Gold Nanorods as Drug Delivery Vehicles for Rifampicin Greatly

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Gold Nanorods as Drug Delivery Vehicles for Rifampicin Greatly Improve the Efficacy of Combating Mycobacterium Tuberculosis with Good Biocompatibility with the Host Cells. Hala R. Ali, Moustafa R.K. Ali, Yue Wu, Salah A. Selim, Hazem F. M. Abdelaal, Essam A. Nasr, and Mostafa A. El-Sayed Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00430 • Publication Date (Web): 05 Sep 2016 Downloaded from http://pubs.acs.org on September 7, 2016

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Bioconjugate Chemistry

Gold Nanorods as Drug Delivery Vehicles for Rifampicin Greatly Improve the Efficacy of Combating Mycobacterium Tuberculosis with Good Biocompatibility with the Host Cells.

Hala R. Ali

∞, Φ, #, ‡

. Moustafa R. K. Ali

∞, ‡

. Yue Wu∞. Salah. A. Selim#. Hazem F. M. Abdelaal Ψ.

Essam A. NasrΓ and Mostafa A. El-Sayed ∞, ∆,*

∞

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-

0400, USA. Φ

Animal Health Research Institute (AHRI), Department of bacteriology and Immunology, Dokki,

Giza, Egypt #

Department of Veterinary Medicine, Cairo University, Giza, Cairo, Egypt.

Ψ

Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI

53706, USA. Γ

Veterinary Serum and Vaccine Research Institute, "Bacterial Diagnostics Research Department"

(Tuberculosis), Abbasia, Cairo, Egypt ∆

Adjunct Professor, School of Chemistry, King Abdul Aziz University, SA

‡

Equal contribution

* Corresponding author: 901 Atlantic Drive, Atlanta, GA, 30332-0400.USA. E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract TB remains a challenging disease to control worldwide. Nanoparticles have been used as drug carriers to deliver high concentrations of antibiotics directly to the site of infection, reducing the duration of treatment along with any side effects of off-target toxicities after systemic exposure to the antibiotics. Herein we have developed a drug delivery platform where gold nanorods (AuNRs) are conjugated to rifampicin (RF), which is released after the uptake into macrophage cells (RAW264.7). Due to the nature of the macrophage cells, the nanoparticles are actively internalized into macrophages and release RF after uptake, under the safety frame of the host cells (macrophage). AuNRs without RF conjugation exhibited an obvious antimicrobial activity. Therefore, the AuNRs could be a promising antimycobacterial agent and an effective delivery vehicle for the antituberculosis drug Rifampicin for use in tuberculosis therapy.


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Figure abstract

Introduction Tuberculosis (TB) is a highly infectious and transmissible disease worldwide. 1-3 It is caused by M. tuberculosis, which is an intracellular pathogen with the alveolar macrophage as the primary site of replication.

(4, 5, )6

) Although effective antimicrobials against M. tuberculosis are

extensively available, such as isoniazid and rifampicin, the two most powerful first-line anti-TB drugs, TB remains a major public health threat.

7, 8

The recent treatments fail either to cross the

macrophage barrier or to reach the infection site in sufficient concentrations to be able to cure. 9 Additionally, the side effects of anti-TB drugs include the harm of hepatocytes and neuronal cells with the desired result of targeting the infected macrophages often being missed.

10-13

Side

effects also cause reduced quality of life for patients, leading many of them to abandon their therapy, which results in the heightened complexity of treatment and the emergence of severely resistant strains of TB.

14, 15

Selective delivery of antibiotics into macrophages has the potential

to greatly increase their therapeutic effect by achieving higher drug concentrations locally where



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the M. tuberculosis replicate while limiting systemic toxicities.

16, 17

Recently, multiple

approaches have worked to encapsulate the anti-tuberculosis drugs within nanocarriers.

18-20

because phagocytic macrophages function by clearing the circulatory system of any foreign substances, nanocarriers are able to accumulate within these cells.

