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Accepted Manuscript Particle engineering for intracellular delivery of vancomycin to methicillin-resistant Staphylococcus aureus (MRSA)-infected macrophages

Yihua Pei, Mohamed F. Mohamed, Mohamed N. Seleem, Yoon Yeo PII: DOI: Reference:

S0168-3659(17)30774-5 doi: 10.1016/j.jconrel.2017.08.007 COREL 8909

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

15 February 2017 30 July 2017 5 August 2017

Please cite this article as: Yihua Pei, Mohamed F. Mohamed, Mohamed N. Seleem, Yoon Yeo , Particle engineering for intracellular delivery of vancomycin to methicillin-resistant Staphylococcus aureus (MRSA)-infected macrophages. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Corel(2017), doi: 10.1016/j.jconrel.2017.08.007

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ACCEPTED MANUSCRIPT Particle engineering for intracellular delivery of vancomycin to methicillin-resistant Staphylococcus aureus (MRSA)-infected macrophages Yihua Pei1 , Mohamed F. Mohamed2 , Mohamed N. Seleem2,3 , Yoon Yeo1,4,* 1

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Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA 2

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Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907, USA Purdue Institute for Inflammation, Immunology, and Infectious Diseases, West Lafayette, IN,

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West Lafayette, IN 47907, USA 4

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Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA

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* Corresponding author:

Phone: 765.496.9608

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Fax: 765.494.6545

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Yoon Yeo, Ph.D.

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Methicillin-resistant Staphylococcus aureus (MRSA) infection is a serious threat to the public health. MRSA is particularly difficult to treat when it invades host cells and survive inside the cells. Although vancomycin is active against MRSA, it does not effectively kill intracellular MRSA due to the molecular size and polarity that limit its cellular uptake. To overcome poor intracellular delivery of vancomycin, we developed a particle formulation (PpZEV) based on a blend of polymers with distinct functions: (i) poly(lactic-co-glycolic acid) (PLGA, P) serving as the main delivery platform, (ii) polyethylene glycol-PLGA conjugate (PEG-PLGA, p) to help maintain an appropriate level of polarity for timely release of vancomycin, (iii) Eudragit E100 (a copolymer based on dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate, E) to enhance vancomycin encapsulation, and (iv) a chitosan derivative called ZWC (Z) to trigger pH-sensitive drug release. PpZEV NPs were preferentially taken up by the macrophages due to its size (500-1000 nm) and facilitated vancomycin delivery to the intracellular pathogens. Accordingly, PpZEV NPs showed better antimicrobial activity than free vancomycin against intracellular MRSA and other intracellular pathogens. When administered intravenously, PpZEV NPs rapidly accumulated in the liver and spleen, the target organs of intracellular infection. Therefore, PpZEV NPs is a promising carrier of vancomycin for the treatment of intracellular MRSA infection.

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Keywords: Nanoparticles; intracellular drug delivery; pH-sensitive; macrophages; intracellular MRSA; vancomycin

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ACCEPTED MANUSCRIPT 1. Introduction

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Bacterial resistance has been identified in every geographic region of the world and posed a significant global public health challenge [1]. Annually, in the United States alone, multidrug resistance pathogens negatively impact the lives of over two million patients at a cost of $20 billion to the healthcare system and result in over 23,000 deaths [2]. Half of these fatalities are attributed to a single bacterial pathogen, methicillin-resistant Staphylococcus aureus (MRSA) [3]. S. aureus can invade and survive in mammalian host cells [4]. Within these safe havens, S. aureus reproduces and forms a repository, often causing chronic and recurrent infections. Infected patients become life-long carriers, chronically suffering from the infection, or die from invasive forms of the disease [5-9]. This suggest that eradicating intracellular S. aureus is the key to clinical success; however, treatment with conventional antimicrobials during the S. aureus intracellular invasion phase is a daunting task [4]. Most antimicrobials are unable to access infected host cells and achieve the optimal therapeutic concentrations within the intracellular replicative niches. As such, the therapeutic value of vancomycin (drug of choice for treatment of MRSA) is often limited, and clinical failures are common in intracellular MRSA infections [9-12]. This high failure rate, which exceeds 40%, is mainly attributed to poor intracellular penetration of the drug [4, 9-12].

