Drug Delivery, 13:245–251, 2006Copyright c© Taylor & Francis Group, LLCISSN: 1071-7544 print / 1521-0464 onlineDOI: 10.1080/10717540500313067

Drug Delivery System Based on Covalently BondedPoly[N-Isopropylacrylamide-co-2-Hydroxyethylacrylate]-Based Nanoparticle Networks

Anu Stella Mathews, Chang-Sik Ha, Won-Jei Cho, and Il KimDepartment of Polymer Science and Engineering, Pusan National University, Jangjeon-dong,Geumjeong-gu, Busan, Korea

The present work is focused on investigating the con-trolled drug release behavior of Poly [N-isopropylacrylamide-co-2-Hydroxyethylacrylate] (P [NIPAm-HEAc])-based hydrogel. Thesynthesis process includes the preparation of monodispersed hy-drogel nanoparticles containing specific functional groups, fol-lowed by cross-linking to neighboring spheres to stabilize the entirenetwork. The color and volume of these crystalline hydrogel net-works can reversibly change in response to external stimuli such astemperature, pH, and other environmental conditions. The feasibil-ity of the hydrogel as a controlled release vehicle for 5-fluorouracilis evaluated.

Keywords Drug Delivery, Hydrogels, Nanoparticles, N-Isopropylacrylamide, pH Sensitive, Thermoresponsive

Carrier-mediated drug delivery has witnessed a drasticchange in the past two decades, where the focus has shiftedfrom uncontrolled release systems to controlled release systems.Swelling controlled release systems are valuable among the con-trolled release systems due to their capability of delivering drugsat constant rates over an extended period of time (Peppas andKhare 1993). Hydrogels, because of their ability to swell or col-lapse reversible in response to external stimuli, is an extensivelystudied swelling controlled release system (Tanaka et al. 1982;Siegel and Firestone 1988; Chen and Hoffman 1995; Beebe et al.2000). In particular, poly [N-isopropylacrylamide] (PNIPAm)gels have attracted much interest owing to its biocompatibilitytogether with thermosensitivity, which leads to a temperature-modulated drug release (Dong and Hoffman 1990; Zhang and

Received 31 March 2005; accepted 15 June 2005.This work was supported by grant no. R01-2003-000-10020-0 from

the Basic Research Program of the Korea Science & Engineering Foun-dation. The authors also are grateful to the National Research Labo-ratory Program, the Center for Ultramicrochemical Process Systems(ERC), the Center for Integrate Molecular Systems (SRC), and theBrain Korea 21 Project.

Address correspondence to Il Kim, Department of Polymer Scienceand Engineering, Pusan National University, Jangjeon-dong, Busan,609-735, Korea. E-mail: ilkim@pusan.ac.kr or ik58@cornell.edu

Peppas 1990). PNIPAm homopolymer exhibits a lower criticalsolution temperature (LCST) around 32◦C in aqueous solutionand its hydrogel swells and shrinks in water below and abovethis temperature.

Chemical modifications have been carried out by incorporat-ing another functional component into PNIPAm chain, for thefurther expansion of applications of the PNIPAm. These hydro-gels can be prepared by copolymerizing two different monomers(Bae, Okano, and Kim 1991a, 1991b): by forming interpenetrat-ing polymer networks (Qui and Park 2001) or by creating net-works with microporous structures (Kabra, Gehrke, and Spontak1998). The hydrogels can be made in bulk or in nano- or mi-croparticle form. The bulk gels are easy to handle but usuallyhave very slow swelling rates. Gel nanoparticles react quicklyto an external stimulus but may be too small for practical appli-cations. Hu and Gao (2001) stabilized PNIPAm nanoparticles inbulk forms by bonding the nanoparticles to form a network. To-gether with thermal responsive character, the nano particles alsoare found to be sensitive to pH, solvent, salt, and concentration(Hu et al. 2004).

Our aim is to investigate the feasibility of these covalentlybonded PNIPAm nanoparticle networks for controlled drug re-lease system. For this purpose, we have synthesized and charac-terized covalently bonded P [NIPAm-HEAc] nanoparticles. Inthis research these hydrogel were loaded with the model drug 5-fluorouracil (5-FU) that has been used as antitumor drug. The re-lease behavior of 5-FU at different temperatures (25◦C to 45◦C)and pHs (3 to 9) were investigated.

MATERIALS AND METHODSNIPAm was recrystallized from a benzene/n-hexane mixture.

N, N′ methylene-bis-acrylamide (MBS) and potassium persul-phate (KPS) were purified by recrystallizing three times frommethanol. 2-Hydroxymethylacrylate (HEAc), sodium dodecylsulphate (SDS), and divenyl sulpone (DVS) were used withoutfurther purifications. 5-FU was purified by recrystallizing fromethanol. All the reactions were performed under a purified ni-trogen atmosphere using standard Schlenk techniques.

