Journal of Pediatric Surgery xxx (2014) xxx–xxx
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Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles☆ Xuelai Liu a,1, Wei Hao a,1, Chun-Nam Lok b, Yue Chun Wang c, RuiZhong Zhang a, Kenneth K.Y. Wong a,⁎ a b c
Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China Department of Chemistry, The University of Hong Kong, Hong Kong, China Department of Physiology, Medical College, Ji Nan University, Guangzhou, China
a r t i c l e
i n f o
Article history: Received 29 August 2014 Accepted 6 September 2014 Available online xxxx Key words: Anti-inflammatory Silver nanoparticles Dendrimer Wound healing Drug delivery
a b s t r a c t Background: Our previous studies revealed that silver nanoparticles (AgNPs) promoted wound healing in part through their anti-inflammatory actions. As recent reports also suggested anti-inflammatory effects of dendrimers, we therefore undertook this study using dendrimer as the delivery system for AgNP to explore any potential synergistic anti-inflammatory efficacy. Methods: Lipopolysaccharide (LPS) was added to cultured RAW264.7 and J774.1 cells to mimic in vitro inflammation condition, followed by the addition of either silver dendrimer nanocomposite (Ag-DNC), AgNPs, or dendrimer. The levels of inflammatory markers TNF-alpha and interleukin-6 were assessed using ELISA assay. Furthermore, in vivo effects such of Ag-DNC, AgNPs, or dendrimer were studied in a burn wound model in mice. Results: Our results confirmed that both naked dendrimer and AgNPs had anti-inflammatory properties. In in vitro study, Ag-DNC was shown to have the best anti-inflammatory efficacy than AgNPs or dendrimer alone. In-vivo experiments also indicated that animals in the Ag-DNC group had the fastest healing time with the least inflammation. Conclusion: Our study would suggest that dendrimer could provide additional anti-inflammatory benefits and might be an excellent delivery system for silver nanoparticles for future clinical application. © 2014 Published by Elsevier Inc.
Silver nanoparticles (AgNPs) are nano-scale-sized pure silver measuring less than 100 nm in diameter [1]. Compared with the other forms of silver, the large active surface of AgNPs will induce unusual physicochemical properties and biological activities when exposed to cells or tissues [2]. Our previous studies have revealed that AgNPs possess significant anti-inflammatory effects [3,4]. Although the exact mechanism of AgNPs-mediated anti-inflammation still remains to be elucidated, some researchers have proposed that AgNPs access into target cells and block relevant inflammatory signaling pathways [5,6]. In clinical practice, the use of AgNPs has been gaining popularity, but mostly restricted to incorporation into woven fabrics in wound dressings. Dendrimers are nano-sized, branched, synthetic polymers with layered architectures first described by Tomalia et al. [7]. They have been shown to be promising carriers in bio-mimicry, diagnostics, and therapeutics. Currently the majority of dendrimer designs have been utilized in the field of drug delivery [8,9]: for anti-inflammatory [10–12], antimicrobial and antiviral drugs [13–18], as well anticancer agents [19]. Some studies have showed that naked dendrimer itself
☆ Disclosure: The authors report no conflicts of interest in this work. ⁎ Corresponding author at: Department of Surgery, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong SAR, China. Tel.: +852 22553486; fax: +852 28173155. E-mail address: [email protected] (K.K.Y. Wong). 1 Contributed equally to this work.
also has anti-inflammatory property [20–22]. We therefore hypothesized that if AgNPs were to be conjugated with dendrimer, as silver dendrimer nanocomposites (Ag DNC), the two nanoscale materials would have synergistic effects in terms of anti-inflammation. The purpose of this study was thus to confirm this hypothesis by comparing the anti-inflammatory actions mediated by Ag DNC with AgNPs, through in vitro and in vivo studies. 1. Materials and methods 1.1. Preparation of silver nanoparticles and dendrimer Synthesis of silver nanoparticles (AgNPs) has been described previously [3,4]. Final concentration of AgNPs solution produced was 1 mM. The mean diameter of silver nanoparticles was 10 nm (ranging from 5 to 15 nm), which was confirmed by transmission electron microscopy. Silver dendrimer nanocomposites (Ag DNC) used were “neutral” at biologic pH and consisted of G5 dendrimer and were kindly provided by Dr. L Balogh (Northeastern University, USA). The polymeric chain in dendrimer provided nitrogen atoms for the coordination and stabilization of AgNPs, and was utilized as delivery system to AgNPs (Fig. 1). Ag DNC used in this study had the following chemical and physical characteristics: acetamide surface; 3.1779 g solution containing 0.024402 g Ag (0) in 0.172945 g Ag DNC solution; pH = 6.72; Ag/Dendrimer molar ratio = 51.69; Ag(0)/composite ratio: 0.13673; The store
http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033 0022-3468/© 2014 Published by Elsevier Inc.
Please cite this article as: Liu X, et al, Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles, J Pediatr Surg (2014), http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033
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X. Liu et al. / Journal of Pediatric Surgery xxx (2014) xxx–xxx
Fig. 1. Schematic representation of the polymeric chains in dendrimer stabilizing AgNPs, to form Ag DNC conjugate
concentration of Ag DNC was 73,500 μM and was dissolved into sterile water for various working concentration. The “naked” dendrimer (G5, 10 w/w percent solution in water) used in this study was purchased from Dendritic Nanotechnologies, (MI, U.S.A.). Stock concentration of dendrimer was 24,000 μM and they were dissolved into sterile water for different working concentration.
concentrations of Ag DNC (ranging from 50 to 400 μM) were added, followed by 10 mL MTT reagent to each well. 100 mL of detergent reagent was added to each well after purple precipitate was clearly visible under the microscope. The plate was sealed and put in the dark for 4 h at room temperature. Absorbance in each well was measured at 570 nm in a microtiter plate reader (Microplate Manager, Bio-Rad Laboratories). The average values from triplicate readings were calculated.
