Journal of Controlled Release 259 (2017) 160–167

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Journal of Controlled Release

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pH-degradable PVA-based nanogels via photo-crosslinking ofthermo-preinduced nanoaggregates for controlled drug delivery

Wei Chen ⁎, Yong Hou, Zhaoxu Tu, Lingyan Gao, Rainer Haag ⁎Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustrasse 3, Berlin 14195, Germany

⁎ Corresponding authors.E-mail addresses: chenwei1984@zedat.fu-berlin.de (W

haag@chemie.fu-berlin.de (R. Haag).

http://dx.doi.org/10.1016/j.jconrel.2016.10.0320168-3659/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 August 2016Received in revised form 19 October 2016Accepted 29 October 2016Available online 31 October 2016

pH-Degradable PVA nanogels, which are prepared by photo-crosslinking thermo-preinduced PVAnanoaggregates in water without any surfactants or toxic organic solvents, are used for intracellular PTX releaseand anticancer treatment. These nanogels fast degraded at mildly acidic conditions with a pH-triggered PTX re-lease, and the degradation products are only native PVA and poly(hydroxyethyl acrylate) (PHEA) as well as ac-etaldehyde without any toxic byproducts. The nanogel sizes could be tailored by different temperatures duringthe crosslinking process. The results of confocal microscopy and flow cytometry revealed that smaller nanogelsexhibited enhanced internalization with MCF-7 cells than the ones treated with larger nanogels, by which thesmaller PTX-loaded nanogels induced a more significant cytotoxicity against MCF-7 cells.Graphic abstract: pH-Degradable PVA nanogels can be prepared by photo-crosslinking of thermo-preinducednanoaggregates with tailored nanogel sizes given their pH-triggered PTX release and fast acid-degradation intonative PVA and cell-compatible poly(hydroxyethyl acrylate) (PHEA) as well as acetaldehyde.

© 2016 Elsevier B.V. All rights reserved.

Keywords:PVA nanogelpH-degradationPhoto-crosslinkingDrug deliveryAntitumor treatment

1. Introduction

Nanomedicines have conferred many advantages for cancer diagno-sis and therapy [1–4]. The preclinical and clinical studies have demon-strated that advanced drug delivery nanosystems provide prolongedcirculation time, efficient tumor-targeted accumulation via the en-hanced permeability and retention (EPR) effect, reduced side effects,and improved drug tolerance, that has resulted in better drug bioavail-ability and therapy [5–9]. To meet the pharmaceutical requirements,biocompatible nanocarriers including liposomes, polymeric nanoparti-cles, micelles, and nanogels have been developed for delivery of varioustypes of drugs for cancer treatment. Compared with other nanocarriers,nanogels with internally crosslinked three-dimensional (3D) structuresshow high water content, desirable chemical and mechanical proper-ties, and a large surface area for multivalent bioconjugation [10–14].They are able to stabilize bioactive compounds such as drugs, pep-tides/proteins, and DNA/RNA in their polymeric networks, and more-over, nanogels actively participate in the drug delivery process due totheir intrinsic properties such as stimuli-responsive behavior, swellingand softness, to achieve a controlled drug release at the target site[15–18].

Poly(vinyl alcohol) (PVA) is a synthetic polymer with OH-hydrophi-licity prepared by radical polymerization of vinyl acetate and followed

. Chen),

with partial hydrolysis. Due to its unique properties includingwater sol-ubility, multiple OH-groups for further decoration, and FDA-approvedbiocompatibility and low toxicity, the use of PVA as a biomaterial hasattracted great attention in biomedical applications such as protein/en-zyme immobilization, cell encapsulation in the form of micro/hydrogelscaffolds [19–22]. However, PVA nanostructures failed to meet the de-mand, especially in the field of nanomedicine area, which is mostlydue to their inhomogeneous interior, high porosity, low drug affinity,and uncontrollable release behavior. The modification of OH-groupson PVA is largely considered to introducemore convenient sites for con-jugation and chain extension using ester, carbamate, ether and acetallinkages [23]. For example: Kupal et al. reported that core–shell PVA-based microgels shielded with hyaluronic acid (HA) were prepared by“click” chemistry and inverse emulsion techniques for targeted local de-livery of doxorubicin to adenocarcinoma colon cells (HT-29) [24]. Werecently developed charge-conversional reducible PVA nanogels bycombining nanoprecipitation and “click” chemistry for an enhanced cel-lular uptake towards universal tumor cells and efficient tumorous cyto-toxicity against human cancer MCF-7 cells [25].

Much effort has been made towards the development of pH-sensi-tive nanocarriers for intracellular drug delivery, since there are naturalpH gradients in the tumor tissue microenvironment (pH 6.5–7.2) aswell as the endosomal/lysosomal compartments of tumor cells(pH 4.0–6.5) [26–32]. It is noteworthy that the strategy of pH-triggereddrug release has been exploited tomeet the challenges of various extra-and intra-cellular barriers towards successful cancer chemotherapy fordrug release nanosystems. The acid-labile acetal linkage has been

