Controlled protein delivery from photosensitive nanoparticles Zhiqiang Jiang,1 Huyan Li,2 Yujing You,1 Xuedong Wu,2 Shuangxi Shao,1 Qun Gu1 1

School of Material Science, Ningbo University of Technology, Ningbo, Zhejiang 315211, China Ningbo Institute of Material Technology & Engineering, Chinese Academy of Sciences, Zhenhai District, Ningbo, Zhejiang 31520, China

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Received 30 December 2013; revised 18 February 2014; accepted 5 March 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35158 Abstract: Light provides a powerful approach for delivery of cargos and study of important biological events. This article reports a series of photosensitive and biocompatible delivery of nanoparticles which released proteins upon light irradiation. The nanoparticles were synthesized by emulsion copolymerization of 2-(dimethylamino) ethyl methacrylate with functional monomers and a photoliable o-nitrobenzyl diacrylate crosslinker. Upon mild UV irradiation (k 5 365 nm, 10 mJ/cm2), the photosensitive crosslinker underwent lightinduced degradation and the sizes of the nanoparticles

increased dramatically. The nanoparticles were uptaken by the cells which is confirmed by flow cytometry analysis. Bovine serum albumin and green fluorescent protein were loaded as model proteins into the nanoparticles and accelerC ated photo-triggered release were achieved. V 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A:00A:000–000, 2014.

Key Words: photosensitive, nanoparticle, controlled drug delivery

How to cite this article: Jiang Z, Li H, You Y, Wu X, Shao S, Gu Q. 2014. Controlled protein delivery from photosensitive nanoparticles. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION 1

Light is a unique source of stimulus for delivery system and optical manipulation of biomacromolecular structures.2 The wavelength and intensity can be easily controlled and the direction can be spatially manipulated. Light except deep UV does not have harmful effects on biomacromolecules and cells.3 It provides a powerful approach for study of important biological events such as protein transportation, enzymatic activity, and cellular signaling. Recently, light-responsive controlled delivery systems which encapsulate guest molecules and release them in response to a specific trigger attract great attention,4–6 for the release of loaded cargos from light-responsive systems can be triggered at the specific time and sites. As light-sensitive delivery system is used for biomedical applications, the photoexcitation of photosensitive moieties including photo-induced transformation or bond cleavage should undergo irradiation in a mild microenvironment to keep the bioactivity of the biomacromolecules. A variety of light sensitive moieties and delivery systems were reported.7–9 Spiropyran undergoes reversible photoexcited transformations (k 5 250 nm) with merocyanine, which induces changes in color and self-assembly structures in solution.10–12 Light (k 5 320 nm) induces Wolf rearrangement in 2-diazo-1,2-naphthoquinone moiety and changes its

solubility.13 And upon light irradiation, the trans isomer of azobenzene (k 5 360 nm) converts into cis form.14–16 A photocleavable nitrobenzyloxycarbonyl (NBOC) moiety, which was sensitive to mild-UV, was reported by Anseth and coworkers.17,18 Light-induced fast degradation of the NBOC derived crosslinker was achieved with cytocompatible wavelengths of low-intensity light irradiation (365–420 nm), which facilitated in situ tuning of crosslinking density in the photodegradable hydrogel in the presence of human mesenchymal stem cells (hMSCs) cells. The NBOC derivative underwent fast fragmentation reactions upon mild photoexcitation, which made it a good candidate for biological study.19,20 Smart delivery systems such as micelles,21 lipids,22 hydrogels,23,24 microgels25,26 and microparticles,27 and nanoparticles28 were developed for the biological applications. Recently, photosensitive nanocaged controlled delivery systems have witnessed considerable research interests. Stimulating photoliable chromophores inside the nanoparticle and releasing biomacromolecular cargos provided a platform to study drug delivery and the biological events. Nanosized drug carriers have advantages including long circulation, selective accumulation at the site of action, and good biocompatibility.29 The size of the delivery system has a critical effect for light intensity decays dramatically with light depth. Photosensitive delivery system at nanoscale

