*Corresponding author.

Biomaterials 22 (2001) 2495}2499

Polymeric microspheres composed of pH/temperature-sensitivepolymer complex

Eun Jung Kim�, Sun Hang Cho�, Soon Hong Yuk��*�Advanced Materials Division, Korea Research Institute of Chemical Technology, P.O. Box 107, Yusung, Taejeon, 305-600 South Korea�Department of Polymer Science and Engineering, Hannam University, 133 Ojeong Dong, Daedeog Ku, Taejeon, 306-791 South Korea

Received 10 July 2000; accepted 11 December 2000

Abstract

A new pH/temperature-sensitive polymer system with transitions resulting both from polymer}water and polymer}polymerinteractions has been demonstrated using the mixture of poly(N,N-dimethylamino)ethyl methacrylate (DMAEMA) and polyethylacrylamide (EAAm). Based on the pH/temperature sensitivity of polymer mixture, the microsphere for pH/temperature-sensitive drugrelease have been designed and characterized. Hydrocortisone was used as a model drug. This gave the control of hydrocortisonerelease in an on}o! manner without considerable lag time. � 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Polymeric microsphere; pH/temperature-sensitive polymer mixture; On}o! release of hydrocortisone

1. Introduction

Much interest has been focused on polymer systemsthat show a phase transition in response to externalstimuli such as temperature, pH, ionic strength, andelectric potential because of their scienti"c or technolo-gical importance [1}5]. In particular, stimuli-sensitivepolymer systems have a potential for application inmodulated drug-delivery because these polymers notonly respond to external stimuli but also control therelease rate of drugs. It has been recognized that theconstant release is not the only way to maximum druge!ect and minimum side e!ects and the assumption usedfor constant release rate sometimes fails its validity forphysiological conditions. To overcome this di$culty, ex-ternally modulated or self-regulating drug-delivery sys-tems have been used as novel approaches to deliveringdrug as required. To achieve this drug delivery system,stimuli-sensitive polymer system has been intensively ex-ploited as a candidate material [6}9].

Various applications of thermosensitivity of polymerrelevant to drug delivery have been reported. Okanoet al. proposed the interpenetrating polymer network(IPN) hydrogel comprising poly(N,N-dimethylacryl-

amide) and polyacrylic acid and demonstrated reversibletemperature dependent swelling of the gel with pulsatile(on}o!) solute (ketoprofen) release; `ona at a higher tem-perature and `o!a at a lower temperature [10]. Thisresearch group identi"ed the main interactions to behydrogen bonding and also pointed out the importanceof the zipper e!ect describing the cooperative nature ofthe interaction between two polymers. Kim et al. re-ported the novel system of a soluble pH/temperature-sensitive polymer and its utility in a protein drug loadingprocess [11]. In their study, beads formed from linearpH/temperature-sensitive polymer, poly(N-isopropylac-rylamide-co-butyl methacrylate (BMA)-co-acrylic acid(AAc)) were evaluated as a potential macromolecularcarrier in terms of loading e$ciency, pH-dependentrelease, and preservation of bioactivity of the loadeddrug [11].

In our previous report, a pH/temperature-sensitivepolymer system with transitions resulting from poly-mer}water and polymer}polymer interactions has beendemonstrated using poly((N,N-dimethylamino)ethylmethacrylate (DMAEMA)-co-ethyl acrylamide (EAAm))[12]. In this study, a new pH/temperature-sensitive poly-mer system composed of the mixture of polyDMAEMAand polyEAAm has been prepared. Based on thepH/temperature sensitivity of polymer mixture, themicrosphere for pH/temperature-sensitive drug releasehave been designed and characterized.

0142-9612/01/$ - see front matter � 2001 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 4 3 9 - 7

Table 1Composition of polymer complexes in the study

Code DMAEMA EAAM M�/10��

g mol% g mol%

PolyDMAEMA 3.5Copolymer I 6.6 80 1.2 20 4.2Copolymer II 5.3 60 2.5 40 4.4Copolymer III 4.6 50 3.2 50 4.6PolyEAAm 4.7

PolyDMAEMA PolyEAAm

g mol% g mol%

Polymer complex I 6.6 80 1.2 20Polymer complex II 5.3 60 2.5 40Polymer complex III 4.6 50 3.2 50

�Measured by laser scattering.