161W. Chen et al. / Journal of Controlled Release 259 (2017) 160–167

widely introduced to fabricate pH-sensitive nanostructures and net-works for intracellular drug delivery, in which the acetal groups are rel-atively stable under physiological conditions, while rapidly hydrolyzedat a mildly acidic pH to release the encapsulated cargo [33–35].Acetalization reactions were usually used to equip PVA chains with ac-rylamide groups and conjugated heterocyclic chromophores, as wellas photoactive groups [36,37]. In this study, we developed pH-degrad-able nanogels based on acetal-linked PVA with defined shape and sizefor encapsulation of PTX and intracellular release (Scheme 1). Thesefunctionalized PVA materials could firstly form nanoaggregates inwater by thermo-trigger, which was followed by photo-irradiation toproduce nanogels with high stability under physiological conditions.These nanogels could degrade fast at amildly acidic pH, andmore inter-estingly, the degradation products are only native PVA and cell-compat-ible poly(hydroxyethyl acrylate) (PHEA) as well as acetaldehydewithout any toxic byproducts. We investigated the thermo-transitionbehavior of the functionalized PVA, the stability and degradation ofthe nanogels, together with the in vitro drug release, size-dependentcellular uptake and tumorous cytotoxicity of PTX-loaded nanogels.

2. Experimental section

2.1. Materials and methods

Ethylene glycol vinyl ether (Aldrich, 97%), acryloyl chloride (Aldrich,97%), triethylamine (Et3N, Acros, 99%), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (I2959, Aldrich, 98%), pacli-taxel (PTX, Sigma, 97%), p-toluenesulfonic acid monohydrate (PTSA,Sigma-Aldrich, 98%) were used as received. Polyvinyl alcohol (PVA,Mowiol 3-85, Mw = 15,000 g/mol) was provided by Kuraray EuropeGmbH (Germany). For cell culture experiments, MCF-7 cells (DSMZ,115) were cultured in RPMI supplemented with 10% fetal calf serum,MEM nonessential amino acids, 1 mM sodium pyruvate, and 10 μg/mLhuman insulin. A549 cells (DSMZ, ACC 107) were cultured in DMEMsupplemented with 10% fetal calf serum, and 2 mM glutamine.

1H NMR spectra were recorded on a Bruker ECX 400. The chemicalshiftswere calibrated against residual solvent peaks as the internal stan-dard. IR measurements were carried out on a Nicolet AVATAR 320 FT-IR5 SXC that was equipped with a DTGS detector from 4000–600 cm−1.The size of nanogels was determined by dynamic light scattering

Scheme 1. Illustration of PTX-loaded PVA nanogels via photo-crosslinking of thermo-preipoly(hydroxyethyl acrylate) (PHEA) as well as acetaldehyde.

(DLS) at 25 °C using a Zetasizer Nano-ZS from Malvern Instrumentsequipped with a 633 nm He–Ne laser. Transmission electron microsco-py (TEM) measurement was performed on Philips EM12 and operatedat 100 kV with a nanogel concentration of 1 mg/mL. The TEM sampleswere prepared by dropping 5 μL of nanogel suspensions on the micro-scopical 200 mesh grids, which were placed on liquid nitrogen. After5 min, the samples were lyophilized for TEM measurement.

2.2. Synthesis of vinyl ether acrylate (VEA)

Ethylene glycol vinyl ether (20.00 g, 0.227 mol) and Et3N (41.6 mL,0.30 mol) were dissolved in dichloromethane (CH2Cl2, 300mL), follow-ed by a drop wise addition of acryloyl chloride (22.5 mL, 0.272 mol) at0 °C. After 8 h, the reaction solution was extracted with NaCO3 aqueoussolution twice, and the organic phase was then dried over MgSO4 andconcentrated by rotary evaporation. The final product was collected bydistillation under reduced pressure. Yield: 27.72 g (86%). 1H NMR(400 MHz, CDCl3): δ 6.60 (1H, CH2_CH–O–), 5.85–6.45 (3H,CH2_CH–C(O)–), 4.40 (2H, –CH2–O–C(O)–), 4.05–4.20 (2H, CH2_CH–O–), 3.94 (2H, –CH2–CH2–O–C(O)–); 13C NMR (400 MHz, CDCl3): δ166.10 (CH2_CH–C(O)–), 151.50 (CH2_CH–O–), 131.40 (CH2_CH–C(O)–), 128.12 (CH2_CH–C(O)–), 87.15 (CH2_CH–O–), 65.77(−CH2–CH2–O–C(O)–), 62.79 (−CH2–CH2–O–C(O)–).

2.3. Synthesis of VEA-functionalized PVA (PVA-VEA)

PVA (1.00 g, 17 mmol of OH group) was dissolved in anhydrousDMSO (100 mL), and then VEA (10, 20, and 40 mol% of OH group inPVA) and a catalytic amount of PTSA were sequentially added to the re-action to prepare PVA-VEA with different functionalities. After 6 h, thereaction was quenched by addition of Et3N. The solvent was removedby dialysis against methanol, and then the polymer solution was con-centrated by rotary evaporation. Finally, the polymer was isolated byprecipitation in diethyl ether, and re-dissolved in water and freeze-dried.

2.4. Preparation of pH-degradable nanogels

PVA-VEA polymer dissolved in water with a concentration of1.0 mg/mL containing 0.05 mg/mL of I2959 photo-initiator was kept

nduced nanoaggregates, and pH-triggered degradation of nanogels to native PVA and

162 W. Chen et al. / Journal of Controlled Release 259 (2017) 160–167

in ice-water bath, and then slowly warmed with argon perfusion. Thepolymer solution was stirred under UV exposure for 10 min. Thenanogels were purified by dialysis in water with a molecular weightcut off (MWCO) of 2000 and collected by freeze-drying. For the prepa-ration of PTX-loaded nanogels, PTX dissolved in ethanol (5.0 mg/mL)was added into PVA-VEA solution (1.0 mg/mL containing 0.05 mg/mLof I2959) with a feeding ratio at 10 wt% of PVA-VEA polymer, and thefollowing steps were performed similarly to the preparation of theblank nanogel. The free drug was removed by centrifugation with aMWCO of 10,000, and PTX-loaded nanogels were collected by freeze-drying.

