Adaptive antimicrobial nanocarriers for the control of infectious biofilms
Liu, Yong
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Liu, Y. (2019). Adaptive antimicrobial nanocarriers for the control of infectious biofilms. University of Groningen.
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5
CHAPTER 5
Dual-Antimicrobial Conjugates
in Leukocyte-like Nanocarriers
to Eradicate Intra-Macrophageal Staphylococci
Y. Liu, Y. Ren, L. Shi, H. C. van der Mei, H. J. Busscher. To be
submitted
5
ABSTRACT
Intracellular pathogens are extremely recalcitrant to conventional antibiotic treatment. Here, we synthesized an amphiphilic binary antimicrobial conjugate which will undergo self-assembly into sub-stable nanoparticles. Subsequently, the sub-stable nanoparticles were encapsulated in leukocyte-like cell membrane. The resulting leukocyte-like nanocarriers possess Toll-like receptors on their surfaces and are internalized by especially by infected leukoytes. Once inside an infected leukocyte, encapsulated antimicrobial conjugated nanoparticles are released to kill intracellular staphylococci. The killing efficacy of ACN-LLNs was evaluated both in vitro and in
vivo. ACN-LLNs showed a synergistic killing efficacy, superior to either single antimicrobials or the bare ACN
with membrane encapsulation. This strategy contributes greatly to current antibiotic therapies to overcome the barriers towards intracellular pathogens.
KEYWORDS
:
Macrophage cell membrane, Intracellular pathogens, Synergy, Toll-like receptor5
INTRODUCTION
Infection is predicted to yield more deaths than cancer and to become the number one cause of death by the year 2050,1 mainly because of the growing number of antibiotic resistant strains and the lack of new antibiotics being brought to the market.1 This is a sobering finding, after the optimism that emerged after the discovery of antibiotics by Alexander Flemming in 1928,2 that continued to exist into the nineteen eighties when the first alarming reports on the threat of antibiotic-resistant strains were published.3 One way infectious bacteria evade antibiotic action and killing by host immune cells is by hiding themselves in mammalian cells. The mammalian cell wall acts as a barrier towards penetration of most common antibiotics,4 which makes the intra-cellular environment a protective shelter for infecting bacteria. Moreover, the diversity of enzymes present in host mammalian cells can inactivate antibiotics to further protect bacteria seeking intra-cellular shelter. Intra-cellular shelter is even provided to infecting bacteria by macrophages intended by nature to facilitate their clearance from the body5 and many intra-cellular bacterial pathogens prefer to replicate in the shelter provided by macrophages.6 After phagocytosis, bacteria initially reside in membrane-bound vacuoles, called phagosomes,7
that fuse with lysomes in which bacteria are killed by reactive oxygen species and cationic antimicrobial peptides. Alternatively, bacteria can remain dormant in the low pH environment of phagosomes. Regulation of gene expression by intra-cellular bacteria to induce escape from phagosomes, block phagosome fusion with lyposomes and resistance to reactive oxygen species and antimicrobial peptides allow intra-macrophageal bacteria to survive.7,8
Thus frequently, extremely large doses of antibiotics are needed to eradicate intra-cellular bacteria. This may lead to severe side effects for the patients, while still insufficient to cure infection.9,10
Yet eradication of intra-cellular bacteria is crucial for the long-term success of antibiotic treatment.11,12
Especially Staphylococcus aureus, traditionally considered to be an extra-cellular pathogen, has been found to cause serious infections once intra-cellularly present and is hard to eradicate.13,14
Therefore in an era of rapidly spreading, multi-drug resistant bacteria, in which the number of antibiotics available to eradicate bacterial infections is shrinking at an alarming rate,15–17
new strategies to eradicate bacterial infections are direly needed. Dual antibiotic treatment is gaining interest and has been clinically applied in local drug delivery systems, like for instance bone cements,18 but as a disadvantage, two unconjugated antimicrobials may have different release rates and penetration abilities into infectious biofilms and infected mammalian cells, including macrophages. This disadvantage can be circumvented by conjugating two antimicrobials, ensuring their simultaneous penetration and availability at their target site. Moreover, dual-antimicrobial conjugates have demonstrated the ability to synergistically kill infectious bacteria resistant to either of the two antibiotics better than two single, unconjugated antibiotics together.19,20
