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Sustainable Release of Vancomycin from Silk Fibroin Nanoparticles for Treating Severe Bone Infections in a Rat Tibia Osteomyelitis Model Negar Hassani Besheli, Fatemeh Mottaghitalab, Masoud Eslami, Mahdi Gholami, Subhas C. Kundu, David L Kaplan, and Mehdi Farokhi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14912 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 21, 2017

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ACS Applied Materials & Interfaces

Sustainable Release of Vancomycin from Silk Fibroin Nanoparticles for Treating Severe Bone Infections in a Rat Tibia Osteomyelitis Model Negar Hassani Beshelia, Fatemeh Mottaghitalabb, Masoud Eslamic, Mahdi Gholamid, Subhas C Kundue,f, David L. Kaplang, Mehdi Farokhih,* a

School of Chemical Engineering, Collage of Engineering, University of Tehran, Tehran, PO Box 11155-4563, Iran b

Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, PO Box 14155-6451, Iran

c

Materials Science & Engineering Department, Sharif University of Technology, Tehran, PO Box 11365-9466, Iran d

Faculty of Pharmacy and Pharmaceutical Science Research Center, Tehran university of Medical Sciences, Tehran, PO Box 14155-6451, Iran

e

Department of Biotechnology, Indian Institute of Technology (IIT) Kharagpur, West Bengal 721302, India f

3Bs Research Group, Headquarters of the European Institute of Excellence on Tissue

Engineering and Regenerative Medicine, University of Minho, AvePark - 4805-017 Barco, Guimaraes, Portugal. g

Department of Biomedical Engineering, Tufts University, 4 Colby St, Medford, MA 02155, USA h

National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, PO Box 1316943551, Iran

Corresponding author: Dr. Mehdi Farokhi, National cell Bank of Iran, Pasteur institute of Iran, PO Box 13164, Tel: +98 21 64112358, E-mail address: [email protected]

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Abstract The successful treatment of bone infections is a major challenge in the field of orthopedics. There are some common methods for treating bone infections, including systemic antibiotic administration, local non-degradable drug vehicles, and surgical debridement and each of these approaches has advantages and disadvantages. In the present study, the antibiotic vancomycin (VANCO) was loaded in silk fibroin nanoparticles (SFNPs) and the complexes were then entrapped in silk scaffolds to form sustained drug delivery systems. The release kinetics of VANCO from SFNPs alone and when the SFNPs were entrapped in silk scaffolds were assessed at two different pH-values, 4.5 and 7.4 that affected the release profiles of VANCO. Disk diffusion

tests

performed

with

pathogens

causing

osteomyelitis

methicillin-resistant

Staphylococcus aureus (MRSA) showed antibacterial activity of the released drug at two different pH-values. Additionally, injecting of 8×106 CFU MRSA in the tibia of rats induced severe osteomyelitis disease. Radiographic and histopathological analyses were performed to evaluate the effectiveness of treatment after 6 weeks. The VANCO loaded silk fibroin nanoparticles entrapped in scaffolds reduced bone infections at the defect site with better outcomes than the other treatment groups. In conclusion, this delivery system with biocompatibility and sustained release properties would be appropriate for further study in the context of osteomyelitis disease. Keywords: Silk, nanoparticle, Vancomycin, Osteomyelitis, Bone infection, Scaffold.


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1. Introduction Many factors are responsible for the increase in severe bone and joint infections and tissue suppurations, including accidents and the greater use of orthopedic devices and joint replacements 1-2. Infections have various impacts on patients, for example, about 37% experience delayed unions and about 14% require amputation3. Therefore, treating osteomyelitis remains a critical challenge for many orthopedic surgeries, as current approaches such as administering antibiotics, hyperbaric oxygen, and surgery4 do not suffice. When infections reach advanced stages, bone necrosis restricts the intravenous delivery of antibiotic to the site of infection due to the shortage in blood supply 5. In addition, the pharmacokinetics of antibiotics in terms of short half-life and systemic toxicity limit the application of high doses of these drugs6. Many studies introduced the local administration of antibiotics as a strategy to improve outcomes and reduce systemic toxicity7-9. Recently, many drug delivery vehicles have been designed with the capability to deliver multiple drugs and control release rates10-13. Different structures with various morphologies, including foams, films, gels, microparticles, and nanoparticles (NPs) have been developed for this purpose. Particulate vehicles can reduce the administration of solubilized drug14-15. The unique characteristics of NPs such as high surface area16, capability to perform as adaptable platforms17, tunable size and surface charge18 make them useful for drug delivery. Many natural and synthetic polymers with various degrees of biodegradability have been used for the delivery of antibiotics to the site of infection, including poly(lactide-co-glycolic acid) (PLGA), polycaprolactone (PCL), chitosan, collagen, and polyhydroxyalkanoates19. Despite the useful properties of these biodegradable polymers, limitations such as harsh processing conditions, rapid degradation, and lower mechanical strength (for natural polymers) remain in some circumstances20-21.