2.5. pH-degradation of PVA-VEA polymer and nanogels

PVA-VEA dissolved in acetate buffer (pH 5.0)with a concentration of0.5 mg/mL was stirred at room temperature. To monitor the degrada-tion of VEA group from PVA backbone, 10 mL of polymer solution wastaken out at the desired time points and freeze-dried. The degradationdegree of PVA-VEA was characterized by 1H NMR. PVA-VEA nanogelswere separately suspended in phosphate buffer (PB, pH 7.4) and acetatebuffer (pH 5.0) with a concentration of 1.0 mg/mL. The samples wereslowly stirred at 37 °C, and the nanogel size was monitored over timeby DLS.

2.6. In vitro release of PTX

The in vitro release of PTX from PTX-VEA nanogels was investigatedat 37 °C in acetate buffer (pH 5.0) and PB (pH 7.4). PTX-loaded nanogelswere divided into two aliquots of 1 mL, and immediately transferred toa dialysis tube with a MWCO of 12,000–14,000. The dialysis tubes wereimmersed into 20 mL of appropriate buffers and shaken at 37 °C. At settime intervals, 5.0 mL of the release medium was taken out from eachexperimental group and replenished with an equal volume of fresh ap-propriate medium. The release medium was freeze-dried and theamount of PTX was determined by HPLC (Knauer, Advanced ScientificInstruments), with UV detection at 227 nm using a 60/40 (v/v) mixtureof acetonitrile and water as a mobile phase. Release experiments wereconducted in triplicate. The results are presented as the average± stan-dard deviation.

2.7. Size-dependent cellular uptake of PVA nanogels

PVA nanogels with different sizes were prepared by UV-crosslinkingof PVA-VEA with the functionality of 6% (PVA-VEA6%) under the ther-mo-trigger ranging from 18 to 30 °C. After that, FITC was linked to thenanogels using an ester condensation reaction between the isothiocya-nate group (NCS) of FITC and the hydroxyl group of PVA nanogels. FreeFITC was removed by centrifugation with a MWCO of 10,000. MCF-7cells were plated on microscope slides in a 24-well plate (1.0 × 104

cells/well) using 1640 culture medium containing 10% FBS. After 24 hincubation, themediumwas replaced by450 μL of fresh culturemediumand 50 μL of prescribed amounts of FITC-labeled nanogel samples. Afterincubation for 3 h, the culture mediumwas removed and the cells werewashed twice with phosphate buffered saline (PBS). The cells werefixed with 4% paraformaldehyde for 20 min, incubated with AlexaFluor 594 (ThermoFisher, Germany) to label the cell membrane at37 °C for 1 h, and the cell nuclei were stained with DAPI. Fluorescenceimages of cells were obtained with confocal laser scanning microscope(CLSM, Leica, Germany) and analyzed by Leica 2.6.0 software.

Cellular uptake of FITC-labeled PVA nanogels was also quantified byflow cytometry analysis. MCF-7 cells were cultured in a 24-well plate(1.0 × 105 cells/well) for 24 h, and then the medium was replaced by450 μL of fresh culture medium and 50 μL of prescribed amounts ofFITC-labeled nanogel samples. After 6 h incubation, the culture mediumwas removed, and the cellswere rinsed thricewith PBS and treatedwithtrypsin. The cell suspensions were washed twice with PBS and re-

suspended in PBS. The quantification of fluorescence was performedby a FACScalibur (BD Accuri C6).

2.8. Cytotoxicity of PTX-loaded PVA nanogels

The cytotoxicity of blank PVA nanogels was studied by the MTTassay using A549 and MCF-7 cells. Cells were seeded into a 96-wellplate at a density of 1 × 104 cells per well in 90 μL of culture mediumand incubated at 37 °C with 5% CO2. After 24 h, 10 μL of blank nanogelsamples at different concentrations in PB (10 mM, pH 7.4) were addedand the cells were incubated for another 48 h. To study the cytotoxicityof PTX-loaded PVA-VEA6% nanogels, 10 μL of prescribed amounts of PTX-loaded nanogel samples were added. The cells were incubated withPTX-loaded samples for another 48 h incubation. After that, 10 μL ofMTT solution (5 mg/mL) was added and the cells were incubated for4 h. The medium was replaced by 150 μL of DMSO to dissolve theresulting purple crystals. The optical densities were measured by a mi-croplate reader at 570 nm. To further study the cytotoxicity of PTX-load-ed nanogels induced by different sizes,MCF-7 cells were incubatedwith10 μL of prescribed amounts of PTX-loaded nanogel samples for 6 h, andthen the mediumwas replaced by fresh medium for another 48 h incu-bation. After that, the cell treatment procedure was carried out asabove-mentioned to test the cytotoxicity value. The experiments wereconducted in triplicate. The results are presented as the average± stan-dard deviation.

