Hyaluronic-Acid-Based pH-Sensitive Nanogels for Tumor-Targeted

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Hyaluronic acid-based pH-sensitive nanogels for tumor targeted drug delivery Shujuan Luan, Yingchun Zhu, Xiaohe Wu, Yingying Wang, Fengguang Liang, and Shiyong Song ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00444 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017

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ACS Biomaterials Science & Engineering

Hyaluronic acid-based pH-sensitive nanogels for tumor targeted drug delivery Shujuan Luan1, Yingchun Zhu1, Xiaohe Wu1, Yingying Wang1, Fengguang Liang2, Shiyong Song∗,1

Corresponding author: Shiyong Song, Email: [email protected]; Telephone and fax numbers: +86-371-23880680 Address: 1

North Jinming Road, Institute of Pharmacy, Pharmaceutical College of Henan University,

Kaifeng, China 475004 2

No. 8 Baobei Street, Orthopedics Department of Huaihe Hospital, Henan University, Kaifeng,

China 475000

ACS Paragon Plus Environment


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ABSTRACT A natural polysaccharide based nanogel has been synthesized and characterized as pH-sensitive drug delivery system for poorly water-soluble anticancer drugs. In this work, methacrylated hyaluronic acid (MAHA) was used to prepared acid degradable nanogels by a surfactant-free polymerization method in water, where 2, 2-dimethacroyloxy-1-ethoxypropane (DMAEP) served as a pH labile cross linker. Nanogels of different cross linking density were prepared and doxorubicin (DOX) was successfully encapsulated into the nanogels with drug loading contents (DLC) ranging from 7.67% to 12.15%. An accelerated DOX release was found in acidic conditions. Cytotoxicity study showed that the DOX loaded nanogels have significantly enhanced cytotoxicity in vitro compared with the non-sensitive ones. Confocal microscopy revealed that there was more DOX in the nuclei of tumor cells when incubated with DOX loaded pH sensitive nanogels for

3 to 12 hours. The

enhanced anticancer activity of DOX loaded pH sensitive nanogels was also verified by in vivo therapeutic study on mice, in which

tumor volume evolution was

measured and tumor tissues cell apoptosis and proliferation was examined. Keywords: nanogel; pH-sensitive; ketal; hyaluronic acid; drug delivery

INTRODUCTION pH-responsive drug carriers have gained extensive attentions due to their promise for tumor targeted drug delivery.1 Numerous pH gradients exist in both normal and pathophysiological conditions and a slightly more acidic pH in tumor tissue compared with blood and normal tissue,2,3 which makes tumors targetable for pH-responsive


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drug delivery. Moreover, the further decrease of pH in

endosomes (pH 5.5-6.0) and

lysosomes (pH 4.5-5.0) will also lead to an accelerated release of drug in the pH-sensitive systems after endocytosis. Micelle is mostly explored in pH-sensitive drug delivery systems. They can be prepared from drug conjugated polymers linked by a pH sensitive linkage.4-6 They can also be made from pH-sensitive groups containing polymers,7,8 where a sufficient structural change will result an accelerated drug release. Acid labile groups such as hydrazone,9,10 acetal,11,12 orthoester,13,14 citraconic amide15 and Schiff base bonds7 et al. were reported. Even great advance have been achieved by pH-sensitive micelles, drug loading capacity and long circulation stability in vivo are still the main obstacles for clinical application. Nanogels have been shown great potentials for nanomedicine for its unique properties. They are biocompatible and capable of carrying a broad spectrum of guest molecules, ranging from small molecule drugs to biomacromolecules, inorganic nanoparticles.16 Nanogels can also be tailored with pH-sensitivity, complying with the acidic condition encountered outside and inside tumor cells. The decrease of pH triggers conformation or structure changes of nanogels, in turn an accelerated drug release. In the pH-responsive nanogels made from poly (methylacryl acid) or amino-polymers, pH-induced charge transformation is employed release.17,

18

to control the drug

Alternatively, nanogels installed with acid-labile groups

were also

widely explored as pH controlled drug carriers. Narain et al.19 fabricated core-shell nanogels with acid degradable cross-linkers via a one-pot radical polymerization.