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One way to overcome poor intracellular delivery of vancomycin is to encapsulate the drug in particulate formulations and take advantage of the inherent ability of phagocytes to internalize the particles. Several studies have used liposomal nanoparticles (NPs) for the delivery of vancomycin to macrophages [13-16]. However, the liposome formulations generally suffer from low drug encapsulation efficiency (drug entrapped/drug added, 10 times higher vancomycin concentration in macrophages than free vancomycin.

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We attempted to quantify vancomycin delivered to J774A.1 macrophages via PpZEV or free vancomycin. Macrophages were treated with PpZEV NPs or free vancomycin (both equivalent to 4.35 µg/mL vancomycin) for 24 h. 5.57 µg of vancomycin was recovered from 2.45  107 macrophages, much greater than the quantity recovered from free vancomycin-treated macrophages (0.408 μg recovered from 1.93  107 cells), consistent with the confocal images. These values translate to 575.5 µg/mL and 53.5 µg/mL, respectively, given that the volume of a single macrophage is 3.95  10-10 mL [28]. Both concentrations would be substantially higher than the reported MIC values of vancomycin against MRSA (0.5-2 μg/mL) [35]. However, these values do not represent exclusively intracellular vancomycin. It is rather likely to include those bound to the macrophage surface, which could not be completely removed by washing due to the high affinity of vancomycin for cell surface proteins.

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3.7. PpZEV NPs enhanced reduction of intracellular MRSA.

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Prior to intracellular microbial activity testing, toxicity of PpZEV NPs to J774A.1 macrophages was evaluated by the MTS assay. The viability of the macrophages was not affected after 24 h incubation with 56 µg/mL and 112 µg/mL of PpZEV NPs, equivalent to 5 and 10 µg/mL vancomycin, respectively (Supporting Fig. 11). This indicates that PpZEV NPs would not affect the viability of macrophages at the concentrations used for evaluation of intracellular antimicrobial activity.

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Antimicrobial activity of different NPs against intracellular bacteria was evaluated in J774A.1 macrophages infected with MRSA USA400, a community acquired MRSA strain predominant in the United States [36]. The MRSA-infected macrophages were incubated with NPs or free vancomycin at an equivalent vancomycin dose (5 µg/mL) for 24 h. Of note, since the tested NPs had different drug loading capacities (Section 3.1, Table 1), the amounts of NPs corresponding to the specific dose of vancomycin were unequal. We assumed that this variation would have minimal influence on macrophage uptake of the NPs given the phagocytic nature of macrophages. After 24 h incubation with the vancomycin treatment, the cells were washed and lysed, and the colony forming units (CFU) of MRSA in the cell lysate were compared. PpZEV NPs that had shown high drug release (Fig. 2a) showed the greatest anti-bacterial activities against intracellular MRSA (Fig. 5a), whereas PEV NPs with the least drug release was virtually ineffective. It is worthwhile to note that PV, PZV and PpZV did not show significant reduction in intracellular MRSA despite the high drug release (Supporting Fig. 3, Fig. 5a). The main difference between PpZEV and these NPs is that the former released less vancomycin in pH 7.4 than the latter. This indicates that intracellular antibacterial activity was driven by the vancomycin released in the cells (i.e., at acidic organelles) rather than the drug released prior to macrophage uptake. Peterson’s correlation analysis shows a positive relationship (r = 0.8313, p = 0.0205) between the intracellular antibacterial activity (Fig. 5a) and the difference in drug releases at two pHs (Figs. 5b, c), indicating that the intracellular drug release was critical to the antibacterial activity in MRSA-infected macrophages.

