Antimicrobial activity of silver nanoparticles encapsulated in poly-N-isopropylacrylamide-based polymeric nanoparticles
Received 7 October 2017
Accepted for publication 21 November 2017
Published 3 January 2018 Volume 2018:13 Pages 235—249
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Prof. Dr. Thomas J. Webster
Muhammad Qasim,1 Nopphadol Udomluck,1 Jihyun Chang,1 Hansoo Park,1 Kyobum Kim2
1School of Integrative Engineering, Chung-Ang University, Seoul, 2Division of Bioengineering, Incheon National University, Incheon, Republic of Korea
Abstract: In this study, we analyzed the antimicrobial activities of poly-N-isopropylacrylamide (pNIPAM)-based polymeric nanoparticles encapsulating silver nanoparticles (AgNPs). Three sizes of AgNP-encapsulating pNIPAM- and pNIPAM-NH2-based polymeric nanoparticles were fabricated. Highly stable and uniformly distributed AgNPs were encapsulated within polymeric nanoparticles via in situ reduction of AgNO3 using NaBH4 as the reducing agent. The formation and distribution of AgNPs was confirmed by UV-visible spectroscopy, transmission electron microscopy, and inductively coupled plasma optical emission spectrometry, respectively. Both polymeric nanoparticles showed significant bacteriostatic activities against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria depending on the nanoparticle size and amount of AgNO3 used during fabrication.
Keywords: pNIPAM, silver nanoparticles, antimicrobial activities, surface charge
Various bacteria and fungi cause postsurgical infections. Such infections are frequently associated with biomedical implants, including prosthetic joints and fracture fixation materials.1–3 Because of the slow progress of such infections, detecting them at an early stage is challenging. In the absence of an effective antibiotic, failure to treat postsurgical infections with classical antibiotics is a serious global issue.1,4,5 A bacterial strain carrying the NDM-1 gene was recently reported to be resistant to strong antibiotics; thus, microbial drug resistance and the lack of effective antimicrobial agents pose great challenges.6
Non-conventional treatments for microbial infections include antimicrobial peptides, small molecule inhibitors, naturopathic therapy, phytotherapy, and metallic particles.7–9 Previously, the use of silver nanoparticles (AgNPs) was suggested for eliminating microbial infections.10 Broad-spectrum antimicrobial activities of Ag against pathogens, including both Gram-positive and Gram-negative bacteria, have been reported.11,12 Ag was complexed with other materials to give them with antibacterial properties. For example, the antimicrobial activity of Ag-impregnated nylon fibers against Staphylococcus aureus and Candida albicans was evaluated for wound dressing.13,14 AgNPs have also been used to treat Escherichia coli because of their ability to damage bacterial cell walls.15 Currently, AgNPs in burn ointments, wound dressings, and Ag-coated medical devices such as catheters, vascular grafts, and endotracheal tubes are used to prevent and treat bacterial infections.16–18
Polymeric nanoparticles are an excellent vehicle for AgNP delivery because they can easily be modified according to the target site; moreover, their size, morphology, and surface charge can be controlled by changing the ratio of monomer to cross-linker during nanoparticle fabrication.19,20 Another advantage of encapsulating AgNPs inside a polymeric nanoparticle is that nanoparticles distributed in a polymeric nanoparticle show low aggregation.21,22 Moreover, polymeric nanoparticles reduce concerns regarding the cytotoxicity of AgNPs because the gel limits the direct exposure of a patient’s cells to AgNPs and release AgNPs slowly.23,24 Previously, it was reported that encapsulation of AgNPs inside a polymeric nanoparticle did not affect the size and morphology of the nanocomposite.25 Polymeric nanoparticles are an attractive vehicle for sustained AgNP release during local therapy because of their tunable size, numerous functional groups, thermo-responsiveness, high loading capacity, good stability, biocompatibility, and anionic charge.21,24,26
In this study, we fabricated three sizes of two polymeric nanoparticle types: poly-N-isopropylacrylamide (pNIPAM) and pNIPAM-NH2. In each polymeric nanoparticle, we encapsulated three different concentrations of AgNPs using a water-soluble reducing agent. We then examined the effects of polymeric nanoparticle surface charge and AgNP concentration on antimicrobial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. Our aim was to increase antimicrobial activity by encapsulating AgNPs inside a polymeric nanoparticle to limit their cell uptake and cytotoxicity. Our AgNP-encapsulated polymeric nanoparticles (AgNP-pNIPAM/pNIPAM-NH2 nanoparticles) showed significant bacteriostatic activities against E. coli and S. aureus that were dependent on nanoparticle size and AgNO3 concentration. The resulting AgNP-pNIPAM/pNIPAM-NH2 polymeric nanocomposite can be developed for antimicrobial bioengineering applications.