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Silk fibroin (SF) has been explored for delivery systems due to its unique characteristics among other natural and synthetic polymers22-24. Suitable biocompatibility, low immunogenic response, low bacterial adhesion, tunable degradability, ambient conditions for processing into devices, tunable architecture, mechanical properties and functionality are all useful features11, 25.In the present study, the goal was to exploit SFNPs to control the release kinetics of vancomycin (VANCO) towards an aim to develop a treatment against severe osteomyelitis disease. VANCO was chosen as a model drug because of its potential against methicillin-resistant Staphylococcus aureus (MRSA) as the major sepsis-causing organism in osteomyelitis infections. SFNPs containing antibiotic, SFNPs with and without VANCO entrapped in SF based scaffolds were compared upon implantation in an animal model with severe osteomyelitis. 2. Experimental 2.1 Materials Most of the materials were purchased from Sigma-Aldrich (USA) including sodium carbonate, lithium bromide (LiBr), 12,000 Da cut off dialysis tubing, Dulbecco Modified Eagle’s Medium (DMEM), Ham’s F-12 Medium, fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin, streptomycin, ketamine/xylazine, collagenase type I, [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide] (MTT), acetone and isopropanol. In addition, ALP’s kit was purchased from Pars azmun, Iran. 2.2 Preparation of SF solution To extract fibroin, Bombyxmori SF fibers were degummed in Na2Co3 (0.02 M) for 30 min and this step was repeated for another 30 min followed by washing in deionized water. Afterwards, the extracted fibroin fibers were dissolved in LiBr (9.3 M) for 4h at 60°C. The prepared solution 4 ACS Paragon Plus Environment

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was then dialyzed against water using 12,000 Da cutoff dialysis tubes for 72h. The remaining purified SF solution (4% w/v) was used for the preparation of NPs. 2.3 Preparation of SFNPs A dissolution method was used for the preparation of SFNPs where 15 mL SF solution, 2% w/v, was added drop wise to 35 mL of acetone. The precipitated SF was then collected using centrifugation at 23,500×g for 10 min. The sample was further centrifuge at 13,400×g for 10 min. The pellet was then dispersed in deionized water by sonication at 30% amplitude for 15 min. The nanoparticles were then lyophilized using a freeze-dryer (Christ alpha, UK) until further use26-27. 2.4 Characterization of SFNPs Dynamic light scattering (DLS; 3000, HS, Malvern, UK) was used for evaluating the polydispersity index (PDI), size, and surface charge of the SFNPs. The lyophilized SFNPs were dissolved in sterile phosphate buffer saline (PBS) and sonicated for 20 min at 70% amplitude. DLS was repeated three times for each measurement at 25°C and 630 nm, with a detection angle of 90°. The morphology of SFNPs was also assessed under field emission scanning electron microscopy (FESEM; TESCAN MIRA3 LMU, Czech Republic) at an accelerating voltage 15 kV. Before imaging, 20 µl of sonicated solution was positioned on aluminum foil and the sample was sputter coated with gold after drying. 2.5 Preparation of VANCO loaded SFNPs (VSFNPs) Various ratios of VANCO and SFNPs were chosen for determining the optimum loading capacity of SFNPs. First, VANCO (1 mg/mL) was loaded in SFNPs solutions with different concentrations (1, 5, and 10 mg/mL) under stirring for 24h. VSFNPs were then collected using 5 ACS Paragon Plus Environment


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centrifugation for 10 min at 4,000 rpm. The amount of loaded VANCO was measured by UV spectrophotometry (Epoch 2 microplate spectrophotometer, USA) at 280 nm. In a second step,

different concentrations of VANCO (0.1, 0.3, 0.5, 0.7, and 1 mg/mL) were added to a constant concentration of SFNPs (5 mg/mL). These samples are referred as A, B, C, D and E, respectively. The loading capacity (LC) and encapsulation efficiency (EE) of VANCO in SFNPs were measured using the following equations: Loading capacity (%)=

