Degradation and Degradation-Induced Re-Assembly of PVP-PCL

Biomacromolecules 2010, 11, 481–488

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Degradation and Degradation-Induced Re-Assembly of PVP-PCL Micelles Yong Hu,† Zhiping Jiang,‡ Rui Chen,‡ Wei Wu,‡ and Xiqun Jiang*,‡ Nanjing National Laboratory of Microstructure, Department of Material Science and Engineering, Nanjing University, Nanjing, 210093, People’s Republic of China, and Laboratory of Mesoscopic Chemistry and Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, People’s Republic of China Received October 25, 2009; Revised Manuscript Received December 26, 2009

The nonenzymatic and enzymatic degradation behaviors of the poly(vinyl pyrrolidone)-poly(ε-caprolactone) (PVPPCL) diblock copolymers micelles in aqueous solution were investigated by dynamic light scattering (DLS), size exclusion chromatography (SEC), and high performance liquid chromatography (HPLC), and the morphology variation of these micelles in the degradation procedure were inspected by transmission electronic microscopy (TEM). It is found that the enzymatic degradation of PVP-PCL micelles is much faster and the degradation rate of PVP-PCL micelles is proportional to the enzyme concentration for a given micelles’ concentration. However, in the nonenzymatic case, the degradation of PVP-PCL micelles is quite slow under neutral condition and fast in acidic or basic medium. Interestingly, morphology transformation from spheres to a necklace and rod-like nanostructure was observed during the degradation procedure under basic condition. This shape reconstruction of PVP-PCL nanoparticles in the degradation process opens a new window to fabricate hierarchical supramolecular structures.

Introduction Biocompatible and biodegradable polymeric micelles with a hydrophobic core and a hydrophilic shell have been of particular interest in pharmaceutical and biomedical applications because the core can encapsulate a hydrophobic drug and release it in a controlled manner, while the shell provides good stability for the micelles in a biological system.1,2 Typically, the core of polymeric micelles is composed of hydrophobic components, such as polycaprolactone (PCL) and polylactide (PLA),3,4 and the shell is a hydrophilic one, such as poly(ethylene glycol) (PEG)4 or poly(vinyl pyrrolidone) (PVP).5 Drugs loaded can be released from these polymeric micelles through the diffusion manner or due to the degradation of the micelles. Because the degradation behaviors of these polymeric micelles affect not only drug release characteristics but also micelle stability in medium and fate in vivo, the investigations on the micellar degradation have received increasing attention in recent years and several degradation mechanisms have been proposed.6-9 For example, in the case of hydrolytic degradation of sphereshaped PEG-PCL block copolymer micelles, the random scission of chains in core followed by micelle destabilization was proposed.6 Whereas, in our previous work, a two-stage degradation mechanism with core-shell interfacial erosion followed by core erosion was deduced.7c The degradation process of worm-shaped micelles made of PEG-PCL was found that the worm micelles were spontaneously shortened to spherical micelles by chain-end hydrolysis of the PCL.8 In addition to hydrolytic degradation, in the enzymatic degradation case, a degradation mechanism of PEG-PCL micelles that the enzyme “ate” the PCL core in a one-by-one manner, was proposed.9 * To whom correspondence should be addressed. Fax: 86-25-83317761. E-mail: [email protected]. † Nanjing National Laboratory of Microstructure. ‡ Laboratory of Mesoscopic Chemistry and Department of Polymer Science and Engineering.

