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Surface-functionalized biodegradable nanoparticles consisting of amphiphilic graft polymers prepared by radical copolymerization of 2-methylene-1,3-dioxepane and macromonomers Taka-Aki Asoh, Takahito Nakajima, Takuya Matsuyama, and Akihiko Kikuchi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b01149 • Publication Date (Web): 05 Jun 2015 Downloaded from http://pubs.acs.org on June 9, 2015

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Surface-functionalized biodegradable nanoparticles consisting of amphiphilic graft polymers prepared by radical copolymerization of 2-methylene-1,3-dioxepane and macromonomers Taka-Aki Asoh†, Takahito Nakajima, Takuya Matsuyama, and Akihiko Kikuchi*

Department of Materials Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan † Present address: Advanced Research Institute for Natural Science and Technology, Osaka City University, 3-3-138 Sugimoto Sumiyoshi-ku, Osaka-shi, 558-8585, Japan Corresponding author: Prof. Akihiko Kikuchi Phone: +81-3-5876-1415; Fax: +81-3-5876-1415; E-mail: [email protected]

Keywords:

2-methylene-1,3-dioxepane,

ring-opening

radical

nanoparticles, hydrolysis

ACS Paragon Plus Environment

polymerization,

biodegradable

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Abstract Biodegradable polyester-based nanoparticles were prepared by precipitation of amphiphilic graft copolymers,

which

were

prepared

by

the

ring-opening

radical

2-methylene-1,3-dioxepane (MDO) and amphiphilic macromonomers.

copolymerization

of

The diameter of the

nanoparticles was controlled by the degree of grafting and the molecular weight of the grafting oligomer. PMDO-g-poly(ethylene glycol) nanoparticles were degraded by the alkaline hydrolysis of the polyester backbone. Although the colloidal stability of nanoparticles was retained due to the re-orientation of the PEG chains during hydrolysis, the size of the nanoparticles decreased with increasing hydrolysis time. We also prepared PMDO-g-poly(N-isopropylacrylamide) nanoparticles, which show aggregation in response to increasing temperature.

2


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Introduction Nanoparticles have found use in a wide range of applications due to their well-defined morphologies and large surface areas. In particular, polymeric nanoparticles have been used in biomedical applications, because the properties of these nanoparticles can be adjusted by designing their chemical structure, surface functionalities, and size.1-8 Therefore, biodegradable polymeric micelles or nanoparticles based on polysaccaride1, 2, poly(amino acid)3-5, and polyester6-8 have been studied as carriers for drug delivery systems. Block copolymers, which are prepared by the ring-opening polymerization (ROP) of ring monomers from hydrophilic macro-initiators often form micelles in aqueous medium9. Amphiphilic graft copolymers were used for the preparation of nanoparticles of hydrophobically modified polysaccharides or polypeptides1, 2, 4, 5 with a hydrophilic backbone and hydrophobic side chains by a self-assembly processes. Although polyesters, such as poly(lactic acid) (PLA)10, poly(ε-caprolactone) (PCL)11, and poly(1,3-trimethylene carbonate) (PTMC)12 have been used as particles, it is difficult to copolymerize a functional vinyl monomer and/or to modify vinyl monomers that are polyester side chains for the preparation of graft copolymers composed of a polyester backbone and hydrophilic graft chains. For example, PCL-g-PEG was prepared by combination of ROP and click chemistry.11 At first, chloro-functionalized poly(ε-caprolactone) was prepared by ring opening polymerization of ε-caprolactone and α-chloro-ε-caprolactone, and then the chloro groups of the polymers were converted into azido groups by NaN3. Finally, a copper-catalyzed cycloaddition reaction was carried out between azido-functionalized poly(ε-caprolactone) and alkyne-terminated poly(ethylene glycol) (PEG) to prepare the PCL-g-PEG required for multi-step reactions. On the other hand, 2-methylene-1,3-dioxepane (MDO) is a well-known radical polymerizable cyclic monomer13-15, and biodegradable plastics were prepared by the copolymerization of hydrophobic vinyl monomers such as styrene and methyl methacrylate16-18. Although micelles with a diameter of a few 3