1.2. Cell culture RAW264.7 and J774.1, both mouse macrophage cell lines, were purchased from ATCC (VA, USA). The above cell lines were the same as those described in our previous study [4], aiming to accurately explore in vitro anti-inflammatory efficacy of Ag DNC and AgNPs. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL) and fetal bovine serum (FBS; 10%) and grown in 37°C incubator with 5% CO2 humidified atmosphere. The culture medium was changed two or three times per week. Upon reaching 80%–90% confluence, cells passage was performed by addition of 0.25% trypsin-EDTA, aspirating and dispensing in new flasks. Passage 5 cells were used in this study. 1.3. In vitro inflammation model Upon reaching 70% confluence of cultured cells, lipopolysaccharide (LPS; Sigma-Aldrich, USA) with 100 ng/mL of working solution concentration was added. Our previous study showed that AgNPs could effectively decrease LPS-induced TNF-α expression [4]. Therefore, in the present study, macrophage cells were incubated either with Ag DNC, AgNPs, dendrimer or no treatment respectively after the addition of LPS. The supernatant in each group was harvested post-incubation 1 h after addition of LPS. The levels of pro-inflammatory cytokines TNF-α and IL-6 were measured using TNF-α and IL-6 (Mono/Mono) ELISA kits (BD Biosciences, San Diego, CA, USA) according to manufacturer’s protocol. 1.4. MTT assay MTT assay kit was purchased from Roche (Germany) to measure cell viability and proliferation. Cultured cells (5 × 105) were plated out in triplicate into 96-wells plate. Three control wells with medium alone were used to provide blanks for absorbance readings. Various
1.5. Animals and burn wound model C57BL/6 N mice, between 14 and 16 weeks old and weighing 40–46 g, were obtained from the Laboratory Animal Unit, The University of Hong Kong. Mice were allowed diet and water ad libitum in a 12-h light, 12-h dark cycled room. Experimental protocol was approved by the Committee of the Use of Live Animals in Teaching and Research, The University of Hong Kong (CULATR 1974-09). The animals were randomly divided into 4 groups: Ag DNC, AgNPs, dendrimer, and untreated group (n=6). Anesthesia was achieved with pentobarbital sodium solution at a dose of 50 mg/kg by intra-peritoneal injection (Abbott Laboratories, USA). Both excisional wound model and burn wound model have been described previously [23,24]. In brief, a full-thickness piece of skin measuring 1.0 × 1.0 cm2 was excised on the back of each mouse using a pair of scissors for the excisional wound model. For the burn wound model, a burn template was established in a plastic 50-mL syringe by cutting a window (3.0 × 2.0 cm2) on one side, while the opposite half side was removed. This would allow a mouse to be laid on the template. The template was then put horizontally into a water bath at 70 °C for 30 s, followed by immediate placement of mouse in iced water bath to stop the burn process. This model would achieve approximately 6.7% deep partial thickness thermal injury of total body surface area, simulating scald injury in children. Dressings with either Ag DNC or AgNPs were used to cover the wound site. In both AgNPs and Ag DNC groups, the silver content in the dressing was 0.04 mg/cm 2[3,23]. In the dendrimer treatment group, the wound was covered with dendrimer-coated dressing. The dendrimer content for each dressing was 5 mg/cm 2, which was the same as Ag DNC groups. In the no treatment group, only medical dressing was covered as control. Postoperatively, all mice were kept warm until fully awake. The dressings were changed every 3 days until day 9. All animals
Please cite this article as: Liu X, et al, Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles, J Pediatr Surg (2014), http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033
X. Liu et al. / Journal of Pediatric Surgery xxx (2014) xxx–xxx
were humanely sacrificed by CO2 asphyxiation after wound was closed completely. 1.6. Morphometric assessment of wound closure For the assessment of wound healing, mice were photographed daily with a digital camera secured on a cantilever at a fixed distance. Wound closure analysis was performed three times in a singleblinded manner. Wound areas (cm 2) were calculated from wound perimeter tracings using Photoshop CS (Adobe, USA) until complete closure. The healing rate was expressed as a percentage of the original dorsal wound area on day 0 (excisional wound: 1.0 × 1.0 cm 2; burn wound: 3.0 × 2.0 cm 2) after wounding.
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Ag DNC was the most effective in reducing LPS-induced inflammation. Our result also supported that dendrimer alone had underlying in vitro anti-inflammatory effect. In our previous studies, we showed that the addition of AgNP did not result in significant apoptotic cell death [4]. In order to confirm if the addition of dendrimer to silver nanaoparticles would not alter the toxicity, MTT assay was conducted to evaluate cell viability. Here, more than 85% of both RAW264.7 and J774.1 survived after exposure to 100 μM concentration of Ag DNC [data not shown]. 2.2. Skin wound models revealed enhanced anti-inflammatory efficacy of Ag DNC
2. Results
The in vitro study indicated an enhancement of anti-inflammatory effect induced by Ag DNC. We next asked whether similar finding could be seen in vivo. As the process of wound healing is characterized by an inflammatory response, it is thus a good model for inflammation research. We first investigated the synergistic effects of Ag DNC in an excisional model. Here, when compared to untreated and dendrimer alone groups, both Ag DNC and AgNPs contributed to faster healing with similar healing times (Fig. 3). In the untreated and dendrimer groups, the mean closure time was 17.0 ± 0.7 and 16.3 ± 0.7 days respectively (p =0.07559), while in the AgNPs and Ag DNC groups, it was 12.1 ± 0.3 and 12.6 ± 0.5 days respectively (p =0.36322). This result was in contrast to our in vitro finding. We hypothesized that the reason why no difference was observed between Ag DNC and AgNPs in the in vivo model was due to minimal inflammation in the excisional wound model. In this case, the additional anti-inflammatory effects exerted by Ag DNC would not have been apparent. As a result, we next turned to a burn wound model, which would offer significantly more post-injury inflammation. In the next experiment, we compared the pro-healing efficacy of Ag DNC, AgNPs and dendrimer using a burn wound model. Here, animals treated with Ag DNC had the fastest healing time of 23.8 ± 0.8 days when compared to AgNPs (27.2 ± 1.2 days), dendrimer (31.5 ± 0.8 days) and untreated (38.0 ± 0.9 days) (p b 0.001) (Fig. 4A). This was also reflected in the rate of healing (Fig. 4B).
2.1. Ag DNC suppresses TNF-α and IL-6 production in vitro
3. Discussion
Activated macrophages play an important role in the initiation of inflammation through production of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin (IL-6). We showed previously that AgNPs were able to suppress the production of TNF-α, IL-6 and TGF-β [3,4]. In this study, we employed the same in vitro inflammation model to assess and compare the effects of Ag DNC, AgNPs and “naked” dendrimer. Here, TNF-α level in Ag DNC group was decreased by 36.6% in RAW264.7 and by 47.4% in J774.1 (p b 0.001). In the AgNPs group, TNF-α level was decreased by 21.2% in RAW264.7 and by 41% in J774.1 (p b 0.01). Reduction of TNF-α was also seen in the dendrimer group: 9.3% in RAW264.7 and 17.7% in J774.1 (p b 0.05) [Fig. 2A&B]. In terms of IL-6 production, the level was decreased in Ag DNC group by 41.1% in RAW264.7 and by 24.6% in J774.1 (p b 0.001). In contrast, AgNPs treatment decreased IL-6 level by 33.6% in RAW264.7 (p b 0.01) and by 18.1% in J774.1. For the dendrimer group, it was decreased by 12.1% in RAW264.7 (p b 0.05) and by 7.7% in J774.1 respectively (p = 0.065) (Fig. 2C&D). Taken together, it would suggest that
Inflammation is a double-edged sword. On one hand, it is a protective mechanism against the potential invasion of a variety of microorganisms. On the other hand, de-regulation can lead to chronic inflammatory conditions. From the point of wound healing, chronic inflammation results in delayed or even non-healing. In children, burn injuries from scalding are commonly seen and can give rise to significant physical and psychological morbidity. Persistent inflammatory response after wounding will also lead to delayed healing and increased complications. Therefore, a more effective treatment to inhibit local inflammatory response may help accelerate wound healing. Inflammation is regulated by a variety of immune cells and cytokines, in which activated macrophages play an essential role in the initiation and amplification of inflammation through the production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL-6, and interleukin-1β (IL-1β) [25]. Our previous studies already revealed that silver nanoparticles (AgNPs) had significant anti-inflammatory effects through the inhibition to macrophage and decreased production of inflammatory cytokines [3,4]. In this regard, Nadworny and Bhol postulated the potential mechanism of AgNPsmediated anti-inflammation to be intracellular blocking of inflammatory pathways and down-regulated pro-inflammatory cytokines [5,6]. With advance in nanotechnology, various nano delivery systems have been used to carry drug molecules and genes. Among these, dendrimer is an important and effective nano carrier platform [26]. It is a hyper-branched, tree-like polymer with a defined structure whose size and molecular weight can be controlled rigorously due to the
1.7. Immunohistochemistry (IHC) The healing skin tissues on day 3 post-wounding were harvested and processed. Endogenous peroxidase activity was quenched by treatment with 3% H2O2/CH3OH for 30 min. Sections were incubated for 1 h at room temperature by using blocking solution containing 5% concentration of normal goat serum (Dako Bioresearch, USA). For antigen retrieval of neutrophil, macrophage, interleukin-6 (IL-6), and tumor necrosis factors-alpha (TNF-α) staining, sections were further blocked against nonspecific binding using 10% normal goat serum before primary antibody (Table 1) was added respectively. The sections were incubated overnight at 4°C and then further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, USA) for 2 h at room temperature. Positive signals were developed by using 3,3’-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin. 1.8. Statistical analysis Statistical analysis was performed using Student’s paired t test and p-value of b 0.05 was considered statistically significant.
