An Enzyme-Responsive Nanogel Carrier Based on PAMAM

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An Enzyme-Responsive Nanogel Carrier Based on PAMAM Dendrimers for Drug Delivery Yao Wang, Yiyang Luo, Qiang Zhao, Zhijian Wang, Zejun Xu, and Xinru Jia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05567 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 17, 2016

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ACS Applied Materials & Interfaces

An Enzyme-Responsive Nanogel Carrier Based on PAMAM Dendrimers for Drug Delivery Yao Wang,†# Yiyang Luo,†# Qiang Zhao,‡ Zhijian Wang,† Zejun Xu,† and Xinru Jia*† †Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Polymer Chemistry and Physics of the Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China, ‡Beijing National Laboratory for Molecular Sciences (BNLMS), State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China. KEYWORDS: PAMAM dendrimer, Enzyme, Nanogel, Crosslink, Drug delivery

ABSTRACT

G4 PAMAM dendrimer molecules were modified via covalently conjugating RGDC, RAADyC and PEG chains on the periphery (Mac-1), by which a nanogel drug carrier with enzymesensitivity (NG-1) was constructed through an oxidation reaction by using NaIO4 to initiate the chemical crosslink of the functional groups on the periphery of dendrimers. Mac-1 and NG-1 both had the sphere-like shape with relatively uniform size of 20 nm for Mac-1 and 50 nm for NG-1 evidenced by TEM, SEM images and DLS measurements. NG-1 showed much higher drug loading capacity as compared with Mac-1 although the cavities in the dendritic structure were used

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to encapsulate drug molecules as reported in many literatures. In addition, the size of NG-1 embedded DOX reduced significantly to 15 nm in the presence of elastase, which indicated the decomposition of the nanogel triggered by enzyme, such leading to the drug release with a sustained manner in vitro. The NG-1 carrier was non-cytotoxic and well biocompatible and it achieved the same cytotoxicity as free DOX when the drug molecules loaded inside. From confocal images, the penetrative process of DOX from nanogel could be clearly observed in 8 h. Such a dendrimer-based nanogel may be a potential nanocarrier for drug delivery in cancer therapy.

INTRODUCTION Nanogels1,2 with three-dimensional hydrophilic networks at nanoscale have been reported as the promising drug carrier with the ability for crossing the cellular barriers and the superior properties for transferring drugs3-5, proteins6,7, DNA8,9, and imaging agents10,11 etc. into cells. Compared with the conventional drug carriers, such as liposomes12, micelles13-15, nanoparticles16-18, and nanotubes19, nanogels as drug carrier have the advantages20-22 of higher stability, larger loading capacity, more efficient cellular uptake rate, increased bioavailability, and safety for drugs in vivo. Hence, this kind of nanosized materials has been rapidly developed. Poly (amido amine) (PAMAM) dendrimers23-25 with highly specific and hierarchical threedimensional architecture have been widely studied in the biological field in recent years due to their analogical characteristics of globular biological macromolecules, including histone, hemoglobin and cytochrome C, etc.26,27. Several examples have proved that the dendrimer-based hydrogels hold the potentials for drug encapsulation and release28-30.The dendritic architecture can be a scaffold as well as an anchor to conjugate multiple ligands with different properties. In


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addition, the optimum molecular level nanostructure and the easily accessible reactivity afford them with unique properties to meet the requirements in biological systems31,32. As well known, the enzyme-responsive peptide materials, small molecule assemblies, nanoparticles, nanocarriers and hydrogels are extremely attractive because of enzymes’ biocompatibility, selectivity and recognizability33-35. Especially, the enzyme-catalyzed processes exhibit superior potential biological applications in vivo36. Elastase is a serine protease with a broad specificity for its ability to cleave the proteins at the carboxyl side of small hydrophobic amino acids such as Ile, Gly, Ala, Ser, Val, and Leu. The study on elastase-sensitive carriers for biological applications has been extensively explored. For example, Jeong et al. constructed a model protein drug delivery system by synthesizing poly(alanine-co-leucine)-poloxamer-poly(alanine-coleucine), in which FITC-labeled bovine serum albumin (FITC-BSA) was encapsulated37. Their results showed that the polymer chains were cleaved by elastase, thus the FITC-BSA was release over one month in vitro. Anseth et al. prepared the poly(ethylene glycol) hydrogels by thiolene photo-initiated polymerization for enzyme-responsive protein delivery, in which elastase was used to degrade the hydrogel in situ38. Ulijn et al. investigated a series of peptide-based hydrogels and summarized different responsive activity of enzymes39-41. An important point in their study was the special high selectivity and sensitivity of elastase to peptide DAAR which applied in our experiment. The cleavage ratio of elastase to peptide DAAR reached more than 99%, while for other enzymes such as chymotrypsin, the cleavage ratio is only 7.7%. There are many reports on nanogel carriers, dendrimer-based delivery systems and enzymeresponsive vehicles. However, the development of efficient drug carriers with desired biocompatibility, circulation time and stability to achieve satisfied clinical outcome remains enormous challenge. Considering the merits of nanogels, dendrimers and enzymes, we aim to


