Preparation, Biocompatibility, and Potential Application as Drug Carriers

May 12, 2014 - peroxodisulfate (KPS, 99.5%) and NaHSO4 were obtained from. Sinopharm Chemical Reagent Co., Ltd. All of the chemicals were used...
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Poly(N‑vinylpyrrolidinone) Microgels: Preparation, Biocompatibility, and Potential Application as Drug Carriers Qing Yang,† Kai Wang,‡ Jingjing Nie,‡ Binyang Du,*,† and Guping Tang*,‡ †

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science & Engineering, Zhejiang University, Hangzhou 310027, China ‡ Department of Chemistry, Zhejiang University, Hangzhou 310027, China S Supporting Information *

ABSTRACT: The biocompatible poly(N-vinylpyrrolidinone) (PNVP) microgels were synthesized via surfactant free emulsion polymerization with N-vinylpyrrolidinone (NVP) as the monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker at 60 °C. The obtained PNVP microgels are spherical in shape with hydrodynamic diameter of approximately 200 nm and narrow size distribution. The PNVP microgels show rough surfaces due to the different reaction rates of monomer NVP and cross-linker EGDMA. The obtained PNVP microgels could well disperse in phosphate-buffered saline (PBS) solution with long-term stability, which make them candidates for bioapplications. The results of 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) tests indicated that the PNVP microgels are biocompatible with low toxicity even at a concentration of 1000 μg/mL. By labeling the PNVP microgels with fluorescein comonomer, it was demonstrated that the PNVP microgels could be uptaken by human embryonic kidney 293 (HEK-293) cells. The experimental results indicated that the release of isoniazid (INH, the hydrophilic model drug) could be well described by a Fickian release, whereas the microgels exhibited burst release for 5-fluorouracil (5-fu, the hydrophobic model drug).

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INTRODUCTION Much attention has been paid on the development of poly(Nvinylpyrrolidinone) (PNVP) related hydrogels because such hydrogels could have potential applications as contact lenses,1,2 cell culture substrates,3−7 drug carriers for controlled-releasing of therapeutic drugs, and so on. Regarding the aspect of using them as drug carriers, for example, Geever et al.8 developed a thermoresponsive poly(N-isopropylacrylamide)-co-poly(N-vinylpyrrolidinone) (PNIPAm-co-PNVP) hydrogel for drug delivery applications, and two model drugs (diclofenac sodium and procaine HCl) were tested. pH-responsive poly(Nvinylpyrrolidinone)-co-poly(acrylic acid) (PNVP-co-PAA) hydrogel was also developed in the same research group for the encapsulation and releasing of aspirin and paracetamol. The authors found that aspirin was released slower than paracetamol in all cases studied, which was attributed to the intermolecular bonding between the PNVP-co-PAA network chains and drug loaded.9,10 PNIPAm/PNVP interpenetrating polymer networks were also synthesized for the delivery and release of lidocaine.11 Furthermore, it would be useful if PNVP-related nanoparticles or microgels could be developed for application as drug-controlled-release systems. Nanoparticles and microgels have particle sizes of tens to hundreds of nanometers and could be well dispersed in aqueous solution and hence injected to any position where drug-controlled-release might be required. The nanometer size of drug-carriers may potentially increase the drug circulation time after intravenous (i.v.) administration.12 © XXXX American Chemical Society

Several PNVP-modified biocompatible and biodegradable polymer nanoparticles have been developed for drug delivery.12−17 For instance, poly(N-vinylpyrrolidinone)-b-poly(ε-caprolactone) (PNVP-b-PCL) nanoparticles have been synthesized to load and deliver paclitaxel for antitumor applications. The drug loading content of paclitaxel in PNVPb-PCL nanoparticles was about 15%. The authors found that the length of PNVP block had a significant influence on cytotoxicity, anti-BSA adsorption, circulation time, biodistribution, and antitumor activity for the nanoparticles.16,17 Polymeric micelles of poly(N-vinylpyrrolidinone)-b-poly(d,L-lactide) (PNVP-b-PDLLA) were synthesized to efficiently solubilize anticancer drugs, such as paclitaxel, docetaxel, teniposide, and etoposide.12 The safety, pharmacokinetics, biodistribution, and antitumor activity of these drug-load PNVP-b-PDLLA micelles were studied and discussed.12 Microgels are three-dimensional cross-linked polymeric colloidal particles with sizes of 1−1000 nm and possess the properties of hydrogels and colloidal particles. Monodispersed thermoresponsive poly(N-isopropylacrylamide) (PNIPAm) microgels were first synthesized by surfactant-free emulsion polymerization (SFEP) at elevated temperature (>55 °C).18 Since then, extensive efforts have been paid on the studies of Received: March 26, 2014 Revised: May 10, 2014

A

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Table 1. Experimental Conditions of a Series of PNVP Microgels Prepared DLS sample code

NVP (mg)

EGDMA (mg)

N-E3.5 N-E6 N-E9 N-E12 E

165 156 156 156 0

11 18 28 38 134

Dh (nm)

PDI

135 192 ± 213 ± 203 ± 249 ±

0.319 0.098 0.071 0.042 0.061

2 1 2 4

Rg/Rh

TEM DT (nm)

ξZ (mV)