Table 1 Primary monoclonal antibodies in this study. Antibody against
Abbreviation
Dilution
Specie
Manufacturer
Neutrophil Macrophage Interleukin-6 Tumor necrosis factors-alpha
PMN F4/80 IL-6 TNF-α
1:10,000 1:200 1:500 1:50
Rabbit Mouse Rabbit Mouse
Sant Crutz Abcam Abcam Abcam
Please cite this article as: Liu X, et al, Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles, J Pediatr Surg (2014), http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033
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Fig. 2. TNF-α expression levels measured by ELISA for (A) RAW264.7 and (B) J774.1cells, as well as IL-6 expression levels measured by ELISA for (C) RAW264.7 and (D) J774.1 cells. Respective cell lines were incubated with 100 μM Ag DNC, AgNPs or dendrimer alone after the addition of LPS stimulation. Values are the mean ± SE for all groups; * p b 0.05, **p b 0.01, and ***p b 0.001.
iterative step-by-step synthesis. These structural features, together with the high density of chemical-reactive functions on the outer shell of these molecules, potentially widen their application in medicine [27]. An unexpected property of dendrimer reported recently was the antiinflammation. Chauhan et al. reported that dendrimers had potential to inhibit inflammation due to bearing of amine or hydroxyl surface groups, and therefore exhibited anti-inflammatory activities in carrageenan-induced paw edema model [21]. In this regard, Fruchon et al. also suggested that dendrimer-activated monocyte had
immunnosuppressive phenotype and could serve as new nano-tools to promote anti-inflammatory and immunosuppressive activation in human monocytes [22]. Furthermore, some conjugated dendrimers for drug delivery were also revealed to exert anti-inflammatory actions [28,29]. Lastly, dendrimer carboxylate salts could carry high local concentrations of silver, due to a large number of active surface groups [30]. Taken together, we hypothesized that if dendrimers were used to carry AgNPs, the synergistic effect might greatly improve the antiinflammatory efficacy.
Fig. 3. The effect of Ag DNC on excisional wound. Excisional wounds were created on the dorsa of C57BL/6 N mice, and they were divided into four groups (n = 6) according to treatment with Ag DNC, AgNPs, dendrimer alone or no treatment as control. The wounds were inspected daily, and the time of healing was recorded.
Please cite this article as: Liu X, et al, Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles, J Pediatr Surg (2014), http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033
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Fig. 4. The effect of Ag DNC on thermal wound. Thermal wounds were created on the dorsa of C57BL/6 N mice and wounds were treated with Ag DNC, AgNPs, dendrimer alone or no treatment as control. (A) Wound appearance and healing times of the four groups. (B) Rate of wound closure at various time points.
In the present study, both in vitro and in vivo studies confirmed that dendrimer had anti-inflammatory properties. Furthermore, when Ag DNC was used in LPS-induced inflammation model in vitro, it showed significantly more inhibition of the production of pro-inflammatory cytokines than when AgNPs was used alone. Extrapolating this to the in vivo setting, we would anticipate a similar synergistic effect between AgNPs and dendrimer. Nonetheless, we noticed no significant difference in wound healing between the two groups in the excisional wound model. This could be due to the fact that the excisional wound, on one hand, was a relatively clean model with little post-injury inflammatory response. On the other hand, the excised area created in the present study was relatively small. As wound size was another important issue which would influence the degree of inflammation and healing time
[31], little inflammation was observed. These could explain why the additional anti-inflammatory effects exerted by Ag-DNC were not apparent, as the use of AgNPs alone already could exert sufficient antiinflammatory effects. Another possible explanation could be that dendrimer in Ag DNC had no anti-inflammatory effect, and their role was only a biological carrier for AgNPs. When we changed to a burn wound model, animals treated with Ag DNC showed the fastest healing time and rate when compared to AgNPs. This would suggest that conjugating AgNPs with dendrimer indeed had synergistic effects and both contributed to better wound healing. So far, reports of explorations to biological and medical effects in dendrimer–metal conjugates are sparse. In terms of Ag DNC, most reports have concentrated on its preparation and physiochemical characteristics [32–36]. Lesniak et al.