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Three proteins were encapsulated into the nanogel and pH responsive release profiles were found during 48 hours. Thayumanavan et al.20 designed a series of acetal and ketal cross linkers with different pH sensitivity. These moieties were incorporated within nanogels, to which the pH-induced variations in encapsulation of guest molecules were evaluated. Beyond the stimuli-responsiveness, biocompatibility and biodegradability are critical for nanogels as drug carriers. There is a risk of body accumulation for the most nanogels made from synthetic polymers, even though they are able to degrade into small polymer fragments so as to accelerate the release of the cargo and eventually to be eliminated easily by

renal .21

Therefore, biodegradable polymers, especially naturally occurred ones, are mostly prefered.22,23 Akiyoshi et al.24 prepared a pH sensitive nanogel, self-assembled from an pullulan derivative, on which acid-cleavable cholesterols were grafted to

to the

pullulan backbone by click reaction. The pullulan nanogels underwent degradation to facilitate drug release under acidic conditions, while kept stable at normal physiological pH. Haag et al.25 fabricated a dual-responsive nanogel with hyper branched polyglycerol, which has redox sensitivity induced by the disulfide containing cross-linker and pH-responsiveness sponsored by an acid-labile hydrazone linker between polyglycerol matrix and DOX via. Hyaluronic acid (HA), a naturally occurred polysaccharide, has found extensive applications in advanced drug delivery systems,

for its unique multifunctional

groups, biodegradability and biocompatibility.26 Besides, HA-based drug carriers also shows unique targeting effect to CD44+ tumor cells.27 Jiang et al.28 prepared


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enzyme-sensitive nanogels with hyaluronic acid for DOX delivery. It was found that these nanogles were able to load a high content of drug (DLC 16%) and penetrate tumor more efficiently . A promoted antitumor efficacy was found in a H22 mice model. While, pH sensitive HA-based nanogel with acid-labile linkers has not been not reported for tumor targeted drug delivery. In this work, a HA-based nanogel with ketal cross linkers was prepared by a one-pot, surfactant-free method. Such well-defined pH-sensitive, biodegradable nanogels are employed to deliver DOX and exhibit a pH-dependent drug release behavior. Subsequently, cytotoxicity assays and cellular uptake were investigated in vitro. The in vivo antitumor efficacy about the nanogels was examined on H22 tumor xenografts mice model. EXPERIMENTAL SECTION Materials. Sodium hyaluronic acid (Mw = 6 kDa) was purchased from Freda Biochem Co. Ltd (Jinan China). 2, 2-dimethoxypropane (DMOP), 2-hydroxyethyl methacrylate

(HEMA),

methacrylic

anhydride

(MA)

and

ethylene

glycol

dimethacrylate (EGDMA) were purchased from J&K Scientific Co. Ltd (Beijing China). Toluene-4-sulfonic acid (ρTSA) is the product of Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Potassium persulfate (KPS) is the product of Kemiou Chemical Reagent Co. Ltd (Tianjin, China) and recrystallized before use. Doxorubicin (DOX) hydrochloride was bought from Dalian Meilun Biology Technology

Co.

Ltd

(Dalian,

China).