Also, a mechanism for the enzymatic degradation of the PEGb-PCL micelles including random PCL core erosion followed by cavitation of micellar core and micellar dissociation was supposed.7b However, most of these investigations on micellar degradation were focused on the molecular weight and chemical structure changes of the copolymers, the micellar size variations and degradation kinetics. Little work on the micellar morphology variation and degradation-induced reassembly of micelles during the degradation process has been addressed. Actually, the morphology of micelles can impact their stability, drug loading, release properties, and cellular internalization as well as in vivo pharmacokinetics and biodistribution.10 Rod-like particles was found to be preferentially internalized by nonphagocytic cells compared with the spherical nanoparticles.11 A high drug loading level and better sustained release of paclitaxel over more than 30 days without significant initial burst were achieved after the formation of fiber-like stereoblock copolymer/paclitaxel complexes.12 The filomicelles could circulate longer time in the bloodstream, which were thought to help these micelles escape from the reticuloendothelial system (RES) and be effectively accumulated in the tumor site by enhanced permeability and retention effect (EPR).13 Worm-like micelles showed better penetration ability in agarose gels compared with spherical nanoparticles.14 To date, although it has been found that the in vivo fate and overall performance of polymeric drug-loaded nanocarriers depend not only on their size and stability but also on their degradation properties and aggregate shape during the degradation procedure,15 their morphology changes during the degradation procedure in vitro or in vivo have not been extensively investigated yet. Here, we report the degradation of PVP-PCL core-shell micelles and their morphology variation during the degradation process. Choosing PVP-PCL micelles as a study model is because PVP is a well-known hydrophilic biocompatible polymer like PEG,16 and the degradation of PVP-PCL

10.1021/bm901211r  2010 American Chemical Society Published on Web 01/14/2010

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micelles has not been well addressed so far. The hydrolytic and enzymatic degradation of PVP-PCL micelles were studied by dynamic light scattering (DLS), high performance liquid chromatography (HPLC), and proton nuclear magnetic resonance (1H NMR). The morphology changes of the PVP-PCL micelles from spherical to rod-like shape during the degradation process were captured by transmission electronic microscopy (TEM). It is plausible to expect that the degradation-induced reassembly of PVP-PCL micelles can go deep into the understanding on the degradation behaviors of polymeric micelles and open a new door to fabricate hierarchical supramolecular structures without precisely controlling the molecular structure and experimental condition.

2. Experimental Section 2.1. Materials. Vinyl pyrrolidone (VP; Aldrich) was purified by distillation under reduced pressure. ε-Caprolactone (Fluka) was dried over calcium hydride for 48 h at room temperature and distilled under reduced pressure. Stannous octoate (Sigma) and 6-hydroxycaproic acid (Alfa Aesar) were used as-received. Lipase AY with the activity of about 2 U/mg (Fluka) was purified by freeze-drying. 2-Mercaptoethanol (ME) was used without further purification. 2,2-Azoisobutyronitrile (AIBN; WAKO) was purified by precipitation into ice water from an acetone solution and dried under vacuum. All other chemicals were of analytical grade and used without further purification. 2.2. Synthesis of Hydroxy-Terminated PVP (PVP-OH). The PVPOH was synthesized by the radical polymerization of VP monomer using ME as a chain transfer agent.5 Polymerization was performed in a 100 mL glass reactor equipped with a glass-vacuum joint and a Teflon-coated stir bar. Briefly, ME (70 µL, 1.0 mmol) and AIBN (0.15 g) were dissolved in VP (10.0 g, 90 mmol) monomer with 20 mL of ethanol in the glass reactor. The dissolved solution was frozen into liquid nitrogen. The reactor was then degassed through connecting it with a vacuum pump. The reactor was sealed off and placed into an oil bath set at 75 °C, and then polymerization reaction was allowed to maintain for 8 h. The reaction was terminated through cooling in a water bath. The reaction product was then dissolved in ethanol, and precipitated with an excess ethyl ether to give a white product. The resulting product was dried under vacuum at 40 °C for 48 h to obtain the final sample. 2.3. Synthesis of PVP-PCL Block Copolymers. The PVP-PCL block copolymers were synthesized through a ring-opening copolymerization. Briefly, a predetermined amount of CL was added into a polymerization tube containing 1.0 g PVP-OH macromolecular initiator and a small amount of stannous octoate (0.1 wt %/wt). The tube was then connected to a vacuum system, sealed off, and placed into an oil bath at 130 °C for 48 h. After the polymerization was ended, the crude copolymers were dissolved with mixture of ethanol and acetone and precipitated into an excess amount of ethyl ether to remove the unreacted monomer and oligomer. The precipitates were then filtered and washed with ethyl ether several times and thoroughly dried at reduced pressure. 2.4. Preparation of PVP-PCL Polymeric Micelles. PVP-PCL block copolymer micelles were prepared by a precipitation method. Copolymer (100 mg) was dissolved in a mixture of 10 mL of acetone and 1 mL of ethanol, and the solution was added dropwise into 100 mL of distilled water under moderate stirring at 25 °C to produce an aqueous suspension. The acetone and ethanol in suspension were then removed under reduced pressure followed by dialysis in water. The suspension was then filtered with a microfilter with pore size 450 nm to remove the polymer aggregates and the larger micelle aggregates. 2.5. Degradation of PVP-PCL Micelles. The degradation behaviors were studied by the nonenzymatic and enzymatic degradations. In the nonenzymatic experiment, in each 25 mL bottle, 10 mL suspensions of PVP-PCL micelles (about 0.5 mg/mL) was added, and the volume was adjusted to 20 mL with buffer solution (KH2PO4, 0.05 M, pH 4.4,