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reported fluorescence of dendrimer/silver composites, and their surface charge, cellular internalization, toxicity, and cell labeling capabilities. Their results and others indicated that the composites had potential application as cell biomarkers [37,38]. On the other hand, Tse et al. explored the effect of dendrimer/silver on human oral epidermoid cancer cells and found that they exhibited a significant reduction in breakdown threshold and thus selectively promoted intracellular laser-induced optical breakdown [39]. Furthermore, atomic/molecular dispersion of the guest in dendrimer host was regarded to have potential to create unique physical and chemical properties [40]. Our current finding supported that silver dispersion in dendrimer had better biological property as well. In summary, we elucidated that dendrimer could be an ideal biological carrier for AgNPs to reduce the in vitro and in vivo inflammation, and could effectively promote wound healing, especially in those with significant inflammation. Regarding future work, we will target dendrimer to further assess the delivery and release efficacy, so as to bring their potential clinical application closer to fruition. Acknowledgments The authors would like to thank Dr. L Balogh (Department of Pharmaceutical Sciences, Northeastern University, Boston, USA) for his kind provision of silver dendrimer nanocomposites in this study. References [1] Wong KKY, Liu XL. Silver nanoparticles—the real “silver bullet” in clinical medicine? MedChemComm 2010;1:125–31. [2] Caruso DM, Foster KN, Blome-Eberwein SA, et al. Randomized clinical study of Hydrofiber dressing with silver or silver sulfadiazine in the management of partial-thickness burns. J Burn Care Res 2006;27:298–309. [3] Tian J, Wong KK, Ho CM, et al. Topical delivery of silver nanoparticles promotes wound healing. ChemMedChem 2007;2:129–36. [4] Wong KK, Cheung SO, Huang L, et al. Further evidence of the anti-inflammatory effects of silver nanoparticles. ChemMedChem 2009;4:1129–35. [5] Nadworny PL, Wang J, Tredget EE, et al. Anti-inflammatory activity of nanocrystalline silver in a porcine contact dermatitis model. Nanomedicine 2008;4:241–51. [6] Bhol KC, Schechter PJ. Effects of nanocrystalline silver (NPI 32101) in a rat model of ulcerative colitis. Dig Dis Sci 2007;52:2732–42. [7] Tomalia DA. Dendrimer research. Science 1991;252:1231. [8] Padilla De Jesús OL, Ihre HR, Gagne L, et al. Polyester dendritic systems for drug delivery applications: in vitro and in vivo evaluation. Bioconjug Chem 2002;13: 453–61. [9] Ihre HR, Padilla De Jesús OL, Szoka Jr FC, et al. Polyester dendritic systems for drug delivery applications: design, synthesis, and characterization. Bioconjug Chem 2002;13:443–52. [10] Asthana A, Chauhan AS, Diwan PV, et al. Poly(amidoamine) (PAMAM) dendritic nanostructures for controlled site-specific delivery of acidic anti-inflammatory active ingredient. AAPS PharmSciTech 2005;6:E536–42. [11] Theoharis S, Krueger U, Tan PH, et al. Targeting gene delivery to activated vascular endothelium using anti E/P-Selectin antibody linked to PAMAM dendrimers. J Immunol Methods 2009;343:79–90. [12] Gajbhiye V, Palanirajan VK, Tekade RK, et al. Dendrimers as therapeutic agents: a systematic review. J Pharm Pharmacol 2009;61:989–1003. [13] Chen CZ, Cooper SL. Interactions between dendrimer biocides and bacterial membranes. Biomaterials 2002;23:3359–68. [14] Chen CZ, Beck-Tan NC, Dhurjati P, et al. Quaternary ammonium functionalized poly (propylene imine) dendrimers as effective antimicrobials: structure–activity studies. Biomacromolecules 2000;1:473–80.
[15] Landers JJ, Cao Z, Lee I, et al. Prevention of influenza pneumonitis by sialic acidconjugated dendritic polymers. J Infect Dis 2002;186:1222–30. [16] Witvrouw M, Fikkert V, Pluymers W, et al. Polyanionic (i.e., polysulfonate) dendrimers can inhibit the replication of human immunodeficiency virus by interfering with both virus adsorption and later steps (reverse transcriptase/integrase) in the virus replicative cycle. Mol Pharmacol 2000;58:1100–8. [17] Gazumyan A, Mitsner B, Ellestad GA. Novel anti-RSV dianionic dendrimer-like compounds: design, synthesis and biological evaluation. Curr Pharm Des 2000;6:525–46. [18] Bourne N, Stanberry LR, Kern ER, et al. Dendrimers, a new class of candidate topical microbicides with activity against herpes simplex virus infection. Antimicrob Agents Chemother 2000;44:2471–4. [19] Quintana A, Raczka E, Piehler L, et al. Design and function of a dendrimer-based therapeutic nanodevice targeted to tumor cells through the folate receptor. Pharm Res 2002;19:1310–6. [20] Navath RS, Kurtoglu YE, Wang B, et al. Dendrimer–drug conjugates for tailored intracellular drug release based on glutathione levels. Bioconjug Chem 2008;19:2446–55. [21] Chauhan AS, Diwan PV, Jain NK, et al. Unexpected in vivo anti-inflammatory activity observed for simple, surface functionalized poly(amidoamine) dendrimers. Biomacromolecules 2009;10:1195–202. [22] Fruchon S, Poupot M, Martinet L, et al. Anti-inflammatory and immunosuppressive activation of human monocytes by a bioactive dendrimer. J Leukoc Biol 2009;85: 553–62. [23] Liu X, Lee PY, Ho CM, et al. Silver nanoparticles mediate differential responses in keratinocytes and fibroblasts during skin wound healing. ChemMedChem 2010;5: 468–75. [24] Cribbs RK, Luquette MH, Besner GE. A standardized model of partial thickness scald burns in mice. J Surg Res 1998;80:69–74. [25] Kim NH, Son Y, Jeong SO, et al. Tetrahydroabietic acid, a reduced abietic acid, inhibits the production of inflammatory mediators in RAW264.7 macrophages activated with lipopolysaccharide. J Clin Biochem Nutr 2010;46:119–25. [26] Gravante G, Caruso R, Sorge R, et al. Nanocrystalline silver: a systematic review of randomized trials conducted on burned patients and an evidence-based assessment of potential advantages over older silver formulations. Ann Plast Surg 2009;63: 201–5. [27] Cloninger MJ. Biological applications of dendrimers. Curr Opin Chem Biol 2002;6: 742–8. [28] Wang B, Navath RS, Romero R, et al. Anti-inflammatory and anti-oxidant activity of anionic dendrimer–N-acetyl cysteine conjugates in activated microglial cells. Int J Pharm 2009;377:159–68. [29] Bosnjakovic A, Mishra MK, Ren W, et al. Poly(amidoamine) dendrimer–erythromycin conjugates for drug delivery to macrophages involved in periprosthetic inflammation. Nanomedicine 2011;7:284–94. [30] Balogh L, Swanson DR, Donald A, et al. Dendrimer–silver complexes and nanocomposites as antimicrobial agents. Nano Lett 2001;1:18–21. [31] Preiser JC, Reper P, Vlasselaer D, et al. Nitric oxide production is increased in patients after burn injury. J Trauma 1996;40:368–71. [32] Chung YM, Rhee HK. Dendrimer-templated Ag-Pd bimetallic nanoparticles. J Colloid Interface Sci 2004;271:131–5. [33] Esumi K, Matsumoto T, Seto Y, et al. Preparation of gold-, gold/silver–dendrimer nanocomposites in the presence of benzoin in ethanol by UV irradiation. J Colloid Interface Sci 2005;284:199–203. [34] Li G, Luo Y. Preparation and characterization of dendrimer-templated Ag-Cu bimetallic nanoclusters. Inorg Chem 2008;47:360–4. [35] Liu Z, Wang X, Wu H, et al. Silver nanocomposite layer-by-layer films based on assembled polyelectrolyte/dendrimer. J Colloid Interface Sci 2005;287:604–11. [36] Castonguay A, Kakkar AK. Dendrimer templated construction of silver nanoparticles. Adv Colloid Interface Sci 2010;160:76–87. [37] Lesniak W, Bielinska AU, Sun K, et al. Silver/dendrimer nanocomposites as biomarkers: fabrication, characterization, in vitro toxicity, and intracellular detection. Nano Lett 2005;5:2123–30. [38] Maly J, Lampova H, Semeradtova A, et al. The synthesis and characterization of biotin–silver–dendrimer nanocomposites as novel bioselective labels. Nanotechnology 2009;20:385101. [39] Tse C, Zohdy MJ, Ye JY, et al. Enhanced optical breakdown in KB cells labeled with folate-targeted silver–dendrimer composite nanodevices. Nanomedicine 2010;7: 97–106. [40] Dastjerdi R, Montazer M. A review on the application of inorganic nano-structured materials in the modification of textiles: focus on anti-microbial properties. Colloids Surg B Biointerfaces 2010;79:5–18.