3. Results and discussion

3.1. Synthesis of PVA-VEA polymer

Vinyl ether acrylate (VEA)was synthesized by acrylation of ethyleneglycol and vinyl ether with a yield over 85% (Fig. S1). It has been dem-onstrated that the acetalization between vinyl ether and hydroxyl orthiol is quite efficient and the formed acetal linkers are labile to mildlyacidic conditions [38]. VEA-functionalized PVA polymers with the VEAfunctionalities of 6%, 3.5%, and 2% (denoted as PVA-VEA6%, PVA-VEA3.5% and PVA-VEA2%, respectively) were synthesized by acetalizationbetween vinyl ether group and hydroxyl in the presence of PTSA(Scheme 2, and Table 1). The degree of VEA functionalities was deter-mined by 1H NMR according to the integral ratio of the signals at δ5.88–6.42 and 2.01, which are attributed to the double bond of VEAand the methyl group on PVA, respectively (Fig. S2A). It is interestingto note that the VEA functional domain is highly flexible for crosslinkngor modification via UV-irradiation or Michael-type addition reaction.Furthermore, the acetal linker between VEA and PVA backbone is pH-degradable, and the UV-crosslinked structure based on PVA-VEA candegrade to native PVA, PHEA, and acetaldehyde without any toxicbyproduct.

3.2. Nanogel formation and stability

The PVA-VEA polymer was readily dissolved in water (1.0 mg/mL)with an average size of 6–10 nm at a low temperature, indicating thatPVA-VEA exists as a unimer (Fig. 1A). However, the clear solutionturned turbid upon increasing the temperature, and dynamic light scat-tering (DLS) showed a lower critical solution temperature (LCST) of 16,22 and 40 °C for PVA-VEA6%, PVA-VEA3.5%, and PVA-VEA2%, respectively(Table 1). The hydrophobic modification with VEA units on PVA couldweaken the intra- and intermolecular hydrogen bonds of adjoiningOH-groups, which endows PVAmaterial with thermo-transition ability.It is interesting to note that the transition temperature of PVA-VEA solu-tion increased as the decrease of VEA functionality, in which the respec-tive sizes of thermo-preinduced nanoaggregates were 220, 201, and170 nm for PVA-VEA6%, PVA-VEA3.5%, and PVA-VEA2%. As the VEA func-tionality was further increased to 8.2%, the polymer couldn't be directlydissolved in water even at low temperature, and formed

Scheme 2. Synthesis of VEA-functionalized PVA (PVA-VEA) in DMSO at room temperature using p-toluene sulphonic acid (PTSA) as a catalyst, and UV-crosslinking of PVA-VEA in thepresence of Irgacure 2959 (I2959) photo-initiator. Both polymer structures can degrade into native PVA at acidic conditions.

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nanoaggregates with the size of 90 nm inwater at 5 °C by the assistanceof methanol in suspending. The nanoaggregates were sequentially ex-posed under UV-light to form nanogels with decreased sizes varyingfrom 125 to 180 nm (Scheme 2), and remarkably PVA-VEA6% nanogelsshowed 180 nm with a particularly narrow size distribution of 0.05(Fig. 1B). The crosslinking reaction of PVA-VEA was confirmed by 1HNMR in deuterated methanol using PVA-VEA6%, in which proton reso-nances at δ 5.88–6.42 attributed to the double bond clearly disappearedafter UV-exposure (Fig. S2A). FT-IR showed that the peak at 1645 cm−1

attributed to C_C group also disappeared after crosslinking (Fig. S2B).To further confirm the stability of the nanogels, PVA-VEA6% nanogelswere measured by DLS at 10 and 25 °C, and the results showed thatthere was no size difference for the crosslinked PVA-VEA6% at both tem-peratures, while the non-crosslinked PVA-VEA6% still presented revers-ible thermo-response (Fig. 1C). The DLS results performed at 25 °C alsoshowed that there was just a little swelling (30–50 nm increase) forPVA-VEA6% nanogels by 1000-fold dilution in water or using methanolfor a suspension, in contrast to the non-crosslinked PVA-VEA6%, whichdissociated by high dilution or dissolved in methanol (Fig. 1D). This in-dicates that these thermo-preinduced nanoaggregates under UV-irradi-ation are good process for preparing degradable PVA nanogels.

3.3. pH-dependent degradation

The pH-dependent acetal hydrolysis of PVA-VEA6% was character-ized by 1H NMR according to the disappearance of acrylate proton reso-nance at 5.88–6.42 ppm (Fig. S3). By calculating the integral ratio of δ5.88–6.42 and 2.01 (proton resonance of acrylate and methyl groupon PVA, respectively), the half-life of acetal hydrolysis at pH 5.0 wasabout 6 h. By contrast, a negligible acetal hydrolysis of PVA-VEAwas ob-served at pH 7.4 over 24 h. This hydrolysis rate of acetal at pHswas sim-ilar to those reported by Fréchet, Zhong, and our group for PEGcopolymers with (tri)methoxybenzylidene acetals attached at the sideor periphery [39–42]. The size change of nanogels in response to acetalhydrolysis was followed by DLS. The placement of nanogels into pH 5.0

Table 1Characteristics of PVA-VEA polymers and nanogels.