21-22

) The nanocarriers,

consequently, may deliver high concentrations of therapeutic agents to the infected sites,

23-26

)

facilitating the sterilization of these sites while also minimizing the incidence of drug resistance, without exposing the patients to the side effects associated with high systemic doses. Although with many advantages for selective delivery and elimination of side effects, nanomaterials for TB treatment have been rarely developed and applied to date, and as such require a great deal of further research. nanoparticles

29-31

27

) So far, only SiO2, Fe2O3 (19) , silver nanoparticles

28

)

and polymer

) have been conjugated or loaded with TB antibiotics, and some toxicity

effects from these particles have been observed. 32, 33Although gold nanoparticles (AuNPs) have not yet been tested for TB treatment, they have been introduced in multiple biomedical applications such as the diagnostics of cancer, plasmonic photothermal therapy (PPTT), and as a platform for targeted delivery of anticancer drugs. 26, 34, 35 This noble metal, on the nanoscale, has shown antimicrobial properties against Gram-negative and Gram-positive pathogens, including multidrug-resistant pathogens.

36

Thus, we hypothesized that conjugation of the AuNPs with

rifampicin might potentiate their efficacy in combating the M. tuberculosis infection in macrophages while having minor effect on the host cells, as AuNPs are widely confirmed to be biocompatible in RAW264.7 and many other cell lines.

37, 38

In this study, we investigate the

activity of AuNPs either in free form or conjugated with rifampicin on extracellular and intracellular TB infections, the uptake of AuNRs by the targeted cells, and the cytotoxicity of this nanocarrier against RAW264.7 cells as a host model for TB. Our experiment showed


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promising results for effectively targeting and killing TB under the safety frame of the host cells. Results and Discussion Characterization of AuNRs We synthesized AuNRs using seedless growth method according to known methods.

39

) TEM

was used to determine the quality, dimensions, and homogeneity of the AuNRs. As shown in Figure 1, the AuNRs were uniformly shaped with an approximate length and width of 25 (± 3) × 5 (± 0.8) nm. Figures 1b show the UV-Vis absorption spectra for AuNRs before and after conjugation. Before conjugation, the AuNRs show an aspect ratio of approximately 5 and exhibit a SPR band that is centered on 800 nm. The nanoparticles were first conjugated with BSA in order to block nonspecific interactions and enable further loading of Rifampicin (RF).

40

To

confirm successful conjugation with BSA and RF, we measured the absorption spectra of the AuNRs before and after conjugation. As shown in Figure 1b, after conjugating with BSA, the SPR band of AuNRs showed an observed red shift of 8 nm. After further conjugation with RF, we observed a new peak, which appeared at 310 nm that belonged to the absorption wavelength of RF. Zeta potential further confirmed the presence of BSA and RF conjugations on gold nanoparticles. AuNRs with CTAB have positive zeta potential of 52.3±11.0 mV. After BSA conjugation, the zeta potential of AuNRs@BSA decrease to -18.4±7.09 mV. The final zeta potential of AuNRs@RF is -21.7±5.55 mV. The number of rifampicin molecules per each gold nanorod has been calculated from their extinction spectra, From the calculation, we found that ~657 rifampicin molecules present on each AuNRs@BSA. The high payload capacity and biocompatibility of the AuNRs@BSA have been attributed to the stabilization of the AuNRs by the protein corona around it.



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AuNSs were also synthesized using the conventional citrate reduction method. The same conjugation was applied. The characterization of AuNSs (including TEM, UV-Vis) indicates the successful formulation of AuNSs@RF, as shown in the Supplementary Information Figure S1.

Figure 1. Characterization of AuNRs (Length 26±3, width 5±0.8 nm). (a) TEM image with 100 nm scale bar. (b) UV-Vis absorbance spectra showing the SPR peaks of AuNRs before and after conjugation with BSA and RF.