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nanometers11,19-22 composed of PMDO or a PCL backbone with PEG grafting have been reported, the ability to control the size of biodegradable nanoparticles is a key requirement for developing technologies for drug delivery systems utilizing particle carriers, because the selection of particles with a size ranging from a few nm to sub-µm scale is required for target cells and/or organs. Moreover, the surface-functionalization of nanoparticles is also required to achieve colloidal stability23,

24

, stealth

properties, for inhibition of an unspecific interaction, and for interaction control of proteins and/or cells in the living body.25-28 In this study, we prepared biodegradable nanoparticles with amphiphilic corona on the nanoparticle surface by a precipitation method of hydrophilic or amphiphilic oligomer grafted PCL (Figure 1). An amphiphilic graft copolymer, such as PMDO-g-PEG, was simply prepared by the free-radical ring-opening copolymerization of MDO and PEG monomethacrylate (PEG-MA). The diameter of the nanoparticles was successfully regulated by controlling the degree of grafting of the PEG units to the PMDO backbone. The colloidal dispersion and chemical stability of PMDO-g-PEG nanoparticles in phosphate buffered saline (PBS) and under alkaline conditions were investigated at 37 °C. Moreover, PMDO-g-poly(N-isopropylacrylamide) nanoparticles were prepared, and the colloidal dispersion stability was modulated by external stimuli for the potential demonstration of surface-functionalized biodegradable nanoparticles.

Experimental Materials Poly(ethylene glycol) monomethacrylate (2,000 and 4,000 g/mol) and chloroacetaldehyde dimethylacetal were purchased from Aldrich and were used as purchased without further purification. In addition, 1,4-butanediol, Dowex 50 (H+), potassium t-butoxide (t-BuOK), tetrahydrofuran (THF), N-isopropylacrylamide (NIPAAm) and 2,2′-azobis(isobutyronitrile) (AIBN) were purchased from Wako Pure Chemical Industries (Osaka, Japan). 2-Methylene-1,3-dioxepane (MDO) was prepared by an acetal 4


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exchange reaction followed by dehydrohalogenation according to a procedure in the literature.16 Briefly,

2-chloromethyl-1,3-dioxepane was prepared by the reaction of chloroacetaldehyde

dimethylacetal and 1,4-butanediol in the presence of Dowex 50 (H+). Then, MDO was obtained from 2-chloromethyl-1,3-dioxepane by a dehydrochlorination reaction in the presence of t-BuOK in THF. PNIPAAm macromonomer was synthesized using a three-step process that was previously described.23, 24

Synthesis of PMDO-g-PEG The copolymerization of MDO and PEG monomethacrylate was carried out in bulk using 1 mol% AIBN as initiator under a nitrogen atmosphere.

PEG monomethacrylate (2000 g/mol) and AIBN were

dissolved in MDO and then polymerization was carried out for 48 h at 70 °C with an MDO: PEG monomethacrylate molar feed ratio of 500:1, 200:1, 100:1, and 50:1.

The resulting polymer was

washed by ethanol, and then the product was obtained by lyophilization after the polymer was dialyzed using a Spectra/Por regenerated cellulose membrane (molecular weight cut-off of 50,000) in distilled water. The chemical structure of PMDO-g-PEGs was confirmed by proton nuclear magnetic resonance (1H-NMR) spectroscopy.

Products are coded as PMDO-g-PEGx, where x represents the grafting degree

of PEG. The molecular weight and polydispersity of the various PMDO-g-PEG polymers were determined by gel permeation chromatography (GPC) at 45 °C using DMF containing 10 mmol/L LiCl as an eluent. Polystyrene was used as a molecular weight standard.