PolymersLCSTa

(°C)

Before crosslinkingb After crosslinkingb PTX loadingd

Size (nm) PDI Size (nm) PDILC(wt.%)

LE(%)

PVA-VEA6% 16 ± 0.8 220.3 ± 2.4 0.08 179.7 ± 1.7 0.05 6.2 59.5PVA-VEA3.5% 22 ± 0.9 201.2 ± 3.5 0.13 173.1 ± 4.6 0.25 2.0 18.4PVA-VEA2% 40 ± 1.3 169.8 ± 5.2c 0.22c 125.3 ± 6.3 0.27 1.2 10.9

a Determined by DLS at polymer concentration of 1 mg/mL.b Determined by DLS at 25 °Cc Measured at 40 °C.d Loading content (LC) and loading efficiency (LE) of PTX calculated by HPLC.

acetate buffer (100 mM) resulted in rapid and remarkable swelling, inwhich the size increased from 180 nm to about 520 nm in 2 h thatreached over 1000 nm after 6 h. It is interesting to notice that after24 h the size of nanogels further decreased to 7–10 nm, which is similarto the size of non-functionalized PVA dissolved in water (Fig. 2A). Incontrast, no change of nanogel size was observed over 24 h at pH 7.4.The degradation of the nanogels remained consistent with degradationof PVA-VEA at pH 5.0 and 7.4. The degradation of PVA-VEA6% nanogelswas also checked by 1H NMR (400 MHz, CD3OD), and the spectrashowed that the nanogels after the treatment at the acidic conditionspresented the new signal at δ 3.60 attributed to the methylene protonsneighboring to the OH-group in the degradation product of PHEA, andthe new signals at δ 8.07 and 2.46 attributed to the acetaldehyde pro-tons (Fig. S4), indicating that the degradation product of PVA-VEAnanogels are PVA, PHEA as well as acetaldehyde, without any toxicside product.

3.4. PTX encapsulation and pH-triggered release

Paclitaxel (PTX) was encapsulated into PVA-VEA nanogels to studythe drug loading capability. Before UV-irradiation, PTX dissolved in eth-anol was added into PVA-VEA aqueous solution with a theoretical drugloading content (DLC: weight of loaded drug/total weight of polymerand loaded drug × 100%); of 10 wt%. The results showed that DLC anddrug loading efficiency (DLE: weight of loaded drug/weight of drug infeed × 100%) of PVA-VEA6% for paclitaxel were approximately 6.2 wt%and 59.5%, respectively (Table 1). The drug loading capabilities ofPVA-VEA3.5% and PVA-VEA2% were very poor, with PTX DLE of only18.4% and 10.9%, respectively,whichweremainly due to the insufficienthydrophobic domains inside the nanogels for PTX interaction. In the fol-lowing, PVA-VEA6% nanogels were further used for an in vitro PTX re-lease study. The in vitro release profile of PTX from nanogels wasinvestigated at pH 7.4 and pH 5.0 at 37 °C. PTX release at physiologicalpH (pH 7.4) was highly restricted with a released amount of only21.2% after 30 h (Fig. 2B). However, the PTX release was enhancedunder a endosomal/lysomal pH condition (pH 5.0) due to the cleavageof acid-labile acetal linker, in which 77.0% of PTX was released in 6 h,and release amount reached 90% in 30 h. PTX release fromPVAnanogelsclearly proceeds in a controlled manner and can be triggered by a lowpH.

3.5. Size-dependent cellular uptake

It is interesting to note that the size of PVA-VEA6% aggregates beforeUV-crosslinking can be tailored by different thermo-triggers rangingfrom160nmto 450nm, followed byUV-crosslinking to produce pH-de-gradable nanogels with different sizes. The average size of the polymernanoaggregates became larger as the temperature increased from 18 to

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Fig. 1. (A) Thermo-dependant size of PVA-VEA aqueous solution (1.0mg/mL)without crosslinking; (B) size distribution of PVA-VEAnanogels after crosslinking determinedbyDLS; and (C,D) stability of non-crosslinked (NCL) and UV crosslinked (CL) PVA-VEA6% (1.0 mg/mL) treated with thermo-trigger, methanol suspension or high-fold water dilution.

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30 °C, which is probably due to Oswald ripening. For example:nanoaggregates were crosslinked at 18–20 °C to form nanogels withan average size of 180 nm (NG-180) determined by DLS and TEM, andas the temperature increased during the UV-crosslinking, the nanogelsizes could reach 324 (NG-324) and 486 nm (NG-486) crosslinked at22–25 °C and 28–30 °C, respectively (Fig. 3). The nanogel size evidentlyaffects the efficiency of cellular uptake and subsequent cellular internal-ization [43–45].We investigated the cellular uptake behavior of FITC-la-beled nanogels with different sizes using MCF-7 cells. By CLSM images,it was found that FITC-labeled NG-180 well distributed in the MCF-7

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cells after 6 h incubation, while NG-324 displayed a weaker FITC inten-sity than that of NG-180, and therewas only a little FITCfluorescence forthe cells incubated with NG-486 under otherwise the same conditions(Fig. 4). Cellular uptake of FITC-labeled nanogels was further quantifiedby flow cytometry analysis. As expected, the flow cytometry resultsshowed that MCF-7 cells following 6 h treatment with NG-180displayed enhanced FITC fluorescence, 1.5–3.5-fold higher than that ofcells incubated with NG-324 and NG-486 (Fig. 5A). Hence, the cellularuptake is highly size-dependent in the order: NG-180 N NG-324 N NG-486, indicating that the smaller nanogels endow the cellular uptake

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Fig. 3. Size distribution of PVA-VEA6% nanogels prepared under different thermo-trigger measured by DLS and TEM.

165W. Chen et al. / Journal of Controlled Release 259 (2017) 160–167

more effectively [46]. The mechanism is supposed to be caused by acombination of factors, i.e. the surface ratio of adhesion, membranestretching, and the bending energy of cell membrane with differentsizes of nanogels, as well as polydispersity of nanogels particles.

