Biological Response of and Blood Plasma Protein Adsorption on

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Bio-interactions and Biocompatibility

Biological Response of and Blood Plasma Protein Adsorption on Silver-doped Hydroxyapatite Kexun Chen, Putu Ustriyana, Francisco Moore, and Nita Sahai ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00996 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Biological Response of and Blood Plasma Protein Adsorption on Silver-doped Hydroxyapatite

Kexun Chen1, Putu Ustriyana1, Francisco Moore2,3, Nita Sahai1,3,4,*

1. Department of Polymer Science, The University of Akron, 170 University Avenue, Akron, OH 44325-3909, United States 2. Department of Biology, The University of Akron, 235 Carroll Street, Akron, OH 443253908, United States 3. Integrated Bioscience Program, The University of Akron, 235 Carroll Street, Akron, OH 44325-3909, United States 4. Department of Geosciences, The University of Akron, Crouse Hall 114, Akron, OH 44325-3909, United States 

Corresponding author: [email protected]

1

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

Hydroxyapatite (HAP) is an extensively used orthopedic biomaterials because of its high biocompatibility and osteoconductivity. Implant-related infection is a major cause of orthopedic device failure. Previous research showed that silver-doped hydroxyapatite nanoparticles (AgHAP NPs) have prominent antimicrobial activity but their biocompatibility and plasma protein response remained unexplored. Here we investigated the effects of synthesis conditions on AgHAP NP antimicrobial (E. coli and S. epidermidis) activity, biocompatibility, and the adsorption of two blood plasma proteins, human serum albumin (HSA) and Fibrinogen (Fib). It was found that synthesis pH affected the Ag content of Ag-HAP NPs and subsequent Ag+ release from the NPs in solution. This, in turn, affected antimicrobial efficiency and cytotoxicity to murine preosteoblast cells (MC3T3-E1). More HSA than Fib was adsorbed on a molar basis. The conformation of HSA changed drastically from predominantly 𝛼-helix and minor 𝛽-sheet content in solution to greater 𝛽-sheet than 𝛼-helix content when adsorbed. Correspondingly, the melting temperature Tm of HSA changed significantly from 76 °C in solution to ~65-66 °C when adsorbed. Fib exhibited a modest decrease in 𝛼-helix content while the 𝛽-sheet content increased modestly upon adsorption and its Tm remained unchanged at ~60 °C. These differences in behavior of HSA and Fib are ascribed to the much smaller size of HSA, which allows a greater molecular packing density on the surface, which induces greater conformational changes. The protein adsorption behavior on Ag-HAP was similar to that on pure HAP. Thus, we show that Ag-HAP NPs have antimicrobial activity without deleterious effects on biocompatibility and blood plasma protein adsorption.

2


Page 2 of 34

Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Keywords: silver-doped hydroxyapatite nanoparticles; blood plasma proteins; adsorption; antimicrobial

3



1. Introduction The prevalence of antibiotic resistant strains of bacteria presents a burgeoning crisis in the healthcare industry.1 Orthopedic implants are highly susceptible to infection. When bacteria are present around an implant, they can adhere to the surface of the devices to form biofilms and lead to implant-associated infection.2 Bacterial cells in biofilms have a much higher antimicrobial tolerance as compared to planktonic cells as well as protection from the host’s immune system and external toxins.3-6 Moreover, the formation of biofilm may cause failures of implant, in more severe cases, leading to mortality of patients.7 Revision implant surgeries, like hip and knee arthroplasty, also cause a financial burden and pain to patients.8 Therefore, it is crucial to develop materials with intrinsic antimicrobial property. Biomimetic synthetic hydroxyapatite (Ca10(PO4)6(OH)2, HAP) nanoparticles (NPs) have been extensively used in orthopedic biomedical applications,9-12 such as in implant coatings, bone fillers, bone cements, drug and gene delivery systems, as a scaffold for bone tissue engineering, and even in wound healing dressing.13-18 An ideal orthopedic implant should have the ability to reduce the attachment and formation of biofilm and promote desired tissue response.19-21 In spite of its high biocompatibility, bioactivity, and osteoconductivity, HAP itself does not have inherent antimicrobial property. In order to overcome this problem, synthetic HAPs doped with several kinds of ions have been developed in attempts to introduce antimicrobial property and also improve osteoconductivity.19 Ag+, Cu2+, and Zn2+ ion-doped HAP have been developed, and amongst them Ag-doped HAP (Ag-HAP) synthesized at alkaline pH provides the most significant antimicrobial effect.22-23 Significantly, it is hard for bacterial cells to adapt resistance against silver.24-25 Because of these advantages of silver, it has been used as an antimicrobial agent for centuries, and silver has been extensively used in catheters and for