± ± ± ± ±

0.844 0.920 0.778 0.723

97 ± 18 114 ± 12 115 ± 5 150 ± 17 161 ± 3

+13.3 +31.9 +31.5 +20.4 +39.6

76 81 98 79 90

4 2 4 1 2

NY, USA.). Fetal bovine serum (FBS) and fetal calf serum (FCS) were obtained from Zhejiang Tianhang Biotechnology Co., Ltd. Potassium peroxodisulfate (KPS, 99.5%) and NaHSO4 were obtained from Sinopharm Chemical Reagent Co., Ltd. All of the chemicals were used as received. The phosphate-buffered saline solution (PBS 0.01 M, pH 7.4) was prepared according to the standard protocols, and Milli-Q ultrapure water was used in the cytotoxicity and cell experiments. Preparation of PNVP Microgels. The PNVP microgels were synthesized via SFEP with NVP as the monomer, EGDMA as the cross-linker, and AIBA as the initiator at 60 °C. Given amounts of NVP and EGDMA were dissolved in 48 mL of deionized water at 60 °C under vigorous stirring. Oxygen was eliminated by bubbling nitrogen through the solution for 30 min. Afterward, 2 mL of initiator AIBA (5 mg/mL) was added into the solution to initiate the polymerization. The reaction was continued at 60 °C for 6 h. The obtained PNVP microgels were then purified by extensive dialysis (MWCO = 14 000) against deionized water for 3 days followed by centrifugation at 12000 r/min with replacement of fresh water three times. Table 1 shows the experimental conditions of a series of PNVP microgels prepared in the present work. The sample codes were named according to the following example: for N-E9, N is NVP, E is EGDMA, and the numeric value of 9 means that the molar fraction of EGDMA in the feedings of EGDMA and NVP was 9%. Preparation of Fluorescent PNVP Microgels. The fluorescent PNVP microgels were synthesized via the same procedure of N-E9 PNVP microgels as described above by additional adding 1 wt % of fluorescent comonomer, fluorescein O-methacrylate 97 (FMA).32 The fluorescent PNVP microgels were then coded as N-E9-FMA. Control Experiments. Several control experiments were also carried out. Linear homopolymer PNVP was synthesized without the presence of any cross-linker. BIS was used to replace EGDMA as the crosslinker for the fabrication of PNVP microgels under the same experimental conditions. However, no PNVP microgels could be obtained, and the resultant solutions were transparent. Initiator KPS was used to synthesize homopolymer polyEGDMA at 60 °C, and a milky suspension was obtained. Oxidation−reduction initiator systems KPS+TEMED and KPS+ NaHSO4 were also used to synthesize homopolymer polyEGDMA at room temperature, and milky suspensions were again obtained. It was further confirmed that these initiator systems, KPS, KPS+TEMED, and KPS+ NaHSO4 could not initiate the copolymerization of NVP and EGDMA. Methods. Composition Characterization. FT-IR spectra were recorded on a Vector 22 Bruker spectrometer. 1H NMR of the PNVP microgels was performed on a 300 MHz Varian Mercury Plus NMR instrument with D2O as solvent and tetramethylsilane (TMS) as internal standard. Elemental analyses were performed on a ThermoFinnigan Flash EA-1112 instrument to determine the content of elements carbon (C), nitrogen (N), and hydrogen (H) in the obtained PNVP microgels. Size and Microstructure of PNVP Microgels. The hydrodynamic diameter (Dh) of the PNVP microgels in both deionized water and PBS solutions were measured at 25 °C by using a 90 Zeta Plus Particle Size Analyzer (Brookhaven Instruments Corp.) at a scattering angle θ of 90°. The wavelength of laser light λ was 635 nm. The Zeta potentials ξZ of the PNVP microgels were also measured by electrophoretic light scattering (ELS) using the same Zeta Plus particle size analyzer. The pH values of sample solutions were measured by a pH meter (FE20, Mettler Toledo).

fabrication, structure, and properties as well as the potential applications of various microgel systems, which might be responsive to various external stimuli, like temperature, pH, and ionic strength.19−21 The potential applications of microgels as drug delivery systems have attracted much attention.22−29 For example, Dadsetan et al.28 developed pH-responsive microgels made from a copolymer of oligo(poly(ethylene glycol)) fumarate (OPF) and sodium methacrylate (SMA) for the delivery of doxorubicin (DOX) and found that the OPF−SMA microgels prolonged the cell killing effect of DOX. Gu et al.25 developed pH-responsive and uniform injectable microgels, which consist of a pH-responsive chitosan matrix, enzyme nanocapsules, and recombinant human insulin, for controlled glucose-responsive release of insulin. The authors demonstrated that these microgels could facilitate insulin release and be used to treat type 1 diabetes mellitus.25 Hence, the PNVP microgels might have superiority for application as drug-controlled-release systems because PNVP microgels could possess both properties of PNVP-related hydrogels and PNVP-related nanoparticles. However, up to now, no PNVP microgels have been synthesized and reported. It is still a challenge to fabricate PNVP microgels because PNVP is fully water-soluble and generally not responsive to external stimuli, like temperature and pH. In the present work, the hydrophobic nature of poly(ethylene glycol dimethacrylate) (polyEGDMA) and the fast reaction rate of ethylene glycol dimethacrylate (EGDMA) were utilized to realize the fabrication of PNVP microgels.30,31 By choosing EGDMA as the cross-linker and N-vinylpyrrolidinone (NVP) as the monomer, PNVP microgels could be synthesized via SFEP of NVP and EGDMA at 60 °C. The composition, morphology, size, and microstructure of the obtained PNVP microgels were characterized by combined techniques of Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (1H NMR), elemental analysis, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and static and dynamic laser light scattering (SLS and DLS). The biocompatibility and cell up-taking of the obtained PNVP microgels were evaluated. Isoniazid (INH) and 5-fluorouracil (5-fu) were chosen as the hydrophilic and hydrophobic model drugs, respectively, to explore the potential applications of the PNVP microgels as drug-nanocarriers.