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prepare a poly (amido amine) (PAMAM)-based nanogel with enzyme sensitivity, by which to construct a nanocarrier with better biocompatibility, higher loading ability and controllable drug release property. Up to date, only several reports addressed the dendrimer-based nanogels as carriers. For example, Nyström and co-workers designed and synthesized the thermo-responsive hydrogels based on PAMAM dendrimer/poly (N-isopropylacrylamide) and studied their structure, swelling properties and drug release behavior 42. They found that the incorporation of dendrimers resulted in a more homogeneous, hydrophilic and expanded nanogel with increased drug uptake and higher drug release ratio. Li and co-workers developed a simple approach to prepare double crosslinked dendrimer/alginate nanogels (AG/G5) by using G5 dendrimer as a co-crosslinker44. They found that G5 dendrimer played a crucial role for the formation of compact nanogels with smaller size and more stable structure under different pH 7.4 and 5.5. Notably, the AG/G5 nanogels showed three times higher DOX loading capacity as compared with AG nanogels, and a sustained way for releasing DOX in vitro. Herein, we report the synthesis of PAMAM dendrimer-based macromonomer (named Mac-1) with the bio-adhesive components (Fmoc-arg-gly-asp-cys-SH) (for short as RGDC) and enzyme responsive components (Ac-arg-ala-ala-asp-D-tyr-cys-NH2) (for short as RAADyC) on the periphery of G4 PAMAM dendrimers. The reason we choose elastase as the responsive component is due to the excess neutrophil elastase (NE), one of the elastases, has been found in tumor tissues43, expect it special high selectivity and sensitivity to the peptide DAAR. In the clinical reports, increased amount of NE has been detected in different types of cancers, and the concentration of NE is associated with the cancer stage, grade, and the survival. For example, Yamashita et al. tested free and α1-antitrypsin-combined forms of NE in 144 non-small-cell lung cancers. NE was detected in 115 of 144 tumors’ extracts with the concentration ranging from 0.21 to 23.35 mg/100


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g protein. The nanogel (named NG-1) was fabricated by the chemical crosslinking of Mac-1 macromonomers in the presence of NaIO4. It was found that the obtained nanocarrier showed the increased drug loading capacity, sustained drug (e.g. DOX) release triggered by enzyme, and the cytotoxicity to C6 cells. To the best of our knowledge, a nanocarrier with the superiorities of nanogel, dendrimer and enzyme sensitivity has rarely been reported.

Experiment section Materials Methoxy PEG succinimidyl carbonate ester (mPEG-NHS, Mw = 2000) was purchased from Biomatrik Inc. (Jiaxing, China). Maleimide-PEG-carbonate-NHS (MAL-PEG-NHS, Mw = 5000) was purchased from NOF Corporation (Japan). H-arg-gly-asp-D-tyr-cys-SH (RGDyC), Ac-argala-ala-asp-D-tyr-cys-NH2 (RAADyC), Fmoc-arg-gly-asp-cys-SH (Fmoc-RGDC) were purchased from CL Biochem (Shanghai, China). Elastase from porcine pancreas and sulforhodamine B (SRB) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). DOX was purchased from Duodian chemical cooperation (Nanjing, China). PBS (Phosphate Buffer Saline) was purchased from Hyclone Company and Paraformaldehyde was purchased from Alfa Aesar Company. Other reagents were purchased from Beijing Chemical Reagents (Beijing, China). All the reagents were used as received and the solvents were purified according to the general procedures before used. 1

H NMR data was collected on Bruker 400 MHz spectrometers operated at room temperature.