4


Page 4 of 34

Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


wound healing dressings.25-27 The Ag-HAP NPs may potentially be used directly as a bone filler or as coatings on implant surfaces. However, the cytotoxicity of such Ag-HAP NPs has not been previously studied. Furthermore, when a surface is introduced into biological fluid, the first event to occur is the adsorption of proteins on the surface.28-29 The host cells “see” this adsorbed layer of proteins. The interaction between proteins and interface may change the conformation of the adsorbed proteins, consequently exposing new epitopes,29 ultimately leading to cellular responses different form the normal response.28, 30-31 Thus, it is critical to understand the interaction between the surface of implants and proteins to gain insight into how cells will respond.28-29 The presence of Ag+ in the Ag-HAP NPs may affect protein adsorption and conformation differently from Ca2+ in HAP, but this effect remains to be examined. In the present study, our goal was (1) to understand how the synthesis method influences the antimicrobial properties and biocompatibility (cytotoxicity) of Ag-HAP, and, (2) whether the presence of Ag affects the amount and conformation of adsorbed plasma proteins, as compared to pure HAP. In order to achieve the above goals, Ag-doped HAP NPs were synthesized at different pHs using the wet chemistry co-precipitation method. Antimicrobial properties were determined by measuring minimum inhibition concentration of the various HAP NPs and estimating the amount of biofilm formation. These antimicrobial estimates were related to the amount of Ag-incorporated into the Ag-HAP NPs during synthesis and the amount of Ag+ released in solutions. A murine preosteoblast cell-line (MC3T3-E1) was used to study the biocompatibility of the Ag-doped materials. The biocompatibility was assessed by cell viability and proliferation assays after treatment with NPs. Blood plasma proteins, human serum albumin (HSA) and fibrinogen (Fib) were chosen because the former protein is the most abundant plasma

5



Page 6 of 34

protein and Fib is involved in cell binding and the blood coagulation cascade. Also, though these two proteins are both negatively charged, they differ significantly in their molecular weight and solution conformation (Table 1), so we hypothesized that they may exhibit quite different adsorption behaviors. Protein adsorption was quantified by obtaining adsorption isotherms on the various HAP NP surfaces. Conformational changes of the proteins were monitored by circular dichroism (CD) in solution and adsorbed on the NPs.

Table 1. Properties of HSA and Fib. IEP

MW (kDa)

Dimensions (nm)

Main protein structure(s) in solution

HSA

4.732

66.533

~834

𝛼-helix

Fib

5.535

34035

~50 x 536

𝛽-sheet, 𝛼-helix, and random coil

2. Experimental Section 2.1. HAP and Ag-HAP Synthesis The synthesis method was adapted from Lim et al.37 with modifications. For the synthesis of HAP, 1852.4 mg of Ca(OH)2 was suspended in 50 mL ultrapure water (18.2 MΩ•cm, Barnstead™ GenPure™ xCAD Plus, Thermo Scientific, Rockford, IN, USA) to reach total concentration of 0.5 M calcium ions. The suspension was stirred for 60 mins. Fifty mL of 0.3 M phosphoric acid was added drop-wise to the Ca solution until pH 11 was reached. Ammonium hydroxide solution was used to maintain the pH value at ~11 (S220 SevenCompact™ pH/Ion, Mettler Toledo, Columbus, OH). The solution was stirred for another 16 h, and the precipitate was aged for 1 week. For Ag-HAP synthesis, 1763.5 mg of Ca(OH)2 was suspended in 50 mL water and stirred for 30 mins, then 203.9 mg of AgNO3 was added into Ca(OH)2 suspension and

