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Drug release property of a pH-responsive double-hydrophilic hyperbranched graft copolymer SUN XiaoYi, ZHOU YongFeng† & YAN DeYue† College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China

In this paper, we report the synthesis and self-assembly of double-hydrophilic hyperbranched graft copolymers of HPG-g-PDMAEMA, which consist of a hyperbranched polyglycerol (HPG) core and several grafted poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) arms. HPG was synthesized by cationic polymerization. Then HPG-Br macroinitiator was obtained by esterification of HPG with 2-bromoisobutyryl bromide, which was subsequently used in the preparation of HPG-g-PDMAEMA graft copolymers through atom transfer radical polymerization (ATRP) of DMAEMA monomers. The molecular structures were studied by 1H NMR and GPC. The pyrene-based fluorescent probe method, 1H NMR and DLS were used to study the self-assembly behavior of HPG-g-PDMAEMA. The drug loading and pH-responsive release properties of HPG-g-PDMAEMA were also investigated by using coumarin 102 as a model drug. The results show that the HPG-g-PDMAEMA micelles can continuously release and re-encapsulate coumarin 102 as the pH continuously changes from 11.5 to 2.5; however, this process is not totally reversible. hyperbranched polymers, double-hydrophilic copolymer, polyglycerol, ATRP, drug delivery

1 Introduction Double-hydrophilic block copolymers (DHBCs) are a new class of amphiphilic macromolecules which consist of two water-soluble blocks with different chemical structures[1]. Typically, the change of external conditions, such as pH and temperature, can induce one hydrophilic block to become hydrophobic, thus double-hydrophilic block copolymers will transform into amphiphilic block copolymers. This phenomenon is called environmentally sensitive micellization. DHBCs have attracted more and more interest in a wide range of application fields, such as novel drug carrier systems and gene therapy[2], crystal growth templates[3], and induced nanoreactors for metal colloid synthesis[4]. Up to now, a great deal of research has been conducted on linear double-hydrophilic block copolymers, especially linear pH-sensitive double-hydrophilic block copolymers. Webber et al.[5] first reported the pH-sensitive micellization of DHBCs. They studied the pH-in-

duced micellization of poly(2-vinylpyridine)-block-poly (ethylene oxide) diblock copolymer (PVP-b-PEO), and found that in a certain alkaline solution, P2VP blocks cannot be protonated but form hydrophobic cores, with the soluble PEO as a shell outside to stabilize the mi― celles. Armes, Liu and coworkers[6 9] have reported several examples of DHBCs that exhibit so-called schizophrenic micellization behavior. These copolymers can self-assemble in dilute aqueous solution in the absence of any organic cosolvent to form two distinct micelle structures. In each case, the individual blocks can be independently tuned to become either hydrophilic or hydrophobic by subtle adjustment of the solution temperature, solution pH or ionic strength. Received July 24, 2009; accepted August 6, 2009 doi: 10.1007/s11426-009-0227-4 † Corresponding author (email: [email protected]; [email protected]) Supported by the National Natural Science Foundation of China (Grant Nos. 20774057 & 50633010), National Basic Research Program (973 Project, Grant No. 2007CB808000), the Basic Research Foundation of Shanghai Science and Technique Committee (Grant No. 07DJ14004), and the Shanghai Leading Academic Discipline Project (Grant No. B202)