Fig. 1. Preparation method of microsphere.

2. Materials and methods

2.1. Materials

DMAEMA monomer was purchased from Aldrich.N,N-azobis(isobutyronitrile) (AIBN) and hydrocortisonewere purchased from Sigma Chemical Co. DMAEMAmonomer was distilled before use. Other reagents wereused as received.

2.2. Synthesis

EAAm was synthesized in our laboratory as describedpreviously [13]. Poly(DMAEMA-co-EAAm), poly-DMAEMA, and polyEAAm were prepared by freeradical polymerization as follows: 7.8 g of distilled mono-mers and 0.02 g of AIBN as an initiator were dissolved in100ml of water/ethanol binary solvent (5/5 by volume)(see Table 1). The ampoule containing the solution wassealed by conventional methods and immersed in a waterbath held at 753C for 15 h. After polymerization, allpolymers were dialyzed against distilled}deionized waterat 43C and freeze-dried.

2.3. Transmittance measurements

The phase transition was traced by monitoring thetransmittance of a 500nm light beam on a Spectronic 20spectrophotometer (Baush & Lomb). The concentrationof the aqueous polymer solution was 5wt%, and thetemperature was raised from 5 to 703C in 2-deg in-crements every 10min. To observe their pH/temperaturedependence, the phase transitions of polymers in citric-

phosphate bu!ered solution versus temperature at twopH values (4.0 and 7.4) were measured.

2.4. Preparation of microsphere usingpH/temperature-sensitive polymers

Using 5ml syringe (26 gauge needle), polymer solutionmixture composed of polyDMAEMA, poly EAAm andmodel drug was transferred dropwise to the phosphatebu!ered solution ((PBS), pH 7.4) as shown in Fig. 1. Thetemperature of PBS should be higher than the LCST ofpolymer for the coagulation of polymer and hydrocor-tisone was used as a model drug. The microsphere waskept in sealed bottle at 403C to maintain the swollen stateand the diameter of swollen microsphere was approxi-mately 2mm. The loading amount is approximately20wt%.

2.5. Release experiment for hydrocortisone

The release of hydrocortisone from the microspherewas measured in response to temperature change.Five hundred milligram of microspheres were introducedinto 200 ml of release medium (pH 7.4 PBS) at desiredtemperature. For measuring the pH-sensitive releaseof hydrocortisone, the citric-phosphate bu!ered solu-tions were used as release media. The amount ofreleased hydrocortisone was measured by taking 1mlof the release medium at a speci"c time, replacingthe total release medium with fresh one to maintainsink conditions and assaying the drug concentrationat 248nm using a UV spectrophotometer (Shimadzu,Japan).

2496 E.J. Kim et al. / Biomaterials 22 (2001) 2495}2499

Fig. 2. LCST of poly(DMAEMA-co-EAAm) (�) and polymer complex(�) in citric-bu!ered solution at pH 4.0 and 7.4. (The number ofexperiment is 3 and LCST of poly(DMAEMA-co-EAAm) was adaptedfrom Ref. [12].)

3. Results and discussion

3.1. pH/temperature-induced phase transition

pH/temperature-induced behavior of poly(DMAEMA-co-EAAm) was reported previously [12].The LCST of poly(DMAEMA-co-EAAm) was shifted toa lower temperature with the increase of EAAm contentas shown in Fig. 2. In general, the LCST should increasewith increasing hydrophilicity of the polymer. However,a LCST shift to a lower temperature was observed withthe incorporation of the hydrophilic EAAm. This is dueto the formation of hydrogen bonds, which protect (N,N-dimethylamino)ethyl groups from exposure to water andresult in a hydrophobic contribution to the LCST. At pH4, no LCST was observed with polyDMAEMA and theLCST of all copolymers was increased compared to thatat pH 7.4. (see Fig. 2) At pH 4.0, (N,N-dimethyl-amino)ethyl groups of DMAEMA are fully ionized. Anincreased electrostatic repulsion between charged siteson DMAEMA disrupts the hydrogen bonds betweenEAAm and DMAEMA. These interfere with the hydro-phobic interactions between (N,N-dimethylamino)ethylgroups above the LCST and the hydrophobic contribu-tion to the LCST due to the hydrogen bonding.