6


Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


stirred for another 30 mins. Then, the same procedure of HAP synthesis was performed. Fifty mL of 0.3 M phosphoric acid was added drop-wise to the Ca plus Ag solution until the desired final pH was reached. The final pHs were maintained at either 9.5 or 11, and the products obtained were labeled as Ag-HAP9.5 and Ag-HAP11, respectively. After aging, the NPs was washed with ultrapure water, then lyophilized (FreeZone 4.5 Liter Console Freeze Dry System, Labconco, Kansas City, Mo, USA). After freeze drying, all samples were pulverized using mortar and pestle. All samples were stored at room temperature in dark prior to use. 2.2. Material Characterization 2.2.1. Transmission Electron Microscopy (TEM): Nanoparticles were suspended in 70% (v/v) ethanol to reach a final concentration of 1 mg･mL-1. The suspension was sonicated for 30 mins, then 8 𝜇L was pipetted onto formvar carbon-coated copper grids (300 mesh, Ted Pella, Redding, CA, USA). After 3 min, the liquid was blotted gently, and the grid was air-dried overnight. The grid was then visualized under TEM (JSM-1230, JEOL, Peabody, MS, USA). The particle size distribution was analyzed using ImageJ software. The distribution was obtained by measuring 100 particles from 10 different images in each sample. Each measurement was repeated three times. 2.2.2. X-ray Diffraction (XRD): Lyophilized powders were scanned in the range of 2θ = 570° using a step width of 0.04° and speed of 1·min-1 (SMART Apex, Bruker, Billerica, MA, USA). A CuKα tube operated at 40 kV and 35 mA was applied to generate X-rays. The crystallite size was obtained by using Debye-Scherrer equation.38-39 2.2.3. Fourier Transform Infra-red (FTIR) Spectroscopy: FTIR spectra were measured by mounting dry NP powder on the sample stage after collecting the background spectrum

7



(Excalibur FTS 3000, PerkinElmer, Shelton, CT, USA). The spectrum was measured from 5004000 cm-1 and 16 scans were collected per sample. 2.2.4. Inductively-Coupled Plasma Optical Emission Spectrometry (ICP-OES): Five percent nitric acid was added to Ag-HAP powder to dissolve the NPs completely achieve a final concentration of 1 mg･mL-1. This sample was diluted 30 before the measurement of silver ion concentration using ICP-OES (ICP-OES 720, Agilent, Santa Clara, CA, USA). The average of three replicates is reported. 2.3. Antimicrobial tests 2.3.1. Minimum Inhibition Concentration (MIC) Test: In order to test the antimicrobial property of different Ag-HAPs, the MIC test was performed on Escherichia coli (ATCC® 25922TM, Manassas, VA, USA) or Staphylococcus epidermidis (ATCC® 38954TM, Manassas, VA, USA). After obtaining bacteria, the pellet was hydrated by recommended media and inoculated overnight in an incubator shaker (Innova 4300, New Brunswick scientific, Edison, NJ, USA) at 37 °C. After overnight incubation, a loopful of bacteria solution was streaked on agar plates to obtain separated colonies. A single colony was picked up to inoculate in 5 mL of tryptic soy broth (TSB) supplied with 6% yeast extract, and incubated at 37 °C at a speed of 130 rpm overnight (~16 h). One hundred 𝜇L of overnight culture was inoculated in 5 mL of TSB with 6% yeast extract, until the bacterial solution reached turbidity (OD600) of 0.4-0.7. The bacterial solution was then diluted using TSB with 6% yeast extract to OD600 ~0.1-0.12. The bacterial working solution was prepared by further diluting the solution by 100 in either TSB with 6% yeast extract (a protein rich medium) or M9 minimal medium (M9MM), resulting in an approximate colony forming unit (CFU) concentration of 1×106 CFU･mL-1.40 Then, 100 𝜇L of bacteria working solution was mixed with 100 𝜇L of Ag-HAP suspended in sterile HEPES buffer