3-(4,

5-dimethyl-2-thiazolyl)-2,

5-diphenyl-2-H-tetrazolium bromide (MTT) and 4', 6-diamidino-2-phenylindole



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(DAPI) were purchased from Sigma (Shanghai, China). All organic solvents were analytical reagents and used as received. The human liver cancer cell line HepG2 and human breast cancer cell line were purchased from Shanghai cell bank of Chinese Academy of Sciences (Shanghai, China). Female Swiss mice were purchased from Medical Experimental Animal Center, Henan Province (Zhengzhou, China). All animal experiments were performed in accordance with the principles of care and use of laboratory animals and were approved by the experimental animal administrative committee of Henan University. Characterization. An AVATAR360 (Nicolet, USA) spectrometer was used to collect Fourier transformed-infrared spectra (FTIR. 1H NMR spectra were recorded on an AVANCE 400 spectrometer (Brucker, Switzerland) operating at 400M Hz. The dynamic light scattering (DLS) size, polydispersity index and Zeta potential of the nanogels were measured at 25 °C on a Zetasizer Nano-ZS90 (Malvern Instruments, UK). The morphology of nanogels were investigated on a Dimension Icon atomic force microscopy (AFM) (Brucker, Switzerland) . Briefly, a pre-cleaned glass wafer (size: 1cm × 1cm) was spin-coated with nanogel dispersion, form which images were collected under automatic mode. Fluorescence measurement was performed using an F-4600(Hitachi, Japan) spectrometer. The images of cellular uptake were obtained on a confocal laser scanning microscope (CLSM710, Zeiss, Germany). Preparation of Ketal Containing Hyaluronic Acid Nanogels. Synthesis of Cross-linker: 2, 2-dimethacroyloxy-1-ethoxypropane (DMAEP). The cross-linker with ketal was prepared following a procedure reported previously .19,29 Typically, 2.0 g


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(19.2 mmol) 2,2-dimethoxypropane(DMOP) was added into a 10 mL anhydrous round-bottom flask, 5.0 g(38.4 mmol) 2-hydroxyethyl methacrylate(HEMA) , 33.0 mg toluene-4-sulfonic acid(ρTSA) and 15 mL methylbenzene were added, kept under a nitrogen atmosphere for 30 min, then the solution was stirred at room temperature overnight. Then, it was rotary evaporated to remove the solvent of methylbenzene. The crude product was purified by silica column chromatography using a mobile phase of hexane: ethyl acetate: TEA (85:14:1, v/v). 1HNMR (400MHz, CDCl3, ppm): δ1.28 (6H, C (CH3)2); δ1.94 (6H, (OCOCCH2CH3)2); δ3.71 and 4.27(8H, (OCH2CH2O) 2); δ5.568 (2H, C=CH2, syn to methyl); δ6.110 (2H, C=CH2, anti to methyl). Synthesis of methacrylated hyaluronic acid (MAHA). MAHA was synthesized according a previously reported method.28, 30, 31 Typically, 0.5 g HA (Mw = 6 KDa) was dissolved in 50 mL of water, then preplanned ratios of methacrylic anhydride (MAA) were added. Then, the

reaction solution was adjusted to pH 8.0 using 5 M

NaOH solution. The reaction was conducted at 4°C by stirring for 24 h. Then, the solution was added dropwise into ethanol to precipitate the product . The precipitate was re-dissolved and dialyzed in deionized water for 72 hours . Finally, a white powder was got by

freeze-drying. The ratio between HA and MAA determines the

degree of substitution (DS) of methacrylate groups, which is confirmed by 1H NMR. 1

HNMR (400MHz, D2O, ppm): δ1.79 (3H, (OCOCCH2CH3)), δ6.02 and 5.59(2H,

(OCOCCH2CH3)). Synthesis of pH sensitive nanogels (pH-NG).The pH sensitive nanogels were



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prepared by radical polymerization. In brief, 50 mg MAHA, 28 mg

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pH-CL and 60

mg NaHCO3 were placed into a three-neck flask which contains 20 mL deionized water and 3 mL isopropyl alcohol. The resulted solution was stirred and refluxed at 70°C under a nitrogen atmosphere.

A half hour later, 2 mL (25 mg/mL) KPS was

added into the flask to initiate polymerization. Four hours later the solution was transferred into a dialysis bag (cut-off-molecular-weight of 8000-14 000), which was placed dialyzed in phosphate buffer saline (pH 7.4) for 48 h to remove the unreacted reagents. The dialysis medium was refreshed every six hours. Three kinds of pH-NG were prepared from different systems containing different ratios of MAHA and DMAEP, e.g. pH-NG1 (nMAHA : n

