Protective Effects and Mechanisms of G5 PAMAM Dendrimers against

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Protective Effects and Mechanisms of G5 PAMAM Dendrimers against Acute Pancreatitis Induced by Caerulein in Mice Yin Tang,†,‡,§ Yingchun Han,†,‡ Lu Liu,†,‡ Wenwen Shen,†,‡ Huayu Zhang,†,‡ Yunan Wang,†,‡ Xin Cui,†,‡ Yuhui Wang,†,‡ George Liu,†,‡ and Rong Qi*,†,‡ †

Peking University Institute of Cardiovascular Sciences, Peking University Health Science Center, Beijing, China Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China § Department of Hematology, China−Japan Friendship Hospital, Beijing, China ‡

S Supporting Information *

ABSTRACT: In this study, generation 5 (G5) polyamidoamine (PAMAM) dendrimers with two different surface groups, G4.5-COOH and G5-OH, were investigated for their protective effects on pancreas injury in a caerulein-induced acute pancreatitis (AP) mouse model. Both dendrimers significantly decreased pathological changes in the pancreas and reduced the inflammatory infiltration of macrophages in pancreatic tissues. In addition, the expression of pro-inflammatory cytokines was significantly inhibited by the two dendrimers, not only in pancreatic tissues from AP mice but also in vitro in mouse peritoneal macrophages with LPS-induced inflammation. G4.5-COOH, which had better in vivo protective effects for AP than G5-OH, led to a significant reduction in the total number of plasma white blood cells (WBCs) and monocytes in AP mice, and its anti-inflammatory mechanism was related to inhibition of the nuclear translocation of NF-κB in macrophages.

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INTRODUCTION Polyamidoamine (PAMAM) dendrimers constructed with a diamine (commonly ethylenediamine) core and branched surface groups have been widely used as carriers for drugs,1 genes,2,3 and diagnostic agents.4,5 Generation 5 (G5) PAMAM dendrimers are the most promising scaffolds due to their spherical and flexible structure, well-defined molecular weight, fine size (approximately 5 nm), and proper amount of surface groups (128 branches) for the conjugation of therapeutics or diagnostic agents.6 Recently, researchers found that low generation (G ≤ 4) dendrimers have certain bioactivities and functions. Majoral et al. found that phosphorus-containing dendrimers could activate human monocytes.7−9 Tomalia et al. reported that G4-NH2 had significant anti-inflammatory activity in an arthritis rat model,10 and its mechanisms involved the inhibition of cyclooxygenases COX-1 and COX-2 and the decrease of nitric oxide (NO) production. In addition, the neutral G4-OH PAMAM dendrimer had unusual targeting to activated microglia and astrocytes; therefore, it could deliver drugs into rabbit brains to treat cerebral palsy.11 Even anionic G3.5-COOH could transport across cell membranes bearing negative charges and inhibit inflammation in mouse microglia BV-2 cells.12 G5 PAMAM dendrimers have a higher degree of branches, which allow for greater surface modification as compared to lower generation dendrimers. This generation also maintains a less congested surface, retains arm flexibility, and is substantially less cytotoxic than higher generations.13 Besides, the larger interior core of G5 material allows a higher drug-loading © 2014 American Chemical Society

percentage and also promotes the absorption and transepithelial transport of the loaded drugs.14 Although this generation is the most intensively studied PAMAM dendrimer for the delivery of therapeutic and diagnostic agents, studies on bioactivities and mechanisms of G5 PAMAM dendrimers are inadequate. Compared with cationic G5-NH2, anionic G4.5-COOH and neutral G5-OH PAMAM dendrimers are more interesting carriers for in vivo drug delivery, because they are more biocompatible, less cytotoxic and hemolytic, and have less protein interactions.15−17 Therefore, in this study, G4.5-COOH and G5-OH were chosen to explore their anti-inflammatory bioactivities and mechanisms in mice. Acute pancreatitis (AP) is a life-threatening inflammatory disease usually caused by excessive uptake of alcohol, gallstones, or other reasons. The developing process of this disease is rapid and brings a series of complications and multiple organ failures.18 In the process of AP, damaged pancreatic cells in the pancreas release abundant trypsin, lipases, and cytokines, which increase autolysis and impairment in the pancreas. The infiltration of inflammatory cells from the circulatory system to the pancreas can cause more severe edema and inflammation.19 In addition, some inflammatory mediators, such as IL-1β and TNF-α, play important roles in AP manifestation and distant organ dysfunction.20 Received: September 18, 2014 Revised: December 4, 2014 Published: December 5, 2014 174