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246 A. S. MATHEWS ET AL.

Synthesis of P [NIPAm-HEAc] NanoparticlesThe precipitation polymerization of NIPAm was carried out

in a flask equipped with a mechanical stirrer and nitrogen feed.NIPAm (3.8 g), MBS (0.066 g), and SDS (0.10 g) were dis-solved in 260 ml deionized water, under continuous stirring.N2 gas is bubbled into the solution for 1 hr. HEAc (0.4 ml)was added dropwise before placing into a 70◦C hot bath. Afterheating the solution to 70◦C under nitrogen for 40 min, 0.17 gKPS was added to initiate polymerization. The reaction wascarried out at 70 ± 1◦C under N2 atmosphere for 5 hr to ensurethat all monomers were reacted. After cooling down to roomtemperature, the final reaction dispersion of P [NIPAm-HEAc]nanoparticles was purified via dialysis by changing stirring wa-ter outside the dialysis tube twice a day for 2 weeks, at roomtemperature. The resultant dispersion was further purified by ul-

FIG. 1. (a) Photographs of covalently bonded poly [N-isopropylacrylamide-co-2-hydroxyethylacrylate] (P [NIPAm-HEAc]) nanoparticle networks at variousparticle concentrations, (wt%), from left to right: 3, 3.5, 3.8, 4.1, 4.7, 5.0, 5.5, 6.0, 7, and 7.9 wt%. (b) Highlights of the crystal phase, from left to right: 2.8, 3.2,3.6, 3.9, and 4.1 wt%.

tracentrifugation to remove all unreacted chemicals and smallmolecules. The concentration of the dialyzed dispersion wascalculated from the weight difference of the solution before andafter drying at 120◦C. The percentage of polymer in dispersionwas calculated from the following:

C = (W/Wo) × 100 [1]

where Wo is the initial weight of the sample and W is the weightof the sample after drying at 120◦C.

Preparation of Covalently Bonded NIPAm-HEAcNanoparticle Networks

Different weight percentages of the pure P [NIPAm-HEAc]solution (3 wt% to 7 wt%) was prepared in deionized water

COVALENTLY BONDED PNIPAm 247

and heated above the phase transition temperature. DVS wasadded dropwise to make it homogeneously dispersed through-out the solution. Then the dispersion was cooled below 25◦Cand NaOH solution at pH13 was added. The resultant hydro-gel gave different color (Figure 1a) at different concentrationsand colored speckles in a very narrow concentration range(3–5 wt%), at 25◦C indicating the formation of large colloidalcrystals (Figure 1b).

Swelling StudiesSwelling behaviors of the nonbonded P [NIPAm-HEAc] gels

(NBG) and covalent bonded P [NIPAm-HEAc] gels (CBG) atdifferent temperatures (25–45◦C) and different pH (1–14) werestudied. The water content of the hydrogels was estimated by thedifference between the weight of fully swollen hydrogel samples(Weq ), after careful wiping of their surface with a soft tissue, andthe weight of the samples after being perfectly dried (Wi ). Themass equilibrium swelling ratio, SR, and the rate of swelling, k(min−1) were calculated using equations (2) and (3).

SR = [(Weq − Wi )/Wi ] [2]

kt = − ln(Weq − Wt ) [3]

where Wt is the sample weight at time t .

UV-Visible Spectroscopic CharacterizationThe environmentally induced color change of the nonbonded

and bonded hydrogels was measured using UV-Visible Spec-trometer (Shimadzu 1650 PC). The spectra of the hydrogels atdifferent temperatures and different concentrations were taken.

5-FU Loading and ReleaseThe covalently bonded particles (CBG) were thoroughly

washed using deionized water and perfectly dried before loading5-FU. A definite mass of the perfectly dried covalently bondedP [NIPAm-HEAc] nanoparticles (CBG) is then allowed to swellin different concentrations (1 mg–7 mg) 5-FU solution in 10 mlbuffer solution at 27◦C. The amount of the drug absorbed to thehydrogel matrix was monitored using UV-Visible spectrometerafter regular intervals of time until a constant value was reached.The 5-FU loading capacity of the hydrogel was calculated usingthe following equation:

LC = [W d/W ] × 100 [4]

Then each loaded hydrogel was rinsed with water and directlyimmersed into vials containing 10 ml pH 7 buffer solution placedin water baths at different temperatures to evaluate the tempera-ture sensitive release behavior. Simultaneously a different set ofdrug loaded hydrogels was immersed into different pH solutions(pH 3–pH 9) at room temperature to study the pH sensitive re-lease behavior of covalently bonded P [NIPAm-HEAc] nanopar-ticle networks. For simulating the in vivo behavior of the system,

release rate also was evaluated when the gel was immersed firstin pH 3 medium, then, after completion of the release, trans-ferred to pH 6 medium, and subsequently to pH 8 solution. Theamount of 5-FU released was measured spectrophotometricallyin periodically taken samples and again placed in the same vesselso that the liquid volume was kept constant. The release profilesbetween 10% and 70% release were characterized by fitting thefollowing equation by nonlinear regression (Peppas et al. 2000).