DMAEP

= 2:1), pH-NG2 (nMAHA : n

DMAEP

=2:2),

pH-NG3 (nMAHA : nDMAEP = 2:3).( DS of MAHA is 16%) As a comparison, the pH insensitive nanogels (NG) were also prepared using EGDMA as cross-linker. pH-triggered Size-Change of Nanogels. pH-triggered change in size of nanogels was monitored by DLS measurement. Briefly, 1 mL freshly prepared nanogels (1 mg/mL) dispersions were added to phosphate buffers( 0.01 M) with different pH values of 5.0, 6.5 and 7.4, respectively After incubation at 37°C for 24 h , the size distribution curve was measured before and after the incubation. Loading and Release of DOX in Nanogels. Doxorubicin (DOX) was loaded in nanogels by a simple incubation method. Firstly, 10 mg DOX·HCl was dissolved in 10 mL deionized water , which in turn was adjusted into a solution of

pH 8.5

using 0.1 M NaOH. Then, the DOX containing solution was added by drop wise into nanogels dispersion. After 24h stirring at 25°C, the mixture was centrifuged at 11000


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r/min for 0.5 h and washed twice using PBS (pH 7.4) . Finally, the supernatant was lyophilized after passing through a 0.22µm syringe filter. Drug loading content (DLC) and drug loading efficiency (DLE) were determined by dissolving the freeze-dried nanogels into a solution mixed with water and DMSO (1:49, V/V). The amount of the loaded DOX was quantified by fluorescence spectrophotometry (excitation wavelength at 481 nm and emission wavelength at 558 nm). The value of DLC and DLE were calculated following the formula below:

DLC (wt %) =

DLE(wt %) =

Weight of loaded drug × 100% Weight of the nanogels

Weight of loaded drug ×100% Weight of the feeding drug

DOX released from the DOX-loaded nanogels was studied in three buffer solutions of different pH: PBS of pH 7.4, 6.5 (0.01M) and acetate buffered solution of pH 5.0 (0.01M). 3 mL of DOX-loaded nanogels solution was placed in a dialysis membrane bag (MWCO 8000-14000 Da) in 40 mL buffer solution (pH 7.4, pH6.5 or pH5.0) at 37°C. At predetermined time intervals, 3 mL of the medium outside the dialysis bag was taken out to determine the amount of released DOX by fluorescence measurement, meanwhile, 3mL fresh buffer solution was replenished. Cytotoxicity Assay and Cellular Uptake. Standard MTT method was utilized to evaluated toxicity of nanogels toward HepG2 and MCF-7 cells. Cells were seeded in a 96-well plate at a concentration of 1×104 cells/well, and cultured for 24 h (37°C, 5% CO2). The cells were treated with blank nanogels, drug-loaded nanogels and free drug at designed concentrations. After 48h, the supernatant was wiped off and 20 µL MTT



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solution (5 mg/mL) per well was added and cultured for another 4 h. Then MTT solution was removed, 100 µL DMSO was added and to dissolve formazan thoroughly by shaking for 10min. Absorbance at 570 nm measured by a microplate reader (EL800, BIO-TEK Instruments Inc., USA). Cellular uptake and intracellular release of the drug were investigated on a confocal laser scanning microscopy (CLSM 710, Zeiss, Germany). HepG2 cells were counted (1×105 cells/well) and then incubated in a six-well plate for 24h (37°C, 5% CO2). DOX-loaded nanogels or free DOX (15 µg/mL) were added and incubated for another 3h, 9h or 12h, the cells were washed three times with PBS. Thereafter, the cells were fixed with 4% (W/V) paraformaldehyde for 10 min and stained the cell nuclei with DAPI (4', 6-diamidino-2-phenylindole). DAPI (blue) and DOX (red) fluorescence were visualized using the CLSM. In vivo Antitumor Efficacy. In vivo antitumor efficacy was evaluated using the H22 tumor bearing Swiss female mice (18-22 g). H22 cells (1×106 in 200 µL PBS for each mouse) were injected subcutaneously in the right armpit. The tumor volume was allowed to grow to about 70mm3 before the first treatment, and then the tumor-bearing mice were randomly divided into four groups (12 for each group) and tail vein injection was applied for the administration of saline and different drugs (5.0 mg/kg DOX eq.) via. All the groups were injected every two days and lasted for 15 days. The day of the first treatment was coded as Day 1, body weight were recorded regularly on an electronic scale. Tumor size was measured using Vernier caliper and tumor volumes were calculated. Histological Analysis. The mice were sacrificed on