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Figure 5. (a) Log-fold reduction of intracellular MRSA USA 400 by different NPs and free vancomycin. *: p < 0.05; **: p < 0.01; ***: p < 0.001; and ****: p < 0.0001 by Tukey's multiple comparisons test. (b) Difference in vancomycin release between pH 7.4 and pH 5 in 1h. *: p < 0.05 by Tukey's multiple comparisons test. (c) Correlation of drug release difference at two pHs and intracellular antibacterial effects. All treatments were equivalent to 5 µg/mL vancomycin.

3.8. PpZEV NPs showed superior antibacterial activities against different intracellular pathogens resident in J774A.1 macrophages. The antibacterial activities of PpZEV NPs were tested against an extended collection of intracellular bacteria and compared with those of free vancomycin at an equivalent dose. PpZEV (equivalent to 5 µg/mL vancomycin) showed superior antibacterial activities to those of free

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vancomycin against most intracellular bacteria (Fig. 6a). In particular, PpZEV exhibited 100 times greater antibacterial effect against intracellular Listeria than free vancomycin. When the dose was doubled (equivalent to 10 µg/mL vancomycin), the difference between PpZEV and free vancomycin was more pronounced in several strains, such as MRSA USA1000, 1100 and S. pneumoniae 51916 (Fig. 6b). Blank carrier (PpZE) did not possess any antibacterial effect on free bacteria or intracellular bacteria (data not shown); therefore, the superior effect of PpZEV is attributable to intracellular delivery of vancomycin.

Figure 6. Intracellular antimicrobial activity of PpZEV against important Gram-positive bacterial pathogens. (a) PpZEV: 56 µg/mL (equivalent to 5 µg/mL vancomycin); *: p < 0.01; ***: p < 0.0001 by Sidak's multiple comparisons test, (b) PpZEV: 112 µg/mL (equivalent to 10 µg/mL vancomycin); *: p < 0.01; **: p < 0.001; ***: p < 0.0001 by Sidak's multiple comparisons test.

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3.9. Intracellular release kinetics of vancomycin may explain incomplete elimination of intracellular MRSA.

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PpZEV showed greater activities against intracellular bacteria than free vancomycin, but it did not completely eliminate them. It was difficult to determine the concentration of exclusively intracellular vancomycin, as described in Section 3.6. Nevertheless, if we consider efficient cellular uptake of NPs evident from the confocal microscopy, we may speculate that the amount of PpZEV delivered into the cells was sufficiently high but intracellular vancomycin release has been suboptimal. Specifically, we noted that macrophages incubated with duallabeled P*pZEV* for 3 h and 24 h showed persistent yellow signals (Fig. 4, Supporting Fig. 12) indicating the colocalization of RhoB-PLGA and BODIPY-vancomycin. This suggests that vancomycin release in the acidic organelles was incomplete, in contrast to the prediction based on in vitro drug release at pH 5 (Fig. 2). We were curious to know if intracellular vancomycin release was more restricted than in release medium due to the limited escape of NPs and the released vancomycin from the phagosomes. To validate this, macrophages were incubated with the dual-labeled P*pZEV* NPs for 3 h or with additional 24 h in NP-free medium and lysed with Triton X100. The phagocytosed NPs were separated from the cell lysate and imaged with confocal microscopy. In parallel, P*pZEV* NPs incubated in buffers (pH 7.4 and pH 5) were also imaged along with fresh NPs. Fresh NPs with no drug release showed bright yellow signals indicating complete overlap of vancomycin (green) and polymer (red) signals (Fig. 7a). The NPs incubated in pH 5 buffer for 1 h showed red polymer signals due to the depletion of vancomycin (Fig. 7c), and those in pH 7.4 buffer intermediate signals indicating partial drug release (Fig. 7b), consistent with the in vitro drug release. On the other hand, the NPs incubated in the macrophages for 3 h or additional 24 h showed all three colors, confirming incomplete intracellular drug release (Figs. 7d and 7e). The incomplete drug release may be caused by the lack of phagosomal escape of PpZEV NPs after phagocytosis, which will result in drug saturation in the organelles and thus hamper further vancomycin release from the NPs.