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Table 1. Characterizations of G5 PAMAM Dendrimersa

Experimental pancreatitis can be induced by alcohol, Larginine, or hormones in animals.21 Caerulein, a hormone, induces AP in rodents by increasing digestive enzyme secretion, which forms cytoplasmic vacuolization and leads to acinar cell necrosis, and causes pancreatic edema.22 In addition, caerulein activates NADPH oxidase, the c-Jun N-terminal kinases (JNK) signaling pathway, and other inflammation inducers to promote pathological changes in the pancreas.23 Macrophages are considered to be crucial cells involved in inflammation and development in the pancreas.24 Migration inhibitory factor (MIF), which is secreted by macrophages, rapidly increases at the beginning of AP and gradually decreases thereafter.25 Pancreatic enzymes, such as trypsin, elastase and lipase, induce TNF-α secretion in peritoneal macrophages in vitro.19 In addition, macrophage depletion by injecting liposome-encapsulated dichloromethylenedi-phosphonate in mice demonstrated protection against virus-induced pancreatitis.26 In this study, to gain deeper insight into the antiinflammatory mechanisms of the G4.5-COOH and G5-OH PAMAM dendrimers in AP mice, macrophage infiltration in the pancreas, inflammatory factor expression in pancreatic tissues and peritoneal macrophages were investigated. In addition, the effects of G4.5-COOH on the total number of inflammatory cells in the peripheral circulation of AP mice and NF-κB nuclear translocation in vitro in peritoneal macrophages were studied.

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theoretical molecular weight generation No. surface groups actual Mn end groups PDI

G4.5-COOH

G5-OH

26252 5 128 25150 111 1.026

28951 5 128 27806 115 1.074

a

Actual Mn (g/mol) was determined by GPC and NMR, and number of end groups was measured via GPC and potentiometrie titration.

Center (Beijing, China). The Laboratory Animal Care Principles (NIH publication no. 85−23, revised 1996) were followed, and experimental our protocol was approved by the Animal Care Committee, Peking University Health Science Center. AP was induced in ICR mice by intraperitoneal (ip) injections of 20 μg/kg body weight (diluting stock solution in saline) caerulein at hourly intervals for a total of seven injections. Mice received peritoneal injections of saline alone in a parallel experiment to serve as normal controls. Protective Effects of the Two Dendrimers on AP. Gabexate is a serine protease inhibitor that is used to treat AP in the clinic.27 In this study, gabexate was used as a drug-positive control with a regimen used in clinic to investigate the in vivo preventive effects of the two dendrimers on AP. Briefly, mice were divided into four groups with five mice per group. Two groups were treated intravenously (i.v.) with G4.5-COOH or G5OH PAMAM dendrimers at a dose of 50 mg/kg q.o.d. for 2 weeks according to a regimen published by literature;28 the third group was treated with saline (NS) with the same regimen used for the two dendrimer-treated groups; the last group, the drug-positive control, was treated intravenously with gabexate (240 mg/kg body weight) with five injections in which the first injection was initiated on the day of AP induction right before the first caerulein injection, and subsequent four injections were complete at 3, 6, 9, and 12 h after the first injection. All mice were housed under a 12 h light/dark cycle with free access to food and water. After a two-week treatment with the two dendrimers, NS, or the first injection of gabexate, all mouse groups were induced to have AP by peritoneal injection with caerulein. After the first injection of caerulein, blood samples from each group were collected at 0, 3, 6, 9, and 12 h for amylase determination and biochemical analysis. The mice were sacrificed and their pancreatic tissues were harvested after 12 h. The obtained tissues were frozen at −80 °C for real-time PCR or fixed in 4% paraformaldehyde in pH 7.4 phosphate-buffered saline (PBS) and embedded in paraffin for pathological analysis. Dose-Dependent Protective Effects on AP. G4.5-COOH was chosen to study its dose-dependent effects on the anti-inflammatory bioactivities of PAMAM dendrimers. Briefly, mice were treated with 50 or 150 mg/kg G4.5-COOH according to the same regimen described above (i.v. q.o.d. for 2 weeks). Afterward, AP was induced in the mice by caerulein (30 μg/kg body weight) according to the same procedure described above. Blood samples and pancreatic tissues of the mice were collected for subsequent studies. Peripheral Hemogram Analysis. To evaluate the effects of dendrimers on the mouse circulatory system, blood samples from G4.5-COOH-treated mice were collected 9 h after AP induction, and a hemogram test was then performed. Parallel experiments were completed in NS pretreated and AP-induced mice (the AP-positive control) and NS pretreated but AP noninduced mice (the normal control). HE and Immunohistological Staining to Pancreatic Tissues. Fixed and embedded mouse pancreatic tissues were sectioned and stained with hematoxylin-eosin (HE). A modified method based on our previous study29 was used to evaluate the severity of pancreatitis in terms of edema, inflammatory infiltration, necrosis, and hemorrhage. The pancreatic sections were scored double-blinded according to a standard list in Table 2.