Synthesis of PMDO-g-poly(N-isopropylacrylamide) The copolymerization of MDO and poly(N-isopropylacrylamide) (PNIPAAm) macromonomer was carried out in bulk using 1 mol% AIBN as initiator under a nitrogen atmosphere. The PNIPAAm macromonomer and AIBN were dissolved in MDO and then polymerization was carried out for 48 h at 70 °C with an MDO: PNIPAAm macromonomer molar feed ratio of 400:1. 5


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Preparation of nanoparticles For particle formation, 10 mg of PMDO-g-PEG was dissolved in 1 mL of THF, and then dropped into 1 mL of water at room temperature.

Nanoparticles were dialyzed using a Spectra/Por regenerated

cellulose membrane (molecular weight cut-off of 50,000) in distilled water, and then concentrated by centrifugation. The size distribution of the nanoparticles was measured by dynamic light scattering (DLS, ELS-Z 1000S, Otsuka Electronics Co., Ltd., Osaka, Japan), and the morphology of the nanoparticles was observed with a scanning electron microscope (SEM, JSM-6060LA, Jeol, Tokyo, Japan).

Degradation of nanoparticles The hydrolysis of PMDO-g-PEG nanoparticles was performed under alkaline conditions as an acceleration test. For the alkaline hydrolysis, the PMDO-g-PEG nanoparticles were dispersed in a 0.01 or 0.05 mol/L NaOH solution, and immersed in a thermostatic water bath at 37 °C for 21 days.

Results and discussion Preparation of PMDO-g-PEG PMDO-g-PEG was prepared by the free-radical ring-opening copolymerization of MDO and PEG monomethacrylate (2000 g/mol) (Figure 2a). Figure 2b shows the FT-IR spectrum of PMDO-g-PEG: the band at 1735 cm-1 was assigned to the ester group, indicating formation of the polyester by the ring-opening reaction of MDO. The polymerization results are summarized in Table 1. The polymerization in bulk at 70 °C was achieved with AIBN as the initiator. The Mn and Mw/Mn ratio increased and the SEC curve (Fig. S1) became multimodal as the grafting degree of the PEG units increased, thereby indicating the introduction of the PEG monomethacrylate (2000 g/mol) into the PMDO backbone. The chemical composition of PMDO-g-PEG was adjusted by controlling the molar 6


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ratio of MDO and PEG monomethacrylate. Figure 2c shows the 1H-NMR spectrum of typical PMDO-g-PEG obtained in this way. The 1H-NMR spectrum showed typical signals marking the methylene protons of PMDO and PEG at 4.06 and 3.64 ppm, respectively. This result indicates that PEG monomethacrylate was introduced into the PMDO backbone. It is well known that branched structures are obtained by the radical ROP of MDO.14, 15 This study confirmed the branch structure and the typical signal of the methyl protons of the branched termini was observed at 1 ppm. The molar ratio of the PEG units to the total units was calculated by obtaining the ratio of the integral intensities of the backbone methylene protons to the methyl protons of the PEG units (Fig. S2). Although it is difficult to determine the copolymerization parameters, the Mn of PMDO-g-PEGs was increased by changing the ratio of the [MDO]/[PEG], and then varying the degree of PEG that was introduced from 0.25 to 2.12 %; hence, the grafting degree that was obtained corresponded with that of the feed.

Preparation of nanoparticles PMDO-g-PEG nanoparticles were prepared by mixing THF as a good solvent and water as a poor solvent for PMDO. These PMDO-g-PEG particles were stable in water, even after removing the THF by dialysis. Figure 3 shows the SEM image of PMDO-g-PEG nanoparticles, and a spherical morphology was confirmed by SEM observation with a smooth surface. PMDO-g-PEGx nanoparticles with a varying number of PEG units were successfully obtained, although PMDO particles were not formed (data not shown). The amount of PEG plays a key role in the stabilization of particles and their stealth properties in water. The MDO units were arranged in the particle core probably because their hydrophobicity was higher than that of the PEG units. Therefore, PMDO-g-PEG0.25 formed a few nanoparticles, however, aggregation was observed because of the lower number of PEG units. The nanoparticle size was determined by DLS measurement and is shown in Figure 4. The diameters of the PMDO-g-PEGx nanoparticles obtained with a 0.44 % grafting degree was about 250±115 nm (Figure 4a) and decreased to 92±46 nm (Figure 4b) when the grafting degree was increased to 0.82 %, leading to the formation of 7