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the last day of treatment and the heart, liver and tumor tissue were excised and fixed in 4% paraformaldehyde buffer. Then the tissues were dehydrated, embedded in paraffin and sliced into 5.0 µm thickness (LEICA RM2235). The slices were photographed under a microscope (OLYMPUS BX43) for observing the histopathological changes after staining with hematoxylin and eosin (H&E). The apoptotic cells of tumor were identified using a terminal transferase dUTP nick-end labeling (TUNEL) assay kit (Roche, Mannheim, Germany). The sections of tumor tissues were observed and photographed under an inverted fluorescence microscope (NIKON, Japan). The fluorescence spots of blue and green refer to cell nuclei dyed by DAPI and the apoptotic cell nuclei dyed by FITC, respectively. Statistical Analysis. Data were presented as mean ± SD. Origin 9.1 and GraphPad Prism 5.0 Software was used for statistical analysis via Student's t-test and one-way ANOVA analysis. Significant differences were considered at P < 0.05 statistically.



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RESULTS AND DISCUSSION

Figure 1. Synthesis of pH-nanogels (pH-NG).

Preparation of Ketal Containing Hyaluronic Acid Nanogels. pH-sensitive nanogels are synthesized by a facile surfactant-free, radical polymerization technique, with methacrylated hyaluronic acid (MAHA) and ketal containing cross linker DMAEP. These nanogels are designed degradable under slightly acidic conditions of tumor, and the degradation rate is determined by both pH and density of cross-linking. As shown in Figure 1, the pH-responsive ketal containing cross-linker is synthesized from two molecules of HEMA and one 2, 2-dimethoxypropane. Then, MAHA is synthesized by reaction between methacrylic anhydride and HA. The successful synthesis of MAHA and DMAEP was verified by 1

H NMR characterization (Figure S1).19, 28 In addition, degree of substitution (DS) of


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MAHA varied with the reactions mole ratio between HA (6k Da) and MA (1:6, 1:4, 1:2, 1:1). According to 1H NMR results, the DS of 30%, 16%, 9% and 0% corresponds to the mole ratios of 1:6, 1:4, 1:2 and 1:1, respectively. It has been reported that the targeting ability of HA to CD44 receptors disappeared when the DS ratio was more than above 25%.27, 32 Thus, MAHA with DS of 16% was selected for the synthesis of nanogels. In the reaction solution, NaHCO3 was added to maintain a basic atmosphere so as to minimize the cleavage of ketal bond on DMAEP throughout the whole process of polymerization, 33 and a small amount of isopropyl alcohol was added to make DMAEP dissolve sufficiently. Three kinds of nanogels with different cross linking density were obtained by varying mole ratio between MAHA and DMAEP, e.g. pH-NG1 (nMAHA : n

DMAEP

= 2:1), pH-NG2 (nMAHA : n

DMAEP

=2:2),

pH-NG3 (nMAHA : nDMAEP = 2: 3). FT-IR spectrum were also collected to confirm the synthesis MAHA, DMAEP and nanogels (Figure S2). It was also found that the HA nanogels have a diameter ranging from 72.52 nm to 154.0 nm (Fig. 2)and zeta-potential around −30.0 mV in aqueous solution. The more mole of DMAEP, the bigger in

size. The small size below 200 nm and negative zeta potentials of nanogels

are beneficial to avoid phagocytosing by the immune system and clearing by renal excretion and resist the adsorption of negatively charged protein.34-37



20 pH-NG1 pH-NG2 pH-NG3

15 Intensity(%)

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10

5

0 1

10

100

1000

10000

Size(nm)

Figure 2. The size of pH-NG with different molar ratio of MAHA and DMAEP.

Figure 3. AFM images of pH-NG3: height image (left) and phase image (right).