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Hyperbranched polymers (HBPs) are a class of quasispherical highly branched macromolecules, with internal cavities and a large number of functional groups[10,11]. Because of their unique characteristics of the structure and properties, HBPs have already become a hot research field. However, there is little research on hyperbranchedbased double-hydrophilic copolymers[12,13], especially the ones with low grafting ratio. pH-sensitive DHBCs are very promising in controllable drug delivery. Generally, under a certain pH condition, polymers of this kind form micelles to encapsulate drug molecules. Once the pH changes, the polymeric micelles disassociate rapidly into molecules again and wholly release the encapsulated drugs simultaneously. This process is called micelle-to-unimer transformation. As the pH recovers, the unimers will reversibly transform into micelles and re-encapsulate the drugs into the hydrophobic parts of micelles. However, such a pHinduced transformation mechanism is concluded merely from linear DHBCs. Since HBPs have a unique molecular structure and property compared with linear polymers, we wonder whether this mechanism can be extended to HBPs. In this work, we synthesized a double-hydrophilic hyperbranched graft copolymer of HPG-g-PDMAEMA, with a hydrophilic HPG core and several pH-responsive PDMAEMA arms. Pyrene-based fluorescence probe method and DLS were used to study the self-assembly behavior of HPG-g-PDMAEMA copolymers, and the results show that they can form aggregations in both acidic and alkaline solutions, which is different from the self-assembly behavior of pH-sensitive linear DHBCs. Coumarin 102 was used as a model drug to study the pH-responsive drug release property of HPG-gPDMAEMA copolymers. The results show that when the solution pH decreases to an acidic condition, the encapsulated drug inside the micelles can be released rapidly, but it does not fully release at one time; when pH increases again, the drug can be partly re-encapsulated into the HPG-g-PDMAEMA micelles. Since both HPG and PDMAEMA have good biocompatibility, HPG-g-PDMAEMA is expected to have potential applications in the fields of medicine and bioengineering.

2 Experimental 2.1 Materials Boron trifluoride diethyl etherate (BF3·OEt2) (from 1704

Shanghai Chemical Reagent Co.) was dried by refluxing over CaH2 for 24 h under nitrogen and then distilled under vacuum. Glycidol (from Aldrich Chemical Co.) was purified in the same way. CHCl3 and pyridine (from Shanghai chemical Reagent Co.) were refluxed with CaH2 and then distilled prior to use. CuBr (from Shanghai Chemical Reagent Co.) was stirred in glacial acetic acid and washed with ethanol, and finally dried in vacuum. 2-(Dimethylamino)ethyl methacrylate (DMAEMA, 99%, Acros) was passed through a column of basic alumina to remove the inhibitor and then vacuum distilled before polymerization. All other reagents (2-bromoisobutyryl bromide, methanol, 2,2′-bipyridine (bpy), hexane, cyclohexane) were purchased from commercial sources and used as received without purification. 2.2 Measurements 1

H NMR and 13C NMR were performed on a Varian Mercury Plus 400-MHz spectrometer using CD3OD as solvent. TMS was used as the internal reference. The molecular weights of the products were measured by GPC at 70℃ on a Perkin-Elmer Series 200 system (100 μL injection column, PL gel (10 μm) 300 mm × 7.5 mm mixed-B columns) equipped with a RI detector. Dimethylformamide containing 0.05 mol/L lithium bromide was used as eluent at a flow rate of 1.0 mL/min. The column system was calibrated by mono-dispersed standard poly(ethylene oxide). The DLS measurements were performed in aqueous solution at 25℃ using a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ = 633 nm. All samples with concentrations of 1.0 mg/mL were measured at a scattering angle of 173°. The fluorescence spectra were recorded on a Perkin-Elmer LS-50B luminescence spectrometer. 2.3 Synthesis of polyglycerol (HPG) HPG was synthesized by cationic polymerization ac― cording to the literature[14 16]. 2.4 Synthesis of HPG-Br macroinitiator In a typical run, 5.0 g HPG (68 mmol hydroxyl groups) was dried by azeotropic distillation with toluene and then dissolved in 100 mL anhydrous pyridine. After the solution was cooled to 0℃, 1.61 g (7 mmol) of 2-bromoisobutyryl bromide was added dropwise under vigorous stirring, subsequently the temperature was allowed to rise to room temperature. The reaction continued