Materials and methods
NIPAM, AgNO3, NaOH, sodium dodecyl sulfate, and NaBH4 were purchased from Sigma-Aldrich Co. (St Louis, MO, USA) and used after further purification through recrystallization. Ammonium persulfate (APS), N,N-methylenebisacrylamide (BIS), and N-(3-aminopropyl) methacrylamide hydrochloride (APMAAHC) were purchased from Polysciences (Warrington, PA, USA). Luria-Bertani (LB) broth medium was purchased from Oxoid (Basingstoke, UK). E. coli (25922) and S. aureus (25923) were purchased from ATCC (Manassas, VA, USA). DMEM containing glucose, phosphate-buffered saline, fetal bovine serum, penicillin G (pen; 10,000 U/mL), streptomycin (strep; 100 μg/mL), and amphotericin B (AmB; 25 μg/mL) were purchased from GE Healthcare Life Sciences (Little Chalfont, UK). Human adipose tissue was obtained from the Korea Cancer Center Hospital under the guidelines of the Institutional Review Board at Chung-Ang University (Seoul, South Korea). MTT and DMSO were purchased from Sigma-Aldrich Co. Deionized water (DW) was used to prepare solutions and for washing. All chemicals were used as received without further purification.
Synthesis of pNIPAM/pNIPAM-NH2 nanoparticles
pNIPAM nanoparticles were fabricated by conventional radical polymerization of NIPAM.28,29 Depending on the NIPAM to cross-linker ratio, three different sizes of pNIPAM nanoparticles (between 100–500 nm) were prepared. Specifically, 0.95 g of NIPAM with different amounts of BIS (0.026 g for 50:1, 0.013 g for 100:1, and 0.0065 g for 200:1) was dissolved in 195 mL of DW, and the resulting solutions were transferred into a three-necked round-bottomed flask with a constant supply of argon gas. Sodium dodecyl sulfate was added in the same ratio as BIS as a surfactant. The solution was heated to 58°C–65°C with a constant supply of argon gas for 1 h. Next, 5 mL of APS solution (0.12 g of APS dissolved in 5 mL of DW) was injected into the solution to initiate polymerization and the reaction was allowed to proceed for 4 h.30 Argon gas was purged through the solution until the end of the reaction to avoid any contact with oxygen, which may intercept radicals and disrupt polymerization. The resulting dispersion was dialyzed in DW using a porous membrane (6,000–8,000 Da molecular weight cutoff) and freeze-dried. The resulting pNIPAM-based nanoparticles are referred to as G1, G2, and G3, representing the nanoparticles prepared using three NIPAM:BIS ratios (0.0065 g BIS for 200:1, 0.013 g BIS for 100:1, and 0.026 g BIS for 50:1, respectively), as shown in Figure 1A. To fabricate polymeric nanoparticles with an amine (NH2) group, 0.065 g of APMAAHC was added to the three different NIPAM:BIS ratio reaction solutions, creating three different sizes of pNIPAM-NH2 nanoparticles. The resulting pNIPAM-NH2-based polymeric nanoparticles were similarly referred to as G1, G2, and G3, as shown in Figure 1B. The sizes of the prepared polymeric nanoparticles were characterized by dynamic light scattering (DLS).
Figure 1 Average sizes of poly-N-isopropylacrylamide (pNIPAM) nanoparticle groups G1–G3 (A), and average sizes of pNIPAM-NH2 nanoparticle groups G1–G3 (B).