Hu et al. and citrate solution). The medium pH can also be adjusted to the required value by addition of 1 M NaOH solution. These bottles were covered with rubber, sealed with parafilm, and insulated from the air. Therefore, the effect of CO2 on this degradation experiment was ignored and not counted in the experiments. After that, these bottles were stored at different temperatures in dark. At determined intervals, samples were taken out from the bottles for analysis. In the enzymatic biodegradation experiment, a proper amount of lipase (AY) was added into the polymeric micelle dispersion to perform biodegradation. The biodegradation was conducted at 37 °C either inside the DLS cuvette to in situ measure the effective diameter and light scattering intensity with time or in a big container to collect the samples for other tests simultaneously. 2.6. Characterization of Copolymers and the Micelle Degradation Products. Size exclusion chromatography (SEC) measurements were performed at room temperature on a Waters 515 system equipped with a Wyatt Technology Optilab rEX refractive index detector. The columns were STYRAGEL HR3, HR4, and HR5 (300 × 7.8 mm) from Waters. HPLC grade tetrahydrofuran (THF) was used as an eluent at a flow rate of 1 mL/min. THF and samples were filtered over a filter with pore size of 0.45 µm (Nylon, Millex-HN 13 mm Syringes Filters, Millipore, U.S.A.). The columns were calibrated by using polystyrene standards with molecular weights in the range from 900 to 1.74 × 106 g/mol. MALLS detector (DAWN EOS) was placed between the absorbance detector and the refractive index detector. The molecular weight and molecular weight distribution were determined by SEC/ DAWN EOS/Optilab rEX. ASTRA software (version 5.3.1.4) was utilized for acquisition and analysis of data. 1H NMR (Bruker MSL300) spectra of the copolymers and the micelle degradation products were recorded in CDCl3 at room temperature. Mean diameter of the PVP-PCL micelles and the light scattering intensity of the micelles suspension were determined by DLS method using a Brookheaven BI9000AT system (Brookheaven Instruments Corporation, U.S.A.). Morphological examination of the micelles was conducted using a JEOL (Japan) JEM-100S transmission electron microscope (TEM). One drop of micelle suspension was placed on a copper mesh covered with nitrocellulose membrane and dried in air before being stained with phosphotungstic sodium solution (1% w/v). Atomic force microscope (AFM; SPI 3800, Seiko Instruments, Japan) was used to study the surface morphology of nanoparticles. A 1 drop aliquot of properly diluted micelles was placed on the surface of a clean silicon wafer and dried under nitrogen flow at room temperature. The AFM observation was performed with a 20 µm scanner in tapping mod. HPLC measurements were carried out on a Shimadzu LC-10AD (Shimadzu) HPLC system using a Lichrospher C-18, 5 µ, 200 × 4.6 mm RP-HPLC analytical column. The mobile phase was consisting of 20/80 acetonitrile (HPLC grade, Merck, Germany)/ultrapure water. The column was eluted at a flow rate of 1.0 mL/min at 35 °C and the working wavelength was 215 nm by UV absorption. The standardization curve was a straight line and the minimum detectable amount was 0.01 mg/mL of 6-hydroxycaproic acid. The degradation products were obtained as follows: the samples were first adjusted to pH 2-3 with 2 M HCl solution and then the undissociated micelles were separated from the aqueous phase by centrifugation (Ultra ProTM 80, Du Pont) at 16000 rpm for 1 h. The solution above the sediment was collected for HPLC measurement, and the sediment was first washed with distilled water three times and then frozen and lyophilized to obtain the dried residual micelle products for 1H NMR measurements. Enzymatic degradation products were treated by acidification with 2 M HCl to stop the degradation at designated time and were centrifugated before injected into the HPLC system.17