3.6. Cytotoxicity of PTX-loaded nanogels

MTT assays using MCF-7 and A549 cells revealed that PVA-basednanogels were practically non-toxic (cell viabilities ≥ 90%) up to a testedconcentration of 1.0 mg/mL (Fig. S5A), confirming that these nanogels

Fig. 4.CLSM images ofMCF-7 cells incubatedwith FITC-labeled PVA-VEA6% nanogelswith differe(blue), cell membrane labeled by Alexa Fluor 594 (red), FITC-labeled nanogels in cells (green),25 μm in all the images.

had a good cell-compatibility. Notably, PTX-loaded NG-180 displayedsignificant tumorous cytotoxicity towardsMCF-7 and A549 cells follow-ing 48 h incubation (Fig. S5B). For example, the half maximal inhibitoryconcentrations (IC50) of PTX-loaded nanogels towards MCF-7 and A549cells were determined to be 0.79 and 1.50 μg PTX equiv./mL, respective-ly, which were close to the values of free PTX (MCF-7 cell: 0.58 μg PTXequiv./mL, and A549 cell: 1.07 μg PTX equiv./mL). To further studysize-dependent tumorous cytotoxicity of PVA-VEA6% nanogels, MCF-7cells were incubated only for 6 h with PTX-loaded nanogels with differ-ent sizes. The culture mediumwas removed and replenishedwith fresh

nt sizes for 6 h. The images show for eachpanel from left to right cell nuclei stained byDAPIoverlays of three fluorescent images, and bright field image. The scale bars correspond to

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Cou

nts

Fig. 5. (A) Flow cytometry profiles of MCF-7 cells after 6 h incubation with FITC-labeled PVA-VEA6% nanogels with different sizes (The cells without any treatment were used as a blankcontrol), and (B) cytotoxicity of PTX-loadedPVA-VEA6% nanogels determinedbyMTT assay usingMCF-7 cells (The cellswere treatedwith PTX-loadednanogels or free PTX for 6 h, then themedium was removed and replenished with fresh culture medium for another 48 h. The data are presented as the average ± standard deviation (n = 3, student's t-test: *p b 0.05,**p b 0.01).

166 W. Chen et al. / Journal of Controlled Release 259 (2017) 160–167

culture medium and the cells were cultured for another 48 h. Interest-ingly,MCF-7 cells treatedwith PTX-loadedNG-180displayed cell viabil-ities of 52.9% and 43.0% with the PTX feeding concentrations of 5 and10 μg/mL, respectively, which was much lower than those incubatedwith PTX-loaded NG-324 (5 μg/mL: 69.6%, and 10 μg/mL: 44.5%) andNG-486 (5 μg/mL: 83.8%, and 10 μg/mL: 70.7%) (Fig. 5B). The resultsdemonstrated that smaller nanogels exhibited enhanced cellular uptakeand more significant tumorous cytotoxicity. The in vitro therapeutic ef-fects of nanogels were determined by complicated reasons, but at leastthis provides an insight that the interactions between particle sizes andbiological effects should be definitely considered in investigating thera-peutic applications.

4. Conclusion

We have successfully developed biocompatible and pH-degradablePVA-based nanogels for intracellular PTX release and anticancer treat-ment, in which thermo-preinduced PVA-VEA nanoaggregates wereformed and UV-irradiated to prepare nanogels without any surfactantsor toxic organic solvents. These nanogels possesswell-defined structureand size with excellent colloidal stability but rapid degradation and apH-triggered drug release under intracellular acidic conditions. Further-more, the size of PVA-VEA aggregates can be tailored by different ther-mo-triggers, followed by UV-crosslinking to fabricate pH-degradablenanogels with different sizes. The smaller nanogels exhibited enhancedinternalization withMCF-7 cells, inducing a higher tumorous cytotoxic-ity. As a promising template, this nanogel system can be further deco-rated with specific ligands for tumor tissue or cell targeting binding,providing a highly potential platform for delivery of various chemother-apeutics and proteins to actively treat different malignant tumors.

Acknowledgements

We thank the German Research Foundation of SFB 1112 for a finan-cial support.We thank Dr. FlorianMummy (Head of Research & Techni-cal Services in Kuraray Europe GmbH) for providing the PVA samples.We also thank Dr. Pamela Winchester for carefully language polishingthis manuscript. Wei Chen is grateful for a research fellowship fromthe Alexander von Humboldt Foundation.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2016.10.032.

References

[1] P. Couvreur, Nanoparticles in drug delivery: past, present and future, Adv. DrugDeliv. Rev. 65 (2013) 21–23.

[2] M.E. Davis, Z. Chen, D.M. Shin, Nanoparticle therapeutics: an emerging treatmentmodality for cancer, Nat. Rev. Drug Discov. 7 (2008) 771–782.

[3] T. Sun, Y.S. Zhang, B. Pang, D.C. Hyun, M. Yang, Y. Xia, Engineered nanoparticles fordrug delivery in cancer therapy, Angew. Chem. Int. Ed. 53 (2014) 12320–12364.

[4] A. Wicki, D. Witzigmann, V. Balasubramanian, J. Huwyler, Nanomedicine in cancertherapy: challenges, opportunities, and clinical applications, J. Control. Release 200(2015) 138–157.

[5] L. Brannon-Peppas, J.O. Blanchette, Nanoparticle and targeted systems for cancertherapy, Adv. Drug Deliv. Rev. 64 (2012) 206–212.