EXPERIMENTAL SECTION

Materials. Generation 5 PAMAM dendrimers G4.5-COOH and G5-OH were purchased from Sigma-Aldrich Chemical Co. (WI, U.S.A.). Caerulein was purchased from Sigma-Aldrich Chemical Co. (WI, U.S.A.) and dissolved in saline (containing 1% BSA) to achieve a concentration of 0.5 mg/mL before AP induction. The Mac-2, NF-κB p65 and goat antirabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology Company (CA, U.S.A.). Anti-Gapdh and anti-Histone antibodies were purchased from Cell Signaling Technology, Inc. (MA, U.S.A.) and Abbkine, Inc. (CA, U.S.A.), respectively. Thioglycolate broth was purchased from Becton and Dickinson Company (NJ, U.S.A.) and dissolved in water to 40 mg/mL for use. The BCA protein assay kit was purchased from the Pierce Company (IL, U.S.A.). Polyvinylidene difluoride membranes were purchased from Amersham Pharmacia Biotech Company (NJ, U.S.A.). Enhanced chemiluminescence system reagents were from Cell Signaling Technology, Inc. (MA, U.S.A.). Hank’s Balanced Salt Solution (HBSS) was purchased from M&C gene technology (Beijing, China). Fetal bovine serum (FBS), SYBR green fluorescence, and RPMI 1640 medium were purchased from Life Technologies Corporation (CA, U.S.A.). TRI-reagent for RNA extraction was purchased from Molecular Research Center, Inc. (OH, U.S.A.), and the RT kit was purchased from the Promega Corporation (WI, U.S.A.). Gabexate and the other chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (WI, U.S.A.). Methods. Dendrimer Purification. In case there are some impurities of small molecules and structure defective dendrimers mixed in the materials, dialysis was done to purify PAMAM dendrimers before the experiments. Briefly, G5-OH and G4.5COOH were dissolved in pure water and dialyzed against distilled water (cutoff: 10 kDa) and then centrifuged with Millipore tubes (Amicon Ultra, cutoff: 5 kDa). Purified PAMAM dendrimers were collected and lyophilized. The Mn (g/mol) of the two investigated dendrimers was analyzed by 1H NMR and GPC, and the number of terminal groups of dendrimers was determined by GPC and potentiometric titration. The results are shown in Table 1. Caerulein-Induced AP in Mice. ICR (CD-1) mice were purchased from the animal department of Peking University Health Science 175

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Table 2. Scoring Standard for Evaluation of the Pancreatic Injury after AP Inductiona score edema inflammatory infiltration necrosis hemorrhage

1 local interlobular swelling 5 foci

a Inflammatory cells were counted as the average number of cells per 10 fields at 200× magnification. The degree of parenchymal necrosis was evaluated as percent area of acinar cell necrosis.