Packaging of existing antibiotics in suitable nanocarriers is another way to kill antibiotic resistant bacteria, and has hitherto only been done with single antibiotics and never with two conjugated antimicrobials. Surface-adaptive, pH responsive single antibiotic-loaded nanocarriers have been demonstrated to break the barriers posed by infectious biofilms to increase the efficacy of selected antibiotics to the extent that multi-drug resistant bacteria can be killed both in vitro and in vivo.21
Inspired by nature, cell membrane coating of nanocarriers has been applied to overcome mammalian cell wall barriers impeding drug delivery into the cell.22
Cell membrane nanocarrier coatings derived from red blood cells,23–25
leukocytes,26–31
platelets,32,33
tumor cells,34–36
and bacteria37
have been applied for delivery of chemotherapeutics,38
contrast agents,39
and anti-inflammatories,40
while photothermal gold-silver nanocages coated with macrophage membranes have been proposed for treatment of osteomyelitis.41
However, cell membrane coated nanocarriers have not yet been used for the delivery of antimicrobials in mammalian cells. Considering the ability of cell membrane coated nanocarriers to enter mammalian cells, they appear promising as well for use as an antimicrobial carrier for eradication of intra-macrophageal bacteria, while moreover such leukocyte-like nanocarriers can circulate
long-5
time in the blood.42
Conjugation of two antimicrobials and packaging them in leukocyte-like nanocarriers suitable to allow intra-macrophageal killing of infecting bacteria, is highly challenging. First of all, because the nanocarrier should be able to cross both the bacterial and macrophageal cell wall, each posing specific difficulties due to their possession of different chemical and structural features. Secondly, optimal conjugates are composed of chemically different antimicrobials, such as a hydrophilic and hydrophobic one, which poses not only a challenge to the conjugation, but also to the packaging. Usually, micelles are most suitable for packaging of hydrophobic drugs in their core, while liposomes can host hydrophilic drugs.43
Packaging a conjugate of a
Scheme 1. Challenges to overcome in the design of dual-antimicrobial conjugates, encapsulated
in leukocyte-like nanocarriers for intra-macrophageal killing of infectious bacteria. Details not drawn to scale. 1. Conjugation of a hydrophobic and hydrophilic antimicrobial and self-assembly into a suitable antimicrobial conjugate nanoparticle (ACN). 2. Isolation of leukocyte membranes.
3. Encapsulation of the antimicrobial conjugate nanoparticle by leukocyte membranes, to yield
a leukocyte-like nanocarrier (LLN). 4. Entry of antimicrobial conjugates encapsulated in LLNs through macrophage cell walls, mediated by their targeting to intra-cellular, infecting bacteria captured in membrane-bound phagosomes. 5. Once inside a macrophage, LLNs release their antimicrobial conjugates, killing infecting intra-macrophageal bacteria.
Leukocyte Cell membrane ACN 1 2 4 5 Antimicrobial conjugate
Conjugation and self-assembly into an antimicrobial conjugate nanoparticle (ACN)
Isolation of leukocyte membranes
Macrophage entry Intracellular antimicrobial conjugate release and
bacterial killing LLN Encapsulation 3 Nucleus Bacteria Nucleus
5
Figure 1. Characteristics of leukocyte-like nanocarriers with dual-antimicrobial conjugates.
(A) Hydrodynamic diameters as a function of storage time in 10 mM phosphate buffer of antimicrobial conjugate nanoparticles (ACNs), and ACNs encapsulated in murine (m-LLNs) or human (h-LLNs) leukocyte-like nanocarriers. Data were expressed as mean ± standard deviations (SD) over triplicate nanocarrier preparations. (B) Transmission electron micrograph of negative-stained (0.5% uranyl acetate) m-LLNs after 4 weeks of storage, showing the leukocyte membrane coatings as dark area around the ACNs. Scale bar indicates 100 nm. (C) Zeta potentials as a function of storage time in 10 mM phosphate buffer of ACNs and ACNs encapsulated in m-LLNs or h-LLNs. Data were expressed as mean ± standard deviations (SD) over triplicate nanocarrier preparations. (D) Conjugate content in wt% of m-LLNs and h-LLNs expressed relative to the initial antimicrobial conjugate content of ACNs in absence of encapsulation. Antimicrobial contents were derived from UV-Vis spectroscopy (see Figure
S4). Data are expressed as mean ± standard deviation over triplicate nanocarrier preparations.
Asterisks above the data points indicate statistical significance at p < 0.05 (*, Students’ T-test) between uncoated ACMs and LLNs. (E) Cumulative antimicrobial conjugate release in wt% from ACNs in absence of encapsulation and encapsulated m-LLNs or h-LLNs as a function of exposure time to a potassium phosphate buffer at pH 7.4. Drug release was measured using UV-Vis absorption spectroscopy. Data are expressed as mean ± SD over triplicate nanocarrier preparations. (F)Same as panel (E), now for antimicrobial conjugate release during exposure to potassium phosphate buffer at pH 5.0.