[6] M. Elsabahy, K.L. Wooley, Design of polymeric nanoparticles for biomedical deliveryapplications, Chem. Soc. Rev. 41 (2012) 2545–2561.

[7] K. Knop, R. Hoogenboom, D. Fischer, U.S. Schubert, Poly(ethylene glycol) in drug de-livery: pros and cons as well as potential alternatives, Angew. Chem. Int. Ed. 49(2010) 6288–6308.

[8] G. Liu, Z. An, Frontiers in the design and synthesis of advanced nanogels fornanomedicine, Polym. Chem. 5 (2014) 1559–1565.

[9] G. Pasut, F.M. Veronese, PEG conjugates in clinical development or use as anticanceragents: an overview, Adv. Drug Deliv. Rev. 61 (2009) 1177–1188.

[10] R.T. Chacko, J. Ventura, J. Zhuang, S. Thayumanavan, Polymer nanogels: a versatilenanoscopic drug delivery platform, Adv. Drug Deliv. Rev. 64 (2012) 836–851.

[11] D.M. Eckmann, R.J. Composto, A. Tsourkas, V.R. Muzykantov, Nanogel carrier designfor targeted drug delivery, J. Mater. Chem. B 2 (2014) 8085–8097.

[12] A.V. Kabanov, S.V. Vinogradov, Nanogels as pharmaceutical carriers: finite networksof infinite capabilities, Angew. Chem. Int. Ed. 48 (2009) 5418–5429.

[13] K.S. Soni, S.S. Desale, T.K. Bronich, Nanogels: an overview of properties, biomedicalapplications and obstacles to clinical translation, 2015. J. Control. Release. , http://dx.doi.org/10.1016/j.jconrel.2015.1011.1009.

[14] X. Zhang, S. Malhotra, M. Molina, R. Haag, Micro- and nanogels with labile crosslinks- from synthesis to biomedical applications, Chem. Soc. Rev. 44 (2015) 1948–1973.

[15] Y. Li, D. Maciel, J. Rodrigues, X. Shi, H. Tomás, Biodegradable polymer nanogels fordrug/nucleic acid delivery, Chem. Rev. 115 (2015) 8564–8608.

[16] W. Chen, M. Zheng, F. Meng, R. Cheng, C. Deng, J. Feijen, Z. Zhong, In situ forming re-duction-sensitive degradable nanogels for facile loading and triggered intracellularrelease of proteins, Biomacromolecules 14 (2013) 1214–1222.

[17] J.-H. Ryu, R.T. Chacko, S. Jiwpanich, S. Bickerton, R.P. Babu, S. Thayumanavan, Self-cross-linked polymer nanogels: a versatile nanoscopic drug delivery platform, J.Am. Chem. Soc. 132 (2010) 17227–17235.

[18] X. Zhang, K. Achazi, D. Steinhilber, F. Kratz, J. Dernedde, R. Haag, A facile approachfor dual-responsive prodrug nanogels based on dendritic polyglycerols with mini-mal leaching, J. Control. Release 174 (2014) 209–216.

[19] B.V. Slaughter, S.S. Khurshid, O.Z. Fisher, A. Khademhosseini, N.A. Peppas, Hydrogelsin regenerative medicine, Adv. Mater. 21 (2009) 3307–3329.

[20] K.S. Lim, M.H. Alves, L.A. Poole-Warren, P.J. Martens, Covalent incorporation of non-chemically modified gelatin into degradable PVA-tyramine hydrogels, Biomaterials34 (2013) 7097–7105.

[21] B.E.B. Jensen, L. Hosta-Rigau, P.R. Spycher, E. Reimhult, B. Stadler, A.N. Zelikin,Lipogels: surface-adherent composite hydrogels assembled from poly(vinyl alco-hol) and liposomes, Nanoscale 5 (2013) 6758–6766.

[22] S.-F. Chong, A.A.A. Smith, A.N. Zelikin, Microstructured, functional PVA hydrogelsthrough bioconjugation with oligopeptides under physiological conditions, Small 9(2013) 942–950.

[23] M.-H. Alves, B.E.B. Jensen, A.A.A. Smith, A.N. Zelikin, Poly(vinyl alcohol) physicalhydrogels: new vista on a long serving biomaterial, Macromol. Biosci. 11 (2011)1293–1313.

167W. Chen et al. / Journal of Controlled Release 259 (2017) 160–167

[24] S.G. Kupal, B. Cerroni, S.V. Ghugare, E. Chiessi, G. Paradossi, Biointerface properties ofcore–shell poly(vinyl alcohol)-hyaluronic acid microgels based on chemoselectivechemistry, Biomacromolecules 13 (2012) 3592–3601.

[25] W. Chen, K. Achazi, B. Schade, R. Haag, Charge-conversional and reduction-sensitivepoly(vinyl alcohol) nanogels for enhanced cell uptake and efficient intracellulardoxorubicin release, J. Control. Release 205 (2015) 15–24.

[26] F. Meng, Y. Zhong, R. Cheng, C. Deng, Z. Zhong, pH-sensitive polymeric nanoparticlesfor tumor-targeting doxorubicin delivery: concept and recent advances,Nanomedicine 9 (2014) 487–499.

[27] M. Kanamala, W.R. Wilson, M. Yang, B.D. Palmer, Z. Wu, Mechanisms and biomate-rials in pH-responsive tumour targeted drug delivery: a review, Biomaterials 85(2016) 152–167.