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Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles☆ Xuelai Liu a,1, Wei Hao a,1, Chun-Nam Lok b, Yue Chun Wang c, RuiZhong Zhang a, Kenneth K.Y. Wong a,⁎ a b c
Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China Department of Chemistry, The University of Hong Kong, Hong Kong, China Department of Physiology, Medical College, Ji Nan University, Guangzhou, China
a r t i c l e
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Article history: Received 29 August 2014 Accepted 6 September 2014 Available online xxxx Key words: Anti-inflammatory Silver nanoparticles Dendrimer Wound healing Drug delivery
a b s t r a c t Background: Our previous studies revealed that silver nanoparticles (AgNPs) promoted wound healing in part through their anti-inflammatory actions. As recent reports also suggested anti-inflammatory effects of dendrimers, we therefore undertook this study using dendrimer as the delivery system for AgNP to explore any potential synergistic anti-inflammatory efficacy. Methods: Lipopolysaccharide (LPS) was added to cultured RAW264.7 and J774.1 cells to mimic in vitro inflammation condition, followed by the addition of either silver dendrimer nanocomposite (Ag-DNC), AgNPs, or dendrimer. The levels of inflammatory markers TNF-alpha and interleukin-6 were assessed using ELISA assay. Furthermore, in vivo effects such of Ag-DNC, AgNPs, or dendrimer were studied in a burn wound model in mice. Results: Our results confirmed that both naked dendrimer and AgNPs had anti-inflammatory properties. In in vitro study, Ag-DNC was shown to have the best anti-inflammatory efficacy than AgNPs or dendrimer alone. In-vivo experiments also indicated that animals in the Ag-DNC group had the fastest healing time with the least inflammation. Conclusion: Our study would suggest that dendrimer could provide additional anti-inflammatory benefits and might be an excellent delivery system for silver nanoparticles for future clinical application. © 2014 Published by Elsevier Inc.
Silver nanoparticles (AgNPs) are nano-scale-sized pure silver measuring less than 100 nm in diameter [1]. Compared with the other forms of silver, the large active surface of AgNPs will induce unusual physicochemical properties and biological activities when exposed to cells or tissues [2]. Our previous studies have revealed that AgNPs possess significant anti-inflammatory effects [3,4]. Although the exact mechanism of AgNPs-mediated anti-inflammation still remains to be elucidated, some researchers have proposed that AgNPs access into target cells and block relevant inflammatory signaling pathways [5,6]. In clinical practice, the use of AgNPs has been gaining popularity, but mostly restricted to incorporation into woven fabrics in wound dressings. Dendrimers are nano-sized, branched, synthetic polymers with layered architectures first described by Tomalia et al. [7]. They have been shown to be promising carriers in bio-mimicry, diagnostics, and therapeutics. Currently the majority of dendrimer designs have been utilized in the field of drug delivery [8,9]: for anti-inflammatory [10–12], antimicrobial and antiviral drugs [13–18], as well anticancer agents [19]. Some studies have showed that naked dendrimer itself
☆ Disclosure: The authors report no conflicts of interest in this work. ⁎ Corresponding author at: Department of Surgery, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong SAR, China. Tel.: +852 22553486; fax: +852 28173155. E-mail address: [email protected] (K.K.Y. Wong). 1 Contributed equally to this work.
also has anti-inflammatory property [20–22]. We therefore hypothesized that if AgNPs were to be conjugated with dendrimer, as silver dendrimer nanocomposites (Ag DNC), the two nanoscale materials would have synergistic effects in terms of anti-inflammation. The purpose of this study was thus to confirm this hypothesis by comparing the anti-inflammatory actions mediated by Ag DNC with AgNPs, through in vitro and in vivo studies. 1. Materials and methods 1.1. Preparation of silver nanoparticles and dendrimer Synthesis of silver nanoparticles (AgNPs) has been described previously [3,4]. Final concentration of AgNPs solution produced was 1 mM. The mean diameter of silver nanoparticles was 10 nm (ranging from 5 to 15 nm), which was confirmed by transmission electron microscopy. Silver dendrimer nanocomposites (Ag DNC) used were “neutral” at biologic pH and consisted of G5 dendrimer and were kindly provided by Dr. L Balogh (Northeastern University, USA). The polymeric chain in dendrimer provided nitrogen atoms for the coordination and stabilization of AgNPs, and was utilized as delivery system to AgNPs (Fig. 1). Ag DNC used in this study had the following chemical and physical characteristics: acetamide surface; 3.1779 g solution containing 0.024402 g Ag (0) in 0.172945 g Ag DNC solution; pH = 6.72; Ag/Dendrimer molar ratio = 51.69; Ag(0)/composite ratio: 0.13673; The store
http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033 0022-3468/© 2014 Published by Elsevier Inc.
Please cite this article as: Liu X, et al, Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles, J Pediatr Surg (2014), http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033
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Fig. 1. Schematic representation of the polymeric chains in dendrimer stabilizing AgNPs, to form Ag DNC conjugate
concentration of Ag DNC was 73,500 μM and was dissolved into sterile water for various working concentration. The “naked” dendrimer (G5, 10 w/w percent solution in water) used in this study was purchased from Dendritic Nanotechnologies, (MI, U.S.A.). Stock concentration of dendrimer was 24,000 μM and they were dissolved into sterile water for different working concentration.
concentrations of Ag DNC (ranging from 50 to 400 μM) were added, followed by 10 mL MTT reagent to each well. 100 mL of detergent reagent was added to each well after purple precipitate was clearly visible under the microscope. The plate was sealed and put in the dark for 4 h at room temperature. Absorbance in each well was measured at 570 nm in a microtiter plate reader (Microplate Manager, Bio-Rad Laboratories). The average values from triplicate readings were calculated.