3. Results and Discussion 3.1. Preparation of PVP-PCL Micelles. In this work, amphiphilic block copolymer composed of PCL block as the

Re-Assembly of PVP-PCL Micelles


Table 1. Properties of the PVP-PCL Block Copolymer PVP-OH

PVP-PCL

sample

feeding ratio VP/CL

Mw

Mn

Mw

Mn

ratio VP/CL

PVP-PCL

1:3

11050

9950

50040

31210

1:3.05a

a

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presence of lipase AY. Thus, the intensity of scattering light (I) can be expressed as

I ≈ NM2

Determined by the SEC results.

hydrophobic part and PVP block as the hydrophilic one was synthesized through ring-opening polymerization with hydroxylterminated PVP as a macromolecular initiator. The major physicochemical properties of obtained copolymer are summarized in Table 1. The ratio of VP:CL determined from SEC is close to the feeding ratio, which means that the ring-opening copolymerization of ε-caprolactone with PVP-OH is almost complete. The chemical structure and composition of PVP-PCL were also confirmed by 1H NMR and FT-IR (data is not shown). The micelles from PVP-PCL block copolymer were obtained through a precipitation method. Obtained micelles have a mean diameter about 163 nm with a narrow size distribution as shown in Figure 1. Their morphology was measured by AFM, and shown in Figure 1b. Most of them have a spherical shape, and their average size was similar to that measured by DLS. Although, the micelles are stacked to some extent in the AFM image due to the drying and their regular spherical shape outline can be clearly figured out. 3.2. Degradation Behavior of PVP-PCL Micelles in the Presence of Lipase AY. Biodegradable amphiphilic micelles are usually completely biodegraded into small molecules inside the body in the action of body fluid, enzyme, and cells. Thus, both in vivo and in vitro studies of the biodegradation of these micelles are important. To study the enzymatic degradation of the PVP-PCL micelles, lipase AY was introduced into the degradation experiment. According to the previous report by Wu7a and us,7b dynamic light scattering was used to measure the degradation behaviors of PVP-PCL micelles in solution because it was a nonintrusive, sensitive, and powerful analytical tool. The changes in light scattering intensity and hydrodynamic size of PVP-PCL micelles in the action of lipase AY were used to reflect the enzymatic degradation of PVP-PCL micelles. Figure 2 shows the variations in the light scattering intensity and micelle size of the samples recorded in situ by DLS during the degradation procedure of PVP-PCL micelles in the presence of different concentrations of lipase AY at 37 °C. From Figure 2a, it can be seen that the light scattering intensity of the sample at lipase AY concentration of zero is almost invariant in the given degradation period, indicating that PVP-PCL micelles are very stable at this condition. When lipase AY is added into the system, the light scattering intensity drops very fast even with smallest lipase concentration (0.1 mg/mL). The decrease rate in light scattering intensity of the sample is also in parallel with the amount of lipase AY added in the micelle solution and all the samples show a quasi-linear decrease in light scattering intensity until the intensity reaches zero. On the other hand, it is interesting that the micelle size of the enzymatic degradation samples keeps almost constant in given degradation time regardless lipase AY concentration as shown in Figure 2b. Based on DLS theories, the intensity of light scattered by a suspension of particles with diameter d is proportional to the number of particles N, the square of the particle mass M, and the particle form factor P(q,d), which depends on particle size, scattering angle, index of refraction, and wavelength for the micelles having constant size and morphology.9b,18 However, in this system, it was found that the size of the PVP-PCL micelles was constant during the degradation procedure in the