[28] E.S. Lee, Z. Gao, Y.H. Bae, Recent progress in tumor pH targeting nanotechnology, J.Control. Release 132 (2008) 164–170.

[29] Y. Zhong, K. Goltsche, L. Cheng, F. Xie, F. Meng, C. Deng, Z. Zhong, R. Haag, Hyaluronicacid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treatCD44-overexpressing human breast tumor xenografts in vivo, Biomaterials 84(2016) 250–261.

[30] Y. Zhao, W. Ren, T. Zhong, S. Zhang, D. Huang, Y. Guo, X. Yao, C. Wang, W.-Q. Zhang,X. Zhang, Q. Zhang, Tumor-specific pH-responsive peptide-modified pH-sensitive li-posomes containing doxorubicin for enhancing glioma targeting and anti-tumor ac-tivity, J. Control. Release 222 (2016) 56–66.

[31] M. Calderón, P. Welker, K. Licha, I. Fichtner, R. Graeser, R. Haag, F. Kratz, Develop-ment of efficient acid cleavable multifunctional prodrugs derived from dendriticpolyglycerol with a poly(ethylene glycol) shell, J. Control. Release 151 (2011)295–301.

[32] E. Fleige, K. Achazi, K. Schaletzki, T. Triemer, R. Haag, pH-responsive dendritic core–multishell nanocarriers, J. Control. Release 185 (2014) 99–108.

[33] E.M. Bachelder, T.T. Beaudette, K.E. Broaders, J. Dashe, J.M.J. Fréchet, Acetal-derivatized dextran: an acid-responsive biodegradable material for therapeutic ap-plications, J. Am. Chem. Soc. 130 (2008) 10494–10495.

[34] W. Chen, F.H. Meng, F. Li, S.J. Ji, Z.Y. Zhong, pH-responsive biodegradable micellesbased on acid-labile polycarbonate hydrophobe: synthesis and triggered drug re-lease, Biomacromolecules 10 (2009) 1727–1735.

[35] D. Steinhilber, M. Witting, X. Zhang, M. Staegemann, F. Paulus, W. Friess, S. Küchler,R. Haag, Surfactant free preparation of biodegradable dendritic polyglycerol

nanogels by inverse nanoprecipitation for encapsulation and release of pharmaceu-tical biomacromolecules, J. Control. Release 169 (2013) 289–295.

[36] R.H. Schmedlen, K.S. Masters, J.L. West, Photocrosslinkable polyvinyl alcoholhydrogels that can be modified with cell adhesion peptides for use in tissue engi-neering, Biomaterials 23 (2002) 4325–4332.

[37] P. Martens, J. Blundo, A. Nilasaroya, R.A. Odell, J. Cooper-White, L.A. Poole-Warren,Effect of poly(vinyl alcohol) macromer chemistry and chain interactions on hydro-gel mechanical properties, Chem. Mater. 19 (2007) 2641–2648.

[38] Y.H. Lim, G.S. Heo, Y.H. Rezenom, S. Pollack, J.E. Raymond, M. Elsabahy, K.L. Wooley,Development of a vinyl ether-functionalized polyphosphoester as a template formultiple postpolymerization conjugation chemistries and study of core degradablepolymeric nanoparticles, Macromolecules 47 (2014) 4634–4644.

[39] E.R. Gillies, T.B. Jonsson, J.M.J. Fréchet, Stimuli-responsive supramolecular assem-blies of linear-dendritic copolymers, J. Am. Chem. Soc. 126 (2004) 11936–11943.

[40] E.R. Gillies, J.M.J. Frechet, A new approach towards acid sensitive copolymermicellesfor drug delivery, Chem. Commun. (2003) 1640–1641.

[41] W. Chen, F. Meng, R. Cheng, Z. Zhong, pH sensitive degradable polymersomes fortriggered release of anticancer drugs: a comparative study with micelles, J. Control.Release 142 (2010) 40–46.

[42] D. Steinhilber, T. Rossow, S. Wedepohl, F. Paulus, S. Seiffert, R. Haag, A microgel con-struction kit for bioorthogonal encapsulation and pH-controlled release of livingcells, Angew. Chem. Int. Ed. 52 (2013) 13538–13543.

[43] H. Liu, T. Liu, L. Li, N. Hao, L. Tan, X. Meng, J. Ren, D. Chen, F. Tang, Size dependentcellular uptake, in vivo fate and light-heat conversion efficiency of gold nanoshellson silica nanorattles, Nanoscale 4 (2012) 3523–3529.

[44] W. Jiang, Y.S. KimBetty, J.T. Rutka, C.W. ChanWarren, Nanoparticle-mediated cellularresponse is size-dependent, Nat. Nanotechnol. 3 (2008) 145–150.

[45] L. Tang, X. Yang, Q. Yin, K. Cai, H. Wang, I. Chaudhury, C. Yao, Q. Zhou, M. Kwon, J.A.Hartman, I.T. Dobrucki, L.W. Dobrucki, L.B. Borst, S. Lezmi, W.G. Helferich, A.L.Ferguson, T.M. Fan, J. Cheng, Investigating the optimal size of anticancernanomedicine, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 15344–15349.

[46] A.L. Sisson, D. Steinhilber, T. Rossow, P. Welker, K. Licha, R. Haag, Biocompatiblefunctionalized polyglycerol microgels with cell penetrating properties, Angew.Chem. Int. Ed. 48 (2009) 7540–7545.