Chemical shifts (δ) are reported in ppm with (CH3)4Si and the residual solvent peak as the reference, respectively. UV-Vis spectra were recorded on the Perkin-Elmer Instruments Lambda spectrometer. Dynamic light

scattering (DLS) experiments were conducted on an


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ALV/DLS/SLS5022F light scattering apparatus, equipped with a 22 mW He - Ne laser (632.8 nm wavelength). The samples were filtered with 450 nm filters. The scattering angle was fixed at 90°. Transmission Electron Micrograph (TEM) was observed by a Jeol JEM 100CX, 80kV and JEM2100, 200kV. Scanning Electron Microscope (SEM) measurement was taken on a scanning electron microscope (Hitachi S4800, 5 kV). Laser Scanning Confocal images were performed on the Nikon A1R-si microscope.

Synthesis Synthesis of G4.0-RGDC-RAADyC (Mac-1) and G4.0-RGDyC (Mac-2) The reaction was done as described before45. Taking Mac-1 as an example, specifically, PAMAM dendrimer (G4, 30.00 mg, 2.11 μmol) was stirred with MAL-PEG-NHS (211.04 mg, 42.21 μmol) for 30 min, then mPEG-NHS (168.84 mg, 84.41 μmol) was added and kept for another 60 min. Finally, Fmoc-RGDC (16.45 mg, 21.11 μmol) was added dropwise into the solvent, and the mixture was stirred for 2 h. Finally, RAADyC (15.59 mg, 21.11 μmol) was added and kept for another 2 h. The whole process was taken place in the solvent of DMSO:H 2O = 1:1. The pure product was collected through freeze-drying after dialysis again water at a MWCO = 8000 dialysis bag.

Methods Preparation of the Nanogels Nanogels with the components of Mac-1 or Mac-2 were prepared by a similar method. Take NG-1 as an example, 7.5 mg Mac-1was added into 50 μL deionized water in an EP tube. Then, 50 μL NaIO4 (1 mg/100 μL) dissolved in deionized water was added into the tube. The solution was


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kept for 48 h and then NG-1 was acquired. For the solid sample, the solvent of NG-1 was transferred into a dialysis bag (MWCO = 2000) for the purification. Finally, NG-1 was obtained by the dry-freezing method. The deionized water was sterilized by filtering through a 0.2 µm nylon syringe filter before used. For the sake of convenience, we named 7.5% w/v crosslinked Mac-1 as NG-1 and 7.5% w/v crosslinked Mac-2 as NG-2. Preparation of the DOX Loaded Nanogels Doxorubicin hydrochloride (DOX) was loaded using an equilibrium dialysis method. Take NG1-DOX as an example, 5 mg DOX hydrochloride was mixed with 15 mg of Mac-1 in 100 µL deionized water and stirred for 30 min in dark. The soution was kept in dark for 48 h before 100 µL NaIO4 (1 mg/100 µL) was added and the solution was kept for another 24 h. The solution was purified by dialyzing in a MWCO = 2000 dialysis bag against water at least 72 h to remove free DOX. The encapsulation percentage of DOX was determined by UV-Vis scanning spectrophotometer at 480 nm in water. The calibration curve was acquired with different DOX concentrations. Drug loading content was calculated according to the following equations: Drug loading content = (weight of loaded drug/weight of nanogel) × 100% Scanning Electron Morphology (SEM) The samples were dropped on the silicon wafers and then dried prior to SEM imaging45,46. Images were captured on a scanning electron microscope (SEM, Hitachi S4800, 5 kV). Transmission Electron Morphology (TEM)