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under stirring for 18 h. A large part of pyridine was distilled under reduced pressure. The residue was washed with cyclohexane twice and then dialyzed against deionized water to remove the impurities. After removal of the water by freeze drying, a pale yellow, viscous product was obtained (HPG-Br). 2.5 Synthesis of HPG-g-PDMAEMA graft copolymer In a typical run, 0.5 g (0.44 mmol Br) HPG-Br macroinitiator, 0.0625 g (0.44 mmol) CuBr, 0.137 g (0.88 mmol) bpy, and 25 mL methanol were placed in a dried reaction flask and degassed for three cycles by pulling a vacuum and back-filling with nitrogen gas. Then 3.46 g (22 mmol) DMAEMA was added by syringe. The polymerization was at room temperature. After 12 h, the reaction was quenched with methanol and exposed to the air. The reaction mixture was diluted with methanol and passed through a column with activated Al2O3 to remove the catalyst. The product was precipitated twice by dissolution/precipitation with tetrahydrofuran/hexane. After dialyzed against deionized water and freeze drying, a white powder of HPG-g-PDMAEMA graft copolymer was obtained. 2.6 Self-assembly of HPG-g-PDMAEMA At room temperature, 20.0 mg HPG-g-PDMAEMA copolymer was dissolved in 2 mL DMF solvent and then 15 mL deionized water was added dropwise under stirring. The appearance of turbidity indicates the formation of the aggregation. The solution was dialyzed against deionized water for 48 h using a dialysis bag (MWCO 1000 Da) to remove DMF. The solution inside the dialysis bag was collected and divided into several aliquots, and then the pH of every aliquot was adjusted to a certain value by adding hydrochloric acid or NaOH solution (1 M). 2.7 Drug loading and release At room temperature, 5.0 mg coumarin 102 and 20.0 mg HPG-g-PDMAEMA were dissolved in 2 mL DMF followed by dropwise addition of 15 mL deionized water under vigorous stirring. The solution was dialyzed against deionized water for 48 h using a dialysis bag (MWCO 1000 Da) to remove DMF, and then the solution inside the dialysis bag was collected. Fluorescence measurements were performed on LS-50B luminescence spectrometer. The excitation wavelength was set at 420 nm, and the intensity of maximum emission peak at 486 nm within 5 minutes

was recorded. In a typical run, 3 mL polymer solution was placed in a 1×1 cm quartz cuvette. The pH of the solution was adjusted to 11.5 using 2 M NaOH solution, and the curve of maximum emission intensity within 5 minutes was recorded. Then the pH of the solution was adjusted to 2.5 using 2 M hydrochloric acid, and again the curve of maximum emission intensity during the 5 min was recorded. The operation was cycled 5 times, and then the curves of fluorescence intensity versus time were drawn. 2.8 Critical micellization concentration (CMC) HPG-g-PDMAEMA sample was dissolved in a 6×10−6 M pyrene aqueous solution and diluted into various desired concentrations. The excitation wavelength was set at 334 nm. The ratio values of emission intensity at 373 nm and 383 nm (I1/I3) were recorded.

3 Results and discussion 3.1 Characterization of HPG-g-PDMAEMA and its precursors The synthetic procedure of HPG-g-PDMAEMA graft copolymer is presented in Figure 1. HPG was synthesized by cationic polymerization according to the litera― ture[14 16]. The degree of branching (DB) and the numberaverage degree of polymerization (DPn) of synthesized HPG is 56% and 37, respectively, according to 13C NMR spectrum. Thus the number-average molecular weight (Mn) was estimated to be 2700 g/mol. The Mn and polydispersity index (PDI) of HPG determined by GPC is 3900 g/mol and 2.10, respectively. The typical 1H NMR spectra of HPG, HPG-Br and HPG-g-PDMAEMA are shown in Figure 2. Comparing the 1H NMR spectra of HPG and HPG-Br, the new signal appeared at 1.92 ppm attributed to the ―C(CH3)2Br group in the spectrum of HPG-Br, which confirms that macroinitiator was synthesized successfully. The conversion of hydroxyl groups can be obtained by comparing the ―C(CH3)2Br signal (peak b) at 1.92 ppm with the scaffold hydrogens of HPG (peak a) at 3.4―4.1 ppm. The result shows that the percentage of esterification is 7.52%, which means average 3 hydroxyl groups of each HPG molecule (37 hydroxyl groups) were esterified. The 1H NMR spectrum of HPG-g-PDMAEMA is shown in Figure 2(1). The signals (c, d, e, f and g) at 0.8―1.2, 1.7―2.0, 2.2―2.45, 2.55―2.75, and 4.0―4.2 ppm are ascribed to the repeated DMAEMA unit. Characteristic

