

facile solvent-exchange method.24 A 0.2 mg sample of lipid(DOPC labeled by 0.5 mol % Rh-PE, red fluorescence) wasfirst dissolved in chloroform (2.0 mg mL−1) and driedovernight under vacuum. The dry lipid film was rehydratedwith a 0.1 mL mixture of 40 vol % ethanol and 60 vol %pNIPAM aqueous suspensions (containing about 1010

pNIPAM particles). Distilled water (1 mL) was then addedto the mixture. A micelle-to-bilayer transition and liposomedeposition occur during this solvent-exchange process. Thebulk solution was centrifuged at 6000 rpm for 10 min. Thewash and centrifugation were repeated three times to removeexcessive lipids. The precipitates were resuspended in 500 μL ofdistilled water for use.2.3. Characterization. The size distribution and ζ potential

of the pNIPAM and lipogel particles were determined using aZetasizer Analyzer (ZETASIZER Nano-ZS90, Malvern Instru-ment Ltd., U.K.). The morphology of the particles wascharacterized on a scanning electron microscopy (SEM)instrument (Raith Pioneer) after the particles were freeze-dried.The optical observation was performed on an inverted

confocal fluorescence microscope (Zeiss, LSM 710) equippedwith a 100× oil objective. Rh-PE and calcein were excited at543 and 488 nm, and fluorescence was collected in the red andgreen channels, respectively. The temperature of the systemwas set and stabilized with the native temperature controlcomponents from Zeiss. All the experiments were carried out atroom temperature (22 °C) except stated otherwise.Before each observation, 500 μL of particle dispersions

(pNIPAM or lipogel) was pretransferred to a homemadesample cell with a PDMS-coated glass coverslip as substrate andstabilized for 2 h for particle immobilization (to a density of∼500 particles per cm2).25 For the calcein release tests, 100 μLof saturated calcein solution was added to the particledispersion in situ directly preceding observation, and theparticles were infiltrated with calcein immediately. The settings,including the laser power and amplifier offset, were maintainedconstant throughout the release period.26 The fluorescenceintensity of calcein integrated from a model calcein-loadedparticle was used to calculate the cumulative calcein releasepercentages: cumulative calcein release at certain time (%) = (1− fluorescence intensity integrated from the calcein-loadedparticle at certain time/fluorescence intensity at initial state) ×100%.

3. RESULTS AND DISCUSSION

3.1. Structural Characterization of Lipogel. At roomtemperature, the native pNIPAM microgel particles have acore−shell structure with a size of 2.7 ± 0.1 μm.20 However,the lipogel, with a size comparable to that of the original

pNIPAM particle (Figure S1, Supporting Information),presents a different sunlike morphology.20 The lipogel can beimaged clearly through the fluorescence channel under confocalmicroscopy because of the presence of the rhodamine-labeledlipid components (Figure 1). An encircling coating layer of

separated lipid assemblies (mostly vesicles or micelles), whichflares brightly in the fluorescence channel, is found adsorbed tothe surface of the pNIPAM scaffold. The existence of these lipidassemblies is also confirmed by SEM (Supporting Information,Figure S2). Beneath a dense layer of hydrophilic pNIPAMpolymers which presents obviously in the transmission channelbut flames feebly in fluorescence, a spherical core of the lipogelcan be distinguished in both the fluorescence and transmissionchannels, indicating a loose aggregate of polymer chains(mostly hydrophobic) incorporated with a large amount oflipid molecules. The lipogels are stable for days under ambientconditions.On the basis of the volume phase transition of pNIPAM

polymer when crossing the LCST of ∼32 °C, the mostpronounced character of the lipogel is its thermo-responsivetransformation.17,18,20 Figure 1 shows the morphologicaltransitions of both a native pNIPAM particle and a lipogelsphere when the temperature was increased from 22 to 37 °C.Both the particle and lipogel contracted to a much smaller sizeof 1.7 ± 0.1 μm (a ∼40% decrease in diameter). When thetemperature was lowered to 22 °C again, the spheres recoveredtheir initial shape and volume and the lipids of the lipogelrecovered their initial fluorescence distributions. For both thepNIPAM and lipogel spheres, such temperature-dependentreversible transformations can be repeated more than 10 times.

3.2. Temperature-Triggered Lipid Release. Besidesbeing an important component of cell membranes, lipids arealso a typical class of biomolecules with potential applicationsfor therapy and diagnosis.27 Additionally, they can be designedto be optimal assemblies for accessory transfer agents for cargodelivery.28 The diversity of lipids in terms of molecularstructure and ligand decorations enables functionalization of

Figure 1. Transmission and fluorescence micrographs of a nativepNIPAM particle (a, b) and a lipogel consisting of pNIPAM particleand lipids (c, d) at 22 (a, c) and 37 °C (b, d). (e) A schematicillustration of the structures of the particle and lipogel showing thetransformations when crossing the LCST of pNIPAM. The redfluorescence of the lipogel originates from the Rh-PE-labeled lipids. Allthe scale bars represent 2 μm.