Preparation of AgNP-polymeric nanoparticles
To encapsulate AgNPs inside the polymeric nanoparticles, 30 mg of each polymeric nanoparticle (ie, G1, G2, and G3 with and without NH2) was first incubated in 10 mM NaBH4 solution in DW (5 mL) for 5 h. Next, the solutions were mixed with freshly prepared AgNO3 solution (pH 7.0, 5 mL) and further incubated for 5 h at room temperature.31 Before use, the pH of the AgNO3 solutions was adjusted to pH 7 with NaOH and a pH meter. The AgNO3 and polymeric nanoparticle solutions were centrifuged at 11,000 rpm for 40 min and then washed with DW. This process was repeated three times to remove unreduced Ag+ or excess AgNPs located outside the polymeric nanoparticle network. Finally, 5 mL of DW was added to each sample before storing it at 8°C. After washing, the AgNP pNIPAM/pNIPAM-NH2 nanoparticle composites were characterized by UV-visible spectroscopy, transmission electron microscopy (TEM), and inductively-coupled plasma optical emission spectrometry (ICP-OES). The overall fabrication scheme was described in Figure 2.
Figure 2 Schematic diagram of the synthesis of silver nanoparticle (AgNP)-encapsulated poly-N-isopropylacrylamide (pNIPAM)-based nanoparticle platforms.
Characterization of AgNP-encapsulating polymeric nanoparticles
The sizes of three fabricated pNIPAM and pNIPAM-NH2 polymeric nanoparticles were determined by DLS using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The mean particle diameter (z-average) and particle size distribution (polydispersity index) were measured at 25°C. The spectral absorption of the three AgNP-encapsulating pNIPAM and pNIPAM-NH2 nanoparticle size groups was assessed using a UV-visible spectrophotometer (Synergy™ HT reader, BioTek Instruments, Winooski, VT, USA) in the range of 300–600 nm.27 All experiments were performed in triplicate to determine mean and SD values. The compositions of the resulting AgNP-encapsulating pNIPAM and pNIPAM-NH2 nanoparticle size groups were determined by ICP-OES. The morphological characteristics of the AgNP-encapsulating pNIPAM and pNIPAM-NH2 nanoparticle groups were observed by high-resolution TEM (JEM 3010, JEOL, Tokyo, Japan). For TEM, a drop of AgNP-encapsulating polymeric nanoparticles solution in DW was placed on a copper grid and allowed to dry at room temperature, and then placed in the sample holder of the TEM instrument and analyzed.
Assessment of antimicrobial activity
The antimicrobial activities of the prepared AgNP-polymeric nanoparticles against E. coli and S. aureus were investigated. All bacterial cultures were performed in LB broth. To promote the transition of bacterial cultures into the exponential growth phase, single colonies of E. coli or S. aureus were transferred to separate flasks containing LB broth and incubated overnight at 37°C with shaking at 175 rpm, and then diluted to 108 CFU/mL based on the OD at 600 nm (OD600=1). To evaluate the antimicrobial activities of the AgNP pNIPAM/pNIPAM-NH2 nanoparticle size groups, 40 μL of each polymeric nanoparticle-AgNP composite (G1, G2, and G3 with and without NH2) were added to 160 μL of bacterial suspension (108 CFU/mL) during the exponential growth phase in 96-well plates, and the samples were incubated at 37°C. Pure cultures were used as positive controls and culture media without cells were used as a negative control. All groups were cultured in triplicate. Bacterial growth was monitored by determining the OD of the bacterial suspension at 600 nm4 and the OD600 values were recorded at 0, 1, 2, 3, 4, 5, and 24 h of incubation.
Plate diffusion method
To investigate the antimicrobial activity of AgNPs encapsulated in nanogels on solid media, E. coli and S. aureus (1×106 cells/mL) were cultured on MH agar plates and the plates were dried at room temperature. A well, 8 mm in diameter, was made in each plate by gel puncture. Next, 1 mg/mL of each nanogel with AgNPs was dissolved in autoclaved distilled water and 0.5 mL of this solution was applied to the plates. Each test was performed in triplicate and a pure bacterial culture was used as a control. The plates were incubated at 37°C for 24 h. Antimicrobial activity was measured as the average diameter (mm) of the zone of inhibition around each well.32
X-ray photoelectron spectroscopy (XPS) analysis
XPS was performed using a Leybold LH Max 200 surface analysis system (Leybold, Cologne, Germany) operated with an Mg Kα source, 200 W. Prior to XPS analysis, all samples were thoroughly dried under vacuum. Data analysis was carried out using the Origin 8 analysis programs. The binding energy scales of the high resolution spectra were calibrated by assigning the most intense C1s high resolution peak a binding energy of 286.08 eV (C−N) and 287.58 eV (C=O). A linear function was used to model the background.