1.2. Cell culture RAW264.7 and J774.1, both mouse macrophage cell lines, were purchased from ATCC (VA, USA). The above cell lines were the same as those described in our previous study [4], aiming to accurately explore in vitro anti-inflammatory efficacy of Ag DNC and AgNPs. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL) and fetal bovine serum (FBS; 10%) and grown in 37°C incubator with 5% CO2 humidified atmosphere. The culture medium was changed two or three times per week. Upon reaching 80%–90% confluence, cells passage was performed by addition of 0.25% trypsin-EDTA, aspirating and dispensing in new flasks. Passage 5 cells were used in this study. 1.3. In vitro inflammation model Upon reaching 70% confluence of cultured cells, lipopolysaccharide (LPS; Sigma-Aldrich, USA) with 100 ng/mL of working solution concentration was added. Our previous study showed that AgNPs could effectively decrease LPS-induced TNF-α expression [4]. Therefore, in the present study, macrophage cells were incubated either with Ag DNC, AgNPs, dendrimer or no treatment respectively after the addition of LPS. The supernatant in each group was harvested post-incubation 1 h after addition of LPS. The levels of pro-inflammatory cytokines TNF-α and IL-6 were measured using TNF-α and IL-6 (Mono/Mono) ELISA kits (BD Biosciences, San Diego, CA, USA) according to manufacturer’s protocol. 1.4. MTT assay MTT assay kit was purchased from Roche (Germany) to measure cell viability and proliferation. Cultured cells (5 × 105) were plated out in triplicate into 96-wells plate. Three control wells with medium alone were used to provide blanks for absorbance readings. Various
1.5. Animals and burn wound model C57BL/6 N mice, between 14 and 16 weeks old and weighing 40–46 g, were obtained from the Laboratory Animal Unit, The University of Hong Kong. Mice were allowed diet and water ad libitum in a 12-h light, 12-h dark cycled room. Experimental protocol was approved by the Committee of the Use of Live Animals in Teaching and Research, The University of Hong Kong (CULATR 1974-09). The animals were randomly divided into 4 groups: Ag DNC, AgNPs, dendrimer, and untreated group (n=6). Anesthesia was achieved with pentobarbital sodium solution at a dose of 50 mg/kg by intra-peritoneal injection (Abbott Laboratories, USA). Both excisional wound model and burn wound model have been described previously [23,24]. In brief, a full-thickness piece of skin measuring 1.0 × 1.0 cm2 was excised on the back of each mouse using a pair of scissors for the excisional wound model. For the burn wound model, a burn template was established in a plastic 50-mL syringe by cutting a window (3.0 × 2.0 cm2) on one side, while the opposite half side was removed. This would allow a mouse to be laid on the template. The template was then put horizontally into a water bath at 70 °C for 30 s, followed by immediate placement of mouse in iced water bath to stop the burn process. This model would achieve approximately 6.7% deep partial thickness thermal injury of total body surface area, simulating scald injury in children. Dressings with either Ag DNC or AgNPs were used to cover the wound site. In both AgNPs and Ag DNC groups, the silver content in the dressing was 0.04 mg/cm 2[3,23]. In the dendrimer treatment group, the wound was covered with dendrimer-coated dressing. The dendrimer content for each dressing was 5 mg/cm 2, which was the same as Ag DNC groups. In the no treatment group, only medical dressing was covered as control. Postoperatively, all mice were kept warm until fully awake. The dressings were changed every 3 days until day 9. All animals
Please cite this article as: Liu X, et al, Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles, J Pediatr Surg (2014), http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033
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were humanely sacrificed by CO2 asphyxiation after wound was closed completely. 1.6. Morphometric assessment of wound closure For the assessment of wound healing, mice were photographed daily with a digital camera secured on a cantilever at a fixed distance. Wound closure analysis was performed three times in a singleblinded manner. Wound areas (cm 2) were calculated from wound perimeter tracings using Photoshop CS (Adobe, USA) until complete closure. The healing rate was expressed as a percentage of the original dorsal wound area on day 0 (excisional wound: 1.0 × 1.0 cm 2; burn wound: 3.0 × 2.0 cm 2) after wounding.
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Ag DNC was the most effective in reducing LPS-induced inflammation. Our result also supported that dendrimer alone had underlying in vitro anti-inflammatory effect. In our previous studies, we showed that the addition of AgNP did not result in significant apoptotic cell death [4]. In order to confirm if the addition of dendrimer to silver nanaoparticles would not alter the toxicity, MTT assay was conducted to evaluate cell viability. Here, more than 85% of both RAW264.7 and J774.1 survived after exposure to 100 μM concentration of Ag DNC [data not shown]. 2.2. Skin wound models revealed enhanced anti-inflammatory efficacy of Ag DNC
2. Results
The in vitro study indicated an enhancement of anti-inflammatory effect induced by Ag DNC. We next asked whether similar finding could be seen in vivo. As the process of wound healing is characterized by an inflammatory response, it is thus a good model for inflammation research. We first investigated the synergistic effects of Ag DNC in an excisional model. Here, when compared to untreated and dendrimer alone groups, both Ag DNC and AgNPs contributed to faster healing with similar healing times (Fig. 3). In the untreated and dendrimer groups, the mean closure time was 17.0 ± 0.7 and 16.3 ± 0.7 days respectively (p =0.07559), while in the AgNPs and Ag DNC groups, it was 12.1 ± 0.3 and 12.6 ± 0.5 days respectively (p =0.36322). This result was in contrast to our in vitro finding. We hypothesized that the reason why no difference was observed between Ag DNC and AgNPs in the in vivo model was due to minimal inflammation in the excisional wound model. In this case, the additional anti-inflammatory effects exerted by Ag DNC would not have been apparent. As a result, we next turned to a burn wound model, which would offer significantly more post-injury inflammation. In the next experiment, we compared the pro-healing efficacy of Ag DNC, AgNPs and dendrimer using a burn wound model. Here, animals treated with Ag DNC had the fastest healing time of 23.8 ± 0.8 days when compared to AgNPs (27.2 ± 1.2 days), dendrimer (31.5 ± 0.8 days) and untreated (38.0 ± 0.9 days) (p b 0.001) (Fig. 4A). This was also reflected in the rate of healing (Fig. 4B).
2.1. Ag DNC suppresses TNF-α and IL-6 production in vitro
3. Discussion
Activated macrophages play an important role in the initiation of inflammation through production of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin (IL-6). We showed previously that AgNPs were able to suppress the production of TNF-α, IL-6 and TGF-β [3,4]. In this study, we employed the same in vitro inflammation model to assess and compare the effects of Ag DNC, AgNPs and “naked” dendrimer. Here, TNF-α level in Ag DNC group was decreased by 36.6% in RAW264.7 and by 47.4% in J774.1 (p b 0.001). In the AgNPs group, TNF-α level was decreased by 21.2% in RAW264.7 and by 41% in J774.1 (p b 0.01). Reduction of TNF-α was also seen in the dendrimer group: 9.3% in RAW264.7 and 17.7% in J774.1 (p b 0.05) [Fig. 2A&B]. In terms of IL-6 production, the level was decreased in Ag DNC group by 41.1% in RAW264.7 and by 24.6% in J774.1 (p b 0.001). In contrast, AgNPs treatment decreased IL-6 level by 33.6% in RAW264.7 (p b 0.01) and by 18.1% in J774.1. For the dendrimer group, it was decreased by 12.1% in RAW264.7 (p b 0.05) and by 7.7% in J774.1 respectively (p = 0.065) (Fig. 2C&D). Taken together, it would suggest that
Inflammation is a double-edged sword. On one hand, it is a protective mechanism against the potential invasion of a variety of microorganisms. On the other hand, de-regulation can lead to chronic inflammatory conditions. From the point of wound healing, chronic inflammation results in delayed or even non-healing. In children, burn injuries from scalding are commonly seen and can give rise to significant physical and psychological morbidity. Persistent inflammatory response after wounding will also lead to delayed healing and increased complications. Therefore, a more effective treatment to inhibit local inflammatory response may help accelerate wound healing. Inflammation is regulated by a variety of immune cells and cytokines, in which activated macrophages play an essential role in the initiation and amplification of inflammation through the production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL-6, and interleukin-1β (IL-1β) [25]. Our previous studies already revealed that silver nanoparticles (AgNPs) had significant anti-inflammatory effects through the inhibition to macrophage and decreased production of inflammatory cytokines [3,4]. In this regard, Nadworny and Bhol postulated the potential mechanism of AgNPsmediated anti-inflammation to be intracellular blocking of inflammatory pathways and down-regulated pro-inflammatory cytokines [5,6]. With advance in nanotechnology, various nano delivery systems have been used to carry drug molecules and genes. Among these, dendrimer is an important and effective nano carrier platform [26]. It is a hyper-branched, tree-like polymer with a defined structure whose size and molecular weight can be controlled rigorously due to the
1.7. Immunohistochemistry (IHC) The healing skin tissues on day 3 post-wounding were harvested and processed. Endogenous peroxidase activity was quenched by treatment with 3% H2O2/CH3OH for 30 min. Sections were incubated for 1 h at room temperature by using blocking solution containing 5% concentration of normal goat serum (Dako Bioresearch, USA). For antigen retrieval of neutrophil, macrophage, interleukin-6 (IL-6), and tumor necrosis factors-alpha (TNF-α) staining, sections were further blocked against nonspecific binding using 10% normal goat serum before primary antibody (Table 1) was added respectively. The sections were incubated overnight at 4°C and then further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, USA) for 2 h at room temperature. Positive signals were developed by using 3,3’-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin. 1.8. Statistical analysis Statistical analysis was performed using Student’s paired t test and p-value of b 0.05 was considered statistically significant.