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Drops of samples were put onto 230 mesh copper grids coated with formvar film. Excess water was removed by filter paper, and the samples were stained and finally allowed to dry in ambient air at room temperature before TEM observation. Dynamic Light Scattering Measurement (DLS) For DLS measurements, the aqueous samples of nanogels were diluted (1 mg/mL) and then were passed through a 0.45 μm syringe filter (Sartorius stedim Biotech, Goettingen, Germany) to remove the dust. DOX Release Drug release was performed in Tris-HCl solution. Take NG-1-DOX as an example, briefly, 4 mg NG-1-DOX was dissolved in 1 mL Tris-HCl buffer solution (pH 8.4). The buffer was selected due to the optimum pH for elastase is between 8.0 - 8.5 according to the product information from Sigma-Aldrich. Then 100 μL (0.1 mg/mL) elastase solution was added. The solution was transferred into a MWCO = 2000 dialysis bag and dialyzed against 80 mL Tris-HCl solution at 37°C. 1 mL dialysis fluid outside the bag was taken every 30 min and the same volume of fresh medium was supplied immediately to keep the same volume. DOX release curve was determined by F-4500 spectrometer at 480 nm in deionized water. Cell Culture C6 glioma cells in this study were cultured in DMEM medium supplemented by 10% heatedinactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin. Cells were maintained at 37oC with 5% CO2. In vitro Cytotoxicity Assay


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C6 cells was seeded into 96-well culture plates at a density of 5×103 cells/well and grown for 24 h. Then Mac-1/2, NG-1/2, free DOX, Mac-1/2-DOX, and NG-1/2-DOX were added into 96well culture plates, respectively. After 48 h, the cell viability was measured by a microplate reader at 540 nm after the SRB (Sulforhodamine B) staining assay28-30 by cell fixation, staining and measurement. For Mac-1 and NG-1 without DOX, the concentration used in the cytotoxicity assay is in the range of 0.01- 1000 μg/mL. After loading DOX, the concentration used in the experiment is the DOX encapsulated in each sample in the range of 0.01 - 200 μg/mL. The following formula was used to calculate the cell survival percentage: Survival% = (A540nm for the treated cells/A540nm for the control cells) ×100%, where the A540nm was the absorbance value. Each assay was repeated for 5 times then dose-effect curves were made. In vitro Cellular Uptake Before the experiment, C6 cells were seeded into achambered coverslips at a density of 5×104 cells/well and incubated for 24 h. After free DOX, Mac-1/2-DOX, and NG-1/2-DOX was added into the cells respectively, the cells were incubated for another 1 h, 4 h and 8 h, respectively. Afterwards, the cells were washed with cold PBS (0.01 M, pH 7.2 - 7.4) for three times, fixed with 4% (v/v) paraformaldehyde and finally stained with Hoechst 33258. The confocal images were then taken by the Nikon A1R-si microscope. Statistics Analysis Data are presented as mean ±standard deviation. One-way analysis of variance (ANOVA) was used to determine the significance among groups following the Bonferroni’s post-test. RESULTS AND DISCUSSION


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Mac-1 was synthesized via several steps as shown in Scheme S1. In order to increase the biocompatibility, dissolution property and to covalently conjugate with RGDC and RAADyC, the exterior of G4 PAMAM dendrimers was partly modified with methoxy-PEG-succinimidyl carbonateester (mPEG2000-NHS) and maleimide-PEG-succinimidylcarbonate ester (MALPEG5000-NHS). The structure of Mac-1 was confirmed and the grafting ratios of the targeting moieties were calculated by 1H NMR measurement (Figure S1). As a result, about 10 RGDC, 10 RAADyC and 40 methyl-PEG (mPEG) were linked on the periphery of per G4 PAMAM dendrimer molecule on average. For a comparison purpose, macromonomer (Mac-2) as a control sample without enzyme-sensitive group in the structure was also synthesized by coupling H-arggly-asp-D-tyr-cys-SH (RGDyC) alone on the exterior of PEGylated G4 PAMAM dendrimers (Figure S2). The nanogel samples NG-1 and NG-2 were prepared by the crosslinking of Mac1and Mac-2 (Scheme 1) with the addition of NaIO4 to the system for inducing the reaction of phenol groups of tyrosine contained in the RAADyC motifs. In such a way, tyrosine was coupled to form 3’3-dityrosine for achieving the crosslinkednetworks47-50. Both of Mac-1 and NG-1 were stable in water, PBS buffer, and at the experimental conditions. No change in appearance and morphologies was observed. They could also be lyophilized and the size remained the same when dissolved in the solvent again.