8


Page 8 of 34

Page 9 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with 100 mM KCl in a 96-well plate. Negative control-medium (NC-m), which contains medium and buffer but no bacteria and no NPs, and negative control-bacteria (NC-b), containing only bacteria with medium and buffer but no NPs, were also added in every well plate. The initial OD600 of the plate was measured before incubation at 37 °C for 24 h. After 24 h, OD600 was measured again and MIC was determined as the lowest concentration of Ag-HAP that prevents bacterial growth. The experiment was repeated in triplicate three times. 2.3.2. Crystal Violet (CV) Assay: Crystal violet assay is a common method to quantify bacterial biofilm formation.41 Specifically, after measuring OD600 to determine MIC, solutions in the 96-well plate were discarded. The plate was then washed by pipetting ultrapure water into each well; this step was repeated 5 times. After washing, the plate was heated in an oven (StabilTherm Gravity, Blue M) at a temperature of 60 °C for 1 h to fix the biofilm. After fixation, the plate was cooled for 10 min and 0.1 wt. % CV solution was then added into each well to stain the biofilm for 15 min. After the staining, the CV solution (Sigma-Aldrich, St. Louis, MO, USA) was discarded and the plate was again washed 5 with water. The plate was heated for 10 min in the oven for drying and altered to cool down for another 10 min. In the final step, 90% (v/v) ethanol was added in each well to solubilize CV for 1 h. The absorbance was then read at 590 nm using a plate reader (Synergy H1 Hybrid Multi-Mode Reader, BioTeck, Winooski, VT, USA). The experiment was repeated in triplicate three times. 2.3.3. Silver Ion Release: Silver ion release experiment was performed in TSB and in M9MM. Nanoparticles were suspended in a specific volume of medium to reach a final concentration of 5 mg･mL-1. The suspension was then vortexed and incubated in an incubator shaker (I 24 Incubator Shaker Series, New Bruswick Scientific, Edison, NJ, USA) for 5 h, 12 h, and 24 h. At every time point, the suspension was centrifuged at 150,000 rpm for 3 min. The

9



supernatant was collected and was digested by 30% HNO3 to achieve a final acid concentration of 5%. The experiment was repeated in triplicate three times. 2.4. Murine Cell Viability 2.4.1. Cell Culture: Mouse preosteoblast MC3T3-E1 cells (ATCC, Manassas, VA, USA) were cultured in alpha-MEM medium with 10% FBS (HyClone™ Fetal Bovine Serum, Thermo Scientific, Rockford, IN, USA), 100 U/mL penicillin, and 100 𝜇g/mL streptomycin (HyClone™ Penicillin Streptomycin 100× solution, Thermo Scientific, Rockford, IN, USA). Cells were maintained at 37 °C in 5% CO2 in a water-jacketed incubator (Forma II, Thermo Scientific). The cells were harvested by using 0.25% trypsin solution, then seeded in 24 well plates with a cell density of 10,000 cells per well for a volume of 800 𝜇L. After allowing cells to attach for 24 h, they were treated with NPs. NP stock was prepared by directly suspending NPs in alpha-MEM. NPs with various concentrations were prepared by performing serial dilution. Then, cells were treated with NPs. Passage 5 cells were used throughout the experiments. The experiment was repeated in triplicates three times. 2.4.2. Cytotoxicity: Cytotoxicity tests were performed by using LIVE/DEAD assay according to the manufacturer’s instruction (Invitrogen, Grand Island, NY, USA). At the end of the incubation time with NPs (Day 1 and 3), the medium was aspirated. After aspiration, the assay reagent was added, and incubated in 37 °C for 10 min, followed by visualization using an inverted fluorescence microscope (Olympus IX51, Olympus American Inc., Melville, NY, USA). 2.4.3. Cell Proliferation: CyQuant Cell Proliferation Assay (Invitrogen, Grand Island, NY, USA) was used to evaluate the proliferation of MC3T3-E1 cells. After 1 or 3 days of incubation with or without NPs, the cells were washed with sterile phosphate buffer saline (PBS) and well plates were frozen at 80 °C until being assayed. For the assay, well plates were firstly thawed at