Encapsulation efficiency (%)=

× 100

Equation (1)

× 100

Equation (2)

2.6 In vitro release study of VANCO from SFNPs Two different pH-values (4.5 and 7.4) were considered to determine the in vitro release of VANCO from SFNPs in PBS at different times. The VSFNPs were dispersed in PBS and dialyzed against 15 mL PBS with shaking (100 rpm, 37°C). Every 24h, 2 mL of PBS was replaced with an equal amount of fresh PBS. The samples were then analyzed spectrophotometrically at 280 nm. All analyses were repeated in

triplicate. The same procedure was also carried out to evaluate release of antibiotic from VSFNPs entrapped in SF based scaffolds.

2.7 Evaluating the interaction between VANCO and SFNPs The interactions between VANCO and SFNPs were evaluated using Fourier transform infrared spectroscopy (FTIR; Shimadzu 8400s; Japan). Before the assessment, lyophilized samples were mixed with KBr and the samples were then assessed in the spectral range of 400 to 4000 cm−1 at room temperature in transmittance mode. 2.8 Fabrication of SF scaffold containing VSFNPs 6 ACS Paragon Plus Environment

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The samples D and E were added to 2 mL SF solution with 4% w/v concentration and the mixture was stirred for 2h to form a uniform solution. The homogenous solution was then poured into 24-well tissue culture plates and freeze-dried for 48h. After freeze-drying, the samples were cross-linked using 90% methanol for 10 min. 2.9 Scaffold characterization Scanning electron microscopy (SEM) was used to evaluate the impact of different pH conditions on the surface morphology of the scaffolds. The crystallinity of SFNPs was also assessed using an X-ray diffractometer (GNR- MPD 3000, Italy) with an X'Celerator counter, a scanning rate of 0.066◦/min, and a scanning region of 2Ɵ= 5-60◦ with Cu Kα radiation (λ = 1.5418A˚). The irradiation conditions were also 40 kV and 40 mA. 2.10 Mechanical testing The compressive strength of two types of scaffolds (with and without SFNPs) were evaluated under dry and wet conditions using Mechanical tester (H10KS, Hounsfield, England). Evaluating the compressive strength under wet environment was performed in order to mimic the in vivo condition. The test was repeated three times for each sample in crosshead speed of 0.5 mm/min under compression mode. 2.11 Biocompatibility An indirect test was used to evaluate the biocompatibility of the scaffolds. The extraction solution from the scaffolds was prepared using ISO 10993-5.Accordingly, the scaffolds with surface area of 1 cm2were soaked in 1 mL of culture medium and then incubated at 37 °C for 3, 7 and 14 days. Rabbit osteoblast cells were used for the MTT assay. Briefly, 1×104 cells were cultured in the extraction solution from the scaffolds in 96-well plates at 24h in 37◦C with 5% CO2. Afterwards, the medium was replaced with MTT solution (0.2 mg/mL) and the plates 7 ACS Paragon Plus Environment


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incubated at 37 °C for 4 h. The precipitated formazan was solubilized in isopropanol for 15 min and the absorbance of the supernatant measured spectrophotometrically at 570 nm. Tissue culture polystyrene (TCPS) was considered as a negative control group. 2.12 Alkaline phosphatase (ALP) activity The amount of ALP, as osteoblast cell marker, was measured using an ALP kit. About 2×105cells/well were seeded in 24-well tissue culture plates and incubated for 24h. The medium was replaced with extraction solution from the scaffolds. After 3 days, 10 µL of rabbit osteoblast cell supernatant was added to 1,000 µL of the ALP kit reagent according to the manufacturer's protocol at 37°C. The absorbance was read at 405 nm. TCPS was used as a negative control group. 2.13 Susceptibility testing In order to evaluate the minimum inhibitory concentration of VANCO to MRSA (ATCC43300) an antibiotic tube dilution method was used in Cation Supplemented Mueller-Hinton Broth. VANCO was diluted serially two-fold in tubes from 512 to 0.125 µg/mL. Each series of tubes was filled with 100 µl MRSA inoculum containing 1.5×106CFU/mL and incubated overnight at 37οC. The minimum inhibitory concentration (MIC) was considered to be the lowest concentration of antibiotic that prevented turbidity after incubation for 16-18h. After MIC was determined, 0.01 mL of each clear tube was added onto the surface of a blood agar plate. The minimum bactericidal concentration (MBC) was the lowest concentration of antibiotic that killed the bacterial colonies on the plate after 24h incubation at 37°C28-29. To estimate the bioactivity of the released VANCO on MRSA, the disk diffusion method was used30. For this, about 20 µL of the released VANCO at different time points (1-30days) was added to 6-mm sterile filter-paper disk. The disks were then placed on Mueller Hinton agar plates and MRSA was seeded on the