5
hydrophobic and hydrophilic drug in a nanocarrier is therewith not trivial. We here describe the design and synthesis of dual-antimicrobial conjugates composed of hydrophobic Triclosan and hydrophilic ciprofloxacin (Scheme 1), that will self-assemble in aqueous solution to form nanoparticles that we encapsulate in leukocyte-like nanocarriers (LLNs) with the aim to target and kill multi-drug resistant bacteria, residing inside macrophages (see also Scheme 1). Efficacy of dual-antimicrobial conjugates in leukocyte-like nanocarriers will be demonstrated both in vitro and in two murine infection models (a peritoneal and an intra-vascular one), using Staphylococcus aureus (one of the most common human pathogens44
) as an infecting organism.
RESULTS AND DISCUSSION
Preparation and Characteristics of Leukocyte-like Nanocarriers with Dual-Antimicrobial Conjugates. Dual-antimicrobial conjugates were synthesized via chloroacetylation of Triclosan and subsequent chloride substitution using ciprofloxacin with an overall yield of 70%. The antimicrobial conjugate composition was confirmed by 1H and 13C NMR (Figures S1 and S2, respectively) and electrospray ionization mass spectrometry (Figure S3). Conjugation was done sacrificing the hydroxyl group of Triclosan and the secondary amine group of ciprofloxacin. These groups were selected because of esterification of the phenol group of Triclosan and alkylation of piperazinyl group of ciprofloxacin, respectively have been demonstrated to have very little negative effect on their antimicrobial efficacy.45,46 For self-assembly of the antimicrobial conjugate into a nanoparticle structure, purified conjugates were dissolved in DMSO, added dropwise
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Figure 2. Protein composition and function in leukocyte-like nanocarriers. (A)The number
of leukocyte membrane-associated and other proteins incorporated on m-LLNs and h-LLNs identified by liquid chromatography–mass spectrometry (LC-MS). The inset shows the percentage of membrane-associated and other proteins. (B) Molecular mass distribution of membrane proteins in murine and human leukocytes and on m-LLNs and h-LLNs by LC-MS. (C) Same as panel (B), now for distribution of isoelectric points of the proteins. (D) The function and percentage occurrence of membrane-associated proteins incorporated on m-LLNs and h-LLNs. (E) The relative abundance of Toll-like receptors involved in bacterial recognition by cell membranes on m-LLNs and h-LLNs, identified by LC-MS. Proteins were classified according to UniProt/GO database.
5
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and internalization of LLNs into macrophages with and without intra-cellular staphylococci. (A) Fluorescent images of one and the same bacterium at different times after exposure to Nile red loaded PC-liposomes (negative control), m-LLNs or h-LLNs (see Figure S7 for an overview image at low magnification showing multiple staphylococci). (B) Fluorescence intensity around single S. aureus bacteria as a function of exposure time to Nile red loaded PC-liposomes, m-LLNs or h-LLNs. Error bars indicate SD values over the 10 staphylococci in triplicate bacterial culture samples. (C) Fluorescent counts as a function of red fluorescence intensity using FACS for S. aureus suspensions exposed during 1 h to PBS, Nile red-labelled PC-liposomes, m-LLNs or h-LLNs. Note virtual absence of red fluorescence during exposure to PBS only. (D) Mean fluorescence intensity counts as a function of fluorescent intensities for S. aureus suspensions exposed during 1 h to PBS, Nile red-labelled
5
into water and centrifuged to remove the organic solvent.21
Next, cell membranes separated from murine macrophages or human monocytes were mixed with the antimicrobial conjugate nanoparticles (ACN), and sonicated to obtain dual-antimicrobial conjugates encapsulated either in murine (m-LLN) or human (h-LLN) leukocyte membranes.29
Freshly-prepared ACNs composed of dual-antimicrobial conjugates had diameters of around 90 nm (Figure 1A) that increased with storage time to around 195 nm, indicative of their aggregation. When encapsulated in murine or human macrophage membranes, LLN diameters remained stable over time
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Figure 4. Antimicrobial efficacy of m-LLNs in vitro. (A) Minimal inhibitory and bactericidal
concentrations (MIC and MBC, respectively) in μg mL–1 of S. aureus WHGFP and S. aureus Xen36 against Triclosan, ciprofloxacin, their combination in equal concentrations or ACNs with or without murine or human leukocyte membrane encapsulation in 10 mM phosphate buffer at pH 7.4. (B) Colony forming units of surviving S. aureus WHGFP inside murine macrophages after 16 h exposure to Triclosan or ciprofloxacin in solution or ACNs with or without murine or human leukocyte membrane encapsulation. Data were represented by mean ± SD over triplicate experiments. (C) Same as panel B, now for S. aureus Xen36 in J774 murine macrophages.