10


Page 10 of 34

Page 11 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


room temperature and 150 𝜇L of lysis buffer was added at room temperature for 1 h. Then 150 𝜇L of CyQuant GR dye was added and incubated in dark at room temperature for 5 min. The solution was transferred to a 96-well plate to measure the fluorescent signal. Plate reader was used for the measurement with excitation at 480 nm and emission at 520 nm. The amount of DNA in each sample calculated using a calibration curve and displayed as ng･ml-1. The experiment was repeated in triplicate three times. 2.5. Protein Adsorption 2.5.1. Adsorption Isotherms: Two model proteins, HSA (lyophilized powder, ≥ 96% agarose gel electrophoresis) and Fib (Type 1-S, 65-85% protein, ≥ 75% of protein is clottable) were used to obtain protein adsorption isotherms and the secondary structure in solution and adsorbed to all three types of NPs. For the adsorption experiment, protein concentration of 0-9 mg･mL-1 in a final volume of 175 𝜇L was prepared. Seventy-five 𝜇L of the solution was diluted twice and utilized in the measurement of initial concentration (C0), while the remaining 100 𝜇L was used in the adsorption experiment. The adsorption experiment was performed by adding 100 𝜇L of NP stock solution into 100 𝜇L of the previously prepared protein solution, yielding a system with 5 mg･mL-1 NP with protein concentrations ranging from 0-4.5 mg･mL-1. Both samples for C0 and adsorption experiments were incubated in an incubator shaker (I 24 Incubator Shaker Series, New Bruswick Scientific, Edison, NJ, USA) at 37 °C for 24 h. After incubation, the adsorbed samples were centrifuged at 15,000 rpm for 3 min. The concentration of protein remaining in the supernatant of the adsorbed samples, or in the samples without NPs was determined by UV-Vis absorbance measurement at 280 nm by plate reader (Synergy H1 Hybrid Multi-Mode Reader, BioTeck, Winooski, VT, USA). The concentration of adsorbed sample was established by using a calibration curve of protein solution (0-5 mg･mL-1). The concentration of protein adsorbed,

11



represented by qe (mg･mL-1), was calculated as the difference between the initial concentration (C0) and protein concentration remaining at equilibrium in solution after adsorption (Ce). The experiment was repeated in duplicate three times. 2.5.2. Secondary Structure of Adsorbed Proteins by Circular Dichroism (CD): Sample preparation for secondary structure measurement was similar to the adsorption experiment. Briefly, the stock solutions were prepared and separated into two solutions for the measurement of initial protein concentration and for adsorption with nanoparticles. Far-UV spectra were collected on a CD spectroscope at 190-250 nm using 0.2 nm data pitch, 2 nm bandwidth, 4 s response time, and 50 nm･min-1 scan speed in a 1 mm cuvette (J/0556, Jasco, Easton, MD, USA). The temperature of the sample holder was set at 25 °C and six accumulations were averaged for each measurement. The experiment was repeated in triplicate three times. The deconvolution follows the procedure used by Felsovalyi et al.42 Briefly, the initial protein concentration associated with each adsorbed fraction was converted to molar ellipticity. All the CD spectra were analyzed using the Contin algorithm in CDPro™ software package. Six reference sets were used: SP37, SP43, SDP42, SDP48, SMP50, SMP56. The values of α-helix, β-sheet, sheet, and random coil were averaged across these six reference sets. 2.5.3. Thermal Denaturing of Adsorbed Proteins by CD: Thermal denaturing experiments were conducted to obtain the melting temperature of the protein, which reflects the stability of the protein. The experiment was performed using CD. Briefly, low concentration of protein solution was introduced into NP suspension (final NP concentration: 5 mg･mL-1) to make sure all proteins were adsorbed on NPs. After 24 h of incubation, the sample was diluted by ultrapure water around 20 times for melting temperature measurement. CD spectrum was collected with a 10-mm cell with a bandwidth of 2, and 4 s response time, and in the temperature range of 10-