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disks. The zones of inhibition were measured with a micrometer after 16–18 h incubation at 37°C. The effective bactericidal activity of released VANCO from VSFNPs was compared to the activity of non-processed VANCO (30 µg) standard disks. Zones ≤ 9 mm were considered resistant, while the zones between 10 and 11 mm had intermediate resistance and those that were ≥12 mm were sensitive to MRSA31. All the experiments were performed triplicate. 2.14 Animal model of severe osteomyelitis Male Wistar albino rats (260–330 g) were chosen for animal studies. All the animal experiments were carried out according to the guidelines of the local Ethical Committee of Pasteur Institute of Iran. Prior to surgery, the animals were anesthetized using Ketamine (60 mg/kg) and Xylazin (6 mg/kg). The anteromedial tibia metaphysis of the animals were exposed by 1.5 cm longitudinal incision. The medullary cavity was then exposed using a dental burr, and a Kirchner wire (1 × 30 mm) was inserted into the cavity. The animals were divided into three groups including a control group that had only Kirchner wire without MRSA injection, a group injected with 40 µl of 1 × 108 CFU/mL MRSA suspension, and the last group injected with 40 µl of 2 × 108 CFU/mL MRSA suspension. Afterwards, the wound was sutured. The body weight and count of blood cells (CBC) of all animals were evaluated every 7 days. Finally, the animals were scarified and bone specimens were taken for histological analysis 3 weeks post-operation. 2.15 Implantation of the scaffolds Following the same incision used for induction of osteomyelitis, the rats with severe osteomyelitis were reoperated and the wires were extracted. The formed encapsulated abscess in the subcutaneous area that was formed in most of the animals after osteomyelitis induction was then removed. Subsequently, the Kirchner hole was extended to 4 mm by using dental burr. The site of infection was then removed and washed with saline4, 32. The animals were categorized into



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4 different groups: 1) animals implanted with VSFNPs loaded in SF scaffolds, 2) animals implanted with SFNPs loaded in SF scaffolds, 3) animals implanted with VSFNPs only, and 4) empty hole without any implant. Lateral radiographs were taken from the animal after six weeks post implantation. Finally, the bone samples were fixed in 4% paraformaldehyde for 12 h and then decalcified in 10% EDTA for 2 weeks followed by embedding in paraffin. The tissue blocks were sectioned at 5µm thicknesses and stained with hematoxylin and eosin (H&E). The scaffolds implantations are shown in Figure 1.

Figure 1. Surgical procedure for scaffold implantations. 3D scaffolds (a), Kirchner wire removal (b), extended cavity (c), and implanted scaffold (d). 2.16 Statistical analysis All of the quantitative data were expressed as mean ± standard deviation. Statistical comparisons were performed using ANOVA version 16.0 (SPSS, USA). Differences were considered statistically significant for p values< 0.05. 3. Results 3.1 Characterization of SFNPs


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The nanoparticles were characterized in terms of size, charge and shape. Based on DLS (Figure 2), the SFNPs were negatively charged (-18 mV) with a uniform size distribution of about 80-90 nm and a PDI of 0.1. SEM micrographs also confirmed the sizes of SFNPs obtained from DLS (Figure 3). The SFNPs had spherical shape with no obvious aggregation based on the SEM analysis.

Figure 2. Size distribution of SFNPs (a) and Zeta potential distribution of SFNPs (b).

Figure 3. Scanning electron micrograph of silk nanoparticles.