Additional Supporting Information may be found in the online version of this article. Correspondence to: Z. Jiang; e-mail: [email protected] or Q. Gu; e-mail: [email protected] Contract grant sponsor: Ministry of Science and Technology of the People’s Republic of China; contract grant number: 1106 Contract grant sponsor: Marie Curie Action of European Commission; contract grant number: Ec Piif-Ga-2010–275302 Contract grant sponsor: Ningbo Science Foundation; contract grant number: 2011A610116

C 2014 WILEY PERIODICALS, INC. V

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enhances fast drug release upon light irradiation. Upon light irradiation, the crosslinkers in the photosensitive nanoparticles would be cleaved which induces fast cargo release. In this article, we report a series of light-sensitive nanoparticles for controlled protein delivery. The nanoparticles were synthesized by the polymerization of 2-(dimethylamino) ethyl methacrylate (DEA) with functional monomers including 2-aminoethyl methacrylate hydrochloride (AMA), poly(ethylene glycol) methacrylate (PEGMA), ally a-D-glucoside (GLC), and a photocleavable NBOC derived diacrylate crosslinker (photocleavable diacrylate crosslinker, PDA). Light-induced degradation of photosensitive nanocarrier was studied. Bovine serum albumin (BSA) and green fluorescent protein (GFP) were used as model cargos and light triggered release from the nanoparticle was investigated. MATERIALS AND METHODS

Materials DEA, poly(ethylene glycol) methyl ether acrylate, ammonium persulfate, BSA, and tetramethylethylenediamine were purchased from Sigma-Aldrich. DEA was distilled before use. AMA was purchased from Acros Organics. GLC was purchased from Discovery Fine Chemicals, UK. Propidium iodide was purchased from Merck Chemicals Ltd. Polyvinyl alcohol (Mw 5 30,000) was purchased from Fisher. GFP and photosensitive o-nitrobenzyl diacrylate crosslinker were synthesized in our lab. Other chemicals were used as received. Synthesis of nanoparticle The nanoparticle was synthesized by oil-in-water emulsion method. For example, DEA (0.04 g), AMA (0.01 g), PDA (0.0125 g in 200 lL methylene chloride) were added in 1 mL of phosphate buffered saline (PBS) in 5-mL glass vial. Polyvinyl alcohol (PVA) (Mw 5 30,000) was used as dispersant. (For the synthesis of P(DEA-PEGMA) and P(DEA-GLC), DEA (0.03 g), PEGMA (0.02 g), and DEA (0.03 g), GLC (0.02 g) were used, respectively.) After the dissolved gas was purged with N2 flow for 30 min, the glass vial was sonicated for 2 min to obtain a homogeneous dispersion. Ammonium persulfate (APS) (0.0005 g in 100 lL H2O) and tetramethylethylenediamine (TMEDA) (0.0001 g in 100 lL H2O) was added to the reaction mixture by syringe. The reaction mixture was kept at 4 C for 2 h under stirring. The product was purified via dialysis by stirring H2O for 2– 3 h in PBS at room temperature. The yield was about 78%. Photolysis of PDA and nanoparticles A solution (1 mL) of PDA (0.1% wt/vol in acetonitrile) in 4-mL vial was irradiated by UV light (k 5 365 nm, 10 mJ/cm2). After irradiation, aliquots of samples were analyzed by a Waters 600E high-performance liquid chromatography (HPLC). The solutions of nanoparticles (1% wt/vol, 1 mL) in 4mL glass vial were irradiated by UV light (k 5 365 nm, 10 mJ/cm2). At each timepoint, an aliquot of the solution was sampled for dynamic light scattering (DLS) analyses.