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SLS Rg (nm)

EXPERIMENTAL SECTION

Materials. Chemicals. NVP (99%) and N,N,N′,N′-tetramethylethylenediamine (TEMED, 99%) were purchased from Acros Organics. 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AIBA, 97%), trypsin (Mw = 23300), 4′,6-diamidino-2-phenylindole (DAPI), methyl thiazolyltetrazolium (MTT) and dimethyl sulfoxide (DMSO) were purchased from Aldrich-Sigma. EGDMA (99%), N,N′-methylenebisacrylamide (BIS), 5-fu, INH, (3-aminopropyl)triethoxysilane (APTES), and fluorescein O-methacrylate 97 (FMA) were purchased from J&K Chemical Ltd. Dulbecco’s modified eagle’s medium (DMEM) was obtained from GIBCO Invitrogen Corp. (Greenland B

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Figure 1. TEM images of (A) E, (B) N-E3.5, (C) N-E6, (D) N-E9, and (E) N-E12. The SLS measurements of the PNVP microgels were carried out by using a commercial Brookhaven BI-200SM in the Department of Polymer Science and Engineering, Soochow University. The SLS measurements were performed at 25 °C, and the sample solutions were equilibrated for 15 min. The range of scattering angle θ used for SLS was from 30° to 130° with a step of 5°. The wavelength of laser light λ was 637 nm. Morphology of PNVP Microgels. TEM measurements were carried out by using a JEOL JEM-1230 electron microscope operated at an acceleration voltage of 60 kV. TEM samples were prepared by dipcoating with Formvar-coated copper grids into the PNVP microgel suspension. The solvent was gently absorbed away by a filter paper. The grids were then allowed to dry in air at room temperature before observation. SEM measurements were carried out by using a scanning electron microscope (SEM) (HITACHI, S-4800) operating at 3 kV. A droplet of the PNVP microgel suspension was cast onto the aluminum foils at room temperature. After a few minutes, the excess suspension was gently absorbed away by a filter paper. The aluminum foils were then allowed to dry in air. All samples were then sputter-coated with an ultrathin layer of gold prior to observation. Several positions of each sample were imaged. MTT Test. The relative cytotoxicity of the PNVP microgels was evaluated by a cell counting 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) against human embryonic kidney 293 (HEK-293) and human breast adenocarcinoma MCF-7 cell lines. The HEK-293 and MCF-7 cells were seeded in 96 well plate at 1 × 104 cells/well and incubated at 37 °C under a 5% CO2 atmosphere in DMEM with FCS and FBS, respectively, for 18 h. Afterward, the culture medium was then removed, and the PNVP microgels in PBS were added into the plates at different concentrations. After 24 h incubation, the medium was removed, and 100 μL MTT solutions (0.5 mg/mL) were added into each well. Finally, 4 h later, the supernatant was removed, and DMSO (100 μL) was added to each well to dissolve the MTT formazan crystals. The absorbance of each well was measured by enzyme-linked microplate reader (BIO-RAD 680, USA) at 570 nm test wavelength. The relative cell viability was then calculated as

relative cell viability (%) = (ODsample − ODwater )/(ODcontrol − ODwater ) × 100%

(1)

where ODsample was obtained in the presence of PNVP microgels and the ODcontrol was obtained in the absence of PNVP microgels and the ODwater was obtained once the cells were all dead. CLSM Measurement. The cell uptaking of PNVP microgels was tested by using HEK-293 cells as model cells and the fluorescent PNVP microgels coded as N-E9-FMA. HEK-293 cells (5 × 104 cells per well) were cultured in DMEM supplemented without FCS (10%) and penicillin−streptomycin (1%) with 50 μg/mL N-E9-FMA microgels for 4 h. The cells were washed with PBS solution three times and then fixed onto a APTES modified glass slide using paraformaldehyde solution (4% w/w) in PBS for 10 min. Additionally, the cells were then stained with DAPI (1 ng/mL PBS, 10 min) after being rinsed with PBS solution three times. The cells were imaged under a confocal laser scanning microscope (CLSM 410, Carl Zeiss, USA.) with a 60× objective to visualize the fluorochromes with the following excitation (Ex) and emission (Em) wavelengths: DAPI (Ex: 350 nm, Em: 470 nm), FMA Green (Ex: 486 nm, Em: 528 nm).33 The glass slides for CLSM measurements were first cleaned with the following procedure: (1) 1 M NaOH solution for 10 min followed by washing with deionized water for 5 min; (2) 1 M HCl solution for 1 h followed by washing with deionized water for 10 min; (3) piranha solution (3:7 mixture of 30% H2O2 and concentrated H2SO4) at 90 °C for 30 min followed by rinsing with copious deionized water and drying in a vacuum oven at room temperature for 1 h. (Caution! Piranha solution is a very strong oxidizing agent and reacts violently with organic compounds. It should be handled with extreme care.) The cleaned glass slides were then immersed in a APTES toluene solution (1 v/v %) for 1 h followed by rinsing with copious toluene and drying in a vacuum oven at 60 °C for 1 h. Drug Loading and Release. In order to study the drug-loading capacity of the obtained PNVP microgels, 5-fu and INH were chosen as the hydrophobic and hydrophilic model drugs, respectively. Briefly, 100 mg freeze-dried PNVP microgels (N-E6, N-E9, or N-E12) were added into an aqueous solution of 5-fu (5 mg/mL) under continuous stirring for 24 h until the PNVP microgels were fully swollen. The C

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Figure 2. SEM images of (A) E, (B) N-E3.5, (C) N-E6, (D) N-E9, and (E) N-E12.