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3.2 FTIR analysis of VANCO loaded SFNPs FTIR spectra revealed the interaction between VANCO and SFNPs. Accordingly, three characteristic bonds at 1629 cm−1, 1518 cm−1, and 1230 cm−1 were observed in the SFNP spectra which were respectively related to amide I (C=O stretching vibration and NH in-plane bending), amide II (NH in plane bending and the CN stretching vibration) and amide III (NH bending and the CN stretching vibration) (Figure 4a) bonds. VANCO hydrochloride also exhibited some typical peaks in FTIR spectra such as 3284 cm-1 (stretching vibration of N-H), 1506 cm-1 and 1647 cm-1 (bending vibration of N-H and C=O and stretching vibration of C-H and C-N), and 1021 cm-1 and 1062 cm-1 (stretching banding of C-N and C-O) (Figure 4b). The conjugation between SFNPs and VANCO was confirmed by the appearance of two peaks including 1057 cm1

(stretching banding of C-OH and C-O-C) and 1378 cm-1 (C-C aromatic) (Figure 4c).

Figure 4. FTIR spectra of SFNP (a), VANCO (b), and VSFNP (c). 12 ACS Paragon Plus Environment

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3.3 Drug loading efficiency and in vitro drug release The data showed that an increase in the content of VANCO increased the drug loading capacity of SFNPs. However, the maximum loading of VANCO of 18.84% was observed in VSFNPs with a 5:1 ratio (SFNPs/VANCO), while the loading was only 1.54 for those samples with 5:1 ratio of SFNPs and VANCO. Most of the samples showed >90% EE (Table 1). Figure 5a and 5b show the release kinetics of VANCO from SFNPs in two different pH-values. Close to 100% of the VANCO was released from samples A and B in pH 4.5 after 3 days. A similar trend of VANCO release was also observed at pH 7.4. After 6 days, the release rates of VANCO from samples D and E reached a plateau at both pH-values. Generally, the release rate of VANCO from all samples in pH 4.5 was slower than at pH 7.4. For instance, the amount of VANCO released from sample E was about 50% and 80% after 8 days at pH 4.5 and 7.4, respectively. The cumulative release of VANCO from SFNPs embedded in silk scaffolds at both pH-values is also represented in Figure 6a and b. After 24h at pH 4.5, about 14.5% of the VANCO was released from the scaffolds containing sample D, while this was about 6.5% for sample E. However, at pH 7.4, the release rate of VANCO from the scaffolds containing samples D and E were 17.7% and 12.9%, respectively. After 30days, the release rates of drug from scaffolds containing sample E was about 89.8% in pH 4.5, while this was about 69.9% at pH 7.4. Table. 1. Actual amount of vancomycin, loading content and encapsulation efficiency of VSFNPs

Sample

Ratio Vancomycin Protein/Vancomycin (mg)

Loading content (LC%)

Encapsulation efficiency (EE%)

A

5:0.1

0.1

1.5

77

B

5:0.3

0.3

5.2

87

C

5:0.5

0.5

9.3

93.8



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D

5:0.7

0.7

13

93

E

5:1

1

18.8

94.2

Figure 5. Cumulative release profile of vancomycin from VSFNPs at pH=7.4 (a), and pH=4.5 (b).

Figure 6. Cumulative release profile of VANCO from SF scaffold at pH=7.4 (a), and pH=4.5 (b). 14 ACS Paragon Plus Environment

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3.4 Evaluation of pH on scaffold structure Based on the XRD spectra (Figure7), two sharp peaks related to the SF scaffolds appeared at 2Ɵ=20.16° and 2Ɵ=24.27° which were related to type II and I silk, respectively. Similar peaks were also observed after incubating the scaffolds at pH 4.5 after 30 days, while at pH 7.4, the intensity of 2Ɵ=20.16° decreased and the peak at 2Ɵ=24.27° disappeared. Moreover, SEM micrographs showed that incubating the SF scaffolds at both pH-values changed the surface morphology and inner structure of the scaffolds over 30 days (Figure 8). These results indicated the degradation of the scaffolds and the loss of interconnected pore structure.

Figure7. X-ray diffraction of SF scaffolds at different pH conditions.



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Figure 8. Scanning electron micrographs of SF scaffold (a), SF scaffold after incubating at pH 7.4 (b), and pH 4.5 (c), inner structure of SF scaffold (d), inner structure of SF scaffold after incubating at pH 7.4 (e), and pH 4.5 (f).