sion of microparticles (20 lg, 1 lL, solid content 20 mg/mL) was diluted in 10% PBS in H2O (1 mL), vortexed, and transferred into either a 4-mL polystyrene cuvette (FB55143, Fisher Scientific) or a 1-mL clear zeta potential cuvette (DTS1060, Malvern). The data were collected and analyzed using Dispersion Technology software 4.20 (Malvern). Drug release The solutions of nanoparticles which were loaded with proteins (1% wt/vol, 10 mL) in 20-mL glass vials were irradiated by UV light (k 5 365 nm, 10 mJ/cm2) for 30 min under stirring. Drug release was performed by incubating nanoparticle solution (10 mg) in 5mL PBS at 37 C under shaking (600 rpm). At each timepoint, an aliquot of the solution was sampled. The removed buffer was centrifuged at 17,500 rpm for 10 min and the supernatant was collected. The quantities of the released BSA were measured by the complex of BSA and Coomassie blue on a UV–vis spectrometer. The quantities of the released GFP were measured by the fluorescent intensities. Cell culture Human cervical cancer (HeLa) cells line was used in this cell culture experiment. Dialyzed nanoparticle solutions (0.1 wt %) and 350 lL Dulbecco’s Modified Essential Medium (DMEM) supplemented with heat inactivated FCS 10% vol/ vol, penicillin (100 units mL21), streptomycin (100 mg mL21), and L-glutamine (2.0 mM) were added into 24-well plates. Cells (40,000) were seeded and incubated at 37 C with 5% CO2. The medium was refreshed every 48 h. Flow cytometry analysis Nanoparticles were added to Hela cells with a concentration of 40 mg/mL and incubated for 24 h at 37 C in serum-free dulbecco’s modified eagle medium (DMED) media supplemented with chlorpromazine (10 lg/mL), sucrose (4% wt/ vol), genistein (200 lM), and filipin (5 lg/mL). After incubation samples were washed twice with PBS, and 80 lL Trypansin solution was used to detach the cells from polystyrene walls. The solutions were filtrated and cellassociated fluorescence was detected using FACS Caliber flow cytometer (Becton Dickinson, Franklin Lake, NJ). Cytotoxicity study HeLa cells were grown in 96-well plates in DMEM medium (Invitrogen, Carlsbad, CA), supplemented with 100 units/mL aqueous penicillin G, 100 mg/mL streptomycin, and 10% FBS at concentrations to allow 70% confluence in 24 h. Added the nanoparticles into each well and incubated for another 24 h. Then cells were washed with PBS and incubated with phenolred free Opti-MEM media for 4 h at 37 C. The cytotoxicity cells were determined by MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay. RESULTS AND DISCUSSION

Dynamic light scattering and zeta potential measurement The hydrodynamic diameter and zeta potential of the particles were determined with a Malvern Zetasizer Nano-ZS. A suspen-

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Synthesis and photolysis of photocleavable diacrylate crosslinker In this study, photodegradable diacrylate crosslinker with a NBOC moiety (Scheme 1) was synthesized. A methoxy group

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SCHEME 1. Synthesis of photodegradable NBOC diacrylate crosslinker.

para to the azo moiety was incorporated to enhance the photocleavage quantum yield under Ultraviolet A irradiation.30–32 The synthesis reaction of PDA started from hydroxyl group protecting of 4-hydroxy-3-methoxybenzaldehyde, nitration of the aromatic ring, deprotecting, and finally acrylation of the photolabile group. The structure of as-synthesized compound is confirmed by NMR. 1H-NMR (400 MHz, DMSO-d6) d: 7.58 (s, 1H), 7.24 (s, 1H), 6.44 (d, 1H), 6.32 (m, 2H), 6.25 (m, 1H), 6.05 (d, 1H), 4.13 (t, 2H), 3.88 (s, 3H), 2.35 (t, 2H) and 1.95 (m, 2H). The photolytic reactions of PDA (0.1 wt/vol % in acetonitrile) under UV irradiation (k 5 365 nm, 10 mJ/cm2) were analyzed by HPLC. The mild light irradiation at 10 mJ/cm2 was used, which enhances the cell viability in biological studies. The NBOC group was cleaved via a photoinduced intramolecular hydrogen abstraction, and was followed by thermal reactions which eventually yield an aldehyde and a carboxylic acid. The HPLC analysis of these reactions recorded the loss of the starting compounds and appear-