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nanoparticles with a smaller size because of the higher PEG content. These results were in good agreement with the SEM observation (Figure 3). Agarwal et al. reported the preparation of biodegradable

and

cross-linkable

micelles

utilizing

amphiphilic

poly[MDO-co-PEGMA-co-7-(2-methacryloyloxyethoxy)-4-methylcoumarin], for which the diameter and PEG content of the micelles ranged from 74 to 38 nm and 29 to 45%, respectively.19 The PEG content in this study was tenfold higher than that in the present study, resulting in the formation of micelles instead of nanoparticles. In this study, the particle diameter of PMDO-g-PEG2.12 was 66 nm (Figure 4c). It is well known that the balance between the hydrophilicity and hydrophobicity of amphiphilic polymer chains decides the size of self-assembled nanomaterials; therefore, we changed the molecular weight of PEG-MA from 2,000 to 4,000 g/mol. A comparison of runs 5 and 8 in Table 1 reveals that PMDO-g-PEG (4,000 g/mol)

0.22

formed nanoparticles with a diameter of ca. 360 nm,

although aggregation was observed in the case of PMDO-g-PEG (2,000 g/mol) 0.25. The diameter of the nanoparticles decreased as the molecular weight of PEG increased (comparing runs 3 and 7 in Table 1), indicating that a longer PEG unit is a more effective dispersant. These results are in good agreement with our previously reported core-corona type of nanoparticles.23 Therefore, we successfully prepared nanoparticles and controlled nanoparticle diameters by modulating the composition of PMDO and PEG.

Degradation of nanoparticles Nanoparticle degradation is one of the most important properties of a DDS carrier. The biodegradability of the PMDO-g-PEG nanoparticles was estimated from the decrease in their molecular weight following hydrolytic degradation. The hydrolysis of PMDO-g-PEG nanoparticles has been investigated by analyzing degraded samples of PMDO-g-PEG using GPC after 8 days. When PMDO-g-PEG nanoparticles were incubated in PBS at 37 °C, the polymer was not observed to hydrolyze after 8 days. Both the colloidal dispersion and chemical stability of the nanoparticles were found to be stable in PBS at 37 °C; aggregation, precipitation, and degradation of PMDO-g-PEG nanoparticles were 8


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not observed under these conditions (Figure 5a). The molecular weight was not observed to decrease, as confirmed by SEC measurements before and after incubation. Therefore, the hydrolysis of PMDO-g-PEG nanoparticles was carried out at 37 °C under alkaline conditions ([NaOH] = 0.05 mol/L) as an acceleration test. As shown in Figure 5b, increasing the incubation time eliminated the turbidity of the nanoparticle suspension, indicating the degradation of hydrophobic PMDO. After 8 days, the average molecular weight of PMDO-g-PEG decreased from 19,000 to 3,800 (Figure 5c). The polymer hydrolysis ratio was higher under alkaline conditions than at pH 7.4, indicating that the hydrolysis of PMDO-g-PEG nanoparticles was accelerated by the addition of NaOH. This result suggested that the PMDO-g-PEG nanoparticles degraded as a result of the hydrolysis of the ester groups in the PMDO backbone. Next, the degradability of nanoparticles during hydrolysis was investigated under mild conditions. Alkaline hydrolysis was carried out at 37 °C in 0.01 mol/L aqueous NaOH solution. The structural change of PMDO-g-PEG nanoparticles was measured during hydrolysis by in situ monitoring of the 1H-NMR spectral changes in a solution of D2O (0.01 mol/L NaOD). Figure 6 shows the 1H-NMR spectra during the degradation of PMDO-g-PEG nanoparticles in NaOD. The CH2 proton signal from the polyester unit in the polymer backbone appeared at 4.0 ppm before hydrolysis, and the signal shifted to 3.6 ppm after 7 days of hydrolysis. Other peaks shift upfield, indicating degradation of the polyester backbone and the appearance of compounds with a low molecular weight. After 21 days of hydrolysis, the majority of the peaks arising from the polymers, except for PEG, disappeared because of the degradation of the nanoparticles. The PMDO-g-PEG0.44 nanoparticles show a broad size distribution with an average diameter, as obtained by DLS, of 248 ±117 nm. After an incubation period of 14 days, the larger nanoparticles over 300 nm disappeared, as shown in Figure 7a, and the average diameter obtained by DLS was 161±33 nm. As shown in Figure 7b, the continuous reduction of the nanoparticle diameter indicates successive hydrolysis from the nanoparticle surface to the core. Interestingly, colloidal dispersity was maintained during hydrolysis of nanoparticles from 0 to 14 days, although hydrophobic PMDO may be exposed due to the disappearance of PEG on the surface resulting from the hydrolysis of 9