The decrease of light intensity can be attributed to the decrease of either the particles number (N) or the mass of the particles (M). For micelles, the enzymatic degradation progress might be quite complex and the degradation process induced by the lipase was so fast that the DLS can only detect the number of the remaining undegraded micelles but not reflect the mass change of the micelles. If the mass of single micelle is constant, the change of the light intensity mainly reveals the variation of the micelle number. The quasi-linear decrease of light intensity suggests that the degradation of the PVP-PCL micelles in the presence of lipase AY may follow a “one-byone” degradation procedure similar to the degradation of PCLPEG nanoparticles.9 That is, the lipase will “eat” one micelle completely before it attacks next micelle. With high lipase concentration, more lipase will attack more micelles in the same time, resulting in faster degradation speed. The initial degradation rate (V0) as a function of lipase concentration can be conveniently calculated from the intensity changing curves. Here, V0 is defined as [dCt(I)/dt]tf0. Figure 3 shows the initial degradation rate V0, determined from Figure 2a. The initial degradation rate (V0) increases linearly with the enzyme concentration and can be fitted by the formula V0 (mg mL-1 min-1) ) 0.0091 × E0, indicating that at given micelles concentration, the initial degradation of PVP-PCL micelles increases linearly with the enzyme concentration. 3.3. Degradation of PVP-PCL without the Lipase. The degradability and stability of these PVP-PCL micelles in aqueous solution are also very important for the application of micelles, which will affect their drug encapsulation and release characteristics. Usually, the PCL block in PVP-PCL micelles will be hydrolyzed in solution due to the ester bond breakage, which leads to the decrease of molecular weight and the change of chemical composition. Therefore, it causes the size change or even disassembly of PVP-PCL micelles during the degradation process. Thus, DLS, SEC, and HPLC were used to investigate the hydrolytic degradation behaviors of PVP-PCL micelles at different conditions, such as different pH media and temperatures. Figure 4a shows the size variation of PVP-PCL micelles as a function of storing period in aqueous solution with different pH values measured by DLS. Interestingly, for all the pH values, the size of the micelles decreases continuously during the testing time except at pH 7.18, at which PVP-PCL micelles maintain their original size. We think that the decrease in the size of micelles during the experimental period is related to the degradation of PVP-PCL micelles. In addition, these micelles show different size reducing rates at different pH values: faster decrease in size is observed at either acidic or basic medium, indicating a rapid degradation. Figure 4b shows the size changes of PVP-PCL micelles in the medium with different temperature at pH 10.88. Obviously, higher temperature results in a greater decrease in micelle size, which indicates the degradation of PVPPCL micelles is highly temperature dependent at this condition. Figure 4c shows the changes of the light scattering intensity recorded by DLS during the degradation procedure of PVPPCL micelles with different pH values. At all pH values, including at 7.18, the light intensity decreases, implying that the degradation of PVP-PCL micelles occurs. Among them, the light intensity decreases fastest at acidic condition, and slowest at

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Figure 1. Size distribution of PVP-PCL micelles measured by DLS (a) and AFM image of PVP-PCL micelles (b).

Figure 2. Degradation of 0.25 mg/mL PVP-PCL micelles with different concentration of lipase AY (E0): (A) degradation time dependence of light intensity ratio (It/I0) at 37 °C, where the subscripts “0” and “t” represent initial time and degradation time, respectively; (b) degradation time dependence of the micellar diameter during enzymatic degradation.

4 °C, revealing that PVP-PCL micelles degraded more rapidly at high temperature in the basic medium.

Figure 3. Enzyme concentration dependence of initial degradation rate, V0.

pH 7.18. According to our previous assumption, the light intensity mainly depends on the number and the size of the micelles. Comparing Figure 4a and c, it is found that the decreases in light intensity are more rapid than in the size, which indicates that both the size and number of the PVP-PCL micelles reduce during the degradation procedure in the absence of enzyme. The light intensity variation of PVP-PCL micelles with different temperature is shown in Figure 4d in the medium of pH 10.88. The light intensity decreases fastest at 37 °C and slowest at