FIGURE 1. Decrease of PDA concentration in function of time under light irradiation (k 5 365 nm, 10 mJ/cm2).

ance of the cleaved products (Supporting Information Scheme S1). The photo-induced degradation of PDA is presented in Figure 1. As shown in Figure 1, 48% mol PDA was cleaved within 30 min of UV irradiation. The crosslinker underwent bond cleavage upon UVA irradiation at mild conditions, which made it good candidate for biological studies. Synthesis of functional PDEA nanoparticles The nanoparticles were synthesized by emulsion polymerization of DEA, PDA, and functional monomers including AMA, PEGMA, and GLC at 4 C for 2 h (Scheme 2). Poly(DEA) has positive charged groups which promote more efficient delivery into the cell.33 And it is easy to form complex with the negatively charged cargos via electrostatic interactions and facilitate the load of cargos. The monomers such as AMA, PEGMA, and GLC were copolymerized with DEA to achieve stable dispersion in the aqueous solutions, reduce toxicity, and enhance cell uptake.34 The size of the nanoparticle can tune chemical compositions and reaction conditions in the polymerization process. The sizes of the original nanoparticles were determined to be around 300–450 nm by DLS (Fig. 2), which was favorable for cell uptake. Photo-degradation of the PDEA nanoparticles Since biological applications require the use of the photoswitch in aqueous solution, the photosensitive properties of the nanoparticles were determined in phosphate buffer solution (pH 5 7.4). Ultraviolet light induces ester bond cleavage and the subsequent change in the sizes of the nanoparticles. The light-induced degradation of PDEA nanoparticles were studied by DLS. After UV irradiation for 30 min, the diameter of the nanoparticles increased dramatically, for example, 425 6 34 nm to 1007 6 146 nm for P(DEA-AMA), 343 6 21 nm to 944 6 101 nm for P(DEAPEGMA), 392 6 26 nm to 873 6 148 nm for P(DEA-GLC), respectively (Fig. 3). In the absence of photocleavable

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SCHEME 2. Synthesis of poly(DEA) based nanoparticles.

crosslinker (butanol diacrylate was used as the control), no significant change of particle size was observed. The f potential of P(DEA-AMA), P(DEA-PEGMA), and P(DEA-GLC) at pH 7.4 was 4.82 6 0.91 mV, 27.82 6 0.13 mV, and 26.84 6 0.55 mV, respectively. After UV shining, the ester bonds were broken into carboxyl groups which had negative charges. Their surface charges turned into slightly negative values, for example, 26.84 6 0.55 mV for P(DEAAMA), 215.10 61.56 mV for P(DEA-PEGMA), and 213.35 6 2.76 mV for P(DEA-GLC) after UV irradiation (Table I). Cell uptake of PDEA nanoparticles To examine whether this nanoparticle can deliver cargos into cells, propidium iodide (PI) was loaded into the nanoparticles. The nanoparticles were incubated with Hela cells for 6 h and then irradiated by UV light (10 min 3 3 irradiation with 20-min interval). To discriminate between cellassociation and actual internalization, extracellular fluorescence was quenched by the addition of 0.4% (wt/vol) Trypan Blue in DMED. Figure 4 shows that the Hela cells had strong fluorescence with nanoparticles loaded with PI while