mean diameters measured from TEM images were 97 ± 18 nm, 114 ± 12 nm, 115 ± 5 nm, and 150 ± 17 nm for N-E3.5, N-E6, N-E9, and N-E12 PNVP microgels, respectively. The N-E3.5 PNVP microgels had smallest particle size with large size distribution, which were mainly attributed to the smallest amount of cross-linker. Clearly, increasing the content of EGDMA could narrow the size distribution of PNVP microgels. The SEM images also indicated that the PNVP microgels with higher content of EGDMA have rough surfaces, which were thought to result from the different reaction rates of monomer NVP and cross-linker EGDMA. It had been confirmed that the reaction rate of EGDMA is much larger than that of NVP.30,31 The copolymerization of NVP and EGDMA would usually result in the highly heterogeneous networks, which contain regions rich in EGDMA connected by long chains rich in NVP.4 Furthermore, the homopolymer polyEGDMA is hydrophobic and cannot dissolve in aqueous solution. As a consequence, polyEGDMA nucleuses will form at the early stage of SFEP, and the hydrophilic monomer NVP will copolymerize and connect with the polyEGDMA nucleuses as well as residual EGDMA in the later stage, leading to the formation of PNVP microgels with heterogeneous structures and rough surfaces. The control experiment was carried out to prepare polyEGDMA at 60 °C. As expected, polyEGDMA nanoparticles (sample E) with TEM diameter of 161 ± 3 nm and rough surfaces were obtained, as shown in Figures 1A and 2A. However, the obtained polyEGDMA nanoparticles were unstable in water and precipitated in 1 week. KPS was used to initiate the homopolymerization of ethylene glycol dimethacrylate at 60 °C. Similarly, the polyEGDMA nanoparticles with similar sizes could be obtained, which were again unstable in water and precipitated in 1 week. Furthermore, the polyEGDMA nanoparticles could be also prepared by using oxidation−reduction initiator system KPS+TEMED or KPS+ NaHSO4 at room temperature. However, the resultant polyEGDMA nanoparticles were unstable and precipitated in 1 day. Note that these initiator systems, KPS, KPS + TEMED,

mixture was then centrifuged at 16000 r/min three times with each time of 30 min to remove the unloaded 5-fu drugs. A similar procedure was applied to load the hydrophilic drug INH with concentration of 10 mg/mL. The drug-loaded PNVP microgels were then lyophilized to constant weight for further characterization. To determine the drug loading content (DLC) and encapsulation efficiency (EE), the absorbance of the centrifuged supernatant was measured by a UV−vis spectrophotometer (Cary 300, Varian Australia Pty Ltd.) at 265 nm for 5-fu and 263 nm for INH, respectively. The mass of the unloaded drug was calculated by the standard calibration curve experimentally obtained with 5-fu/H2O and INH/H2O solutions (see Supporting Information). The DLC and EE were then given as

DLC (wt%) =

EE (wt%) =

mass of drug loaded in microgels × 100% mass of drug‐loaded microgels

mass of drug loaded in microgels × 100% the initial mass of drug

(2)

(3)

To investigate the in vitro drug release, the drug-loaded PNVP microgels were put into dialysis bags (MWCO = 3500) and dialyzed against 10 mL PBS solutions at pH 7.4. The release process was kept at 37 °C in a shaker with shaking frequency at 100 r/min. Periodically, aliquots of 2 mL of buffer solution outside the dialysis bag were removed for UV−vis analysis and replaced with the same volume of fresh PBS solution in order to hold the volume of solution constant. The amounts of drug released from the PNVP microgels were measured by UV−vis absorbance at 265 nm for 5-fu and 263 nm for INH, respectively.

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RESULTS AND DISCUSSION The PNVP microgels were synthesized via SFEP with NVP as the monomer, EGDMA as the cross-linker, and AIBA as the initiator at 60 °C. Figures 1 and 2 show the TEM and SEM morphologies of the obtained PNVP microgels, respectively. It could be seen that the dried PNVP microgels are spherical in shape with narrow size distribution except of sample N-E3.5. The sizes of dried PNVP microgels were less than 200 nm and slightly increase with increasing the feeding content of crosslinker EGDMA from 3.5% to 12%, as shown in Table 1. The D

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and KPS + NaHSO4, could not initiate the copolymerization of NVP and EGDMA. BIS was further used to replace EGDMA as the cross-linker for the fabrication of PNVP microgels under the same experimental condition. However, no PNVP microgels could be obtained with BIS as cross-linker, and the resultant solutions were transparent. The above results suggest that the PNVP microgels were obtained by SFEP at 60 °C with NVP as the monomer and EGDMA as the cross-linker. First, the fast reaction of EGDMA and the hydrophobic nature of polyEGDMA formed the nucleuses of microgels at the early stage of SFEP, which determine the shape of resultant microgels. Second, the lower reaction rate of NVP and the hydrophilic nature of PNVP network chains gave rise to the rough surface and long-term stability of microgels in aqueous solutions. Furthermore, the initiator AIBA resulted in the positive surface charges of the obtained PNVP microgels (cf. Table 1), which further stabled the microgels in aqueous solution. This is, to the best of our knowledge, the first example of PNVP microgels with sizes of hundred nanometers and narrow size distribution that may be suitable as drug-controlledrelease materials. To further confirm the composition of obtained PNVP microgels, FT-IR and 1H NMR measurements were carried out. Figure 3 shows the FT-IR spectra of homopolymer polyNVP,

Figure 4. 1H NMR spectra of (a) E, (b) N-E6, (c) N-E9, and (d) NE12 in D2O.