PC-liposomes, m-LLNs or h-LLNs (data derived from panel C. Data were expressed as mean ± SD over triplicate experiments with separately cultured staphylococci. Asterisks above the data points indicate statistical significance at p < 0.0001 (****, Students’ T-test). (E) CLSM images illustrating intra-macrophageal presence of green-fluorescent S. aureus (arrows) with attached Nile red loaded, red-fluorescent PC-liposomes, m-LLNs or h-LLNs into murine macrophages with or without intra-cellular S. aureus WHGFP. (F) Red fluorescence intensity of Nile red loaded, red-fluorescent PC-liposomes, m-LLNs or h-LLNs after entry in murine macrophages with or without intra-cellular S. aureus. Data were expressed as mean ± SD over triplicate experiments with separately cultured bacteria and macrophages. Asterisks above the data points indicate statistical significance at p < 0.001 (***, Students’ T-test).
5
up to at least four weeks, while being slightly larger than of ACNs in absence of membrane encapsulation. Note (see the electron micrograph in Figure 1B), that the bi-layered structure of encapsulated LLNs remained intact after storage. Freshly prepared, bare ACNs had negatively charged surfaces (zeta potential, -41 ± 6 mV) (Figure 1C), that became less negatively charged over time, concurrent with their increase in diameter. When encapsulated in leukocyte membranes, zeta potentials of ACNs were less negative (around -25 mV) than of bare ACNs without significant changes over time. Based on UV-Vis absorption spectroscopy and setting the antimicrobial conjugate content of ACNs in absence of encapsulation at 100% (see Figure S4), it can be seen that additional antimicrobial conjugates were captured in LLN-s during the encapsulation process (Figure 1D), which occurs in presence of antimicrobial conjugates in suspension. Capture of additional conjugates was irrespective of whether murine or human leukocyte membrane encapsulation was involved.
Exposure of ACNs and LLNs to buffer at pH 7.0 only yielded partial release of less than 50 wt% of the conjugate during 80 h with a slightly inhibiting effect of murine and human macrophage membrane encapsulation (Figure 1E) with respect to unencapsulated ACNs. Exposure to buffer at pH 5.0 initially also yielded lower release of encapsulated ACNs, but after 20 h, conjugate release of bare ACNs and membrane encapsulated LLNs became identical (Figure 1F), likely because the conjugate diffuses faster under acidic conditions.
Characterization of Protein Composition and Function in LLNs. Molecular masses of the proteins present in leukocyte membranes and in m-LLNs and h-LLNs were first determined using SDS-PAGE gel electrophoresis. Molecular mass distributions on LLNs were similar as in the corresponding murine and human leukocytes (Figure S5). More extensive liquid chromatography–mass spectrometry (LC-MS) analysis
26,42
indicated that the great majority of proteins found on m-LLNs and h-LLNs are membrane proteins (79% and 63% for m-LLNs and h-LLNs, respectively, see Figure 2A). Membrane protein composition was hardly affected by ACN-loading of the LLNs (Figure S6). Molecular mass distributions in leukocyte membranes
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S. aureus :+*)3Figure 5. Antimicrobial efficacy of m-LLNs assessed in a murine, peritoneal infection model. (A)
Schematics of the murine, peritoneal infection model used. Peritonitis was induced by peritoneal injection of 200 μL of a S. aureus WHGFP
suspension (CFU/mL), followed by peritoneal injection of 200 μL saline, or saline with ciprofloxacin, ACNs or m-LLNs, all at 1 mg mL–1
at day 1 after infection. (B) The number of CFUs retrieved from 5 mL peritoneal fluid in peritoneal fluid, extracted 2 days after intra-peritoneal antimicrobial injection. Data are presented as geometric means with 95% confidence intervals over 6 mice per group. Asterisks above the data points indicate statistical significance at p < 0.05 (*), p < 0.01 (**) and p < 0.0001 (****, Students’ T-test).