Table 3. Primer Sequences Used for Quantitative RT-PCR gene

sense primer

antisense primer

18S IL-1β IL-6 TNF-α TGFβR-1 IL-10 TGFβ-1

5′-GGAAGTGCACCACCAGGAGT-3′ 5′-AGGCTCCGAGATGAACAA-3′ 5′-TTCTTGGGACTGATGCTG-3′ 5′-CTGTGAAGGGAATGGGTGTT-3′ 5′-ATGATATGACAACATCAGGGTC-3′ 5′-ACCTGGTAGAAGTGATGC-3′ 5′-GGCGGTGCTCGCTTTGTA-3′

5′-TGCAGCCCCGGACATCTAAG-3′ 5′-AAGGCATTAGAAACAGTCC-3′ 5′-CTGGCTTTGTCTTTCTTGTT-3′ 5′-CAGGGAAGAATCTGGAAAGGTC-3′ 5′-TCGCCAAACTTCTCCAAA-3′ 5′-AAGGAGTTGTTTCCGTTA-3′ 5′-TCCCGAATGTCTGACGTATTGA-3′

RT-PCR product 163 464 380 384 119 366 201

bp bp bp bp bp bp bp

Extraction of Cytoplasmic or Nuclear Proteins in Macrophages. To extract cytoplasmic proteins, mice peritoneal macrophages were first treated with buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.5 mM DTT, 5 mM MgCl2, and 1 mM PMSF) containing a protease inhibitor cocktail and then centrifuged at 1000 × g for 15 min at 4 °C to isolate supernatants from cell pellets. The obtained supernatants were incubated on ice for 10 min and then centrifuged at 100000 × g for 1 h at 4 °C to extract cytoplasmic proteins for NF-κB detection by Western blot. To extract nuclear proteins, cell pellets from macrophage homogenates were first washed twice with the buffer A and then resuspended with buffer B (1.3 M sucrose, 1.0 mM MgCl2, and 10 mM potassium phosphate buffer, pH 6.8). Pellets were obtained by centrifuging the suspensions at 1000 × g for 15 min, they were then resuspended in buffer B with a final sucrose concentration of 2.2 M and centrifuged again at 100000 × g for 1 h. The resulting pellets were washed once with a solution containing 0.25 M sucrose, 0.5 mM MgCl2, and 20 mM Tris-HCl (pH 7.2), and then centrifuged at 1000 × g for 10 min. The obtained pellets were solubilized with a solution containing 50 mM Tris-HCl (pH 7.2), 0.3 M sucrose, 150 mM NaCl, 2 mM EDTA, 20% glycerol, 2% Triton X-100, 2 mM PMSF, and the protease inhibitor cocktail. The mixture was maintained on ice for 1 h with gentle stirring and then centrifuged at 12000 × g for 30 min. The resulting supernatants containing nuclear proteins were used for Western blot analysis to detect NF-κB. Detection of NF-κB Expression in Macrophages. Western blotting was used to detect NF-κB expression in the cytoplasm and nucleus of macrophages. The protein concentration of each sample was determined by a BCA protein assay kit. Samples containing 20 μg protein were loaded in each lane on a 10% SDS-PAGE gel for electrophoresis. Proteins were separated and transferred to polyvinylidene difluoride membranes using the wet transfer method (220 mA, 90 min). Membranes was first blocked with 5% nonfat milk in Tris-buffered saline-Tween 20 (TBST; 25 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 2 h and then incubated with an anti-p65, anti-Gapdh, or anti-histone antibody at 4 °C overnight. Antirabbit or mouse peroxidase-conjugated IgG was used to detect primary NF-κB antibody. After treatment with enhanced chemiluminescence system reagents, photographic films were exposed to visualize NF-κB bands. Statistical Analysis. All results are presented as the mean ± SEM. Data from quantitative PCR were analyzed with the Student-NewmanKeul’s test, and pathological scoring was analyzed by the Mann− Whitney rank-sum test. A P value < 0.05 was considered statistically significant.