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Figure 1

Synthetic scheme of HPG-g-PDMAEMA copolymer.

estimated by 1H NMR analysis is 15800 g/mol. Figure 3 presents the typical GPC curves of HPG and its related HPG-g-PDMAEMA copolymer. The symmetric peak was observed, which means no coupling termination between different grafted copolymers occurred. The GPC results show that the Mn and PDI of obtained HPG-g-PDMAEMA is 17100 g/mol and 1.74, respectively.

Figure 2 1H NMR spectra of (1) HPG, (2) HPG-Br and (3) HPG-gPDMAEMA, with CD3OD as the solvent.

resonance of HPG scaffold hydrogens is also present in the 1H NMR spectrum of the graft copolymer. These data illustrate the presence of both HPG and DMAEMA units in the polymer chain. The Mn of HPG-g- PDMAEMA 1706

Figure 3

Typical GPC traces of (1) HPG and (2) HPG-g-PDMAEMA.

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The 1H NMR and GPC data prove that HPG-gPDMAEMA has been successfully synthesized via ATRP. Each HPG core was grafted 3 PDMAEMA arms, with an average arm length of 27. 3.2 Self-assembly of HPG-g-PDMAEMA Figure 4 shows the 1H NMR spectra recorded for HPGg-PDMAEMA copolymer in D2O at different pH. At pH 4.0, all the signals expected for each component of HPG-g-PDMAEMA are visible, though the progressive protonation of PDMAEMA chains causes a gradual shift in the signals. In addition, the PDMAEMA signals increase significantly, indicating enhanced hydrophilicity of PDMAEMA chains in acid media. With the increase of pH, the PDMAEMA signals are attenuated in evidence, while the HPG signals keep invariable, indicating that the PDMAEMA chains partly collapse and become hydrophobic. Thus HPG-g-PDMAEMA changes from double-hydrophilic polymer to amphiphilic polymer and undergoes micellization behavior in alkaline media. As shown in Figure 4, PDMAEMA signals have not disappeared completely even at pH 12.0, which indicates PDMAEMA chains are still partly hydrated in the high alkaline media.

Figure 5

Molecular structure of coumarin 102.

the fluorescence intensity will decrease. Therefore, we can monitor the encapsulation and the release process of drugs in the cuvette according to the change of fluorescence intensity[17]. In this work, the drug loading and releasing properties of HPG-g-PDMAEMAs at different pH were evaluated by using hydrophobic C102 as a model drug. We encapsulated C102 into HPG-g-PDMAEMA micelles, and then tracked the release of C102 by measuring the emission intensity of 486 nm (excited at 420 nm). We adopted a “pulse” stimulation to reversibly change the pH of the solution from 11.5 to 2.5 (Figure 6(a)), through the addition of trace sodium hydroxide solution or hydrochloric acid.

Figure 4 1H NMR spectra recorded for the HPG-g-PDMAEMA copolymer in D2O at different pH.

