Elsevier

Journal of Controlled Release

Volume 267, 10 December 2017, Pages 133-143
Journal of Controlled Release

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

https://doi.org/10.1016/j.jconrel.2017.08.007Get rights and content

Abstract

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.

Introduction

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], [6], [7], [8], [9]. This suggests 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], [10], [11], [12]. This high failure rate, which exceeds 40%, is mainly attributed to poor intracellular penetration of the drug [4], [9], [10], [11], [12].

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], [14], [15], [16]. However, the liposome formulations generally suffer from low drug encapsulation efficiency (drug entrapped/drug added, < 20%) [14], [15], [16], [17]. Polymeric NPs based on poly(lactic-co-glycolic acid) (PLGA) or chitosan have also been used to encapsulate vancomycin, but they can only afford 4–6 wt% loading efficiency (drug/NPs) due to the high hydrophilicity of the drug [18], [19]. Both cases are undesirable: the low encapsulation efficiency increases the production cost, and the low drug loading necessitates the administration of a large quantity of excipients, which can possess undesirable biological activities. Moreover, the high fraction of carrier components increases the concentration of NPs, which may cause aggregation of the particles.

In this study, we aim to develop a polymeric particle formulation that can efficiently encapsulate and deliver vancomycin to intracellular pathogens. The particle formulation consists of polymers with distinct functions: poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol-PLGA conjugate (PEG-PLGA), Eudragit E100 (E100, a copolymer consisting of dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate at a ratio of 2:1:1), and a chitosan derivative with a pH sensitive charge profile (Supporting Fig. 1). PLGA serves as the main delivery platform as a biodegradable and biocompatible polymer [20], in which the inclusion of PEG-PLGA helps maintain an appropriate level of polarity for timely release of vancomycin. E100, a cationic copolymer soluble at < pH 5 [21], is used to enhance vancomycin encapsulation. E100 is typically used in oral dosage forms for taste masking and transdermal patches for mucoadhesion [21], [22]. In the current formulation, E100 is blended as a minor fraction of polymer matrix to retain the drug via non-covalent interactions. Finally, a chitosan derivative we previously developed [23], called zwitterionic chitosan (ZWC), is included to trigger the drug release after cellular uptake of the particles. ZWC is negatively charged at pH 7.4 but positively charged in weakly acidic environment. ZWC is thus expected to neutralize the cationic charge of E100 at physiological pH to mitigate potential toxicity but enhance the drug release in acidic milieu via electrostatic repulsion of E100-bound vancomycin. The NPs are produced in the range of 500–1000 nm to promote preferential uptake by macrophages while avoiding embolism [24], [25].

On the basis of the size and the interplay between the components, we hypothesize that the particles will readily be phagocytosed by macrophages, release vancomycin in the acidity of endo/lysozymes, and effectively kill intracellular pathogens. Here, we produce and characterize multi-component NPs with high capacity to encapsulate vancomycin and test their antimicrobial activities against different strains of Gram-positive clinical isolates resident in macrophages. NPs prove to be a unique delivery platform of vancomycin, which is superior to free vancomycin in reducing intracellular pathogens.

Section snippets

Materials

Vancomycin hydrochloride, ninhydrin, hydrindantin, fetal bovine serum (FBS) and Mueller-Hinton broth (MHB) were purchased from Sigma-Aldrich (St. Louis, MO). PLGA (50:50 LA:GA, 0.15–0.25 dL/g, acid-terminated) was purchased from LACTEL® (Denver, CO). Methoxy PEG-PLGA (PEG-PLGA, PEG- 5 kDa, PLGA 50:50 LA:GA 4 kDa) and RhoB-PLGA (LA:GA 50:50, ester-terminated, 30 kDa) were purchased from Akina, Inc. (West Lafayette, IN). Eudragit E100 was a gift from Evonik (Darmstadt, Germany). Ethanol,

Eudragit E100 facilitates vancomycin encapsulation in PLGA NPs

The formulation, yield, and drug loading capacity of different NPs are summarized in Table 1. Vancomycin was initially loaded in PLGA NPs (PV). Due to the hydrophilic nature of vancomycin, PV showed a limited drug loading capacity (3.8 wt%). When ZWC was added (PZV), the drug loading was further decreased to 2.4 wt%. In general, NPs containing ZWC were porous in SEM, most likely due to the heterogeneity of the primary emulsion (Supporting Fig. 2). The pores might have served as water channels

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,

Acknowledgments

This work was supported by NSFDMR-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.

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