To evaluate macrophage infiltration in mouse pancreatic tissues, Mac-2 immunohistological staining was performed on sections using a primary Mac-2 antibody (1:200) and goat antirabbit IgG-HRP. Isolation of Mice Peritoneal Macrophages. ICR mice received a peritoneal injection of 2 mL of 4% thioglycolate broth 3 days prior to harvest, their peritoneal macrophages were lavaged with cold Hank’s balanced salt solution (HBSS) containing 5% FBS, and they were then collected by centrifugation at 300 × g for 10 min. The cell pellets were then resuspended with macrophage adhesion medium (MAM) and seeded onto 12-well plates at a cell density of 2.5 × 106/mL following a 4 h incubation at 37 °C to allow the macrophages to attach to the wells of the plates. Afterward, MAM was replaced with RPMI 1640 medium containing penicillin, streptomycin, and 10% FBS. Stimulation of Inflammation in Macrophages. The mouse peritoneal macrophages were first incubated for 6 h with G4.5COOH, G5-OH, or PBS and then stimulated with 1 μg/mL lipopolysaccharide (LPS) for 12 h to generate inflammation. The inflammation-activated macrophages were collected for RNA extraction and RT-PCR to detect the expression of inflammatory cytokines. RNA Extraction and Quantitative PCR. Total RNA from pancreatic tissues and peritoneal macrophages was extracted by utilizing TRIzol reagent, and first-strand cDNA was generated by a RT kit. Quantitative real-time PCR of the inflammatory factors was performed with the primer sequences shown in Table 3. The amplifications were performed in 35 cycles by an optic continuous fluorescence detection system (MJ Research) with SYBR green fluorescence. Each cycle comprised heat denaturation for 30 s at 95 °C, annealing for 30 s at 58 °C and extension for 30 s at 72 °C. All samples were quantitated using a comparative CT method to evaluate relative gene expression, and results were normalized to 18 S. Observation of NF-κB Nuclear Translocation in Macrophages by Confocal Microscopy. Peritoneal macrophages were seeded onto 24well plates at a cell density of 1 × 105 cells per well in RPMI 1640 complete medium and grown to 70−80% confluence. Then, the cells were treated with PBS or G4.5-COOH at the indicated concentrations and incubated at 37 °C for 6 h. The cells were then rinsed with PBS three times and treated with LPS (1 mg/mL) for 30 min. Afterward, the cells were fixed with 4% formaldehyde for 15 min. For immunostaining, the fixed cells were first permeabilized with 0.1% Triton X-100 for 10 min followed by three washes with PBS, and they were then blocked with 5% BSA for 30 min at room temperature. Afterward, the macrophages were incubated with an anti- NF-κB p65 polyclonal antibody (1:400) at 4 °C overnight. The cells were washed with PBS three times and then incubated with antirabbit IgG Alexa Fluor 488 for 2 h at room temperature, followed by three washes with PBS. NF-κB fluorescence in the macrophages was observed by confocal microscopy. 176

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Figure 1. Plasma amylase (A), pancreatic morphological changes (B), and pathological scores (C, D) of caerulein-induced AP mice. Four groups of mice treated with NS (AP-positive control), gabexate (drug-positive control), G4.5-COOH or G5-OH were induced to have AP. Amylase activity in the plasma was measured at 0, 3, 6, 9, and 12 h time points after the first injection of caerulein. Statistical analysis was performed using the one-way ANOVA test (n = 5, *p < 0.05, **p < 0.01, gabexate-treated mice compared with NS-treated mice; #p < 0.05, G4.5-COOH-treated mice compared with NS-treated mice). Pancreatic sections from NS-, gaxetate-, G4.5-COOH-, or G5-OH-treated mice were stained with HE (B, magnification, 100×) and scored based on four parameters: edema, inflammatory infiltration, necrosis, and hemorrhage (C). Total pathological scores (D) were derived from integrative evaluation of the four parameters. Statistical analysis was performed using the Mann−Whitney test (n = 5, * p < 0.05, compared with NS-treated mice). All results are presented as the mean ± SEM.

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RESULTS Plasma Amylase Activity. Plasma amylase activity is one of the indexes reflecting the severity of pathological changes in the pancreas. Compared with NS-treated AP-positive control mice, mice treated with gabexate significantly reduced the amylase released in plasma at the 3, 6, 9, and 12 h time points (represented by * in Figure 1A). However, the G4.5-COOHtreated mice only significantly reduced plasma amylase activity 12 h after AP induction (represented by # in Figure 1A). The G5-OH-treated mice showed no obvious inhibition in amylase release at any time point (Figure 1A). Pathological Changes in Pancreas. Compared with NStreated AP-positive control mice, mice treated with either G4.5-