The cytotoxicities of the AgNP-polymeric nanoparticles to human adipose stem cells (hASCs) were determined by the MTT assay. The study was approved by the ethical review board of Chung Ang University and prior informed consent was signed by the tissue donor. The hASCs were cultured in DMEM containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution (pen/strep/AmB) at room temperature under a 5% CO2 atmosphere in a humidified incubator. A cell suspension (80 μL; 5,000 cells/well) was mixed with 20 μL of AgNP-polymeric nanoparticles on a 96-well plate for the MTT assay. A suspension of hASCs (80 μL) with 20 μL of DW and no nanoparticle was seeded as a positive control. In a different plate, hASC suspensions containing 20 μL of each size group of the pNIPAM and pNIPAM-NH2 AgNP-nanoparticles were seeded. Each size group (G1, G2, and G3 with and without NH2) was tested in triplicate.
According to the instructions from the MTT assay supplier (Sigma-Aldrich Co.), after incubating the cultures for 48 h, 10 μL of AmB solution was added to each well and incubated for 4 h. Next, 0.1 mL of isopropanol with 0.04 N hydrochloric acid was added to each well. All solutions in the wells were thoroughly mixed by repeated pipetting with a multichannel pipette. Absorbance was recorded using a Synergy™ HT microplate reader (BioTek Instruments) at 570 and 690 nm for quantification.
Absorbance data of the cytotoxicity of the AgNP-polymeric nanoparticles were expressed as the mean ± SD (n=3) in Figure 3. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was performed to determine statistical significance, by using Graphpad Prism software (La Jolla, CA, USA). A value of p<0.05 was considered to denote statistical significance as compared with the positive control group.
Characterization of AgNP encapsulating pNIPAM and pNIPAM-NH2 polymeric nanoparticles
The sizes of the prepared pNIPAM/pNIPAM-NH2 polymeric nanoparticles were determined by DLS. The average sizes of the G1, G2, and G3 pNIPAM nanoparticles were 131±78, 312±71, and 483±35 nm, respectively (Figure 1A), while the average sizes of the G1, G2, and G3 pNIPAM-NH2 nanoparticles were 135±59, 174±10, and 532±48 nm, respectively (Figure 1B).
UV-visible absorption spectrum exhibited a proper encapsulation of AgNP in polymeric nanoparticles (Figures 4 and 5). Previous studies showed that UV-visible absorption spectrum results are sensitive to the formation of AgNPs and that the absorption peaks depend on particle diameter and shape.25,34 The AgNP absorption band is in the 400–500 nm range.35 Previous results have suggested that AgNPs of smaller diameters are obtained at larger absorption doses.36 Figures 4A and 5A show the UV-visible spectra of AgNPs encapsulated in the pNIPAM and pNIPAM-NH2 AgNP-nanoparticles. The results confirmed that the AgNP fabrication process was consistent in terms of the spectral absorbance of the gels. The UV-visible spectra of our AgNP-polymeric nanoparticles showed a maximum absorbance at 437 nm, as shown in Figures 4A and 5A, which confirms the encapsulation of AgNPs inside the polymeric nanoparticles. In previous studies, AgNPs (2–5 nm) embedded in polymer microgels exhibited the same absorption bands in poly(methylmethacrylate-co-butyl acrylate-co-acrylic acid), polyethylene imine, and silica gels.34,37,38 In the present study, all absorption peaks were similar and all AgNP-polymeric nanoparticles showed a maximum absorption at 437 nm (Figures 4A and 5A), suggesting that the AgNPs encapsulated within the AgNP-polymeric nanoparticles are similar in size and morphology. Furthermore, the absence of a peak at 560 nm confirms that there was no AgNP aggregation or cluster formation inside the AgNP-polymeric nanoparticles.39
Figure 4 (A) UV-visible spectra of silver nanoparticle (AgNP)-encapsulated poly-N-isopropylacrylamide (pNIPAM) nanoparticle groups G1–G3, and (B) Ag peak absorbance at 435 nm in gels containing 5 and 10 mM concentrations of AgNO3.