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the nanoparticle surface. In fact, aggregation of nanoparticles was observed during hydrolysis with a 0.05 M NaOH aqueous solution. These results indicate that grafted PEG units were cleaved at an early stage compared with the PMDO backbone in the case of higher NaOH concentrations and the absence of PEG units on the surface of the nanoparticles destabilized their dispersion. The Mn of nanoparticles measured by SEC before and after hydrolysis for 14 days is summarized in Table 2 and was 19,000 and 13,000, respectively, but the Mw/Mn ratio decreased during degradation.

To investigate the chemical

composition during nanoparticle hydrolysis, the nanoparticles were completely dissolved in CDCl3 and the chemical composition of the remaining nanoparticles was measured by1H-NMR. Before hydrolysis, the grafting degree of PEG of PMDO-g-PEG was 0.44%, and this changed to 0.35 % after 14 days of hydrolysis (Table 2). This result suggests that PMDO-g-PEG nanoparticles were initially formed with PEG content increasing as a gradient from core to surface, such that PEG remained inside the nanoparticles. The inner and outer surfaces of nanoparticles had undergone a lower and higher degree of grafting of PEG, respectively, after which the Mw/Mn value decreased during degradation due to the low degree of grafting of PEG, because the initial high value of Mw/Mn depended on a higher degree of grafting of the PEG units. Therefore, the colloidal stability of nanoparticles remained due to the re-orientation of PEG during hydrolysis, although the size of the nanoparticles decreased with increasing hydrolysis time (Figure 7c). Presently, the nanoparticles do not meet the requirements of clinical applications due to their wide size distribution, but the results strongly indicate the possibility that size control and degradation of nanoparticles can be achieved. Moreover, the enzymatic degradation of PEG-grafted PMDO has been reported by other researchers21, and changing the size and colloidal stability of nanoparticles during enzymatic degradation is important for further application. We believe that the present design of the nanoparticles provides further insight into the development of drug carriers. Precise control of the preparation and enzymatic degradation of the nanoparticles is now in progress.

Preparation of thermoresponsive nanoparticles 10


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The biodegradable nanoparticles were simply prepared by the copolymerization of MDO and hydrophilic macromonomer; therefore, we then introduced stimuli-responsiveness to biodegradable nanoparticles. Poly(N-isopropylacrylamide) (PNIPAAm) is a well-known thermoresponsive polymer, which exhibited a thermo-reversible phase transition in aqueous solution at a lower critical solution temperature (LCST). The block copolymer micelles composed of a degradable hydrophobic polymer segment and a PNIPAAm segment show the thermoresponsive hydrophilic/hydrophobic changes of the micelle surfaces around the LCST of PNIPAAm25,