Mt/M∞ = ktn [5]

where Mt/M∞ is the fraction of drug released at time t. Allrelease measurements were carried out in duplicate for eachhydrogel and the average values were plotted.

RESULTS AND DISCUSSION

Characterization of HydrogelsThe swelling ratio is one of the most important parameters

for evaluating hydrogels. Figure 2 shows the temperature depen-dence of the swelling ratio of the hydrogels. The swelling ratioof the covalently bonded nanoparticles (CBG) is smaller thanthat of the nonbonded particles (NBG) at the temperature belowLCST. When the temperature is raised, NBG showed a sharpdecrease in the swelling rate at 32◦C indicating phase transition.But CBG showed the phase transition at 40◦C showing higherthermal stability than NBG. As the temperature increases, bothgels shrink due to the intrachain hydrophobic interaction. Fromthe DVS cross-linking less water can be absorbed in CBG andthus its swelling ratio is less than that of NBG.

FIG. 2. The temperature-dependent swelling ratio of nonbonded (NBG) andcovalent bonded (CBG) P [NIPAm-HEAc] nanoparticles.

248 A. S. MATHEWS ET AL.

FIG. 3. pH dependence of the equilibrated swelling ratio of nonbonded (NBG) and covalent bonded (CBG) P [NIPAm-HEAc] nanoparticles; photos indicate pHresponsive color and size changes of completely swelled hydrogels in different pH solutions. The inset is the perfectly dried hydrogel cut in the shape of butterfly(the red bar represents 1 cm).

The sensitivity of the nanoparticles toward pH is shownin Figure 3. The pH-dependent swelling curve shows that thewater uptake of the hydrogel networks at lower pH values(pH 1–6) is less than that at higher pH solutions (pH 8–14). Inbasic solutions H+ of HEAc comes off and combines with OH−

to form H2O. Charge is compensated by the cations that enterthe gel together with another OH-. Thus, the charge neutralityis maintained. The increased cation concentration gives rise toan osmotic pressure that causes the gel to swell (Sudipto et al.2002). In acidic solutions the gel shrinks to maintain the concen-tration balance (see the photos inside Figure 3). The data revealthat this behavior becomes pronounced as the cross-linking oc-curs, due to the reduced mobility of functional groups in HEAc.

Figure 4 shows the UV-Visible spectra of the nanoparticlesbefore and after cross-linking with DVS at different concentra-tions. The bonded hydrogels exhibit a shoulder shaped curvecorresponding to the color (Hu et al. 2004). This shoulder di-minishes according to the raise in the temperature and disap-pears above LCST (Figure 5a). The covalent linkage betweennanoparticles also results in the remarkable thermal stability ofthe hydrogels. The iridescent pattern at room temperature be-

came invisible when the sample was heated at 40◦C, at whichpoint the gel became cloudy due to phase separation. The non-bonded hydrogels showed the color only up to 32◦C and abovethat temperature phase separation occurred, which is evidentfrom the opaqueness of the system (Figure 5b).

5-FU Loading and ReleaseTo investigate the controlled release behavior of bonded P

[NIPAm-HEAc] nanoparticles, the dried hydrogels were im-mersed in a known concentration of 5-FU solution in pH 7 buffersolutions at 27◦C. The rate of absorption of the drug was mon-itored using UV-Visible spectroscopy. We observed that ∼40%of 5-FU in the solution was incorporated into the hydrogel ma-trix, within 24 hr. The release of 5-FU from the CBG networksat different temperatures is shown in Figure 6. Fast drug releaseis observed up to 1 hr. The hydrogel exhibits initial burst re-lease of 5-FU and about 45–50% of the total amount of drug isreleased during the first 30 min. The maximum release was ob-served within 5 hr. Drug release from a gel is controlled by twofactors: the diffusivity of the drug and the swelling of the poly-mer. When a gel is placed into a solution at constant temperature

COVALENTLY BONDED PNIPAm 249

FIG. 4. UV-Visible spectra of nonbonded (NBG) and covalently bonded(CBG) P [NIPAm-HEAC] nanoparticle networks at different concentrations.

and pH, water molecules begin to diffuse through the gel sur-face and enter the medium. Thus, the first burst release probablyis due to the fast diffusion of the drug accumulated. The drugrelease behavior of CBG at different temperatures reveals thatthe maximum release (96%) occurs at 27◦C within 7 hr.