To further investigate the nonenzymatic degradation behavior of PVP-PCL micelles in aqueous solution, HPLC was used to analyze the final degradation products and the degradation extent. On day 84, 6-hydroxycaproic acid (6-HPA), the complete degradation monomer product of PCL was detected through HPLC and the content of 6-HPA in each sample is listed in Figure 5. From Figure 5, it can be seen the concentration of 6-HPA increases with the increase of the medium pH values at 37 °C and smallest 6-HPA concentration is found in the acidic degradation medium, which means more PVP-PCL micelles would completely degraded into 6-HPA in higher pH medium. However, this result conflicts to the variation of light intensity result as shown in Figure 4c, in which PVP-PCL micelles show fastest decrease in light intensity at acidic medium, indicating a greatest degradation rate in lower pH medium. Therefore, it is reasonable to hypothesize that in the acidic medium, PVPPCL micelles have the fastest degradation rate, but the degradation is a hydrolysis process of “random scission” in the PCL chains and produces various degradation products, including 6-HPA monomer, oligomer of CL, and short PCL segments, which reduce the size and light intensity of PVP-PCL micelles. However, compared to acidic condition, although PVP-PCL micelles have a slower degradation rate in the basic condition, the 6-HPA, which is the complete degradation monomer product in the solution, exhibits a higher concentration and is predominant in hydrolysis products, suggesting that degradation of PCL



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Figure 4. Variation of size (A) and light intensity (C) of PVP-PCL micelles with the extending storing time in the medium with different pH values; variation of size (B) and light intensity (D) of PVP-PCL micelles with the extending storing time at different temperatures in the medium with pH 10.88. Table 2. Molecular Weight and Molecular Weight Distribution of the Degraded PVP-PCL Micellesa

Figure 5. Yields of 6-hydroxycaproic acid in the degradation product of PVP-PCL at different experimental conditions. The last two columns represent the samples incubated in the pH 10.88 medium at room temperature and 4 °C, respectively.

segments may follow a “chain-end cleavage” mechanism in the basic condition. The degradation of PVP-PCL micelles also leads to the decrease in molecular weight of the constituting copolymer. Table 2 lists the molecular weight and molecular weight distribution of copolymer before and after degradation determined by SEC. Compared to the raw materials (sample 1), the molecular weight of PVP-PCL copolymer decreases during storing period at all conditions, which confirms the degradation of PVP-PCL micelles in the solution. The samples have the smallest average molecular weight in the acidic medium at day 84 (samples 2 and 3) and the smaller change in molecular weight is found for the PVP-PCL micelles at neutral condition

sample

Mn

Mw

d (Mw/Mn)

1 2 3 4 5 6 7 8

31210 11460 14830 29680 26520 17180 16780 29080

50040 18180 27910 42110 37630 40070 20720 47330

1.60 1.59 1.88 1.42 1.42 2.33 1.23 1.60

a Sample 1, raw PVP-PCL copolymer; samples 2-6, degradation products of PVP-PCL micelles at 37 °C in the medium with different pH values: 2.34, 3.79, 7.18, 9.21, and 10.88, respectively; samples 7 and 8, degradation products of PVP-PCL micelles at pH 10.88 at different temperature (room temperature and 4 °C), degradation time: 84 days.

(sample 4). These results are well agreement with the variation of light intensity (Figure 4c) that PVP-PCL micelles has fastest degradation rate in the acidic medium. When PVP-PCL micelles were incubated in basic medium (pH 10.88) with different temperatures, it was found that the molecular weight of copolymer almost had no variation at 4 °C compared to that of initial copolymer but fast decreases in molecular weight at room temperature. Besides, an unexpected phenomenon is observed for the sample 6, with the degradation temperature at 37 °C, higher than that of sample 7 (room temperature), it has a higher molecular weight than sample 7, which is not in agreement with the results obtained from Figure 4b,d, where higher storing temperature leads to a faster degradation of PVP-PCL micelles. Thus, the confliction results from light intensity and SEC suggest to further investigate this system by another technology such as TEM.

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Figure 7. TEM images of the degraded PVP-PCL micelles at pH 10.88 with different temperature: a, 4 °C; b, 25 °C; c 37 °C at day 84.

Figure 6. TEM images of the degraded PVP-PCL micelles in the different pH media at 37 °C: a, pH 2.34; b, pH 3.79; c, pH 7.18; d, pH 9.21 at day 84.