COOH or G5-OH showed preventive effects against pancreas impairment caused by AP induction, although they were not as efficient as gabexate (Figure 1B). Compared with NS-treated mice, gabexate-treated mice demonstrated an expected decrease in edema, inflammatory infiltration, and necrosis in the mice pancreas (P < 0.05, Figure 1C). Treatment with G4.5-COOH significant reduced inflammatory cells in the pancreas of AP mice (P < 0.05, Figure 1C), and achieved lower total pathological scoring (P < 0.05, Figure 1 D). G5-OH also presented some protective effects against pancreatic injury and resulted in a significant decrease in total pathological scoring (P < 0.05, Figure 1D). 177

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Figure 2. Pathological changes in the pancreas and peripheral hemograms from mice pretreated with different doses of G4.5-COOH. (A) Pancreatic sections from AP mice with different pretreatments. From left to right: NS, 50 and 150 mg/kg G4.5-COOH. The pancreatic sections were stained with HE (magnification, ×200). (B−D) Counts of WBCs, monocytes, and the percentage of monocytes in mouse peripheral blood, respectively. From left to right: the normal control, NS (AP-positive control), 50 and 150 mg/kg G4.5-COOH pretreated and AP-induced mice. Results are expressed as the mean ± SEM. Statistical analysis was performed with the one-way ANOVA test (n = 5, *p < 0.05, compared with the normal control mice; #p < 0.05, ##p < 0.01, compared with NS-treated mice).

Dose-Dependent Protective Effects of G4.5-COOH. Figure 2A shows that the mice treated with a high dose (150 mg/kg) of G4.5-COOH compared with those treated with a low dose (50 mg/kg) had more significant decrease in edema and inflammatory cell infiltration into pancreatic tissues after AP induction. In addition, compared with NS-treated mice, mice treated with a high dose of G4.5-COOH (150 mg/kg) significantly reduced the total number of white blood cells (WBCs; Figure 2B), and monocytes (Figure 2C,D) in peripheral blood. Treatment with a low dose of G4.5-COOH (50 mg/kg) also inhibited the count and percentage of monocytes in the peripheral blood of the mice, but it did not affect their total WBC number. Macrophage Infiltration in Pancreas. Compared with NS-treated AP-positive control mice, pancreatic tissues from G4.5-COOH-treated mice demonstrated significant inhibition in macrophage infiltration. G5-OH also decreased the infiltration of macrophages in mouse pancreases, but it was less efficient than G4.5-COOH (Figure 3). Expression of Inflammatory Factors in Pancreas. The expression of several inflammatory factors in the pancreatic tissues of mice was measured by RT-PCR. Figure 4 shows that the expression of inflammatory factors was significantly increased in AP-positive control mice compared with normal control mice. The expression of the pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and TGFβR-1 was dramatically decreased in G4.5-COOH- and G5-OH-treated mice. However, the expression of the protective cytokines IL-10 and TGF-β did not change much in pancreatic tissues from dendrimer-treated mice, compared with that from NS-treated AP-positive control mice. Expression of Inflammatory Factors in Peritoneal Macrophages. After preincubation with PBS, G4.5-COOH, or G5-OH, peritoneal macrophages were stimulated with LPS

Figure 3. Macrophage infiltration in the mice pancreas after AP induction. Paraffin sections of the pancreatic tissues from NS- (A), G4.5-COOH- (B), or G5-OH-treated (C) mice were stained with antiMac-2 antibody to detect macrophages in pancreas. The original magnification was 40× (left) and 100× (right).

to induce inflammation. Compared with PBS pretreated cells, preincubating the macrophages with G4.5-COOH or G5-OH significantly inhibited the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and TGFβR-1), but it did not have an influence on the expression of the protective cytokines IL-10 and TGF-β (Figure 5). These results are consistent with the PCR results from mouse pancreatic tissues. 178

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Figure 4. Quantification of cytokine mRNA levels in pancreatic tissues from AP mice. Pancreatic mRNA expression of pro-inflammatory cytokines: IL-1β (A), IL-6 (B), TNF-α (C), and TGFβR-1 (D), and anti-inflammatory cytokines: IL-10 (E) and TGF-β (F) in pancreatic tissues from NS, G4.5-COOH, or G5-OH pretreated and AP-induced mice were measured and compared with that of normal control mice. Results are expressed as the mean ± SEM. Statistical analysis was performed with the one-way ANOVA test (n = 5, *p < 0.05, compared with normal control mice, #p < 0.05, ##p < 0.01, compared with NS-treated AP-positive control mice).