copolymerization of NVP and EGDMA. To further determine the composition of the PNVP microgels, elemental analyses were carried out for the samples with codes of E, N-E6, N-E9, and N-E12. The contents of elements carbon (C), nitrogen (N), and hydrogen (H) in the PNVP microgels are summarized in Table 2. It can be seen that the actual content of N in the Table 2. Contents of Elements Carbon (C), Nitrogen (N), and Hydrogen (H) in the PNVP Microgels content of elementa (%) sample code

Nt

Ne

Ct

Ce

Ht

He

N-E6 N-E9 N-E12 E

12.37 11.73 11.16 2.16

9.89 9.30 9.01 1.98

62.84 62.73 62.63 58.86

58.42 57.85 58.37 56.79

7.97 7.92 7.88 7.09

8.46 8.25 8.26 6.86

The subscript “t” presents the theoretical content of the element. The subscript “e” means the actual content of the element determined by elemental analysis. a

PNVP microgels was smaller than that of theoretical value, suggesting that the conversion rate of monomer NVP was less than 100%. From the content of element nitrogen, the conversion rate of NVP might be estimated to be less than 85%. For the sample E without the presence of NVP, the content of C was slightly smaller than the theoretical value, indicating that the conversion rate of EGDMA was also less than 100% after 6 h of reaction. Note that the element nitrogen (N) of sample E came from the initiator AIBA. The hydrodynamic diameters (Dh) of the obtained PNVP microgels and polyEGDMA nanoparticles in aqueous solutions were measured at 25 °C by dynamic light scattering (DLS) and shown in Table 1. The hydrodynamic diameters (Dh) of NE3.5, N-6, N-E9, and N-E12 PNVP microgels and E were approximately 135 nm, 192 ± 2 nm, 213 ± 1 nm, 203 ± 2 nm, and 249 ± 4 nm, respectively. The PNVP microgels showed narrow size distribution with low polydispersity index (PDI) except of N-E3.5 PNVP microgels. The N-E3.5 PNVP microgels had large size distribution with a large PDI value of 0.319, which was consistent with the observations of TEM and SEM. The hydrodynamic diameters of PNVP microgels were larger than the corresponding dried diameters measured from TEM images, which indicated that the PNVP microgels swelled

Figure 3. FTIR spectra of (a) polyNVP, (b) E, (c) N-E6, (d) N-E9, and (e) N-E12.

polyEGDMA (sample code E), and the PNVP microgels, i.e., N-E6, N-E9, and N-E12. Absorption band at 1728 cm−1, which could be assigned to the CO stretching vibration of EGDMA, was observed for polyEGDMA and all of the PNVP microgels investigated here. The polyNVP showed characteristic absorption bands at 1288 cm−1 for C−N stretching vibration and 1663 cm−1 for CO stretching vibration, which were again observed for all PNVP microgels. Figure 4 shows the 1H NMR spectra of polyEGDMA and PNVP microgels. The proton signals of polyEGDMA were very weak because of the hydrophobic nature and cross-link network of polyEGDMA, which could shield most of the signals from the inner protons. The PNVP microgels could be swollen in D2O so that the signals from the ring methylene protons and −CH− of PNVP could be clearly observed and assigned:34,35 δ3.2 (cCH2), δ2.2 (e CH2), δ1.9 (d CH2), δ1.6 (a CH2), and δ3.5 (b CH2). The FT-IR and 1H NMR results clearly indicated that the PNVP microgels resulted from the cross-linking E

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in water. The radius of gyration ⟨Rg⟩ of the obtained PNVP microgels in aqueous solutions was also measured at 25 °C by static light scattering (SLS). The ⟨Rg⟩ of PNVP microgels was calculated by fitting the SLS data with the Guinier equation (Figure S1 in Supporting Information) and is shown in Table 1. It is known that the value of ⟨Rg⟩/⟨Rh⟩ could reflect the conformation and architecture of a polymer chain in solution or the cross-linking density distribution of the microgels.20 Note that ⟨Rh⟩ = Dh/2 was the hydrodynamic radius of the polymer chain or microgels in solution. For example, ⟨Rg⟩/⟨Rh⟩ values varied in the range of 1.50−1.78 for random polymer coils, depending on the solvent quality. For uniform hard spheres,⟨Rg⟩/⟨Rh⟩ was 0.778. For thermosensitive PNIPAm microgels with inhomogeneous cross-linking network structures, ⟨Rg⟩/⟨Rh⟩ usually had a value of 0.55−0.6.36 The ⟨Rg⟩/ ⟨Rh⟩values were calculated to be 0.844, 0.920, and 0.778 for NE6, N-E9, and N-E12 PNVP microgels, respectively. For the polyEGDMA nanoparticle (sample code E), the ⟨Rg⟩/⟨Rh⟩value is 0.723. These values might suggest that the PNVP microgels had more compact structures. Since the linear homopolymer PNVP is approved by the U.S. Food and Drug Administration (FDA) to be safe for bioapplications, the obtained PNVP microgels with sizes of hundred nanometers could then have promising applications as drug-delivery and controlled-release systems. However, the PNVP microgels obtained here were also composed of crosslinker EGDMA. Therefore, the cytotoxicity of the PNVP microgels was evaluated by an MTT viability assay against HEK-293 and MCF-7 cell lines. Note that cytotoxicity is an important factor when choosing an appropriate drug delivery vector. Before the MTT viability assay, the stability and hydrodynamic diameter of the PNVP microgels in PBS solutions were first investigated. The PNVP microgels could well disperse in PBS solution with long-term stability for at least several months (Figure S2). However, polyEGDMA nanoparticles (sample E) precipitated immediately in PBS solution (Figure S2). The DLS results showed that the hydrodynamic diameters of the N-E6, N-E9, and N-E12 PNVP microgels in PBS solutions at 25 °C were 203 ± 2 nm, 236 ± 3 nm, and 304 ± 10 nm (Figure 5), respectively, which were larger than the corresponding values in water (cf. Table 1). These results indicated that the PNVP microgels were more swollen than those in water. The PNVP microgels in PBS solution also exhibited narrow size distribution, as shown in Figure 5. Note