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lipids, and consequently the lipogel, for advanced biochemicalapplications such as targeted drug release and genetherapy.5−7,28,29 Thus, in our experiments the lipid is alsoviewed as model drug. Figure 2 shows the dynamic release

process of lipids from the lipogel at 22 and 37 °C. At roomtemperature, the fluorescence intensity of the lipogel slightlychanges over time (with only 20% release after 8 h and thenstays constant for days), indicating that the most incorporatedlipids still stay inside the lipogel (Figure 2a). However, at 37 °C(above the LCST), the fluorescence intensity of the lipogeldecreases with time quickly (i.e., the cumulative lipid releaseincreases) in an approximately linear manner, and reachesequilibrium after about 7 h (Figure 2b). Not much quenchingof rhodamine occurs under similar experimental conditions;26

therefore, this phenomenon corresponds to a dynamic processof lipid release from the lipogel.3.3. Controlled Calcein Loading and Release. To

further explore the ability of the lipogel as an advancedmultidrug carrier, the loading and release of calcein were alsoinvestigated. As one of the generally used fluorophores, calceinhas been intensively used as a model drug in related studiesbecause of its water-soluble and membrane-impermeableproperties.30

3.3.1. Release of Calcein at Different Temperatures. Figure3 shows the release of calcein from the native pNIPAMparticles at 22 °C. It is noted that the calcein in pNIPAMparticles can be released completely within 30 min. The finalfluorescence intensity of calcein in the pNIPAM particle is evenlower than that of the bulk solution. Such instability of drugencapsulation, even at room temperature, explains whypNIPAM has rarely been used in particle form for drugimmobilization and delivery.31,32 In sharp contrast, little leakage

of calcein from the lipogel was found even after 2 h (Figure 4).In our experiment, the construction of such a lipogel withencapsulated calcein was imaged through different channelsunder confocal microscopy (Figure 4c). Calcein (green) waslocated and blocked within the encircling coating of lipidassemblies (red) surrounding the pNIPAM scaffold of thelipogel. This formulation demonstrated stability over time, withlittle release or leakage of either lipid or calcein. Obviously, theincorporation of the amphiphilic lipids significantly improvesthe loading capacity and prolongs the retention of hydrophilicsubstances such as calcein within the lipogel, especially in thecentral portion. The calcein-loaded capacity of the lipogel isestimated to be 466.9 μg g−1 (Supporting Information).When the temperature was increased to 37 °C (above the

LCST of pNIPAM), the release of calcein from both the nativepNIPAM and lipogel systems was significantly promoted. Thecalcein in the native pNIPAM particles was completely releasedwithin 10 min (along with the obvious volume contraction ofthe particle, Figure 5a,b), consistent with previous reports.33 Instriking contrast, for the lipogel system, the calcein releaseprocess was significantly prolonged to about 2 h under thesame conditions (Figure 5c and Supporting Information, FigureS3). The release profile can be roughly divided into two stageswith distinctly different release rates. The initial burst releasemight result from the temperature-triggered phase transitionand volume contraction of the lipogel, which induce theexpelling of much of the encapsulated water (with the water-soluble calcein) from the lipogel to bulk solution. After that, thesecond stage is a slow release of the remaining calcein untilequilibrium is reached. An initial burst release followed by asustained slow release provides the potential benefit ofachieving the best possible therapeutic effect.13 This indicatesthat the lipogel sphere can function as a slow release vehicle forentrapped aqueous species at body temperature; the pNIPAMscaffold serves as a drug carrier and the lipids act to slow downthe release of drug molecules. Although prolonged drug releasehas also been accomplished in previous studies in whichmacromolecules such as polyethylene glycol and chitosan wereapplied to control the release from pNIPAM polymers,34,35 therelease kinetics was still largely determined by the volume ofthe bulk hydrogel. For our lipogel, in view of the stable calcein

Figure 2. Release kinetics of lipids from the model lipogels at 22 (a)and 37 °C (b). Release profiles of lipids in (c) were obtained byintegrating the residual fluorescence intensity of the lipogel atpredetermined times. The white circles in (a) and (b) represent thearea for integration. The red fluorescence originates from the Rh-PE-labeled lipids.

Figure 3. (a) Release kinetics of calcein from a native pNIPAMparticle at 22 °C. (b) Corresponding intensity profiles of fluorescencesignals across the lipogel, marked by red arrows in (a).

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encapsulation at room temperature, such temperature-triggeredcalcein release of the lipogel (including an initial burst releasefollowed by a slow release process until equilibrium) indicatesthat the lipogel is an attractive candidate for addressing theproblem of premature drug release.