Figure 5 (A) UV-visible spectra of silver nanoparticle (AgNP)-encapsulated poly-N-isopropylacrylamide (pNIPAM)-NH2 nanoparticle groups G1–G3, and (B) Ag peak absorbance at 435 nm in gels containing 5 and 10 mM concentrations of AgNO3.
Using the current method, we obtained a homogeneous distribution of AgNPs throughout each polymeric nanoparticle. Moreover, the Ag salts loaded in the cross-linked AgNP-polymeric nanoparticles were extensively reduced by NaBH4, after which the color of the solution quickly changed to opaque brown.40 These results indicate that the AgNPs were completely encapsulated within the AgNP-polymeric nanoparticles with strong localization and stabilization provided by the three-dimensional network within the polymeric nanoparticles. In contrast, polymer functional groups such as −OH, −CONH, and −COOH promote the stabilization of AgNPs through surface adsorption.20 Figures 4B and 5B show the changes in absorbance for different concentrations of AgNO3 (5 and 10 mM) in the pNIPAM and pNIPAM-NH2 AgNP-nanoparticles. The color of the AgNP polymeric nanoparticle solution was dependent on the concentration of AgNO3. With the higher AgNO3 concentration (10 mM), the color of the solution changed from light brown to dark brown, and the UV-visible spectrum peak at 437 nm was larger. The absorption peaks at 435 nm, as shown in Figures 4A and 5A, represent the characteristic peaks of surface plasmon resonance absorption by AgNPs.
Surface morphology of the resulting nanoparticles was evaluated using TEM (Figures 6 and 7). The images indicated that the morphologies for pNIPAM and pNIPAM-NH2 AgNP-nanoparticles prepared with 5 or 10 mM AgNO3 clearly depicted the spherical surfaces of the nanoparticles. AgNPs ranging from 1 to 35 nm in diameter were visible inside the polymeric nanoparticles (Figure S1A and B). The size of the AgNPs changed with the concentration of AgNO3 used during fabrication, which affects the nanoparticle’s internal network and surface charge.4 The pNIPAM-NH2 AgNP-nanoparticles contained larger AgNPs than the pNIPAM AgNP-nanoparticles due to the extra positive charge over the pNIPAM-NH2 surface as shown in Figures 6 and 7.
To confirm functionalization of amine group on pNIPAM polymer, XPS analysis was performed. The XPS spectra of pNIPAM-functionalized samples are shown in Figure 8. The high resolution C1s, O1s, N1s spectra (to determine molecular bonding environment), were obtained, and done with curve fitting function in Origin software. For the C1s core-level, the typical characteristics of the amide groups comprising the backbone of the pNIPAM were recorded at 286.08 eV (C−N) and 287.58 eV (C=O), corresponding with the record at 531.08 eV (C=O) in the O1s core-level, and for the N1s core-level peaks at 399.48 eV (C−N). For pNIPAM-NH2 nanogels, the amine group (secondary) was confirmed by the record in N1s core-level peaks 401.48 eV (−NH2+−). From the XPS data, it has been confirmed that pNIPAM has been successfully functionalized with amine group. XPS analysis confirmed the presence of NH2 on pNIPAM polymer by detecting the typical amine peak composing the backbone of the pNIPAM chain.