3.5 Mechanical testing Figure 9 shows the mechanical strength of scaffolds under dry and wet conditions. The compressive modulus of the SF scaffolds with and without SFNPs under dry condition were 2.56 and 2.31 MPa, respectively. This data shows that adding SFNPs has no significant effect on the mechanical strength of SF scaffolds due to addition of low amount of SFNPs to the polymeric matrix. However, the difference between the compressive modulus of these samples might be related to the stress transfer from the polymeric matrix to the NPs. The compressive modulus of SF scaffolds with and without SFNPs under wet condition were 1.134 MPa and 0.9764 MPa, respectively. It seems that placing the samples for 30 min in SBF might be the possible reason


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for decrease in the modulus in wet condition in comparison to dry condition. However, no significant difference in mechanical strength was observed between these samples.

Figure 9. Compressive modulus of SF scaffolds with and without SFNPs under dry and wet conditions. 3.6 Cell viability and ALP activity Figure 10a shows the comparable viability of rabbit osteoblast cells the different scaffolds to TCPS as a control group. Some groups also showed lower but not significant differences in cell proliferation rate in comparison to the control group. Therefore, the fabrication processes and VANCO had no harmful effect on cell viability. ALP production in extract from all three types of the scaffolds showed no significant differences compared to TCPS over time (Figure 10b). However, after 7 and 14 days, higher ALP activity was observed in all samples compared to day



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3. Moreover, the amounts of ALP production at all time intervals were similar to the control group.

Figure 10. MTT assay results of osteoblast cells on the various scaffolds at various incubation times (a), and ALP production by osteoblast cells on the various scaffolds up to 14 days (b). 3.7 Susceptibility testing The MIC and MBC of VANCO for the MRSA used in this study were 1 and 256 µg/mL, respectively. The KB disk diffusion method was also used to evaluate the bioactivity of the eluted VANCO from sample E embedded in SF scaffolds. The positive control, a standardized clinical pathology control containing 30 µg VANCO, showed a zone inhibition of 20 mm, while the negative control (scaffold without VSFNPs) showed a zone of inhibition of 0 mm (data not shown), confirming the assay performance. At all time points, the mean diameter of the zone of inhibition was about 12 mm to suggest sensitivity to MRSA (Figure 11). Moreover, an increase in the elution time frame did not decrease inhibitory zone diameter, significantly. These data indicated that the released VANCO was still bioactive after30days; however, the bioactivity of drug decreased with time (Figure 12).


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Figure 11. S. aureus inhibition zone assay using antibiotic release assay from different time points (day 25-29) pH=7.4 (a), and pH=4.5 (b).

Figure 12. Zones of inhibition measured for VANCO released from SF scaffold.



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3.8 Severe osteomyelitis model Inducting osteomyelitis by injecting1 × 108 and 2 × 108 CFU/mL into a bone defect. The induction of osteomyelitis was confirmed as a severe infection by evaluating body weight, CBC, and H&E staining. The body weight of the infected rats decreased without significant differences between the groups in comparison to the control group. Moreover, a high CBC count was observed in two different injection groups (Figure 13). In all groups, WBC level was in the normal range before surgery. However, this level was significantly increased after seven days post injections. The animal received high doses of bacteria showed higher WBC level. Moreover, no change in WBC level was observed in control group that received no bacterial injection. Many animals which received 2 × 108 CFU/mL bacterium, showed extensive pus at the infected site (Figure 14d). The effectiveness of osteomyelitis induction was further confirmed by histological assessment. The control group showed no histological evidence of infection after 3 weeks (Figure14a). However, bone destruction and a large obsess was observed in both experimental groups for severe conditions, including the 2×108 CFU/mL injected group (Figure14b and c).


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Figure 13. The WBC level before and after bacterial injections. Group 1: control without injection, Group 2: the animals received 1×108 bacteria, Group 3: the animals received 2×108 bacteria.

Figure 14. Histological examination of rat bones in control group (a), 108 CFU/mL and 2*108 injected groups (b & c) and existence of pus at the infected site in 2 x108 injected group (d). Osteocytes (white arrow), blood vessels (white arrowhead), inflammatory cells (black arrow) and pus (black dash line).

3.9 Radiographic Data Lateral radiographs were taken before and after implantation. As mentioned earlier, sequestral bone formation and the rate of destructed bone were used as radiographic criteria for the osteomyelitis models. Furthermore, the success in treating the animals suffering from



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osteomyelitis was also assessed by radiography. Figure15a shows the radiographic image of healthy bone without any intervention. Figure15b shows the tibia bone after injection of 2×108 CFU/mL MRSA without any treatment. As observed, destruction of bone and lysis of the medullary area with blurred cortex are seen as translucent parts at the upper segment of tibia. Similar observations were found in groups implanted with scaffolds containing SFNPs (without antibiotic) (Figure15c). However, the translucency of the infected bone was not clear in groups that received VSFNPs and scaffolds containing VSFNPs, while some infections remained. Moreover, the cavity of implanted scaffolds in tibia was open and was not filled with new bone (Figure15d and 15e).