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Scheme 1. Schematic description of the crosslinked NG-1 networks and the collapsed sketch triggered by enzyme. TEM, DLS and SEM measurements were performed to understand the sizes and the morphologies of the resulting nanogels. Figure 1 (a) and (b) exhibit the TEM images of Mac-1 and NG-1, respectively. It can be seen that both of the dimension size of Mac-1 and NG-1 are uniformly dispersed without obvious derivation. Most of Mac-1 particles are in the size of 20 - 30 nm, while NG-1particles without DOX increase to the size of 50 - 60 nm (Figure 1c, d).


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Figure 1. TEM images of (a) Mac-1 and (b) NG-1; SEM images of (c) NG-1 and (d) NG-2. Scale bar for (a, b): 200 nm, (inset a, b): 50 nm, and (c, d): 1 μm, respectively. We speculate that the particle size increasing is related to the crosslink of the macromonomers Mac-1, which was further confirmed by DLS analysis with the same size change from Mac-1 to NG-1 (Figure S3). The nanogel NG-2 exhibited the similar size with NG-1 evidenced by DLS and TEM measurements (Figure S4). The SEM images of NG-1 and NG-2 (Figure 1c and 1d) clearly show that the nanogel particles are well dispersed in the solvent and the size is similar to the TEM images. This result was consistent with our previous studies on the dendrimer-based hydrogels45,46 composed of G4 PAMAM dendrimers and PLA-PEG-PLA linear polymer or eight-armed PEGDOPA. We found that the second component, such as the linear polymers or armed-PEG-DOPA, was critical in the formation of hydrogels. No hydrogels, but nanogels were obtained without the


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second component in the system due to the possible intramolecular and/or intermolecular crosslink of the dendrimer molecules with the active groups on the exterior.

Figure 2. TEM images of (a) Mac-1 loaded with DOX; and (b) NG-1 loaded with DOX. Scale bar for (a, b): 200 nm, and (inset a, b): 50 nm, respectively. We encapsulated DOX in Mac-1, NG-1 and observed their size change by TEM and DLS for evaluating the drug loading property. From TEM images, we found that the size of Mac-1 with DOX was around 30 nm in average (Figure 2a), and most of the nanogel particles remained at smaller radius with the peak around 10 nm from DLS analysis (Figure S5a). For the average diameter of NG-1 loaded with DOX, a large amount of smaller size particles existed in the system with the diameter around 50 - 100 nm (Figure 2b, S5b) though larger size particles were observed at 400 nm (Figure S5c), which might be ascribed to the unintended aggregation. NG-2 yet exhibited the larger size upon loading DOX (Figure S6). Besides, we monitored UV-Vis spectrum of DOX after being encapsulated in the nanogel and the addition of oxidant, which showed no change for the typical absorption band (Figure S7). These results suggest that the nanogel has been successfully generated in the presence of DOX without variation of the properties. The drug loading capacity of the nanogels was further evaluated quantitively by UV-Vis measurement. Firstly, the calibration curve was acquired by monitoring the absorption of different


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concentrations of DOX at 480 nm. Then we measured the absorption of the nanogels, examined the corresponding drug contents and further calculate the DOX encapsulation rate. The nanogel of NG-1 showed much higher drug loading capacity as compared with Mac-1. The loading amount of NG-1 achieved 6.93 ± 1.55%, which was dramatically enhanced to more than five times than the DOX embedded in Mac-1 (1.48 ±0.59%). The similar loading capacities of Mac-2 and NG-2 were also observed as listed in Table S1. Although the cavity of PAMAM dendrimers and the out layers of PEG could provide space for drugs as reported in many literatures25-30, the nanogels showed stronger capacity of drug encapsulation with more storage space for DOX.

Figure 3. DLS graphs (a, c) and TEM images (b, d) of NG-1 with the addition of elastase (a, b) and NG-2 with the addition of elastase (c, d). Scale bar is (b) 100 nm, (b inset) 10 nm, (d) 1 μm and (d inset) 200 nm, respectively.