Antimicrobial activity of AgNP-polymeric nanoparticles
The antibacterial activities of the pNIPAM and pNIPAM-NH2 AgNP-nanoparticles were evaluated against E. coli (Figures 9 and 10) and S. aureus (Figures 11 and 12). Briefly, for liquid culture, we added 40 μL of each AgNP-pNIPAM/pNIPAM-NH2 nanoparticle to 160 μL of bacterial suspensions in LB broth during the exponential growth phase and monitored further bacterial growth by measuring the OD600 at 0, 1, 2, 3, 4, 5, and 24 h of culture. To evaluate antimicrobial activity on solid culture, a 1×106 cells/mL suspension of bacterial cells was cultured on an MH agar plate. An 8 mm punch was made on agar gel and 0.5 mg of (1 mg/mL) AgNP-pNIPAM/pNIPAM-NH2 nanocomposite was loaded in the punch. The zone of inhibition for each set of composites was recorded after 24 h of incubation at 37°C. Our AgNP-pNIPAM/pNIPAM-NH2 nanoparticles showed significant antimicrobial activities against both tested strains, but appeared more potent against E. coli (Figures 9 and 10) than previously reported.41,42 The overall antimicrobial activities of all size groups (G1, G2, and G3) of the pNIPAM and pNIPAM-NH2 AgNP-nanoparticles in liquid culture against E. coli were similar (Figures 9 and 10A–C), but in the solid culture, the difference between the zone of inhibitions was more size-dependent (Figures 9 and 10D). The average diameter of the zones of inhibition caused by the pNIPAM-AgNP G1, G2, and G3 groups against E. coli were 48±2, 36±6.08, and 28±2 mm and were higher than those against S. aureus (24±2.64, 20±4.58, 16±2.64 mm). In contrast, for pNIPAM-NH2 AgNP with E. coli, the zones of inhibition for the G1, G2, and G3 groups were 58±8.71, 50±4.35, and 30±1 mm, respectively, while against S. aureus they were 27±2, 22±1, and 20±4.35 mm. The overall and pNIPAM-NH2 AgNP antimicrobial effects were slightly higher, and differences in the antimicrobial activity at the last time point were observed because of their different AgNP concentrations and additional NH2 group over the surface of the nanocomposite (Table 1). In addition, the antimicrobial effects of both types of AgNP-pNIPAM/pNIPAM-NH2 nanoparticles against S. aureus (Figures 11 and 12) were lower than those for E. coli.
In vitro cytotoxicity of AgNP-polymeric nanoparticles
One of the purposes of this study was to reduce the cytotoxicity of AgNPs in human cells. To evaluate the cytotoxicity of AgNP-polymeric nanoparticles, MTT assays with hASCs were performed. Figure 3 shows the in vitro cytotoxicities of pNIPAM and pNIPAM-NH2 AgNP-nanoparticles in three size groups (G1, G2, and G3) against hASCs after 48 h. The results showed that smaller AgNP-polymeric nanoparticles had higher cytotoxicity (Figure 3). The cytotoxicities of the pNIPAM and pNIPAM-NH2 AgNP-nanoparticles were higher in the AgNP-nanoparticles fabricated with the highest concentration of AgNO3 (10 mM). The largest (G3) AgNP-polymeric nanoparticles showed the lowest cytotoxicity, as smaller AgNP-polymeric nanoparticles have a greater ability to penetrate biological membranes because of their high surface area to charge ratio.32 In addition, pNIPAM-NH2 AgNP-nanoparticle groups showed higher cytotoxicities than pNIPAM AgNP-nanoparticles, despite their lower Ag+ concentration (Table 1). This may be because of the addition of the NH2 surface charge as confirmed by XPS surface analysis (Figure 8).
Characterization of AgNP-polymeric nanoparticles
AgNP-encapsulating pNIPAM/pNIPAM-NH2 polymeric nanoparticles were synthesized by two step processes: 1) synthesis of pNIPAM/pNIPAM-NH2 nanoparticles via polymerization of NIPAM with the aid of BIS and APS, and 2) encapsulation of AgNPs inside of resulting polymeric nanoparticle networks using AgNO3 solutions. DLS analysis revealed that an increase of NIPAM:BIS ratios increased the particle size of both pNIPAM and pNIPAM-NH2 nanoparticles (Figure 1). The differences in size between the pNIPAM and pNIPAM-NH2 nanoparticles were caused by the presence of the comonomer APMAAHC.32 It is also speculated that addition of an NH2 group to pNIPAM-NH2 by using the APMAAHC comonomer resulted in extra hindrance and repulsion between consecutive NH2 groups over the surface of nanogels, slightly increasing the nanogel size.33 These results indicate that polymeric nanoparticle size can be modulated by changing the monomer to cross-linker ratio.