The release rate decreases with the increase of temperaturedue to the aggregation of the polymer chains occurring as aresult of the hydrophobic interaction driving force among thehydrophobic groups (Zhang et al. 2004). The hydrogel was foundto release 89% of drug incorporated into its matrix at 37◦C,which hints at the fact that it can be an effective temperaturesensitive release system.

The pH responsive drug release of the NBG is representedin Figure 7. The data show that the drug release rate is maxi-mum at pH 7 (96% within 7 hr), In a lower pH (pH3–6), thehydrogel shrinks with a minimum release and in alkaline pH therelease rate is higher because the increased cation concentra-tion gives rise an osmotic pressure that causes the gel to swell.As a result the release of 5-FU from the covalently bonded P[NIPAm-HEAc] hydrogels can be modulated by changing thetemperature as well as the pH.

In our study we paid special attention to what extent the pHhistory of the hydrogel may alter its release properties. Theseparticularly important aspects for predicting the in vivo behav-ior have been rarely reported (Alvarez-Lorenzo and Concheiro2002). With the aim of simulating the evolution of the physiolog-ical pH when a formulation is orally administered, each hydrogelfirst was immersed in medium at pH 3, then transferred to pH 6solution, and finally to pH 8, all at 37◦C. As shown in Figure 8,

(a)

(b)

FIG. 5. (a) UV-Visible spectra of nonbonded P [NIPAm-HEAc] nanoparticlesat different temperatures. The temperatures (◦C) of the curves from left to right:25◦, 27◦, 30◦, 31◦, 32◦, 33◦, and 35◦C. UV-Visible spectra of covalently bondedP [NIPAm-HEAc] nanoparticles at different temperatures. The temperatures(◦C) of the curves from left to right: 25◦, 27◦, 31◦, 35◦, 37◦, 41◦, and 45◦C.

5-FU is not released at acidic pH. The behavior at pH 6, af-ter adjusting from pH 3, is totally different from that observedwhen the hydrogel was directly immersed in the pH 6. This sug-gests that when a strong hydrophobic interaction is promoted(at pH 3), the drug is completely ionized. The release exponent

250 A. S. MATHEWS ET AL.

FIG. 6. Cumulative release of 5-FU from covalently bonded P [NIPAm-HEAc] nanoparticle networks as a function of time at various temperatures.

n (Eq. 5) of the CBG, when directly immersed in pH 6 solutionat 37◦C, was lower (n = 0.25 ± 0.05) than that immersed intopH 6 solution after adjusting from pH 3 (n = 0.45 ± 0.05). Inaddition, for the systems at pH 8 the release mechanism seemsto be affected by the presence of ions. The relative exponent nwas significantly higher (n = 0.72 ± 0.05) for the hydrogels

FIG. 7. Cumulative release of 5-FU from covalently bonded P [NIPAm-HEAc] nanoparticle networks as a function of time in various pH solutions.

FIG. 8. Effect of pH on 5-FU release from covalently bonded P [NIPAm-HEAc] nanoparticles at 37◦C.

immersed in pH 8 solution after immersing in pH 3 and pH6 media than that immersed directly into pH 8 solution (n =0.4±0.05), which may be due to a different balance of the elec-trostatic and the hydrophobic interactions. Values of n below 0.5may be related to the combined diffusion partially through theswollen matrix and partially through water filled pores (Alvarez-Lorenzo and Concheiro 2003). There for, the pH history ofthe hydrogel may govern its release behavior under in vivoconditions.

We considered, therefore, that the loaded covalently bondedP [NIPAm-HEAc] nanoparticles would prevent 5-FU release inthe stomach allowing drug release at higher pH of the gastroin-testinal tract. These facts make the CBG a suitable candidate forpH sensitive drug delivery systems.

CONCLUSIONThe release rate of 5-FU from DVS cross-linked P [NIPAm-

HEAc] nanoparticles at different temperatures and pHs wasinvestigated. The synthesized nanoparticles were found to bedual-stimuli–responsive hydrogels. The temperature responsiverelease rate showed a maximum at 27◦C and the degree of re-lease was 90% at 37◦C. pH-stimulated drug release showed amaximum at pH 7. The 5-FU release rate was higher in al-kaline mediums than in acidic mediums. Drug release studiesalso showed the importance of carrying out the assays chang-ing the pH of the medium sequentially. This dual–stimuli re-sponse behavior of the covalently bonded P [NIPAm-HEAc]may suggest the potential application in oral drug deliverysystems.

COVALENTLY BONDED PNIPAm 251

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