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We examined the enzyme responsive behavior of NG-1 by adding elastase into the solution. TEM images (Figure 3a) show that the size of NG-1 reduced dramatically to 15 nm due to the catalysis of enzyme. Similarly, a peak at about 10 nm appears in the DLS profile (Figure 3b), indicating the collapsibility of the networks of NG-1. On the contrary, NG-2 maintained stable even with enzyme in the system (Figure 3c, d). We assumed that the peptide was decomposed triggered by enzyme, which induced the collapsing of nanogel structures, thus leading to the decrease in size. This result was consistent with the report of Ulijn et al., reflecting the high selectivity and cleavage efficiency of enzyme39-41. The whole process of enzymatic cleavage is in 4 h according to the product information by the company. According to the DOX release test in our experiment, we found that the enzymolysis began within 10 min and the release of DOX reached in balanced after 24 h. In vitro drug release behavior of NG-1 loaded with DOX was detected in buffer at 37oC (Figure S8). Compared with the burst release of free DOX (Figure S8b), it showed a well-controlled sustaining manner with the release amount of DOX achieved 40.63 ± 2.45% in the existence of elastase in 32 h, while low release rate was observed without enzyme in the system (Figure S8a). In comparison, NG-2 loaded DOX exhibited low releasing behavior whether there was enzyme in the system or not (Figure S8c), suggesting the inactivity of elastase to NG-2 and the high specificity of NG-1 to elastase.


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Figure 4. The cell viability of the C6 cells (a) after incubated in Mac-1, Mac-2, NG-1 and NG-2 and (b) Mac-1, Mac-2, NG-1 and NG-2 loaded DOX and free DOX for 48 h. the concentration in (a) means the weight for each material in buffer, and in (b) the concentration means the weight of DOX loaded in buffer. Data were presented as the mean ±standard deviation. Excellent biocompatibility is one of the most significant characteristics for a nanocarrier to deliver drugs. C6 glioma cells was chosen as the typical tumor cells to measure the cytotoxicity of free DOX, Mac-1, Mac-2, NG-1 and NG-2 by the sulforhodamine B (SRB) assay. Mac-1, Mac2 and the nanogels were non-toxic when embedded in the C6 cells (Figure 4a), indicating their well biocompatibility attributed to the grafting of PEG and peptides on the surface of G4 PAMAM dendrimers. The cytotoxicity of NG-1 was also measured in the murine brain microvascular endothelial cells (BMVECs). It showed non-toxic when embedded in BMVECs (Figure S9). While the nanogel NG-1 loaded DOX exhibited the similar cytotoxicity to free DOX under the same conditions, demonstrating that NG-1 loaded DOX could efficiently penetrate membrane into C6 cells. In our previous study, we found that RGD is an effective factor for promoting the cell adhesion51. Specifically, we have reported two hydrogel systems in which mouse bone marrow mesenchymal stem cells (mMSCs) were encapsulated45,46. The experiment results showed that the


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cell viability from the hydrogel modified by RGD is much better than the one without RGD, indicating the introduction of RGD is crucial for enhancing the biocompatibility. We further monitored the distribution of DOX in the C6 cells with different formulations in different time’s incubation. As Figure S10 shown, free DOX appeared in the C6 cell nucleus in 1h, indicating a passive diffusion way of free DOX to directly enter the cell nucleus. However, DOX loaded in NG-1 performed time-dependent releasing behavior during 8 h cell uptake (Figure 5). Most of DOX in NG-1 were in the cell plasma but not in the nucleus of C6 cells in 1 h. After 4 h intake, DOX were observed gradually entering the cell nucleus since some DOX were found there. While after 8 h, DOX were almost existed in the nucleus. The whole uptake and release process could be visibly captured by the confocal images. The results of cell intake combining with the drug release in vitro demonstrated that the drug release was controlled in a sustain manner by using NG-1 nanogelas a nanocarrier.


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Figure 5. Confocal images of the C6 cells with NG-1 loaded DOX incubated in DMEM medium. For each panel, images from left to right showed the cells with DOX fluorescence, with nuclear staining by Hoechst 33258, with light and overlays of the images above (scale bar = 50 μm).