Figure 15. Lateral radiographs of healthy bone (a), control group 6 weeks after injection of 2x108 CFU/mL MRSA (b), treatment group with SF scaffold containing SFNPs(c), treatment group with VSFNPs (d), and treatment group with SF scaffold containing VSFNPs after 6 weeks (e). 3.10 Histopathological analysis Histological analysis performed 6-weeks after implantation confirmed the preservation of osteomyelitis in those animals treated with scaffolds without VSFNPs and those animals without


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implants (Figure16a and 16b). The animals in these groups showed severe osteomyelitis with polymorph nuclear leukocytes infiltration, fibrotic tissue with a high amount of proliferative lymphocytes and some plasma cells, necrotic cells in the center, sequestral bone formation, and destruction of bone. The animals treated with VSFNPs showed necrotic cavity with decreased infiltration of inflammatory cells compared with previous groups (Figure16c). However, those animals treated with the scaffolds containing VSFNPs showed reduced infection in comparison to other groups (Figure16d). Moreover, the structure of normal bone was preserved and the inflammation was decreased.

Figure 16. Histological examination of rat bones for animals without implantation (a), animals treated with scaffolds without VSFNPs (b), animals treated with VSFNPs (c) and SF scaffold containing VSFNPs (d). Pus (white arrowhead), inflammatory cells (black arrow) and osteocytes (white arrow).



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4. Discussion Nanotechnology has opened a new avenue in designing novel delivery vehicles with targeted and sustained release properties for a broad range of biomolecules such as proteins, peptides, small molecules, genes, and antibiotics11. Generally, delivery systems can be prepared with different structures including dendrimers, liposomes, micelles, quantum dots, fullerenes, ferritin, micro and nanofibers and NPs, among other systems23,

33-36

. NPs based on natural polymers have

received focus for drug delivery due to their biocompatibility and biodegradability. In recent years, many studies have focused on using SFNPs for drug delivery applications. The tunable characteristics of SF such as biodegradability, biocompatibility, simple processing, ability to stabilize different drugs, and compatibility to be sterilized with different methods have introduced this natural protein as a potential drug delivery vehicle37. The current study provides a delivery system based on SFNPs for VANCO for treatment of severe osteomyelitis. VANCO hydrochloride was used due to the high water solubility and potency against MRSA. In previous studies, poly(methyl methacrylate) (PMMA), as a non-degradable polymer, was used for VANCO hydrochloride delivery which showed low drug release efficiency38-39. We found that increasing the drug content would be useful in increasing the loading capacity of the SFNPs. For example, a maximum LC% (18.8%) and EE% (94/2) were obtained for 1 mg/mL of VANCO loaded in 5 mg/mL of SFNPs. FTIR spectroscopy revealed the interaction between VANCO and SFNPs. The extra bands appeared at 1057 cm-1 and 1378 cm-1, indicative of spontaneous binding of VANCO with SFNPs. These spectral signatures may be due to electrostatic interactions between negatively charged SF and positively charged VANCO. The potential of SFNPs to bind by electrostatic interactions with antibiotics was also confirmed in other studies. For example,


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Sharma et al. reported gentamicin binding with SFNPs via electrostatic interactions30. Moreover, the release kinetics of VANCO was evaluated at two different pH-values. Mechanism energy metabolism by bacteria such as S. aureus can often occur under anaerobic conditions40. In this situation, the metabolism of glucose and other carbohydrates to pyruvateand subsequent degradation products such as lactic acid, mixed acid and butyric acid to affect the pH41. We hypothesized that acidic conditions may impact the release kinetics of VANCO from SFNPs. Thus, the release rates of VANCO at two different pH-values, 4.5 and 7.4 were assessed and only sample D and E had acceptable release rate at both pH-values; at pH 4.5 the release of sample D and E were 79.43% and 58.92%, respectively. At pH 7.4, the release rates of sample D and E were 93.12% and 100%, respectively. The isoelectric point (pI) of VANCO is about 8.30 and the compound is positively charged in acidic solution42. Therefore, we suggest that the repulsive forces between the positive charges of VANCO and high protonation in acidic solution could limit the release rate of VANCO at pH 4.5. Moreover, embedding VSFNPs in SF scaffolds decreased the release rate of VANCO after 30 days. Surprisingly, the samples immersed in acidic solution had higher release kinetics than pH 7.4.The data from SEM and XRD revealed that the pH could affect the structure of the SF scaffold. For example, the porous structure of the scaffolds containing VSFNPs changed, and the intensity of peaks at 2θ=20.16o and 2θ=24.27o were attenuated after incubation in pH 7.4. Therefore, it is suggested that the impact of different pH conditions on the structure and crystallinity of SF could affect the release rate of VANCO. In a previous study, we showed that the peak at 2θ=20° disappeared after 12 weeks incubation of SF based scaffold in PBS43. It was also shown in another study that increasing amount of silk II structure prompted more burst release from SFNPs44. In the present study, higher release of