In addition, the strongly negative surface charges enables individual dispersion of nanogels by repulsive interaction.38 The morphology of the nanogels was characterized by AFM. As shown in Figure 3, the spherical morphology


and

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nanoscale size of around 70 nm were observed for pH-NG3. While the size detected by AFM is smaller than that of DLS result, due to the dehydration induced shrinkage of nanogels for AFM measurements.39 The incorporation of ketal group into the pH nanogels will endow them with pH sensitivity. The size changes of nanogels with or without ketal bonds in response to acidic stimuli were monitored by DLS measurements. However, there was no changes in size of both nanogels under acidic conditions (Figure S3). Meanwhile, the intensity of the DLS decreased upon the degradation in acidic solution. Similar phenomenon was found by Jiang et al.28 for the enzymes degradation of enzyme-sensitive nanogels, of which size kept almost unchanged with time. That was attributed to one by one fashion in the enzymatic degradation process of nanogels. In our case the pH sensitive nanogels were formed only when there is cross linker DMAEP. So, the cleavage of ketal bonds could destroy the framework of nanogel one by one. The decrease of cross linked network and destroy of nanogel will accelerate the release of encapsulated cargo. Table 1. Particle size, particle size distribution index(PDI), drug loading content(DLC) and drug loading efficiency(DLE) of DOX-loaded pH-NG and NG. pH-NG1-DOX

pH-NG2-DOX

pH-NG3-DOX

NG-DOX

Size (nm)

87.65

126.5

170.8

88.65

PDI

0.072

0.184

0.062

0.124

DLC (%)

7.67

8.52

12.15

10.23

DLE (%)

42.21

51.46

76.73

63.49



Drug Loading and Release from Nanogels. As shown in Table 1, the loading of DOX into nanogel caused an increase in size of each nanogel, which might be the encapsulation of hydrophobic DOX into the network of

nanogels. DLC and

DLE increased along with the increase in cross linking density of nanogels. DOX was loaded into the nanogels with DLC ranging from 7.67% to 12.15%, and DLE ranging from 42.21% to 76.73%. The results also showed that nanogels have superior drug loading capacity over the other nano-particulate drug delivery systems, such as liposomes,40 micelles,41 polymericnanoparticles,42 and dendrimers.43 It should be the specific porous network inside nanogels that provide them with the ability of higher drug loading.44

a

100 pH=7.4 pH=6.5 pH=5.0

80 Cumulative release(%)

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60

40

20

0 0

10

20

30

40

50

60

Time(h)


70

80

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b 100 pH=7.4 pH=6.5 pH=5.0

80 Cumulative release(%)

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60

40

20

0 0

10

20

30

40

50

60

70

80

Time(h)

Figure 4. In vivo release profiles of DOX from pH-NG3-DOX (a) and NG-DOX (b).

The ketal bonds inside the nanogels are very sensitive to acid and the cleavage of them will result in an accelerated drug release. Compared to normal physiological pH of 7.2-7.4, tumor extracellular microenvironment (pH 6.5-7.0) and tumor intracellular lysosome and endosomes (pH 4.5-6.5) are more acidic.2,45-47 The pH-dependent drug release behaviors of the ketal containing nanogels was studied in a simulated pH conditions (5.0, 6.5, 7.4). Even though different in DLC, similar pH-dependent profiles of drug release were found for all pH-sensitive nanogels (Figure S4 and S5,). The release curve of DOX-loaded pH-NG3 are shown in Figure 4a. At pH 7.4, less than 30% of DOX were released for 72h, which signified that pH-NG3-DOX is relatively stable under physiological condition. While significantly accelerated release



of DOX from pH-NG3-DOX was found at pH 6.5 and 5.0, with ca. 50% and ca. 83% in 72 hours, respectively. As a control, pH-insensitive nanogels NG-DOX were also studied under the same conditions (Figure 4b). At the physiological condition, similar amount of DOX, ca. 25% was released in 72 hours. In acidic environment of pH 6.5 and pH 5.0, the drug release from NG-DOX was obviously slowed down, only 35% DOX at pH 6.5 and ca. 53% DOX at pH 5.0 in 72 hours. A slight pH-dependent release of NG-DOX was also shown and should be originated from the pH-sensitivity of DOX itself.48 Compared to the pH insensitive counterparts, the pH-triggered drug release of pH-NG-DOX should be to the result of the faster degradation of the pH sensitive cross linkers. The ketal linkage inside pH-NG can break easily at acidic conditions and result faster drug release.33, 49

a

120

pH-NG NG

100

Cell viability(%)