3.3 Drug release property of HPG-g-PDMAEMA Coumarin 102 (C102) is a commonly used fluorescence dye. The chemical structure of C102 is shown in Figure 5. C102 shows a strong emission in the hydrophobic environment, while its emission intensity decreases drasticcally in water, since C102 is almost insoluble in water. It can be predicted if C102 is encapsulated into micelles, strong fluorescence will be detected; once it is released,

Figure 6 Plots of (a) pH versus time and (b) emission intensity of coumarin versus time.

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Figure 6(b) shows the change of the fluorescence intensity under “pulse” type pH conditions. When the pH decreased from 11.5 to 2.5, the fluorescence intensity decreased dramatically, indicating the quick release of C102. When the pH returned from 2.5 to 11.5, the fluorescence intensity recovered, but was lower than the previous value at pH 11.5. We found that after each cycle the fluorescence intensity cannot be fully reversible, and is lower than that in the previous cycle at the same pH. Figure 7 displays the graph of the emission intensity at different pH versus cycles. It shows that the fluorescence intensity of coumarin linearly decreases with the increase of release cycles at pH 2.5; so is the relationship between the fluorescence intensity and the re-encapsulation cycles at pH 11.5. The fluorescence intensity decreases to a very low value after 5 cycles. The results show that the HPG-g-PDMAEMA micelles can release C102 molecules in acid aqueous solution rapidly and the released drug can be re-encapsulated into the micelles at pH 11.5. However, this process is only partly reversible and the reversibility is related to the number of cycles.

Figure 7 cycles.

Plots of emission intensity of coumarin versus the number of

In addition, we found that the reversibility is also related to the pH of the solution. If we adjust the “pulse” type pH conditions, changing the pH from 10.5 to 3.5, a very different drug release curve is obtained, as shown in Figure 8. When the pH decreases from 10.5 to 3.5, the fluorescence intensity decreases quickly, but the reduced scale is smaller than that when pH changes from 11.5 to 2.5; however, when the pH returns from 3.5 to 10.5, the fluorescence intensity almost remains unchanged. It can be seen that the release property of HPG-g-PDMAEMA micelles is related to the pH of the acidic solution and 1708

the re-encapsulation ability is related to the pH of the alkaline solution. We think this is due to the pH-responsibility of PDMAEMA chains. The 1H NMR results, as shown in Figure 4, have demonstrated that in acidic conditions, PDMAEMA chains are protonated, and the degree of protonation increases as the solution becomes more acidic. Thus the PDMAEMA chains become more hydrophilic, which is more conducive to the release of hydrophobic drugs. When the solution changes from acidic condition to alkaline condition, PDMAEMA chains will be deprotonated and become hydrophobic. At pH 10.5, the PDMAEMA is weakly hydrophobic, making it difficult for the micelles to re-encapsulate the released hydrophobic drugs; however, when the pH reaches 11.5, it is hydrophobic enough for the micelles to re-encapsulate the drugs. Figure 6(b) and Figure 8 show an interesting phenomenon. When the solution changes from acidic condition to alkaline condition, the fluorescence intensity decreases rapidly, and stays at a certain value instead of reaching zero. PDMAEMA is a pH responsive polybase[18,19], exhibiting hydrophobicity at high pH conditions, thus the HPG-g-PDMAEMA can self-assemble into micelles. While at low pH, PDMAEMA chains become hydrophilic due to the protonation, and HPG-gPDMAEMA should transform into double-hydrophilic copolymers. From this standpoint, the drug will be rapidly released at acid condition, for the HPG-g-PDMAEMA micelles dissociate into unimers, and the fluorescence intensity will directly decrease to a very low level. However, our results do not agree with this conclusion. We speculated that at lower pH, HPG-g-PDMAEMAs did not change into unimers, and may retain the micellar

Figure 8 Plots of emission intensity of coumarin versus time with pH changing between 10.5 and 3.5.