3.3.2. Controlled Release Kinetics of Calcein by Lipids in aLipogel. As described in Release of Calcein at DifferentTemperatures, lipids strongly influenced the loading and releaseof calcein from a lipogel. Thus, the amount of lipids in thelipogel undoubtedly affects the release behavior of calcein.Although an excessive amount of lipids was always needed forthe preparation of lipogels during the lipogel fabricationprocess, the relative quantity of lipid molecules incorporatedin each lipogel can be modulated through a salt-induced lipidrelease process of the lipogel, which controls the loadingcapacity and release kinetics of calcein.In one of our previous studies, we demonstrated that

adjusting the salt content of solution would lead to a controlledcycle of lipid release from the lipogel into bulk solution.20 Thatis, a salt incubation would trigger an immediate release of thecoating lipid assemblies of the lipogel into bulk solution. Afterthat, a water incubation would induce the recovery of thelipogel morphology to the initial state. Such salt-induced lipid-release processes can be repeated for more than five cycles,during which the fluorescence intensity of the lipogel,indicating the relative amount of lipids left within the lipogel,decreases successively. As a result, in this work we selectedlipogel samples individually at three different stages during thisprocess: the initial lipogel sphere (named Lipogel-A) and thelipogels after the first (Lipogel-B) and second (Lipogel-C)incubation cycles (Figure 6a). The distribution of thefluorescence intensities of the three lipogels proves that therelative amount of retained lipids reduces sequentially (Figure6b). We took the lipogels as drug carriers and mixed themseparately with the same amount of calcein for the loading.After that, the dynamic releasing processes of calcein from thecorresponding carriers at the same temperatures weremonitored.

Figure 4. (a) The release of calcein from lipogel spheres over time at 22 °C. (b) Corresponding intensity profiles of fluorescence signals across thelipogel, marked by red arrows and dashed squares in (a). (c) Confocal micrographs, including fluorescence, transmission, and overlaid images, of alipogel (with encapsulated calcein). Red, Rh-PE-labeled lipids; green, calcein.

Figure 5. (a) Release kinetics of calcein from a model pNIPAMparticle at 37 °C. (b) Corresponding intensity histograms offluorescence signals from the calcein loaded in the pNIPAM particle,marked by red arrows in the graphs above. (c) Release profile ofcalcein from a model calcein-loaded lipogel at 37 °C.

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Figure 6c presents the normalized calcein releasing profiles ofthe three types of lipogels. It is found that the release rate ofcalcein becomes lower from A to B and C, and the time neededto achieve equilibrium is ∼120, ∼60, and ∼30 min for A, B, andC, respectively. Moreover, the absolute quantity of calcein thatis released cumulatively also follows the order of A > B > C.Such observations are reasonable considering that with thedecrease in lipid content from A to B and C, the ability of thelipogels to encapsulate and retain calcein decreases, and as aresult, it becomes easier for the calcein in the lipogels to reachequilibrium. This result demonstrates that by adjusting thequantity of lipids within a lipogel, we can effectively modulatethe loading capacity and release kinetics of calcein (Figure 6d).

4. CONCLUSIONS

We demonstrated the temperature-triggered release of bothlipids and a hydrophilic model drug, calcein, from a lipogelmade from pNIPAM microgel particles and lipids; moreover,the thermo-responsive property makes the lipogel act as an on/off switch by blocking calcein leakage because of the presenceof lipids when the temperature is below the LCST. Lipids act asdrug holders on the pNIPAM scaffold within the lipogel. As aresult, adjusting the relative amount of lipids incorporated inthe lipogels leads to effective modulation of the loading capacityas well as the release kinetics of calcein. Our work suggests that

such lipid-incorporated lipogels might be excellent drug carriersfor combined delivery and controlled drug release for treatmentof diseases.

■ ASSOCIATED CONTENT*S Supporting InformationSEM images of pNIPAM and lipogel spheres, ζ potential profileof pNIPAM, and release kinetics of calcein from lipogel overtime. This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel/fax: 86 512 65220239 (B.Y.), 86 25 83592900 (Y.M.). E-mail: [email protected] (B.Y.), [email protected](Y.M.).NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was financially supported by the National ScienceFoundation of China (91027040, 31061160496, 21106114,11104192, and 21204058), the National Basic ResearchProgram of China (2012CB821500), and the Natural ScienceFoundation of Jiangsu Province of China (BK2012177). K.Y.

Figure 6. (a) Incubation series of a model lipogel in salt (0.3 M Na2CO3) and water, successively. The three types of lipogels, Lipogel-A, -B, and -C,were marked referring to the corresponding stages. (b) Fluorescence intensity distributions indicating the relative amount of lipids within the threelipogels. (c, d) Normalized calcein-releasing profiles and schematic images of the calcein-loaded lipogels corresponding to Lipogel-A, -B, and -C at 37°C.

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thanks the support of the Key Project of Chinese Ministry ofEducation (210208) and the Applied Basic Research Program(2010CD091). The authors thank Prof. Zexin Zhang (SoochowUniversity) for the pNIPAM synthesis.

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