To prepare pH/temperature-sensitive polymer com-plex, the mixtures of polyEAAm and polyDMAEMA inappropriate ratio with that of copolymer were preparedand their pH/temperature-induced behaviors were ob-served as shown in Fig. 2. The LCST of polyDMAEMAwas shifted to the lower temperature with the addition ofpolyEAAm and the LCST of all the polymer complexeswas increased with decreasing pH, which was observed inthe aqueous copolymer solution.

3.2. Formation of microsphere

Firstly, poly(DMAEMA-co-EAAm) was used for thepreparation of microsphere based on the preparationmethod presented previously. However, the formation ofmicrosphere was not observed. The formation of mico-sphere was observed with the mixture of polyDMAEMAand polyEAAm.

To understand this phenomenon more in detail,temperature-induced phase transition of polymer wasstudied in the various forms of polymer networks. Aspresent previously [12], the LCST of linear (noncrosslin-ked) poly(DMAEMA-co-EAAm) was shifted to a lowertemperature with the increase of EAAm content. This isdue to the formation of hydrogen bonds, which protect(N,N-dimethylamino)ethyl groups from exposure towater and result in a hydrophobic contribution to theLCST. With the formation of crosslinked network, thepolymer gel exhibited the temperature-induced phasetransition which was quite di!erent from that of polymeraqueous solution [14]. The transition temperature ofpoly(DMAEMA-co-EAAm) gel between the shrunkenand swollen was shifted to the higher temperature withthe increase of EAAm content, which was contrary to theLCST change of poly(DMAEMA-co-EAAm) aqueoussolution [14]. This indicates that polymer}polymer inter-action via hydrogen bond contributed hydrophobicallyto the LCST of poly(DMAEMA-co-EAAm) aqueoussolution and it contributed hydrophilically to thetemperature-sensitive swelling behavior of poly-(DMAEMA-co-EAAm) gel [14]. With the formation ofgel network, the degree of freedom of polymer chain issigni"cantly decreased and this leads to the decrease ofthe hydrogen bond between DMAEMA and EAAm.From this result, the temperature-induced phasetransition of poly(DMAEMA-co-EAAm) is highly de-pendent on the change of hydrogen bond due to thestructural di!erence. From this perspective, we cansuggest that there exists the di!erence in the temperature-induced phase transition between poly(DMAEMA-co-EAAm) and the mixture of polyDMAEMA andpolyEAAm. This may arise from cooperative poly-mer}polymer interactions (zipper-like e!ect) [10] in themixture of polyDMAEMA and polyEAAm as shownschematically in Fig. 3. Based on this characteristic ofpolymer complex, we can expect that the formation ofmicrosphere using temperature-induced phase transitionis observed with the polymer complex.

3.3. pH/temperature-sensitive release of hydrocortisonefrom microsphere

Fig. 4 shows the temperature-sensitive release of hy-drocortisone from the microsphere with various poly-DMAEMA/polyEAAm compositions. The high releaserate was observed at lower temperature due to the

E.J. Kim et al. / Biomaterials 22 (2001) 2495}2499 2497

Fig. 3. Schematic description of intra/intermolecular interaction incopolymer and polymer complex.

Fig. 4. Temperature-sensitive release of hydrocortisone as a function ofpolyEAAm content in the microsphere; (A) microsphere composed ofpolymer complex III, (B) microsphere composed of polymer complex II,(C) microsphere composed of polymer complex I.

Fig. 5. The release of hydrocortisone from the microsphere composedof polymer complex I in response to pulsatile temperature change. (Thenumber of experiment is 3.)

Fig. 6. The release of hydrocortisone from the microsphere composedof polymer complex I in response to pulsatile pH change at 403C. (Thenumber of experiment is 3.)

dissociation of temperature-sensitive intermolecularinteraction in the polymer complex. The transition tem-perature between high and low release rates was shiftedto the lower temperature with the increase of polyEAAmcontent in the microsphere, which is in accordance withthe LCST change of polymer mixture with the variation

of polyEAAm content. When we applied a step functionof temperature to the microsphere, hydrocortisone wasreleased in a stepwise manner as shown in Fig. 5.