FIGURE 2. Particle sizes of poly(DEA) based nanoparticles. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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the control cell did not. PI is membrane impermeant and excluded from viable cells. The existence of PI inside the cell suggested the P(DEA)-based nanoparticle can penetrate the cell membrane and enter the cells which could have potential applications in protein and gene delivery into the cells. Protein release from the nanoparticles Light triggered protein release from the nanoparticles were studied and the release profiles of BSA and GFP from P(DEA-AMA) nanoparticles are presented in Figure 5. BSA was used as model protein, which was loaded into P(DEAAMA) nanoparticles by adding the solution of BSA into the reaction medium. The reaction conditions of low reaction temperature (4 C) and short reaction time (2 h) were used to prevent BSA from denaturing. The nanoparticles were purified by centrifugation and the radicals and other small molecules were dialyzed out. Upon UV irradiation BSA was released quickly, which was quantified by Coomassie brilliant blue assay. As shown in Figure 5(a), 842.8 6 94.6 mg of BSA was released from the nanoparticles (10 mg) within 2 days, whereas only 50.7 6 22.1 mg BSA was released in the first day and followed by little additional release

FIGURE 3. Changes of nanoparticle sizes before and after UV irradiation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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TABLE I. Changes of Particle Sizes and Zeta Potentials After Light Irradiation Particle Sizes (nm)

Zeta Potentials (mV)

Samples

Before Irradiation

After Irradiation

Before Irradiation

After Irradiation

P(DEA-AMA) P(DEA-PEGMA) P(DEA-GLC)

425 6 34 343 6 21 389 6 26

10076 146 944 6 101 873 6 148

4.82 6 0.91 27.82 6 0.13 26.84 6 0.55

26.84 6 0.55 215.10 6 1.56 213.35 6 2.76

without UV irradiation. The structural integrity of BSA was preserved during the release (Supporting Information Fig. S3). BSA has a molecular weight of 66.5 kDa, and the initial release without UV irradiation possibly because of small amount of BSA adhered to the surface of the nanoparticles. The photo-triggered release of GFP is also studied. GFP is a marker for gene expression which is composed of 238 amino acid residues (26.9 kDa). When used in cell study, GFP are much less harmful when illuminated in living cells while small fluorescent molecules such as fluorescein isothiocyanate (FITC) are strongly phototoxic. The quantities of released GFP were measured by the fluorescent intensities. As shown in Figure 5(b), accelerated release of GFP was observed in the first 24 h, and 480.2 6 33.7 mg of GFP was released within 4 days after UV irradiation.

at 80 mg/mL) (Fig. 6). Cationic polymers PDEA is cytotoxic and with the incorporation of PEGMA and GLC in the copolymer, the charge density decreased which may lead to lower cytotoxicity.

CONCLUSIONS

A series of biocompatible nanoparticles of P(DEA-AMA), P(DEA-PEGMA), and P(DEA-GLC) were synthesized with a photocleavable diacrylate crosslinker. The PDA underwent bond cleavage under mild UV irradiation. These nanoparticles had optimized surface properties and zeta sizes which are beneficial for cell uptake. Under mild UV irradiation, the sizes of the nanoparticles increased, which induced the accelerated release of BSA and GFP. These properties suggest that the

Cytotoxicity of the nanoparticles HeLa cell was used to assess the cytotoxicity of the nanoparticles. After incubation with the nanoparticles for 24 h, cell viability was determined by the MTT assay.35 The P(DEA-PEGMA) and P(DEA-GLC) nanoparticles did not cause significant cytotoxicity while P(DEA-AMA) nanoparticles showed cell cytotoxicity at high concentrations (76.6 6 6.5%

FIGURE 4. Flow cytometry analysis of Hela cells loaded with nanoparticles after 24 h incubation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 5. Release profiles of BSA (a) and GFP (b) in P(DEA-AMA) under different conditions: without irradiation (!); with UV irradiation (~).

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FIGURE 6. Cell viability studies of Hela cells exposed to nanoparticles. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

nanoparticles are promising light-responsive release systems for protein delivery. ACKNOWLEDGMENTS

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