Figure 6 shows the morphology and microstructure of the degradation products of PVP-PCL micelles at day 84 in the different pH media. It can be seen that, at pH 2.34, no micelles are observed and only flower-like particles are present. For the degradation in pH 3.79 medium, irregular particles with a mean diameter smaller than 100 nm are found. In the medium of pH 7.18, the micelles with integrated spherical morphology are observed, and their size is similar to that of as-prepared PVPmicelles. Further increasing the pH value to 9.21, the sizereduced anamorphic PVP-PCL micelles are found in the TEM images. These TEM images clearly exhibit that the degradationinduced dissociation and deformation rate of PVP-PCL micelles in different pH media is in the order of acidic > basic > neutral medium, and they are relatively stable in the neutral medium (pH 7.18). However, when PVP-PCL micelles were incubated in pH 10.88 medium with different temperatures for 84 days, different morphologies are observed by TEM. At 4 °C, the spherical morphology of integrated PVP-PCL micelles, similar to the initial shape, with mean diameter about 150 nm, is clearly shown in the image (Figure 7a), indicating a slow degradation. When being stored in the medium at 25 °C, PVP-PCL micelles show a serious aggregation and some of the micelles are connected each other as a necklace (Figure 7b), but the spherical outline of each micelle can still be clearly identified. The inset shows the microstructure of these degraded PVP-PCL micelles with higher magnification, and a necklace structure of PVPPCL micelles degraded is clearly shown. Continually increasing the storage temperature to 37 °C, the PVP-PCL micelles show a rod-like morphology and most of these rods are composed of core-shell micelles smaller than 100 nm (Figure 7c). The coexistence of rod-like and spheres structures in Figure 7c indicates PVP-PCL micelles are not only degradable but also can automatically reassembly into nanorods (or nanowires) depending on the storage temperature in the medium of pH 10.88. In addition, these rod-like nanostructures have also been measured by AFM as shown in Figure 7d. By comparison of Figure 7d and Figure 1b, it is clearly that these rod-like nanostructures do not arise from the aggregation of PVP-PCL

Figure 8. TEM images of PVP-PCL micelles degraded in the medium pH 10.88 at 42 °C with different time: (a) as-prepared PVP-PCL nanoparticles; (b) 1 week; (c) 3 weeks; (d) 4 weeks.

micelles due to the solvent evaporation but are the new morphologies. For a single rod, it was composed of PVP-PCL micelles by the fusion each other. Therefore, the shape reconstruction during the degradation of PCP-PCL micelles in pH 10.88 medium at 37 °C might responsible for the retarding of the molecular weight decrease as shown in Table 2. To further probe such degradation-induced reassembly properties of PVP-PCL micelles, the degradation experiment at higher temperature (42 °C) was conducted in pH 10.88 medium, and the TEM images of PVP-PCL micelles at different degradation stages are shown in Figure 8. A spherical structure for as-prepared PVP-PCL micelles at 42 °C is clearly visualized in Figure 8a. After a 1 week incubation in the medium, PVPPCL micelles aggregate together and most of them are connected to each other like a necklace (Figure 8b). The inset is the TEM image of an individual degraded PVP-PCL micelle with higher



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Scheme 1. Illustration of the Morphology Transformation of PVP-PCL Micelles in the Degradation Procedure

magnification. It clearly shows the PVP-PCL micelle has a core-shell structure with PCL as the core and PVP as the shell. However, the shell at this stage is not intact and some PVP chains in the shell are detached from the core due to the ester bond degradation occurring in the core-shell interface of PVPPCL micelles. After 3 weeks incubation, these necklace-like micelles are transformed to the rod-like structures (Figure 8c). These rod-like structures are long and stable even after 8 weeks incubation. The coexistence of PVP-PCL micelles and rod-like nanostructures is also observed during the degradation procedure (Figure 8d), which indicates that the shape reconstruction of PVP-PCL micelles is concomitant with the degradation simultaneously during the incubation period. The morphogenesis of amphiphilic block copolymer systems in aqueous media has been identified in many studies.8,19 Diverse morphologies have been found in the aggregates made from diblock copolymers depending on the factors, including the chemical structure and concentration of the copolymers, solvent polarity, salt concentrations, and the presence of small molecule solubilizates.20,21 It was revealed that morphology transformation is determined by the force balance among three factors: the stretching of the core-forming blocks, the interfacial tension between the micelle core and the solvent, and repulsion among the corona chains.20a,22 Therefore, we proposed in this study that the free energy of a single PVP-PCL micelle (Emicelle) is also the sum of three factors: the stretching ability of PCL chain (Ecore), which is determined by the structure of the PCL and its hydrophobic property; the repulsion force of PVP corona (Ecorona) and the interfacial energy between the PCL core and water (Einterface).