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In this regard, we note that the current practice of in vitro release kinetics carried out in a large volume of medium does not necessarily reflect this challenge and may lead to an overestimation of the intracellular drug activities. For promoting the phagosomal escape, strategies used in gene delivery may be considered, including the use of fusogenic lipids [37] and cell penetrating peptides (CPPs) [38] with the endosomolytic activity, such as dfTAT and GALA peptides [39, 40]. Since these peptides can disrupt the phagosomal membranes, vancomycin confined in the phagosome will be released to the cytosol and help release the remaining vancomycin from the NPs by providing an unsaturated environment [39, 41].

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Figure 7. Confocal images of dual-labeled P*pZEV* NP at different conditions: (a) Fresh NPs, (b) NPs incubated in pH 7.4 buffered saline for 1 h, (c) NPs incubated in pH 5 buffered saline for 1 h, (d) NPs recovered from cells after 3 h incubation, and (e) NPs recovered from cells after 3 h incubation and additional 24 h incubation in fresh medium. Scale bars = 50 µm. (f) Semi-quantitative assessment of the extent of drug release based on image analysis by Fiji software , based on 2-3 images per condition.

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3.11. PpZEV NPs accumulated in the liver and spleen in 10 min.

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We next turned our attention to evaluate the tissue distribution of NPs in vivo. BALB/c mice were injected with PpZEV NPs via tail vein and imaged over time. For whole body fluorescence imaging, PpZEV NPs were loaded with DiR near infrared fluorescent dye. DiR is well retained in the particles suspended in 50% serum, and free DiR is not intensely fluorescent in aqueous medium due to aggregation and also rapidly eliminated via the RES organs and the kidneys [42]. Therefore, DiR encapsulated in PpZEV is suitable for representing the NPs in vivo. As shown in Fig. 8, strong DiR signal was observed in the liver and spleen in 10 min. Those organs showed persistent signals till 3 h post-injection and gradually decreased over the next 93 h. Ex vivo images of organs at 3 h and 96 h post-injection confirmed the preferential accumulation of NPs in the liver and the spleen and very little in other organs including blood. Rapid accumulation of NPs in these organs is desirable for two reasons: First, the liver and the spleen are the target organs where infected macrophages are predominantly located. Second, NPs spend little time in circulation prior to the tissue distribution; thus, the premature drug release is less concerning than in other applications requiring long-term circulation. At present, we do not

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know whether the decreasing fluorescence signal over time indicates the elimination of NPs from the organs or slow release of DiR from the NPs. It also remains to be seen how rapidly the resident macrophages will take up the accumulating NPs, although in vitro observation suggests that this process be quick (Supporting Fig. 5).

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Figure 8. (a) Whole body imaging of BALB/c mouse that received DiR-loaded PpZEV NPs by intravenous injection. V: ventral view; D: dorsal view. See Supporting Fig. 13 for all 3 animals. (b) Ex vivo imaging and fluorescence intensity of major organs at 3 h or 96 h post-injection. From the top: heart (H), lung (Lu), liver (L), spleen (S), kidneys (K), gastrointestinal tract (GI), and blood (B) (n = 3 per time point).

4. Conclusions

We developed vancomycin- loaded pH-sensitive PpZEV NPs with polymers providing distinct functions: PLGA as the main delivery platform, PEG-PLGA to facilitate drug release, Eudragit E100 to enhance vancomycin loading, and ZWC to trigger lysosomal vancomycin release. PpZEV NPs were superior to free vancomycin in killing intracellular MRSA and other intracellular pathogens due to their ability to facilitate the cellular uptake of vancomycin and its delivery to the intracellular bacteria. However, their phagosomal escape remains to be further improved. In mice, intravenously administered PpZEV NPs rapidly accumulated in the liver and spleen, the target organs of intracellular infection.

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ACCEPTED MANUSCRIPT Acknowledgments This work was supported by NSF DMR-1410987. The authors also acknowledge support from the Ronald W. Dollens Graduate Scholarship and Purdue Research Foundation Research Grant. The authors thank Liang Pang and Ning Han for the help with in vivo imaging. References

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