Similar to a previous study, the intensity of the spectral peaks in our study increased with the increase in AgNO3 concentration, but the absorption peak positions remained at the same wavelengths.43 In addition, broad peaks were observed for the AgNP-polymeric nanoparticles with larger AgNPs (Figures 4A and 5A). Our results are similar to those in a recent study that clearly revealed the formation of broad absorption peaks with an increase in the size of either pure AgNPs or AgNPs encapsulated in NIPAM-N,N-methylene-bis-acrylamide (NIPAM-MBA) gels.26 In contrast, a plasmon absorption increment in the UV-visible spectrum indicates the formation of a greater number of nanoparticles.25 In our study, the pNIPAM AgNP-nanoparticles (Figure 4B) exhibited slightly higher absorption values than the pNIPAM-NH2 AgNP-nanoparticles (Figure 5B). These differences might be due to the differing retention of AgNPs inside the AgNP-nanoparticles after washing and the differences in Ag concentrations, as shown in the ICP-OES characterization in Table 1.
The histograms in Figure S1A and B indicate variations in AgNP size; however, the diameters of approximately 85% of the AgNPs were in the range of 1–10 nm. The TEM micrographs also show that the AgNPs were highly dispersed inside the AgNP-polymer nanoparticles and were mostly spherical in shape. Hence, optimization of the experimental conditions, including pH, the concentration of AgNO3, nanoparticle size, and surface charge can achieve monodispersity and uniformity of shape.44 Our aim was to obtain a uniform size distribution of AgNPs inside the AgNP-polymeric nanoparticles, and the method used produced ultra-small AgNPs with a well-controlled size distribution using NaBH4 as the reducing agent. The chemical reaction for the NaBH4 reduction of AgNO3 is:
AgNO3 + NaBH4 → Ag + 1/2H2 + 1/2B2H6 + NaNO3
The main advantage of this in situ AgNP fabrication technique is that it provides size-controlled, uniformly sized, and homogeneously distributed nanoparticles inside a polymeric nanoparticle without the addition of a further stabilizer.8 This is confirmed in Figures 5 and 6, which show that the nanoparticles were evenly distributed within the polymeric chain network. As expected, we observed homogeneous distributions of AgNPs throughout the AgNP-polymeric nanoparticles, even though there was a rapid reduction of Ag+ into AgNPs by NaBH4.25
Changing the size or alignment of AgNPs may be possible by modifying the polymeric nanoparticle network architecture.13,45 This could be achieved by varying the ratio of monomer to cross-linker used when preparing the nanoparticle. Our results showed that the AgNPs formed in AgNP-polymeric nanoparticles with different monomer:cross-linker ratios were uniformly spherical and well dispersed, but they had different sizes depending on the size of the polymeric nanoparticle.
Most orthopedic infections are attributed to Staphylococcus spp., and S. aureus is the leading pathogen causing biomedical implant-related infections. S. aureus is often carried on the skin or in the nose of healthy people and readily adheres to host proteins (eg, fibrinogen, fibronectin) on biomaterials.4 Such adherence can lead to the formation of a biofilm that protects the pathogen against antimicrobial agents. S. aureus is among the most frequently reported pathogens causing deep infections in hospitals. E. coli represents a diverse group of bacteria that reside in the intestines. Many E. coli strains are harmless, but some can cause diarrhea or illness outside the intestinal tract. In this study, we selected both E. coli and S. aureus to test the broad-spectrum antibacterial activities of our AgNP-pNIPAM/pNIPAM-NH2 nanoparticles.