Table 1 Primary monoclonal antibodies in this study. Antibody against
Abbreviation
Dilution
Specie
Manufacturer
Neutrophil Macrophage Interleukin-6 Tumor necrosis factors-alpha
PMN F4/80 IL-6 TNF-α
1:10,000 1:200 1:500 1:50
Rabbit Mouse Rabbit Mouse
Sant Crutz Abcam Abcam Abcam
Please cite this article as: Liu X, et al, Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles, J Pediatr Surg (2014), http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033
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Fig. 2. TNF-α expression levels measured by ELISA for (A) RAW264.7 and (B) J774.1cells, as well as IL-6 expression levels measured by ELISA for (C) RAW264.7 and (D) J774.1 cells. Respective cell lines were incubated with 100 μM Ag DNC, AgNPs or dendrimer alone after the addition of LPS stimulation. Values are the mean ± SE for all groups; * p b 0.05, **p b 0.01, and ***p b 0.001.
iterative step-by-step synthesis. These structural features, together with the high density of chemical-reactive functions on the outer shell of these molecules, potentially widen their application in medicine [27]. An unexpected property of dendrimer reported recently was the antiinflammation. Chauhan et al. reported that dendrimers had potential to inhibit inflammation due to bearing of amine or hydroxyl surface groups, and therefore exhibited anti-inflammatory activities in carrageenan-induced paw edema model [21]. In this regard, Fruchon et al. also suggested that dendrimer-activated monocyte had
immunnosuppressive phenotype and could serve as new nano-tools to promote anti-inflammatory and immunosuppressive activation in human monocytes [22]. Furthermore, some conjugated dendrimers for drug delivery were also revealed to exert anti-inflammatory actions [28,29]. Lastly, dendrimer carboxylate salts could carry high local concentrations of silver, due to a large number of active surface groups [30]. Taken together, we hypothesized that if dendrimers were used to carry AgNPs, the synergistic effect might greatly improve the antiinflammatory efficacy.
Fig. 3. The effect of Ag DNC on excisional wound. Excisional wounds were created on the dorsa of C57BL/6 N mice, and they were divided into four groups (n = 6) according to treatment with Ag DNC, AgNPs, dendrimer alone or no treatment as control. The wounds were inspected daily, and the time of healing was recorded.
Please cite this article as: Liu X, et al, Dendrimer encapsulation enhances anti-inflammatory efficacy of silver nanoparticles, J Pediatr Surg (2014), http://dx.doi.org/10.1016/j.jpedsurg.2014.09.033
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Fig. 4. The effect of Ag DNC on thermal wound. Thermal wounds were created on the dorsa of C57BL/6 N mice and wounds were treated with Ag DNC, AgNPs, dendrimer alone or no treatment as control. (A) Wound appearance and healing times of the four groups. (B) Rate of wound closure at various time points.
In the present study, both in vitro and in vivo studies confirmed that dendrimer had anti-inflammatory properties. Furthermore, when Ag DNC was used in LPS-induced inflammation model in vitro, it showed significantly more inhibition of the production of pro-inflammatory cytokines than when AgNPs was used alone. Extrapolating this to the in vivo setting, we would anticipate a similar synergistic effect between AgNPs and dendrimer. Nonetheless, we noticed no significant difference in wound healing between the two groups in the excisional wound model. This could be due to the fact that the excisional wound, on one hand, was a relatively clean model with little post-injury inflammatory response. On the other hand, the excised area created in the present study was relatively small. As wound size was another important issue which would influence the degree of inflammation and healing time
[31], little inflammation was observed. These could explain why the additional anti-inflammatory effects exerted by Ag-DNC were not apparent, as the use of AgNPs alone already could exert sufficient antiinflammatory effects. Another possible explanation could be that dendrimer in Ag DNC had no anti-inflammatory effect, and their role was only a biological carrier for AgNPs. When we changed to a burn wound model, animals treated with Ag DNC showed the fastest healing time and rate when compared to AgNPs. This would suggest that conjugating AgNPs with dendrimer indeed had synergistic effects and both contributed to better wound healing. So far, reports of explorations to biological and medical effects in dendrimer–metal conjugates are sparse. In terms of Ag DNC, most reports have concentrated on its preparation and physiochemical characteristics [32–36]. Lesniak et al.