12


Page 12 of 34

Page 13 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90 °C. The temperature was raised by 1 °C per minute and was allowed to equilibrate for 30 s before spectrum collection.

3. Results and Discussion 3.1. Nanoparticle Characterization The crystal phases and purity of materials was determined by various methods. The XRD patterns of all three types of NPs (HAP, Ag-HAP9.5, and Ag-HAP11) showed the same diffraction peak positions and all the peaks can be assigned to standard HAP XRD pattern (ICDD card number: 00-024-0033), which means that all the materials were single phase HAP without the presence of any other calcium phosphate- or silver-related crystals (Figure S1(a)). By applying the Debye-Scherrer equation, average particle size was estimated as 19.87 ± 0.98, 16.42 ± 4.54, and 19.69 ± 1.36 nm, respectively, for pure HAP, Ag-HAP9.5, and Ag-HAP11. No statistical difference was found between the three types of NPs (Figure S1 d). Thus, the addition of Ag to HAP did not affect particle size as determined by this method. The FTIR spectra showed a small band at 3572 cm-1, which can be assigned to hydroxyl groups in HAP (Figure S1(b)).43-45 Peaks at 1087, 1032, 962, 601, and 561 cm-1 are due to the presence of phosphate groups.43, 45-46 Carbon dioxide from the atmosphere can be dissolved in the basic solution during the synthesis of HAP and result in the formation of carbonate ionsubstituted HAP. Carbonate in the crystal lattice showed characteristics bands at 1550, 1415, and 875 cm-1.43, 45, 47 Hydroxyapatite synthesized by wet chemistry method will usually lead to water associated with the product, which was shown as a very broad band between 3800 and 3000 cm1,

and also a band at 1630 cm-1.48-50 Data from FTIR and XRD, thus, revealed that the

synthesized products were all carbonate ion-substituted single-phase HAP. This result suggested

13



that all the materials synthesized in this study are biomimetic HAP since biological apatite is also carbonate-substituted HAP.13 TEM was used to characterize the morphology and size of the HAP NPs (Figure S1(c)). All three types of NPs had similar needle-like morphology and similar particle size distribution with most crystals lying between 20-40 nm. These primary crystals were aggregated by end-to-end attachment to form larger sizes of ~ 100 nm length. (Figure S1e). The results were similar for all three types of NPs. Thus, the addition of Ag to HAP did not influence the NP shape, size, or state of aggregation. The silver content of Ag-HAP NPs was determined by ICP-OES (Figure S1(f), (g)). The results showed that Ag-HAP9.5 and Ag-HAP11 had a silver content of 2.4 and 0.6 wt. %, respectively (Table 2). Thus, different silver content in Ag-HAP could be achieved simply by controlling the synthesis pH. Furthermore, the amount of Ag released was related to the silver content of the Ag-HAP NPs (Table 2, Figure S1(f), (g)).