26

, and temperature-induced drug release27 or

intracellular uptake of micelles28 have been investigated. Previously we prepared PNIPAAm modified polystyrene nanoparticles, which were dispersed in cool water, but they were observed to exist as aggregates in hot water21. Dispersion and aggregation of nanoparticles were found to occur to a major extent around the LCST of PNIPAAm. Therefore, we prepared PNIPAAm modified PMDO nanoparticles; PMDO-g-PNIPAAm was prepared by the copolymerization of MDO and PNIPAAm macromonomer (Figure 8a) instead of poly(MDO-co-NIPAAm)29. When the grafting degree of PNIPAAm was 0.14 %, the diameter of PMDO-g-PNIPAAm nanoparticles was 840±310 nm, and the spherical morphology was confirmed by SEM observation (Figure 8b). As shown in Figure 3c, PMDO-g-PNIPAAm nanoparticles show colloidal dispersion stability at the lower temperature of PNIPAAm’s LCST (Figure 8c left; 25 ° C). However, the drastic aggregation of nanoparticles was observed above the LCST of PNIPAAm (Figure 8c right; 40 °C), These results indicate that dispersion and aggregation of PMDO-g-PNIPAAm nanoparticles was controlled by modulating the temperature as shown in Figure 8d.

Conclusions In conclusion, biodegradable nanoparticles were prepared from PMDO-g-PEG, which was prepared by the free-radical ring-opening copolymerization of MDO and PEG monomethacrylate. PMDO-g-PEG nanoparticles with varying PEG unit numbers were successfully prepared by the precipitation method. 11


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The diameter of the nanoparticles was controlled over a wide range by the grafting degree of the PEG units, which led to the formation of nanoparticles of a smaller size as the PEG content was increased. PMDO-g-PEG nanoparticles were degraded under alkaline conditions due to the hydration of the PMDO ester groups, although degradation, aggregation, and precipitation were not observed in PBS at 37 °C. During hydrolysis, the colloidal stability of nanoparticles was retained due to the re-orientation of PEG, although the size of the nanoparticles was found to decrease with increasing hydrolysis time. Thermoresponsive PMDO-g-PNIPAAm nanoparticles were also prepared by copolymerization of MDO with the PNIPAAm macromonomer, which led to the temperature-induced aggregation of nanoparticles. These nanoparticles are expected to be useful as drug delivery carriers, because of the introduction of the desirable attribute to the biodegradable nanoparticles, namely the selection of comonomers and macromonomers.

Acknowledgement Part of this work was supported by the Program for Development of Strategic Research Center in Private Universities by the Ministry of Education, Culture, Sports, Science and Technology, Japan (2010) “Physical Pharmaceutics”. We are grateful to Mr. Takuya Koriyama, and Mr. Syuuhei Komatsu, Tokyo University of Science for assistance with the preparation of the manuscript.

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Xu,

J.;

Liu,

Z.-L.;

Zhuo,

R.-X.

Synthesis

and

Biodegradability

Evaluation

of

2-Methylene-1,3-dioxepane and Styrene Copolymers. J. Appl. Polym. Sci. 2007, 103, 1146–1151. [19] Jin, Q.; Maji, S.; Agarwal, S. Novel Amphiphilic, Biodegradable, Biocompatible, Cross-linkable Copolymers: Synthesis, Characterization and Drug Delivery Applications. Polym. Chem. 2012, 3, 2785– 2793. [20] Delplace, V.; Tardy, A.; Harrisson, S.; Mura, S.; Gigmes, D.; Guillaneuf, Y.; Nicolas, J. Degradable and Comb-Like PEG-Based Copolymers by Nitroxide-Mediated Radical Ring-Opening Polymerization. Biomacromolecules 2013, 14, 3769−3779 [21] Cai, T.; Chen, Y.; Wang, Y.; Wang, H.; Liu, X.; Jin, Q.; Agarwal, S.; Ji, J. Functional 14