Effect of the AuNPs on Macrophage cells Macrophage cells are the host cells for TB. In order to develop a more successful treatment for TB, we need to avoid or decrease the negative effects of the anti-TB treatment on the macrophage cells. RAW264.7 cells were used as a model of macrophage host for TB. After incubation with RAW264.7 cells, a clear uptake of AuNRs was observed under dark field microscopy (Figure 2a). To examine the AuNRs effect on host cells, we tested the cell viability


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of macrophage cells after 24 h incubation with AuNRs under different concentrations. AuNRs@RF at 5 nM concentrations maintained a cell viability above 90% (Figure 2b), while at 10 nM concentration, the AuNRs@RF decrease to 73% (Figure 2b). This decrease in cell viability is likely due to the different cellular uptake, as RF conjugation has been reported to increase the rate of endocytosis to nanoparticles.

40

For AuNSs@RF, both 5 nM and 10 nM

nanoparticle concentration achieved over 90% cell viability (Figure S2). The cytotoxicity of AuNRs is a little higher than AuNSs, which is possibly due the residual CTAB on AuNRs

41

or

difference in nanoparticle uptake, as the uptake of nanorods by macrophages was reported to be more efficient than that of nanospheres.

42

The slight cytotoxicity effect of 10 nM AuNRs@RF

was also confirmed with apoptosis/necrosis assay (Figure S3). The RF conjugation was observed to slightly increase the cytotoxicity of AuNPs to host cells, compared with only BSA coated AuNPs (Figure S4). The introduction of free RF (same amount of RF as conjugated on AuNPs) does not induce any apoptosis, and didn’t cause obvious cell viability change. Results show that 5 nM AuNRs and 10 nM AuNSs has good biocompatibility. When using 10 nM concentration, slight toxicity was observed for AuNRs@BSA and AuNRs@RF.



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Figure 2. (a) Dark field images of macrophage cells (RAW264.7) incubated with AuNRs@RF for 24 h; (b) Cell viability after incubation with AuNPs@RF under different concentration for 24 h. 5 and 10 nM concentrations were selected in our experiments (pink color). AuNPs effects on extracellular TB To examine the anti-TB effects of AuNPs, we first incubated extracellular TB (Mycobacterium tuberculosis without host cells, H37Ra strain type) with 5 and 10 nM of AuNPs. We tested the effects of gold nanoparticles on the extracellular TB by exposing the bacteria to AuNPs for 7 days and then cultivating in Lowenstein Jensen (LJ) media and Middlebrook agar and incubated for 3 weeks, as shown in Figure 3. After cultured on LJ medium, M. tuberculosis appeared as brown and granular colonies, showing in Figure S6 as the greener vial color indicates smaller TB population presented. Results indicate that all the AuNPs showed clear inhibition effect on the TB growth (Figure 3), especially AuNRs, which showed much greater anti-mycobacterial activity than AuNSs (Figure S5). The results revealed that the shapes of AuNPs played an important role in the anti-


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mycobacterial activity. The RF conjugation improved the anti-TB effect, as shown in Figure S6. After treating with 5 nM AuNRs and AuNRs@RF for 7 days, the AuNRs and AuNRs @RF reduce the mycobacterial growth to 5 colony forming units (CFU) and 2 CFU, respectively. We observed a disappearance of TB growth after 10 nM AuNRs incubation, while for the 5 nM concentration, though with great inhibition effect, the growth of TB colonies can be still observed (Figure S6). Therefore, the following experiments were conducted under the optimized concentration (10 nM AuNRs). The same experiment was done in Middlebrook agar plates (Figure 3), which also confirms the conclusion that AuNRs, especially AuNRs@RF, play a huge effect in killing extracellular TB while the effect of AuNSs is minor.