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80 60 40 20 0 1

5

10 25 50 Concentration(mg/mL)


100

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b

120

pH-NG NG

Cell viability(%)

100 80 60 40 20 0 1

5

10 50 25 Concentration(ug/mL)

100

Figure 5. Cell viability of MCF-7(a), HepG2 (b) cell lines after incubation with empty pH-NG and NG for 48h (n=3).

a

100 pH-NG-DOX NG-DOX Free DOX

80 Cell viability(%)

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60

40

20

0 0.1

10.0 5.0 1.0 Concentration(ug/mL)


15.0


b

100

pH-NG-DOX NG-DOX Free DOX

80 Cell viability(%)

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60

40

20

0 0.1

1.0 10.0 5.0 Concentration(ug/mL)

15.0

Figure 6. In vitro cytotoxicity of DOX-loaded pH-NG, DOX-loaded NG and Free DOX against MCF-7(a), HepG2 (b) cell lines after 48 h incubation (n=3).

Cytotoxicity and Cellular Uptake. Biocompatibility of the blank nanogels was assessed on MCF-7, HepG2 cells lines by MTT assay (Figure 5). After incubated with pH-NG and NG for 48h, above 90% cell viability was found when the concentration of blank nanogels increased to 100 µg/mL. It was revealed that both pH-NG and NG were safety and favorable biocompatibility as a drug delivery vehicle. To evaluate the in vitro toxicity of pH-NG-DOX，NG-DOX and free DOX, MTT method was used to MCF-7 and HepG2 cells(Figure 6). Cell viabilities were decreased

when

the concentration of drugs increased, which showed the dose

dependent behaviors of cell proliferation inhibition. The viability of cell was the lowest in free DOX group compared with DOX-loaded nanogels. This could be


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explained by that DOX as small molecule drug could got into cell nuclei by passive diffusion, while DOX-loaded nanogels get into cells via endocytosis and released DOX to arrive the cell nuclei.50 It is noteworthy that cell viabilities of pH-NG-DOX under various drug concentrations are lower than that of NG-DOX in both HepG2 and MCF-7 cell lines, which indicates that pH-NG-DOX has a higher toxicity to MCF-7 cells than NG-DOX. Indicated by the results of in vitro drug release, the superior tumor-killing capacity of pH-NG-DOX might come from the accelerated drug release in acidic atmosphere after being internalized by cancer cells.

Figure 7. CLSM images of HepG2 cells treated with DOX-loaded pH-NG, DOX-loaded NG and free DOX(C) for 3 h, 6 h and 12 h. DOX concentration is 15mg/L for any formulation.



Cellular uptake of free DOX and DOX-loaded nanogels were investigated by CLSM after incubation for 3h, 6h and 12h. For pH-NG-DOX, the fluorescence intensity of DOX increase slowly with increased incubation time (Figure 7). While, the accumulation of pH-NG-DOX is slower than free DOX that because free DOX can pass cell membrane quicker by passive diffusion51 whereas nanogels are internalized into cells via slower endocytosis52. However, cells treated with pH-NG-DOX shows stronger DOX fluorescence than NG-DOX for the same incubation time, due to the accelerated release of DOX from pH-sensitive nanogels when encountering acidic environment. The faster intracellular release of DOX by pH-NG explains the higher inhibition efficacy of tumor cell than inert NG.

a Tumor volume(mm3)

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10000

Saline NG-DOX pH-NG-DOX Free DOX

8000 6000 4000

***

2000

* 0 1

3

5

7

9

11

13

Time(d)


15

**

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b

50

Saline NG-DOX pH-NG-DOX Free DOX

40

Weight(g)

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30

**

20 10 0 1

3

5

7

9

11

13

15

Time(d) Figure 8. In vivo antitumor efficacy of the DOX loaded nanogels (n=12). H22 tumor growth curves (a) and body weight variation (b) of mice after intravenous injection of different formulations of DOX. (*p

Hyaluronic-Acid-Based pH-Sensitive Nanogels for Tumor-Targeted

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