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structure, where the drug molecules can still be loaded. To address the question, we measured the critical micelle concentration (CMC) and particle size to characterize the self-assembly of HPG-g-PDMAEMA at lower pH. 3.4 Critical micelle concentration (CMC) Pyrene has been widely used as a probe to determine solvent environments, for its fluorescence emission ― spectrum is very sensitive to the solvent’s polarity[20 23]. The pyrene probe fluorescence spectrometry is a recognized method for obtaining the information of aggregate formation and determining the critical micellization concentration (CMC). A typical emission spectrum of pyrene has 5 peaks at 373, 379, 384, 394 and 480 nm. The intensity ratio of the first to the third emission peak I1/I3 is sensitive to the local microenvironment of pyrene. In the curves of I1/I3 ratio versus polymer concentration obtained from fluorescence spectrum of pyrene solution, the intersection of the two straight lines is considered as the CMC value. We used pyrene as a fluorescent probe to determine the critical micelle concentration of HPG-g-PDMAEMA at different pH. Figure 9 shows the relationship between emission intensity ratio I1/I3 and the HPG-g-PDMAEMA copolymer concentration at different pH. It can be seen that the CMC value of HPG-g-PDMAEMA in aqueous solution at pH 2.5, 8.0 and 11.6 was 0.25, 0.20 and 0.27 mg/mL, respectively. The CMC measurements have provided a clear evidence that the self-assembly of HPG-g-PDMAEMA can occur in acidic, neutral and alkaline aqueous solution. DLS was used to determine the size distribution of self-assembly particles at different pH. As shown in Figure 10, the size is 194 nm and 206 nm at pH 11.5 and 6.7, respectively. When the pH was adjusted to 2.5, the

particle size increased to 234 nm. The CMC and DLS results at different pH show that the self-assembly structure of HPG-g-PDMAEMA may be the same in acidic, neutral and alkaline solutions. There may be two main factors which drive the selfassembly of HPG-g-PDMAEMA in solutions. One is the existence of weak “hydrophobic interaction” between the PDMEMA chains at room temperature[24]; the other is that the low grafting ratio of the synthesized HPG-gPDMAEMA leads to a large number of residual hydroxyl groups on the surface, which can form strong hydrogen bonds to stabilize the assembly structure. The assembly structure may be a class of large multimolecular micelles[23,24], for its size is more than 100 nm. In addition, it can be seen from Figure 10 that at pH 2.5 the size of micelles is a little larger than that in neutral and alkaline media. We think this is due to the protonation of PDMAEMA chains and electrostatic repulsion among them in acidic condition, which lead to a more extended molecular chain and a looser micellar structure. It also explains the rapid release of drugs in the acidic condition. The self-assembly structures of HPG-g-PDMAEMA at different pH are under further study.

Figure 10 DLS plots of HPG-g-PDMAEMA at different pH. Polymer concentration is 1.0 mg / mL.

4 Conclusions

Figure 9

The CMC curves of HPG-g-PDMAEMA at different pH.

In this paper, a double-hydrophilic hyperbranched graft copolymer of HPG-g-PDMAEMA was synthesized by combination of cationic polymerization and ATRP. 1H NMR and GPC analysis show that each HPG core was grafted 3 PDMAEMA arms, with an average arm length of 27. The pyrene-based fluorescent probe method, 1H NMR and DLS were used to study the self-assembly behavior of HPG-g-PDMAEMA in aqueous solution. It

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shows that the self-assembly of HPG-g-PDMAEMA could occur in acidic, neutral and alkaline aqueous solution. Coumarin 102 was used as a model drug to study the drug loading and releasing properties of HPG-gPDMAEMA under pH stimulation. It was found that HPG-g-PDMAEMA micelles can continuously release and re-encapsulate the drug molecules when pH is con-

1

tinuously changed between 11.5 and 2.5. However, the process is not totally reversible and the reversibility is related to the pH of the solution, which is greatly different from that of the linear double-hydrophilic block copolymers. The finding presented here may provide a new insight on the structures and properties of hyperbranched polymers.

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