that the N-E3.5 PNVP microgels were not further investigated due to its large size distribution. Figure 6 shows the cell viability of HEK-293 and MCF-7 cells after treating with N-E6, N-E9, and N-E12 PNVP microgels with various concentrations. It can be seen that with increasing the concentration of PNVP microgels from 1 μg/mL to 1000 μg/mL, the cell viability decreased gradually. For the PNVP microgels with concentrations below 100 μg/mL, the cell viability was higher than 80% for both HEK-293 and MCF-7 cells. Even with 1000 μg/mL of microgel concentration, the cell viability was still higher than 60%. Such high cell viability suggested that the PNVP microgels had low cytotoxicity toward HEK-293 and MCF-7 cells. The MTT results indicated that the PNVP microgels had good cytocompatibility for normal and cancer cells with concentration less than 1000 μg/mL. With concentration above 1000 μg/mL, the PNVP microgels would become cytotoxic. 5-fu and INH were then chosen as the hydrophobic and hydrophilic model drugs to study the drug-loading and releasing of the obtained PNVP microgels, respectively (see Experimental Section). The hydrophobic polyEGDMA domains in PNVP microgels were thought to be capable of loading the hydrophobic drugs, whereas the hydrophilic PNVP networks could encapsulate the hydrophilic drugs. It is wellknown that hydrophobic drugs can be physically incorporated and stabilized in the hydrophobic domains via the hydrophobic interaction.37 The −NH− groups of hydrophilic drug INH might form hydrogen bonds with −CO−NH− groups of PNVP network chains, leading to the encapsulation of INH in PNVP microgels. The DLC and EE of INH and 5-fu were then determined and shown in Table 3. The N-E6 PNVP microgels exhibited the highest EE and DLC for both of INH and 5-fu. For N-E6 PNVP microgels loading with INH, EE and DLC were 6.2 and 12.4 wt %, respectively, while for N-E6 PNVP microgels loading with 5-fu, EE and DLC were 4.2 and 4.2 wt %, respectively. Increasing the contents of cross-linker EDGMA led to the decrease of EE and DLC for loading of both hydrophobic and hydrophilic model drugs. The PNVP microgels exhibited higher EE and DLC values for loading with hydrophilic INH drug because of the hydrophilic nature of PNVP network chains. The in vitro drug releases of INH and 5-fu from the PNVP microgels were then investigated by placing the drug-loaded PNVP microgels into the dialysis bags and dialyzing against the PBS solutions (pH 7.4) at physiological temperature of 37 °C. The cumulative release curves of INH and 5-fu from the drugloaded N-E6, N-E9, and N-E12 PNVP microgels are shown in Figure 7. For the INH, sustained release was clearly observed for releasing times up to 3000 min, although there was a slight burst release at the initial time (Figure 7A). The final drug release percentages were 16%, 28%, and 46% for N-E6, N-E9, and N-E12 PNVP microgels, respectively. Possibly, the PNVP microgels with less hydrophobic EGDMA contents and lower cross-linking density exhibited more stronger interactions among the PNVP network chains and INH so that the release of INH was more difficult, leading to the lower percentage of final drug release. The release profiles of INH from PNVP microgels could be well described by an exponential heuristic equation:38,39 Mt = kt n M∞

Figure 5. Distribution of hydrodynamic diameter (Dh) of PNVP microgels in PBS solution. F

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Figure 6. Cell viability of (A) HEK-293 and (B) MCF-7 cells in the presence of PNVP microgels with various concentrations ranging from 1 to 1000 μg/mL, as measured by MTT tests.

E12 PNVP microgels, respectively (Figure 7B). It was consistent with the release results of INH that the largest final release percentage was obtained for releasing of the hydrophobic drug 5-fu from N-E6 PNVP microgels with lower hydrophobic EGDMA contents and cross-linking density. With increasing the cross-linking density of PNVP microgels, the release of 5-fu from the PNVP microgels decreased. The N-E6 PNVP microgels exhibited the fastest release of 5-fu in PBS solution. However, the initial burst release of 5-fu was serious, and it achieved the balance of release in short time. Therefore, the PNVP microgels might be not suitable as drug-controlled release vehicles for hydrophobic drug 5-fu. In order to act as drug delivery and controlled-release vehicles in a living body, the question of whether PNVP microgels could be uptaken by living cells is crucial. Therefore, the cellular uptaking of PNVP microgels should be studied. The fluorescent labeled PNVP microgels were then synthesized by additional incorporating 1 wt % of fluorescent comonomer, fluorescein O-methacrylate 97 (FMA). The fluorescent PNVP microgels were coded as N-E9-FMA. After fluorescein labeling, the N-E9-FMA microgels had a hydrodynamic diameter of approximately 245 nm (Figure S3) and exhibited green fluorescence under a confocal laser scanning microscope (CLSM 410, Carl Zeiss, USA) as shown in Figure S4 (see Supporting Information). The fluorescent N-E9-FMA microgels were then freeze-dried and redispersed in DMEM medium. The N-E9-FMA microgels in DMEM medium were added into the HEK-293 cells, followed by incubation for 18 h at 37 °C (see Experimental Section for details). The medium and

Table 3. DLC and EE of N-E6, N-E9, and N-E12 PNVP Microgels for Loading the INH and 5-fu Model Drugs drugs INH 5-fu

EE (wt %) DLC (wt %) EE (wt %) DLC (wt %)