CONCLUSIONS In conclusion, an enzyme-stimuli nanogel based on G4 PAMAM dendrimers was constructed as a nanocarrier for drug delivery. The nanogel showed much higher drug loading capacity and the sustained DOX release properties triggered by enzyme as compared to free DOX with a burst releasing manner in vitro. The nanocarriers were non-toxic and well biocompatible, while the NG1 loaded with DOX achieved the same cytotoxicity as free DOX. Such a dendrimer-based nanogel may be as a potential nanocarrier for the drug delivery in cancer therapy. In the future, more enzyme-sensitive systems based on dendrimer for drug release need to be developed and the experiments in vitro and in vivo are necessary for the applications.

ASSOCIATED CONTENT Supporting Information Detailed characterization of monomers Mac-1 and Mac-2, DLS graphs, drug encapsulation rate and drug release curves are in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *Address correspondence to [email protected]. Author Contributions # Y. W. and Y.-Y. L. contributed equally to this work. ACKNOWLEDGMENT This work is financially supported by the National Natural Science Foundation of China (21274004) to X.-R. Jia. REFERENCES 1. Chacko, R. T.; Ventura, J.; Zhuang, J.; Thayumanavan, S., Polymer Nanogels: A Versatile Nanoscopic Drug Delivery Platform. Adv. Drug Deliver. Rev. 2012, 64, 836-851. 2. Sasaki, Y.; Akiyoshi, K., Nanogel Engineering for New Nanobiomaterials: From Chaperoning Engineering to Biomedical Applications. Chem. Rec. 2010, 10, 366-376. 3. Oh, J. K.; Drumright, R.; Siegwart, D. J.; Matyjaszewski, K., The Development of Microgels/Nanogels for Drug Delivery Applications. Prog. Polym. Sci. 2008, 33, 448-477. 4. Du, J.-Z.; Sun, T.-M.; Song, W.-J.; Wu, J.; Wang, J., A Tumor-Acidity-Activated ChargeConversional Nanogel as an Intelligent Vehicle for Promoted Tumoral-Cell Uptake and Drug Delivery. Angew. Chem., Int. Edit. 2010, 49, 3621-3626. 5. Kabanov, A. V.; Vinogradov, S. V., Nanogels as Pharmaceutical Carriers: Finite Networks of Infinite Capabilities. Angew. Chem., Int. Edit. 2009, 48, 5418-5429.


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Table of Contents


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Scheme 1. Schematic description of the crosslinked NG-1 networks and the collapsed sketch triggered by enzyme. 141x64mm (150 x 150 DPI)


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Figure 1. TEM images of (a) Mac-1 and (b) NG-1; SEM images of (c) NG-1 and (d) NG-2. Scale bar for (a and b): 200 nm, (inset a, b): 50 nm, and (c, d): 1 µm, respectively. 151x101mm (220 x 220 DPI)



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Figure 2. TEM image of (a) Mac-1 loaded with DOX, and (b) NG-1 loaded with DOX. Scale bar for (a, b): 200 nm, and (inset a, b): 50 nm, respectively. 232x82mm (150 x 150 DPI)


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Figure 3. DLS graphs (a, c) and TEM images (b, d) of NG-1 with the addition of elastase (a, b) and NG-2 with the addition of elastase (c, d). Scale bar is (b) 100 nm, (b inset) 10 nm, (d) 1 µm and (d inset) 200 nm, respectively. 172x125mm (150 x 150 DPI)



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Figure 4. The cell viability of (a) the nanocarriers as Mac-1, Mac-2, NG-1 and NG-2 and (b) Mac-1, Mac-2, NG-1 and NG-2 loaded DOX and free DOX by the C6 cells after 48 h. For (a), the concentration means the weight for each material in buffer, and for (b) the concentration means the weight of DOX loaded in buffer. Data were presented as the mean ± standard deviation. 261x102mm (150 x 150 DPI)


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Figure 5. Confocal images of the C6 cells with NG-1 loaded DOX incubated in DMEM medium. For each panel, images from left to right showed the cells with DOX fluorescence, with nuclear staining by Hoechst 33258, with light and overlays of the images above (scale bar = 50 µm). 431x271mm (150 x 150 DPI)


An Enzyme-Responsive Nanogel Carrier Based on PAMAM

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