The release of hydrocortisone in response to pulsatilepH change was observed as shown in Fig. 6. The experi-ment was performed above the LCST of the polymercomplex of microsphere to maintain the aggregate state.At acidic condition, drastic increase in release rate wasobserved due to the dissociation of polymer complex.As presented in the pH/temperature-induced phasetransition of polymer complex (see Fig. 2), the decrease ofpH increased the LCST of polymer complex of micro-sphere and this led to the dissociation of aggregate poly-mer complex, thus the release of hydrocortisone instepwise manner.

2498 E.J. Kim et al. / Biomaterials 22 (2001) 2495}2499

4. Conclusions

pH/temperature-sensitive polymer systems withfunctions resulting from both polymer}water and poly-mer}polymer interactions was demonstrated usingpolymer mixture of polyDMAEMA and polyEAAm. Thee$cient hydrogen bonding in the polymer mixture,which may arise from the zipper-like e!ect, plays animportant role in the formation of microsphere. Based onthe pH/temperature-sensitivity of hydrogen bond in themicrosphere, pH/temperature-sensitive release of hydro-cortisone was demonstrated and this gave hydrocor-tisone release control in an on}o! manner withoutconsiderable lag time.

Acknowledgements

This work was supported in part by a grant fromKorea Ministry of Health and Welfare (HMP-98-G-2-051-B).

References

[1] Heskins M, Guillet JE. Solution properties of poly(NIPAAm).J Macromol Sci-Chem 1968;A2(8):1441}55.

[2] Chen GH, Ho!man AS. Graft copolymers that exhibit temper-ature-induced phase transitions over a wide range of pH. Nature1995;373:49}52.

[3] Drummound WR, Knight ML, Brannon ML, Peppas NA.Surface instabilities during swelling of pH-sensitive hydrogels.J Control Rel 1988;8:179}85.

[4] Ricka J, Tanaka T. Phase transition in ionic gels induced bycopper complexation. Macromolecules 1985;18:83}5.

[5] Kwon IC, Bae YH, Kim SW. Electrically erodible polymer gel forcontrolled release of drugs. Nature 1991;354:291}4.

[6] Ilman F, Tanaka T, Kokufuta E. Volume transition in a gel drivenby hydrogen bonding. Nature 1991;349:400}1.

[7] Nagasaki Y, Honzawa E, Kato M, Kataoka K, Tsuruta T. Novelstimulus-sensitive telechelic oligomers. pH and temperature sensi-tivities of poly(silamine) oligomers. Macromolecules 1994;27:4848}50.

[8] Stayton PS, Shimoboji T, Long C, Chilkoti A, Chen GH, HarrisJM, Ho!man AS. Control of protein}ligand recognition usinga stimuli-responsive polymers. Nature 1995;378:472}5.

[9] Yuk SH, Bae YH. Phase-transition polymers for drug delivery.Crit Rev Ther Drug Carrier Systems 1999;16:385}423.

[10] Aoki T, Kawashima M, Katono H, Sanui K, Ogata N, Okano T,Sakurai Y. Temperature-responsive interpenetrating polymer net-works constructed with poly(acrylic acid) and poly(N,N-dimethylacrylamide). Macromoecules 1994;27:947}52.

[11] Kim YH, Bae YH, Kim SW. pH/temperature-sensitive polymersfor macromolecular drug loading and release. J Control Rel1994;28:143}52.

[12] Yuk SH, Cho SH, Lee SH. pH/temperature-responsive polymercomposed of poly((N,N-dimethylamino)ethyl methacrylate-co-acrylamide). Macromolecules 1997;30:6856}9.

[13] McCormick CL, Nonaka T, Johnson CB. Water-solublecopolymers: 27. Synthesis and aqueous solution behaviour ofassociative acrylamide/N-alkylacrylamide copolymers. Polymer1988;29:731}9.

[14] Cho SH, Jhon MS, Yuk SH. Temperature-sensitive swelling be-havior of polymer gel composed of poly(N,N-dimethylaminoethylmethacrylate) and its copolymers. Eur Polym J 1999;35:1841}5.

E.J. Kim et al. / Biomaterials 22 (2001) 2495}2499 2499