Inhibition of NF-κB Nuclear Translocation in Macrophages. Dual-color images of Alexa Fluor-labeled NF-κB (red) and DAPI-labeled nuclei (blue) in macrophages were detected by confocal microscopy. As shown in Figure 6A, the red fluorescence of NF-κB was mainly found in the cytoplasm in normal control macrophages. After stimulation with LPS for 30 min, the NF-κB red fluorescence in the nucleus of macrophages increased. By comparison, NF-κB nuclear translocation was partially blocked by preincubating the macrophages with G4.5COOH (Figure 6A). The NF-κB expression in the nucleus and cytoplasm of macrophages after LPS stimulation for 0, 5, 15, and 30 min was detected by Western blot. As shown in Figure 6B, compared with PBS pretreated and LPS-stimulated macrophages, pretreating the macrophages with G4.5-COOH decreased the nuclear NF-κB expression in macrophages at 15 and 30 min after LPS stimulation. However, at the same experimental conditions, pretreatment with G5-OH did not obviously inhibit NF-κB nuclear translocation in the macrophages, especially at

Figure 5. Quantification of cytokine mRNA levels in LPS-stimulated mouse peritoneal macrophages. The macrophages were preincubated with PBS, G4.5-COOH, or G5-OH and then stimulated by LPS. The mRNA levels of the pro-inflammatory (A) and the anti-inflammatory (B) cytokines in the macrophages were measured. The results are expressed as the mean ± SEM. Statistical analysis was performed with the one-way ANOVA test (n = 5, *p < 0.05, **p < 0.01, compared with PBS pretreated group).

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toxicity.31,32 The biocompatibility and acute toxicity of the G4.5-COOH (150 mg/kg) or G5-OH (50 mg/kg) were also evaluated briefly in this study. The main organs of the dendrimer-treated mice including heart, liver, kidney, and spleen were sectioned and stained by HE for morphology study, no obvious changes and acute toxicity were observed in the organs of the mice under this regimen (i.v. q.o.d. for 2 weeks). In addition, blood and enzyme chemistry after treating the mice with 150 mg/kg G5-COOH (i.v. q.o.d. for 2 weeks) were also analyzed and no significant difference were observed between dendrimer- and saline-treated mouse groups, except for monocyte and WBS count (Figure 2B−D). In caerulein-induced AP mice, plasma amylase activity could increase from 500 to 3000 U/L, which impairs pancreatic tissues and causes inflammatory cell infiltration in the pancreas. In our experiments, G4.5-COOH and G5-OH did not significantly inhibit amylase release from the impaired pancreases into blood, which is in contrast with gabexate in AP mice (Figure 1A); however, pretreatment with the two dendrimers significantly reduced necrosis and inflammatory cell infiltration and resulted in less severe pancreatic damage compared with NS-treated mice (Figures 3 and 1), indicating that the protective effects of the two dendrimers were not due to inhibition of amylase secretion. The protective effects of G4.5-COOH against pancreatic injury were dose-dependent (Figure 2). Compared with low dose (50 mg/kg) treatment, high dose G4.5-COOH (150 mg/ kg) presented much greater inhibition of edema and inflammation infiltration in the pancreas and better inhibition of the WBC number in the peripheral blood of AP mice. G5-OH, which has the same core, branches, generation, and molecular size as G4.5-COOH but bears different terminal groups and surface charges, was found could selectively target to microglia and deliver anti-inflammatory drugs to treat retinal neuroinflammation.33 In this study, G5-OH was less efficient than G4.5-COOH in inhibiting macrophage infiltration (Figure 3). The longer in vivo clearance time of anionic G4.5-COOH in the blood circulation compared with neutral G5-OH16 may contribute to its better inhibitory effects on inflammatory cell infiltrating into pancreatic tissues. It was reported that glucosamine conjugated PAMAM dendrimer could reduce IL-6 and IL-8 expression in a rabbit shigellosis model.34 Manno-dendrimers, a series of poly(phosphorhydrazone) dendrimers grafted with mannose units, were found significantly inhibited neutrophil influx and TNF-α expression in the mice with acute lung inflammation.35 Our PCR results from pancreatic tissues showed that the secretion of the pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, and TGFβR-1, was significantly inhibited by pretreating the mice with either G4.5-COOH or G5-OH, because macrophages can be activated by pancreatitis-associated proteins36 and secrete the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, which aggravate pancreatic damage and cause systemic inflammation during the AP developing process.37 LPS, a component of cell walls, can stimulate macrophages in vitro and result in a significant secretion of pro-inflammatory cytokines (as observed in the NS-treated cells in Figure 5A), which activate the macrophages during the process of inflammation.38 Consistent with the results of the pancreatic tissues from AP mice (Figure 4), in vitro results demonstrated that preincubating macrophages with either G4.5-COOH or G5-OH dramatically inhibited the expression of pro-inflammatory cytokines in LPS-activated peritoneal macrophages (Figure