14


Page 14 of 34

Page 15 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Silver content (wt. %) of Ag-HAP9.5 and Ag-HAP11, and concentration of Ag+ released (ppm) from Ag-HAP9.5 and Ag-HAP11 after incubation at 37 °C for 24 h in different growth media, as well as silver concentration in TSB after filtration with a 3 kDa cutoff membrane. Error bars represent standard deviation of the average of three independent experiments. Silver Release in Media (ppm) Synthesis

Silver

Growth

pH

Content

Medium

24h 5h

12h

24h (filtered)

Ag-HAP9.5

Ag-HAP11

9.5

11

TSB

8.08 ± 1.30

8.46 ± 1.37

8.46 ± 1.37

0.50 ± 0.11

M9MM

0.29 ± 0.03

0.29 ± 0.04

0.31 ± 0.10

N/A

TSB

1.23 ± 0.25

1.78 ± 0.67

1.78 ± 0.67

0.11 ± 0.04

M9MM

0.25 ± 0.01

0.28 ± 0.03

0.28 ± 0.05

N/A

2.4 wt. %

0.6 wt. %

3.2. Antimicrobial Tests 3.2.1. MIC Test: In order to confirm the antimicrobial activity of Ag-HAP NPs with different silver contents, MIC test and crystal violet assay were performed. The MIC test is usually used to measure the lowest concentration of an antibiotic that can inhibit the growth of a bacteria.51 For the MIC test in this study, two different media were used, TSB with 6% yeast extract, which is a protein-rich medium, and M9MM, which is a protein-free minimal medium. The two media provide insight into how the presence of protein in the medium may influence the estimated antimicrobial property of Ag-HAP.

15



Figure 1. MIC determined in different media. Increase of OD600 after incubation of (a) E. coli in TSB, (b) E. coli in M9MM, (C) S. epidermidis in TSB, (d) S. epidermidis in M9MM at 37 °C for 24 h with respect to the initial OD600. NC-m represents negative control, which is only buffer and growth medium; NC-b contains buffer and medium with bacteria. Error bars represent standard deviation of triplicate in three experiments.

As the NP concentration increased, the OD600 gradually decreased (Figure 1). The MIC values were determined by a steep drop in the OD600 values and the NP concentration at which OD600 was close to the negative control (NC-m). The MIC values are summarized in Table 3. In TSB, 256 𝜇g/mL of Ag-HAP9.5 can sufficiently inhibit the growth of E. coli; however, the same

16


Page 16 of 34

Page 17 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


effect can only be achieved at 512 𝜇g/mL for S. epidermidis. The much thicker peptidoglycan layer probably makes these gram-positive bacteria less susceptible to Ag+ as compared to the gram-negative bacteria. This result was in good agreement with those of Jadalannagari et al.,52 who obtained MIC values of Ag-HAPs against E. coli and Staphylococcus aureus in both protein-rich and protein-free media. Their results show that gram-positive bacteria S. aureus has a higher MIC value as compared to gram-negative bacteria E. coli.

Table 3. MIC values of Ag-HAP9.5 and Ag-HAP11 in TSB and M9MM. E. coli

S. epidermidis

TSB

M9MM

TSB

M9MM

Ag-HAP9.5

256

1

512

0.5

Ag-HAP11

>512

4

>512

2

Note: The unit of each value is g/mL.

In M9MM, S. epidermidis was shown to be less resistant to Ag-HAP, likely because M9MM is a less suitable medium for its growth than for E. coli. This influence is indicated by the less turbid appearance of the negative control (NC-b) of S. epidermidis in M9MM as compared to E. coli. The MIC value of in TSB was nearly 250 times greater than in M9MM, for both E. coli and S. epidermidis (Table 3) showing that the presence of growth medium proteins can significantly decrease the estimated antimicrobial activity of Ag-HAP. In detail, TSB medium is an enriched medium that contains nutrients (proteins), which can allow the bacteria to grow a lot of biofilm. The biofilm acts to suppress the antimicrobial activity of the Ag-HAP by either preventing direct bacterial cell contact with the Ag-HAP or by complexing the soluble Ag+ ions released from the 17



Ag-HAP. In comparison, M9MM medium is a minimal medium, which means that it does not contain protein nutrients, so biofilm production is limited and, hence, exposure to antimicrobial Ag-HAP is higher.