N-E6

N-E9

N-E12

6.2 12.4 4.2 4.2

1.4 2.8 2.8 2.8

1 2 2.7 2.7

where Mt/M∞ is fractional drug release, Mt is the amount of drug released at time t, M∞ is the maximum amount of drug released at time ∞, t is the release time, k is a rate constant of kinetic release, and n is the diffusion exponent, characteristic of the drug release mechanism. For n < 0.5, it indicated that the drug release follows the Fickian diffusion, whereas the nonFickian drug release process had a value of n between 0.5 and 1. The fitting values of n were 0.10, 0.11, and 0.07, for N-E6, NE9, and N-E12 PNVP microgels, respectively, indicating that the release mechanism of INH from the PNVP microgels was the Fickian diffusion. The rate constant values k were approximately 6.67 × 10−2, 11.8 × 10−2, and 27.0 × 10−2 for N-E6, N-E9, and N-E12 PNVP microgels, respectively, indicating that the releasing rate of INH increased with increasing the content of hydrophobic cross-linker E. These results suggested that the PNVP microgels were suitable as drug-controlled release vehicles for hydrophilic drug INH. For the release of 5-fu, a quick release within 250 min was observed for the three PNVP microgels, and the final release percentages were about 37.6%, 24.3%, and 20.8% for N-E6, N-E9, and N-

Figure 7. Cumulative release of (A) INH and (B) 5-fu from (■) N-E6, (●) N-E9, and (▲) N-E12 PNVP microgels as a function of time. G

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could well disperse in PBS solution with long-term stability and be up taken into the cytoplasm regime of HEK-293 cells without entering the nucleus. The drug-loading and release experiments indicated that the PNVP microgels would be suitable for the sustained release of hydrophilic model drug isoniazid (INH), which exhibited a Fickian release mechanism.

unbound microgels were rinsed away with PBS solution, and the live cells were imaged using a confocal laser scanning microscope after being stained. Figure 8 shows that the N-E9-

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ASSOCIATED CONTENT

* Supporting Information S

SLS data of PNVP microgels, DLS data of N-E9-FMA microgels, photographs of N-E9 PNVP microgels and polyEGDMA nanoparticles in PBS solutions, CLSM fluorescent images of controlled cell uptaking experiments with HEK293 cells, UV absorbed standard calibration curves of 5-fu and INH in water and PBS solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly thank Prof. Yingfeng Tu at Soochow University for his help with laser light scattering experiments. The authors thank the National Natural Science Foundation of China (Nos. 21274129 and 21322406), the Fundamental Research Funds for the Central Universities (2014XZZX003-21), the third level of the 2013 Zhejiang Province 151 Talent Project, and Zhejiang University K. P. Chao’s High Technology Development Foundation for financial support.

Figure 8. CLSM fluorescent images of HEK-293 cells incubated with 50 μg/mL of N-E9-FMA microgels for 4 h. In all images, the nuclei of the HEK-293 were stained with DAPI blue dye. (A) The blue image of cell nucleus recorded with a DAPI filter. (B) The green image of NE9-FMA microgels recorded with a FAM filter. (C) The constructive image by overlapping the corresponding fluorescent images of cell nucleus and N-E9-FMA microgels. (D) The constructive image by further overlapping image C with the normal optical image of HEK293 cells.

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REFERENCES

(1) Perera, D. I.; Shanks, R. A. Polym. Int. 1996, 39, 121−127. (2) Bruining, M. J.; Edelbroek-Hoogendoorn, P. S.; Blaauwgeers, H. G. T.; Mooy, C. M.; Hendrikse, F. H.; Koole, L. H. J. Biomed. Mater. Res. 1999, 47, 189−197. (3) Francois, P.; Vaudaux, P.; Nurdin, N.; Mathieu, H. J.; Descouts, P.; Lew, D. P. Biomaterials 1996, 17, 667−678. (4) Smith, L. E.; Rimmer, S.; MacNeil, S. Biomaterials 2006, 27, 2806−2812. (5) Turturro, M. V.; Sokic, S.; Larson, J. C.; Papavasiliou, G. Biomed. Mater. 2013, 8, 025001. (6) Hong, Y.; Chirila, T. V.; Fitton, J. H.; Ziegelaar, B. W.; Constable, I. J. Bio-Med. Mater. Eng. 1997, 7, 35−47. (7) Lapworth, J. W.; Hatton, P. V.; Goodchild, R. L.; Rimmer, S. J. R. Soc. Interface 2012, 9, 362−375. (8) Geever, L. M.; Cooney, C. C.; Lyons, J. G.; Kennedy, J. E.; Nugent, M. J. D.; Devery, S.; Higginbotham, C. L. Eur. J. Pharm. Biopharm. 2008, 69, 1147−1159. (9) Devine, D. M.; Devery, S. M.; Lyons, J. G.; Geever, L. M.; Kennedy, J. E.; Higginbotham, C. L. Int. J. Pharm. 2006, 326, 50−59. (10) Devine, D. M.; Higginbotham, C. L. Eur. Polym. J. 2005, 41, 1272−1279. (11) Akdemir, Z. S.; Kayaman-Apohan, N. Polym. Adv. Technol. 2007, 18, 932−939. (12) Le Garrec, D.; Gori, S.; Luo, L.; Lessard, D.; Smith, D. C.; Yessine, M. A.; Ranger, M.; Leroux, J. C. J. Controlled Release 2004, 99, 83−101. (13) Sharma, D.; Chelvi, T. P.; Kaur, J.; Chakravorty, K.; De, T. K.; Maitra, A.; Ralhan, R. Oncol. Res. 1996, 8, 281−286. (14) D’Souza, A. J. M.; Schowen, R. L.; Topp, E. M. J. Controlled Release 2004, 94, 91−100.