Emicelle ) Ecore + Einterface + Ecorona The energy balance among these three terms controls the morphology transformation of these PVP-PCL micelles during the degradation procedure. The term that contributes most to the free energy in the system will have the greatest influence on the energy balance and control the formation of morphology with lowest energy. In this system, PVP constructed the shell of PVP-PCL micelles, which provides the steric repulsions between PVP chains and PVP-PCL micelles. Therefore, in this case, for the as-prepared spherical PVP-PCL micelles, because PVP provides a dense brush of polymer chains, which perfectly protects the PCL core and minimized the interfacial free energy between the water and PCL core, the stability of PVP-PCL is mainly affected by the PVP chains (Ecorona). At this stage, PVPPCL micelles have the lowest energy. During the degradation procedure of PVP-PCL micelles at higher temperature, some of the PVP chains would break off from the core-shell interface of PVP-PCL micelles and release into the water, similar to the

behavior of PEG in the degradation of PCL-PEG micelles that we previously observed.7c Thus, due to the partial loss of PVP chains, an increase of Ecorona will appear during the degradation for the PVP-PCL micelles. On the other hand, more and more PCL chains in the PCL cores would directly expose to the water due to the detachment of PVP, which consequently increase the interfacial energy between the PCL core and water (Einterface). Therefore, the free energy of the degraded PVP-PCL micelles increases. These degraded micelles with higher free energy (Emicelle) are instable in the solution and would reassembly to form the new morphology to minimize their free energy, such as nanorods. From these results, it is proposed that the possibility of a morphological transition from spheres to rod-like structures upon the degradation of PVP-PCL micelles can be illustrated as Scheme 1. Specifically, for the as-prepared PVP-PCL micelles, they show integrated spherical morphology with a core-shell structure, where PCL cores are surrounded by PVP chains with minimum free energy. As the degradation proceeded, some of the PVP chains are detached from the core-shell interface of PVP-PCL micelles, and leave parts of the PCL core to expose directly to water, which increases the free energy of Einterface and Ecorona (Scheme 1B; some of the PCL core would also partially degrade in this process). In this stage, these PVP-PCL micelles are not stable and prefer to connect with each other to reduce their surface area to minimize their free energy. However, due to the hydrophobic property of PCL chains, the exposed PCL will attract each other to form larger necklace-like nanoaggregates (Scheme 1C). The newly formed nanoaggregates have a PCL core coated with PVP chains, which lowers system free energy (Einterface and Ecorona) as compared to the incomplete PVP-PCL micelle system. After the complete fusion of PCL cores, these necklace-like nanoaggregates would transform into rod-like structure (or wire; Scheme 1D). This shape transformation step prevents the further degradation of PVP-PCL chains, which explains the less molecular weight loss in higher degradation temperature at pH 10.88 for PVP-PCL micelles.

Conclusion In this work, PVP-PCL micelles with a core-shell structure were prepared by the precipitation method and the degradation behavior of the micelles in aqueous solution was investigated. These micelles might degrade “one-by-one” in the presence of Lipase AY. However, without the presence of enzyme, the PVPPCL micelles would randomly degrade in the acidic medium and hydrolyzed from the end by “chain-end cleavage” in the basic medium. Furthermore, at higher degradation temperature (42 °C) in the pH 10.88 medium, it is found that shape of these PVP-PCL micelles will gradually change from integrated

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spheres to broken spheres, necklace and rod-like structure depending on the degradation time. This morphology transformation could be explained by the detachment of PVP from and the fusion of PCL cores in those incomplete PVP-PCL micelles. Acknowledgment. This work is supported by the National Natural Science Foundation of China (Nos. 50625311, 20874042, and 50603008,), National 863 Project No. 2007AA100704, and by the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (No. 707028).

Hu et al.

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Degradation and Degradation-Induced Re-Assembly of PVP-PCL

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