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2-Methylene-1,3-Dioxepane Terpolymer: A Versatile Platform to Construct Biodegradable Polymeric Prodrugs for Intracellular Drug Delivery. Polym. Chem. 2014, 5, 4061–4068 [22] Cai, T.; Chen, Y.; Wang, Y.; Wang, H.; Liu, X.; Jin, Q.; Agarwal, S.; Ji, J. One-Step Preparation of Reduction-Responsive Biodegradable Polymers as Efficient Intracellular Drug Delivery Platforms. Macromol. Chem. Phys. 2014, 215, 1848–1854. [23] Matsuyama, T.; Shiga, H.; Asoh, T.; Kikuchi, A. Thermoresponsive Nanospheres with a Regulated Diameter and Well-Defined Corona Layer. Langmuir 2013, 29, 15770–15777. [24] Matsuyama, T.; Kimura, A.; Asoh, T.; Kikuchi, A. Transformable Core-corona Nanoparticles: Simultaneous Change of Core Morphology and Corona Wettability in Response to Temperature. Colloids Surf. B 2014, 123, 75–81. [25] Kohori, F.; Sakai, K.; Aoyagi, T.; Yokoyama, M.; Sakurai, Y.; Okano, T. Preparation and Characterization

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Figure1. Surface-functionalized nanoparticle prepared by amphiphilic graft polymers consisting of degradable hydrophobic backbone and amphiphilic graft chains.

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Figure 2. (a) Synthesis of the PMDO-g-PEG by ring-opening radical copolymerization of MDO and poly(ethylene glycol) monomethacrylate. 1

(b) FT-IR and (c) 1H-NMR spectra of PMDO-g-PEG0.25.

H-NMR was measured in CDCl3.

Figure 3. SEM images of (a) PMDO-g-PEG0.44 and (b) PMDO-g-PEG0.82 nanoparticles.

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Figure 4. Size distribution of (a) MDO-g-PEG0.44, (b) PMDO-g-PEG0.82, and (c) PMDO-g-PEG2.12 nanoparticles in water.

Inset pictures are optical images of dispersed nanoparticles in water. Green line

in (c) was laser light, indicating the Tyndall phenomenon.

Figure 5. (a, b) Scattering observation of nanoparticles before (left) and after (right) incubation for 8 days. PMDO-g-PEG nanoparticles were incubated in (a) PBS or (b) 0.05 mol/L NaOH aqueous solution 19


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at 37 °C. (c) SEC traces before (circle) and after (triangle) hydrolysis for 8 days

Figure 6. 1H-NMR spectra of PMDO-g-PEG0.44 nanoparticles during hydrolysis in D2O. Hydrolysis was carried out for 21 days in 0.01 mol/L NaOD aq. solution at 37 °C. 20


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Figure 7. Size distribution of (a) PMDO-g-PEG0.44 nanoparticles before and after hydrolysis. Particle diameter of PMDO-g-PEG0.44 during hydrolysis from 0 to 14 days.

(c)

(b)

Illustration of

proposed mechanism of nanoparticle degradation with re-orientation of PEG inside PMDO core during hydrolysis.

Figure 8. (a) Chemical structure of PMDO-g-PNIPAAm and (b) SEM observation of their nanoparticles. (c) Colloidal stability of PMDO-g-PNIPAAm nanoparticles at (left) 25 and (right) 40 °C. White arrow indicates aggregation of nanoparticles. (d) Illustration of temperature-induce aggregation of nanoparticles 22


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in water.

Table 1. Syntheses of PMDO-g-PEG in bulk at 70 °C.a

a

AIBN (1 mol% to monomer) was used as the initiator and polymerization was carried out for 48 h.

Molecular weight of PEG-MA was 2,000 g/mol.

b

The molecular weight and polydispersity were

determined by GPC. c Grafting degree of PEG was calculated from 1H-NMR measurement in CDCl3. d

The particles were prepared by precipitation onto water and the diameter and standard deviation (S.D.)

of nanoparticles was measured by DLS.

e

PEG monomethacrylate (4,000 g/mol) was used instead of

2,000g/mol. f Particles were not formed.

Table 2. Characterization of PMDO-g-PEG0.44 nanoparticles during hydrolysis 23


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Hydrolysis of nanoparticles was carried out in 0.01 mol/L NaOH or NaOD aqueous solution at 37 °C. b

The molecular weight and polydispersity were determined by GPC. calculated from the 1H-NMR measurement.

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c

Grafting degree of PEG was

Surface-Functionalized Biodegradable ... - ACS Publications

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