The results in Figures 9–12 exhibited the antimicrobial effects of our nanoparticle system against both E. coli and S. aureus. Interestingly, a higher antimicrobial effect against E. coli than S. aureus might be due to the structural difference in bacterial cell walls. The difference in the effect of Ag on Gram-negative and Gram-positive bacteria may lie in the structure of their cell walls. Gram-negative bacteria are encased in a thin, negatively charged outer lipopolysaccharide layer (7–8 nm thickness), whereas Gram-positive bacterial cell walls consist of a thick, highly cross-linked rigid peptidoglycan layer (20–80 nm thickness). The higher protection afforded by the Gram-positive cell wall may inhibit or prevent the bactericidal effect of AgNP-pNIPAM/pNIPAM-NH2 nanoparticles. The greater susceptibility of Gram-negative bacteria to AgNP-pNIPAM/pNIPAM-NH2 nanoparticles may also involve effects on bacterial signal transduction (ie, phosphorylation). Moreover, negative charges on lipopolysaccharides are attracted to the weak positive charge on AgNP-pNIPAM/pNIPAM-NH2 nanoparticles.46 Independently of bacterial type, the antibacterial properties of AgNP-pNIPAM/pNIPAM-NH2 nanoparticles increased at higher concentrations of AgNO3. However, polymeric nanoparticle size had a greater effect on antibacterial activity than AgNO3 concentration, and thus the smallest AgNP-pNIPAM/pNIPAM-NH2 nanoparticles had the greatest antibacterial activities against both bacterial strains (Figures 9–12).
The antibacterial properties of our AgNP-polymeric nanoparticles were size-dependent. Smaller nanoparticles have a greater surface area to volume ratio and can more easily penetrate biological surfaces.47,48 Our results showed that smaller G1 and G2 AgNP-polymeric nanoparticles had better antimicrobial activities than G3 AgNP-polymeric nanoparticles. The smaller AgNP-polymeric nanoparticles enable Ag-NPs to easily penetrate the cell wall, and their large surface area per mass brings a large number of atoms into contact with the ambient environment, resulting in an antibacterial material that is readily available to react with the components of bacterial cells.40,32
In particular, AgNP-polymeric nanoparticles with a high Ag content showed increased antimicrobial properties toward E. coli and S. aureus. The pNIPAM AgNP-nanoparticles showed a higher retention of Ag content after washing than the pNIPAM-NH2 AgNP-nanoparticles (Table 1). The antimicrobial properties of AgNP-polymeric nanoparticles allow researchers to tune their use for specific applications. For instance, to prevent biofilm formation, a highly diffusive bactericidal agent loaded into a polymeric nanoparticle may be the most appropriate means of infection prevention. This tunable feature of AgNP-polymeric nanoparticles enables optimization of Ag release for specific clinical uses and infection types, allowing targeted drug delivery that can minimize complications with the use of antimicrobial agents.49 Based on our results, AgNP-polymeric nanoparticles exhibit antimicrobial properties and may be useful in various biomedical applications.
In vitro cytotoxicity
The differences in cytotoxicity among AgNP-polymeric nanoparticles may arise from their different concentrations of Ag and different surface charges; eg, the high cytotoxicity of the AgNP polymeric nanoparticle containing 10 mM AgNO3 is governed by its high concentration of Ag+ (Table 1). This is supported by the observation that pNIPAM and pNIPAM-NH2 nanoparticles do not, on their own, exhibit cytotoxicity against hASCs.10 The use of a lower concentration of AgNO3 (5 mM) in both types of AgNP-polymeric nanoparticles resulted in a significantly higher survival of hASCs. Thus, our AgNP-polymeric nanoparticles developed using 5 mM AgNO3 are suitable for contact with human tissues (Figure 3). The results suggest that cytotoxicity can be further reduced by optimizing the surface charge, in this case by adding NH2 surface charge.
In conclusion, chemical synthesis methods for preparing AgNPs have been investigated in recent studies, with particular attention given to modifying their size and shape distributions. In the present study, the average sizes of the pNIPAM AgNP-nanoparticles were 131±78, 312±71, and 483±35 nm, while those of the pNIPAM-NH2 AgNP-nanoparticles were 135±59, 174±10, and 532±48 nm for small, medium, and large size groups, respectively. Based on our results, AgNPs can be encapsulated within a polymer network. The AgNP-polymeric nanoparticles developed in this study showed antibacterial activities against E. coli and S. aureus that were dependent on the size and amount of AgNPs within the polymeric nanoparticle and polymeric nanoparticle’s surface charge. These results suggest that our AgNP-polymeric nanoparticles can be used for antibacterial treatment to prevent postsurgical infections of biomedical implants.
This research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (HI15C1744). This research was also supported by the Chung-Ang University Graduate Research Scholarship in 2016.
The authors report no conflicts of interest in this work.
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Figure S1 (A, B) Average diameter of AgNP inside the nanogels.
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