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reported fluorescence of dendrimer/silver composites, and their surface charge, cellular internalization, toxicity, and cell labeling capabilities. Their results and others indicated that the composites had potential application as cell biomarkers [37,38]. On the other hand, Tse et al. explored the effect of dendrimer/silver on human oral epidermoid cancer cells and found that they exhibited a significant reduction in breakdown threshold and thus selectively promoted intracellular laser-induced optical breakdown [39]. Furthermore, atomic/molecular dispersion of the guest in dendrimer host was regarded to have potential to create unique physical and chemical properties [40]. Our current finding supported that silver dispersion in dendrimer had better biological property as well. In summary, we elucidated that dendrimer could be an ideal biological carrier for AgNPs to reduce the in vitro and in vivo inflammation, and could effectively promote wound healing, especially in those with significant inflammation. Regarding future work, we will target dendrimer to further assess the delivery and release efficacy, so as to bring their potential clinical application closer to fruition. Acknowledgments The authors would like to thank Dr. L Balogh (Department of Pharmaceutical Sciences, Northeastern University, Boston, USA) for his kind provision of silver dendrimer nanocomposites in this study. References [1] Wong KKY, Liu XL. Silver nanoparticles—the real “silver bullet” in clinical medicine? MedChemComm 2010;1:125–31. [2] Caruso DM, Foster KN, Blome-Eberwein SA, et al. Randomized clinical study of Hydrofiber dressing with silver or silver sulfadiazine in the management of partial-thickness burns. J Burn Care Res 2006;27:298–309. [3] Tian J, Wong KK, Ho CM, et al. Topical delivery of silver nanoparticles promotes wound healing. ChemMedChem 2007;2:129–36. [4] Wong KK, Cheung SO, Huang L, et al. Further evidence of the anti-inflammatory effects of silver nanoparticles. ChemMedChem 2009;4:1129–35. [5] Nadworny PL, Wang J, Tredget EE, et al. Anti-inflammatory activity of nanocrystalline silver in a porcine contact dermatitis model. Nanomedicine 2008;4:241–51. [6] Bhol KC, Schechter PJ. Effects of nanocrystalline silver (NPI 32101) in a rat model of ulcerative colitis. Dig Dis Sci 2007;52:2732–42. [7] Tomalia DA. Dendrimer research. Science 1991;252:1231. [8] Padilla De Jesús OL, Ihre HR, Gagne L, et al. Polyester dendritic systems for drug delivery applications: in vitro and in vivo evaluation. Bioconjug Chem 2002;13: 453–61. [9] Ihre HR, Padilla De Jesús OL, Szoka Jr FC, et al. Polyester dendritic systems for drug delivery applications: design, synthesis, and characterization. Bioconjug Chem 2002;13:443–52. [10] Asthana A, Chauhan AS, Diwan PV, et al. Poly(amidoamine) (PAMAM) dendritic nanostructures for controlled site-specific delivery of acidic anti-inflammatory active ingredient. AAPS PharmSciTech 2005;6:E536–42. [11] Theoharis S, Krueger U, Tan PH, et al. Targeting gene delivery to activated vascular endothelium using anti E/P-Selectin antibody linked to PAMAM dendrimers. J Immunol Methods 2009;343:79–90. [12] Gajbhiye V, Palanirajan VK, Tekade RK, et al. Dendrimers as therapeutic agents: a systematic review. J Pharm Pharmacol 2009;61:989–1003. [13] Chen CZ, Cooper SL. Interactions between dendrimer biocides and bacterial membranes. Biomaterials 2002;23:3359–68. [14] Chen CZ, Beck-Tan NC, Dhurjati P, et al. Quaternary ammonium functionalized poly (propylene imine) dendrimers as effective antimicrobials: structure–activity studies. Biomacromolecules 2000;1:473–80.
[15] Landers JJ, Cao Z, Lee I, et al. Prevention of influenza pneumonitis by sialic acidconjugated dendritic polymers. J Infect Dis 2002;186:1222–30. [16] Witvrouw M, Fikkert V, Pluymers W, et al. Polyanionic (i.e., polysulfonate) dendrimers can inhibit the replication of human immunodeficiency virus by interfering with both virus adsorption and later steps (reverse transcriptase/integrase) in the virus replicative cycle. Mol Pharmacol 2000;58:1100–8. [17] Gazumyan A, Mitsner B, Ellestad GA. Novel anti-RSV dianionic dendrimer-like compounds: design, synthesis and biological evaluation. Curr Pharm Des 2000;6:525–46. [18] Bourne N, Stanberry LR, Kern ER, et al. Dendrimers, a new class of candidate topical microbicides with activity against herpes simplex virus infection. Antimicrob Agents Chemother 2000;44:2471–4. [19] Quintana A, Raczka E, Piehler L, et al. Design and function of a dendrimer-based therapeutic nanodevice targeted to tumor cells through the folate receptor. Pharm Res 2002;19:1310–6. [20] Navath RS, Kurtoglu YE, Wang B, et al. Dendrimer–drug conjugates for tailored intracellular drug release based on glutathione levels. Bioconjug Chem 2008;19:2446–55. [21] Chauhan AS, Diwan PV, Jain NK, et al. Unexpected in vivo anti-inflammatory activity observed for simple, surface functionalized poly(amidoamine) dendrimers. Biomacromolecules 2009;10:1195–202. [22] Fruchon S, Poupot M, Martinet L, et al. Anti-inflammatory and immunosuppressive activation of human monocytes by a bioactive dendrimer. J Leukoc Biol 2009;85: 553–62. [23] Liu X, Lee PY, Ho CM, et al. Silver nanoparticles mediate differential responses in keratinocytes and fibroblasts during skin wound healing. ChemMedChem 2010;5: 468–75. [24] Cribbs RK, Luquette MH, Besner GE. A standardized model of partial thickness scald burns in mice. J Surg Res 1998;80:69–74. [25] Kim NH, Son Y, Jeong SO, et al. Tetrahydroabietic acid, a reduced abietic acid, inhibits the production of inflammatory mediators in RAW264.7 macrophages activated with lipopolysaccharide. J Clin Biochem Nutr 2010;46:119–25. [26] Gravante G, Caruso R, Sorge R, et al. Nanocrystalline silver: a systematic review of randomized trials conducted on burned patients and an evidence-based assessment of potential advantages over older silver formulations. Ann Plast Surg 2009;63: 201–5. [27] Cloninger MJ. Biological applications of dendrimers. Curr Opin Chem Biol 2002;6: 742–8. [28] Wang B, Navath RS, Romero R, et al. Anti-inflammatory and anti-oxidant activity of anionic dendrimer–N-acetyl cysteine conjugates in activated microglial cells. Int J Pharm 2009;377:159–68. [29] Bosnjakovic A, Mishra MK, Ren W, et al. Poly(amidoamine) dendrimer–erythromycin conjugates for drug delivery to macrophages involved in periprosthetic inflammation. Nanomedicine 2011;7:284–94. [30] Balogh L, Swanson DR, Donald A, et al. Dendrimer–silver complexes and nanocomposites as antimicrobial agents. Nano Lett 2001;1:18–21. [31] Preiser JC, Reper P, Vlasselaer D, et al. Nitric oxide production is increased in patients after burn injury. J Trauma 1996;40:368–71. [32] Chung YM, Rhee HK. Dendrimer-templated Ag-Pd bimetallic nanoparticles. J Colloid Interface Sci 2004;271:131–5. [33] Esumi K, Matsumoto T, Seto Y, et al. Preparation of gold-, gold/silver–dendrimer nanocomposites in the presence of benzoin in ethanol by UV irradiation. J Colloid Interface Sci 2005;284:199–203. [34] Li G, Luo Y. Preparation and characterization of dendrimer-templated Ag-Cu bimetallic nanoclusters. Inorg Chem 2008;47:360–4. [35] Liu Z, Wang X, Wu H, et al. Silver nanocomposite layer-by-layer films based on assembled polyelectrolyte/dendrimer. J Colloid Interface Sci 2005;287:604–11. [36] Castonguay A, Kakkar AK. Dendrimer templated construction of silver nanoparticles. Adv Colloid Interface Sci 2010;160:76–87. [37] Lesniak W, Bielinska AU, Sun K, et al. Silver/dendrimer nanocomposites as biomarkers: fabrication, characterization, in vitro toxicity, and intracellular detection. Nano Lett 2005;5:2123–30. [38] Maly J, Lampova H, Semeradtova A, et al. The synthesis and characterization of biotin–silver–dendrimer nanocomposites as novel bioselective labels. Nanotechnology 2009;20:385101. [39] Tse C, Zohdy MJ, Ye JY, et al. Enhanced optical breakdown in KB cells labeled with folate-targeted silver–dendrimer composite nanodevices. Nanomedicine 2010;7: 97–106. [40] Dastjerdi R, Montazer M. A review on the application of inorganic nano-structured materials in the modification of textiles: focus on anti-microbial properties. Colloids Surg B Biointerfaces 2010;79:5–18.
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