Figure 6. NF-κB nuclear translocation in macrophages. Macrophages were preincubated with PBS or G4.5-COOH and then stimulated for inflammation with LPS. A confocal assay (A) was performed 30 min after LPS stimulation. Alexa Fluor labeled NF-κB protein is shown in red, and DAPI-labeled cell nuclei are shown in blue. After LPS stimulation for 0, 5, 15, and 30 min, cytoplasmic and nuclear proteins were extracted from the macrophages, and the expression of NF-κB protein (B) in the cytoplasm and nucleus of macrophages was detected by Western blot and the results were quantified. Statistical analysis was performed with the one-way ANOVA test (n = 3, *p < 0.05, compared with PBS pretreated group).

the peak time point of 15 min after LPS stimulation (Supporting Information, Figure 1).

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DISCUSSION Toxicity of the PAMAM dendrimers is a big concern for their biomedical applications.30 The regimen of the dendrimers used in this work derived from a work of Thomas et al.28 There are some references demonstrate that negative-charged (−COOH) and neutral (−OH) PAMAM dendrimer with a generation up to 6.5 or 7 have no in vivo toxicity, only the cationic dendrimers at high generations and in high doses show some in vivo 180

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5), indicating that the protective effects of the two dendrimers against pancreatic injury in AP mice were related to the macrophages. NF-κB is a major transcription factor that regulates genes related to the innate and adaptive immune response39 and also plays a critical role in acute pancreatitis.40 NF-κB can regulate the expression of TNF-α, IL-6, and other inflammatory factors caused by an immune response.41 Inhibition of NF-κB nuclear translocation attenuates caerulein-induced pancreatitis.42 Our results show that G4.5-COOH significantly inhibits the translocation of NF-κB from the cytoplasm to the nucleus in inflammation-activated macrophages (Figure 6), which explains why the expression of the pro-inflammatory cytokines in macrophages is significantly inhibited by G4.5-COOH. The combined effects of the inhibition of NF-κB nuclear translocation in macrophages, decrease of macrophage infiltration in the pancreas, and reduction in the inflammatory cells in the peripheral circulation contributed to the protective effects of G4.5-COOH against pancreatic impairments caused by caerulein-induced AP. G5-OH did not inhibit NF-κB nuclear translocation in the macrophages (Supporting Information, Figure 1) but reduced macrophage infiltration in the injured pancreas, therefore, also showing some protective effects on AP, even less effective than G4.5-COOH.

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CONCLUSIONS G4.5-COOH and G5-OH PAMAM dendrimers have protective effects against pancreatic impairment in caerulein-induced AP mice. Inhibition of NF-κB nuclear translocation in macrophages and a reduction in inflammatory cells in pancreas and peripheral circulation contribute to more intensive protection effects of G4.5-COOH than G5-OH against pancreatic injury in AP mice. The anti-inflammatory activities of the naked G5 PAMAM dendrimers, especially G4.5-COOH, could be applied not only in the drug development, but also as a potently therapeutic agent in clinic.

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ASSOCIATED CONTENT

* Supporting Information S

The effects of G4.5-COOH and G5-OH on NF-κB nuclear translocation in macrophages was included in supplemental data as supplemental Figure 1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86 10 8280 5164. Fax: +86 10 8280 2769. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (Nos. 30971241, 81270368). REFERENCES

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