3.2.2. CV Assay: Besides killing bacterial cells, biomaterials with antimicrobial property should ideally also inhibit the formation of biofilm. The CV assay is often used to quantify the amount of biofilm formed under specific conditions.41 Results showed that both Ag-HAP9.5 and Ag-HAP11 significantly inhibited the formation of biofilm in both TSB and M9MM compared to pure HAP (Figure 2). With increasing NP concentration, less biofilm was formed, except in the case when S. epidermidis was grown in TSB. In this case, low and intermediate concentrations of Ag-HAP were not able to inhibit the formation of biofilm.

18


Page 18 of 34

Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. CV assay to test the ability of different NPs to inhibit the formation of biofilm. (a) E. coli in TSB; (b) E. coli in M9MM; (c) S. epidermidis in TSB; (d) S. epidermidis in M9MM. NCm represents negative control, which is only buffer and growth medium; NC-b contains buffer and medium with bacteria.

3.2.3. Silver Release: The effect of synthesis method on the amount of Ag+ released from Ag-HAP NPs in TSB and in M9MM was determined using ICP-OES (Figure S1(d), (e); Table 2). Significant differences were observed in the amount of silver released by Ag-HAP9.5 and Ag-HAP11 in TSB. Ag-HAP9.5 released 8-9 ppm Ag+ compared to 1-2 ppm released from AgHAP11. This explains the lower MIC values and reduced biofilm production in the presence of Ag-HAP9.5 compared to Ag-HAP11. Silver ion release in M9MM at ~0.2-0.3 ppm from both the Ag-HAP NPs is much lower than in TSB. These results indicate that the presence of proteins in TSB increases total solubility of Ag+ probably by binding the thiol groups in the proteins.53 The hypothesis was further confirmed by filtering proteins out after 24 h of ion release experiment (Table 2). After filtration, only 5% of silver ions remain in the solution. However, it appears that the protein-bound silver ions in TSB are not able to kill the bacteria, so the MIC values are higher in TSB than in M9MM. The low MIC values in M9MM compared to TSB are ascribed to the fact that the former is a minimal medium, as discussed above.

3.3. Mouse Cell Culture 3.3.1. Cytotoxicity: The cytotoxicity of Ag-HAP NPs to murine MC3T3-E1 cells was assessed by using LIVE/DEAD assay, as shown in Figure 3. The green dots represent the living

19



cells and the red dots indicate dead cells. On day 1, none of the NPs were toxic to cells, even at the highest NP concentration of 1024 𝜇g･mL-1. On day 3, only a small number of dead cells were seen in the presence of HAP and Ag-HAP11 up to high NP concentrations. However, at high Ag-HAP9.5 NP concentrations of >256 𝜇g･mL-1 most of the cells were dead, which indicates that Ag-HAP9.5 has a significantly higher cytotoxicity to MC3T3-E1 cells than HAP and Ag-HAP11. This is consistent with the greater release of Ag+ from Ag-HAP9.5 than AgHAP11 (Figures S1(d), (e); Table 2).

Figure 3. LIVE/DEAD assay of MC3T3-E1 cells on (a) day 1 and (b) day 3 with different NPs in various concentration. 20


Page 20 of 34

Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.3.2. Cell Proliferation: The amount of DNA was significantly lower when cells were treated with Ag-HAP9.5 as compared to HAP (Figure 4). Combining the results of the LIVE/DEAD assay and cell proliferation assay, we concluded that Ag-HAP9.5 has a higher cytotoxicity than Ag-HAP11. In summary, Ag-HAP11 emerges as the material with optimum balance of antimicrobial activity and biocompatibility.

Figure 4. Effect of NPs at different concentration on the proliferation of MC3T3-E1 cells (a) at day 1, (b) at day 3. Cell proliferation was measured by the CyQuant proliferation assay (n=3, * indicates significant difference with HAP, p

Biological Response of and Blood Plasma Protein Adsorption on

Recommend Documents