Figure 3 Middlebrook agar plates inoculated with 100 µl of mycobacterial suspension treated for 7 days with 10 nM of different AuNPs@BSA, AuNPs@RF and free rifampicin. AuNPs effects on Intracellular TB Next, we co-cultured the TB with macrophage cells and applied our AuNPs to the system. The macrophage monolayer was grown on a coverslip for 24 hours, and then incubated with antibiotics and serum free DMEM containing the tubercle bacilli bacteria (107 of mycobacterial cells) for 4 hours. The successful infection of TB on macrophage cells was tested by Zheil Neelsen staining (Figure 4e). Then, the infected cells were treated with AuNPs @RF for 3 days. The internalization of AuNPs was imaged under dark field microscope (Figure 4F) without obvious changes in cell morphology, which is consistent with The longer term (3 days) toxicity



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study of gold nanoparticles as reported in the same macrophage cell line before37. This results are also consistent with the previous reports that the microscopic images show that after treatment with AuNPs, the macrophages were still alive and maintained a normal shape (Figures 4c and d).

Figure 4. The microscopic image of RAW264.7 monolayers a) before infection; b) after infection with H37Ra; c) after infection with M. Tuberculosis and treated with AuNSs@RF (10 nM); d) after infection with M. Tuberculosis and treated with AuNRs@RF (10 nM). e) Microscopic image of RAW 264.7 macrophage cells monolayer stained with Zheil Neelsen confirming their infection of TB bacteria (multiple red bacilli). f) Dark field image for macrophage cells with AuNSs@RF showing the internalization of nanoparticles. The Middlebrook agar plates was then inoculated with lysates of murine macrophage cells infected with H37Ra and treated with 10 nM of AuNSs@BSA, AuNSs@RF, AuNRs@BSA,


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AuNRs@RF for 3 days (Figure 5a). CFU was counted, as shown in the Figure 5b. (average of 3 experiment). Results show a near complete clearance of H37Ra colonies after incubation with AuNRs@RF, which is in agreement with the anti-TB effect AuNRs@RF proved to have in the extracellular TB experiment. AuNPs is known to effective destruction both Gram-negative and Gram-positive pathogens, including multi-drug-resistant pathogens,

36,43-44,

meanwhile exhibited

low toxicity to mammalian cells. In our study, we also found similar results that AuNRs has a strong inhibition towards TB, and biocompatible to the host macrophage cells, which is in agree with the former publications. AuNRs@RF showed the best efficacy among all the particles. Herein we have developed a drug delivery platform where AuNPs are conjugated to RF which is released after the uptake into macrophage cells. Due to the nature of the macrophage cells, the AuNPs are actively internalized into macrophages and release RF after uptake. RF is physically absorbed on the surface of BSA coated AuNPs. The intracellular environment encourages RF to be released from the surface and plays a role in creating an anti-TB environment. In addition, the proteolytic enzymes produced by lysosomes and mesosomes hydrolyze BSA that promotes RF release. In order to test if RF is released from the AuNRs endocytosis, we decreased the pH of solution that disperses particles (Figure 5c) and kept overnight. Then AuNRs were centrifuged and the UV-Vis absorption spectra of both the supernatant and AuNRs were compared with the one of normal pH. The UV-Vis spectrum of isolated AuNRs displayed a loss in the absorption wavelengths of RF after lowering the pH. These results may suggest that after cellular internalization, RF is released from the AuNRs enabling it to inhibit the TB bacteria and enhance the efficacy.



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Figure 5 a) Middlebrook agar plates inoculated with lysates of Murine macrophage (RAW264.7) cells infected with H37 Ra and treated with 10 nM of AuNSs@BSA, AuNSs@RF, AuNRs@BSA, AuNRs@RF for 3 days. b) The histogram shows the mycobacterial CFU versus the different AuNPs@BSA and AuNPs@RF, free rifampicin. Statistical signiﬁcance has been indicated by * (pvalue

Gold Nanorods as Drug Delivery Vehicles for Rifampicin Greatly

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