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VANCO from VSFNP-loaded scaffolds was observed in acidic pH, which may be attributed to the existence of more silk II structure. Susceptibility tests were also performed in order to assess the effect of pH on the bioactivity of VANCO. The bacterial zone of inhibition (killed bacteria) is related to the quantity and efficacy of the released antibiotic. In this study, VANCO showed high bioactivity at both pH-values. The diameter of the zones were >10mm after 3 weeks in agar plates, which suggests significant antibacterial activity. At low pH, VANCO showed a higher release rate from the scaffolds, while the bioactivity of VANCO at both pH-values was similar. Thus the acidic conditions had no significant impact on VANCO bioactivity. For inducing osteomyelitis, a rat model was chosen due to costs and the potential of these animals to tolerate surgical trauma and administration of high doses of antibiotics. To induce100% infection in osteomyelitis models, about 106 to 108 CFU are required; in many studies about 2×106 CFU was used to stimulate osteomyelitis4, 45-46. We used 4×106 and 8×106 CFU to develop severe osteomyelitis disease, based on evaluating the delivery systems in treating severe osteomyelitis. Based on gross bone pathology analysis, extensive destructive lesions were observed in soft tissues with draining skin fistulas that had pus at the injured site. The body weight of animals decreased in both bacterial injected groups, while the CBC increased after bacterial injection. Based on histological analysis, large abscesses associated with osseous necrosis were observed. These results were significant in the groups injected with 8×106 CFU. In order to evaluate the efficacy of the developed drug delivery system in vivo, histopathological and radiology were performed. Based on histopathological assessment, reduced infection was observed in the tibias of animals treated with VSFNPs loaded SF scaffolds. Moreover, no improvement was detected in animals implanted with scaffolds without VSFNPs


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or in the control group (without scaffold). The bone abscesses were reduced in the VSFNPs group in comparison to other groups to suggest the preservation of VANCO activity in vivo. However, the signs of osteomyelitis were more detectable in this group compared with the VSFNPs loaded scaffold. These phenomena may be due to the loss VSFNPs without scaffolds present from the infected bone, thus loss from the defect site. Therefore, the prepared SFNPs showed to be potential in treating chronic osteomyelitis disease. Since osteomyelitis has a high incident of recurrence with S.aureus strains, it is essential to optimize the characteristics of SFNPs in terms of degradation, drug loading, and release behavior in order to prepare a system that completely remove the bacterial strains in the site of infection. Although our study demonstrated improvement through this approach with the scaffold addition, there was no stimulation of bone regeneration at the same time. Therefore, it is necessary to design an appropriate scaffold with the ability to enhance osteogenesis at the defected site while also controlling the infection. 5. Conclusions An effective system based on VSFNPs loaded SF scaffolds for treating an experimental model of severe osteomyelitis caused by MRSA was demonstrated. The antibiotic showed continuous release from SFNPs and VSFNPs loaded scaffolds over 14 and 30 days, respectively. The release of VANCO was related to pH, however pH did not affect bioactivity of the released VANCO. After implanting VSFNPs loaded scaffolds in a rat model, the scaffolds containing VSFNPs reduced infections relative to the untreated control, scaffold alone and VSFNPs only groups after 6 weeks. In conclusion, the designed drug delivery system could be a potential candidate for treating contaminated bone defects.



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Acknowledgments This work has been financially supported by Iran National Science Foundation (INSF) grant number 93030058 and was approved by the Pasteur Institute of Iran.

Conflict of interest The authors claim no conflict of interest.

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