FMA microgels were readily internalized by the HEK-293 cells and localized in the cytoplasm region without entering into the nucleus. The uptaken N-E9-FMA microgels distributed around the nucleus of HEK-293 cells, which were stained with DAPI blue dye and appeared to be fluorescent blue (Figure 8A), whereas the fluorescent N-E9-FMA microgels appeared to be fluorescent green (Figure 8B). By overlapping the fluorescent images of Figure 8A and 8B, it could be seen that the N-E9FMA microgels locate in the cytoplasm regimes of the HEK293 cells (Figure 8C). When further overlapping the normal optical image of HEK-293 cells, the outline of the HEK-293 cells was clearly observed, confirming the uptaking of fluorescent N-E9-FMA microgels into the cytoplasm regime of HEK-293 cells without entering the nucleus (Figure 8D). These results indicated that the PNVP microgels could be taken up into the HEK-293 cells via nonreceptor-mediated endocytosis.40

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CONCLUSIONS Biocompatible PNVP microgels were synthesized via surfactant free emulsion polymerization with NVP as the monomer and EGDMA as the cross-linker at 60 °C. The obtained PNVP microgels were spherical in shape with hydrodynamic diameter of approximately 200 nm and narrow size distribution. The obtained PNVP microgels were biocompatible with low toxicity even at concentration of 1000 μg/mL. The PNVP microgels H

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(15) Benahmed, A.; Ranger, M.; Leroux, J. C. Pharm. Res. 2001, 18, 323−328. (16) Zhu, Z. S.; Li, Y.; Li, X. L.; Li, R. T.; Jia, Z. J.; Liu, B. R.; Guo, W. H.; Wu, W.; Jiang, X. Q. J. Controlled Release 2010, 142, 438−446. (17) Zhu, Z. S.; Xie, C.; Liu, Q.; Zhen, X.; Zheng, X. C.; Wu, W.; Li, R. T.; Ding, Y.; Jiang, X. Q.; Liu, B. R. Biomaterials 2011, 32, 9525− 9535. (18) Pelton, R. H.; Chibante, P. Colloids Surf. 1986, 20, 247−256. (19) Chen, T. Y.; Cao, Z.; Guo, X. L.; Nie, J. J.; Xu, J. T.; Fan, Z. Q.; Du, B. Y. Polymer 2011, 52, 172−179. (20) Kuckling, D.; Vo, C. D.; Adler, H. J. P.; Volkel, A.; Colfen, H. Macromolecules 2006, 39, 1585−1591. (21) Grifantini, R.; Sebastian, S.; Frigimelica, E.; Draghi, M.; Bartolini, E.; Muzzi, A.; Rappuoli, R.; Grandi, G.; Genco, C. A. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 9542−9547. (22) Malmsten, M.; Bysell, H.; Hansson, P. Curr. Opin. Colloid Interface Sci. 2010, 15, 435−444. (23) Smeets, N. M. B.; Hoare, T. J. Polym. Sci., Polym. Chem. 2013, 51, 3027−3043. (24) Sun, L.; Zhang, X. G.; Zheng, C.; Wu, Z. M.; Xia, X. T.; Li, C. X. RSC Adv. 2012, 2, 9904−9913. (25) Gu, Z.; Dang, T. T.; Ma, M. L.; Tang, B. C.; Cheng, H.; Jiang, S.; Dong, Y. Z.; Zhang, Y. L.; Anderson, D. G. ACS Nano 2013, 7, 6758−6766. (26) Ghugare, S. V.; Mozetic, P.; Paradossi, G. Biomacromolecules 2009, 10, 1589−1596. (27) Zhang, H.; Mardyani, S.; Chan, W. C. W.; Kumacheva, E. Biomacromolecules 2006, 7, 1568−1572. (28) Dadsetan, M.; Taylor, K. E.; Yong, C.; Bajzer, Z.; Lu, L. C.; Yaszemski, M. J. Acta Biomater. 2013, 9, 5438−5446. (29) Hoare, T.; Young, S.; Lawlor, M. W.; Kohane, D. S. Acta Biomater. 2012, 8, 3596−3605. (30) Shao, W.; Yin, X. N.; Stevenson, W. T. K. Polym. Int. 1992, 27, 81−85. (31) Bork, J. F.; Coleman, L. E. J. Polym. Sci. 1960, 43, 413−421. (32) Ahmad, H.; Dupin, D.; Armes, S. P.; Lewis, A. L. Langmuir 2009, 25, 11442−11449. (33) Gollavelli, G.; Ling, Y. C. Biomaterials 2012, 33, 2532−2545. (34) Sesta, B.; Segre, A. L.; Daprano, A.; Proietti, N. J. Phys. Chem. B 1997, 101, 198−204. (35) Parambil, A. M.; Puttaiahgowda, Y. M.; Shankarappa, P. Turk. J. Chem. 2012, 36, 397−409. (36) Senff, H.; Richtering, W. Colloid Polym. Sci. 2000, 278, 830− 840. (37) Choi, C.; Chae, S. Y.; Nah, J. W. Polymer 2006, 47, 4571−4580. (38) Korsmeyer, R. W.; Lustig, S. R.; Peppas, N. A. J. Polym. Sci., Part B: Polym. Phys. 1986, 24, 395−408. (39) Wang, Q.; Zhang, J. P.; Wang, A. Q. Carbohydr. Polym. 2009, 78, 731−737. (40) Chang, I. P.; Hwang, K. C.; Chiang, C. S. J. Am. Chem. Soc. 2008, 130, 15476−15481.

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