5
and LLNs derived from LC-MS (Figure 2B) confirmed similarity in membranes and membrane coatings, with minor differences in molecular mass distribution between murine and human protein membrane masses. Overall, more than 58% of all proteins on murine and human macrophage membranes and LLNs were found to be low molecular weight proteins (molecular weight < 60 kDa), while less than 5% of the proteins were found to be high molecular weight (> 150 kDa). Based on iso-electric points (pI; Figure 2C), it can be seen that the percentages of positively (pI > 7) and negatively (pI < 7) charged proteins were equal for murine and human leukocyte membranes as well as for m-LLNs and h-LLNs. Only few (less than 9%) highly negatively charged proteins (pI < 5) were identified, while more than 20% of the proteins carried a high positive charge (pI > 9). Functional classification of the membrane proteins identified obtained from the UniProt/GO database and literature,40
indicated that averaged over murine and human macrophage membranes (Figure 2D) the majority of the proteins were involved in transport (33-40 %) and signaling (32%), in line with previous reports.42
In addition, Toll-like receptors involved in recognition of bacterial lipopolysaccharides and lipoteichoic acids, i.e. TLR 2, TLR3, TLR4 and TLR9,47–49
were abundantly present both on m-LLNs and h-LLNs (Figures 2E). Taken together, these results confirm that the membrane proteins of murine and human leukocyte sources were successfully transferred onto LLNs.
LLN Targeting of S. aureus and Staphylococcal-Induced Macrophage Internalization In Vitro. To demonstrate targeting of LLNs towards S. aureus, LLNs were loaded with red-fluorescent Nile red. For comparison, Nile red loaded phosphocholine (PC) liposomes were included as a negative control.40
CLSM micrographs indicated minor interaction of staphylococci with PC-liposomes and more extended interaction with both m-LLNs and h-LLNs (Figure 3A). Further quantification of the red fluorescence intensity around single staphylococci (Figure 3B) demonstrated significantly more extensive interaction of staphylococci with both types of LLNs (no significant differences between m-LLNs and h-LLNs) than with PC-liposomes within 10-20 min after exposure. FACS analyses of staphylococcal suspensions exposed to Nile red loaded liposomes or LLNs confirmed the superiority of LLNs interaction with S. aureus as compared with PC-liposomes (Figures 3C and 3D).
Next, to demonstrate bacteria-induced internalization of LLNs inside macrophages, macrophages with or without intra-cellular green-fluorescent S. aureus WHGFP
were exposed to suspensions of Nile red loaded nanocarriers and imaged using CLSM. First, in order to obtain macrophages with intra-cellular S. aureus, overnight cultures of staphylococci and murine macrophages were grown to internalize staphylococci inside the macrophages, while washing out extra-cellular bacteria and exposing possible remaining extra-cellular bacteria to gentamycin, an antibiotic unable to penetrate mammalian cells.9,50
Intra-macrophageal presence of staphylococci was clearly indicated in CLSM images of green-fluorescent staphylococci inside macrophages (see Figure 3E). PC-liposomes had no affinity for the macrophages neither with nor without internalized S.
aureus (Figures 3E, F), likely due to the absence of targeting ligands on the liposome surfaces. However, LLNs
showed a bacteria-induced internalization into macrophages (Figures 3E, F), that was not observed in absence of intra-macrophageal staphylococci. This points to an attraction of LLNs to intra-cellular staphylococci that acts across the macrophage cell wall and ensures that in case of in vivo or clinical application, LLNs will not enter macrophages without internalized bacteria.
Antimicrobial Efficacy of LLNs In Vitro. Dual-antimicrobial conjugates with or without leukocyte membrane encapsulation exhibited lower minimal inhibitory and bactericidal concentrations (MIC and MBC, respectively) towards two multi-drug resistant17
S. aureus strains than either triclosan or ciprofloxacin in solution or a solution with equal concentrations of both antimicrobials (Figure 4A). MICs and MBCs of encapsulated ACNs were lower or similar, depending on the strain considered, than of bare ACNs, likely owing to their stronger targeting to staphylococcal surfaces (Figure 4A). The lower MICs and MBCs of the nanocarriers with both antimicrobials conjugated as compared with the ones of the antimicrobials in single
5
solutions, points to a correct choice of the triclosan or ciprofloxacin sites sacrificed for conjugation.
Next, murine macrophages with internalized staphylococci were exposed either of the two antimicrobials, or their conjugates with or without macrophage encapsulation, while using PBS as a negative control. Depending on conjugate concentration, ACNs and LLNs demonstrated two to three log-units better staphylococcal
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Figure 6. Antimicrobial efficacy of m-LLNs assessed in a murine, intra-vascular infection
model. (A) Schematics of the murine, intra-vascular infection model used. Organ infection was induced by intra vascular injection of 200 μL of a suspension of macrophages with intra-cellular S. aureus WHGFP
, followed after 2 h by intra-vascular injection of 200 μL saline, or saline with ciprofloxacin, ACNs or m-LLNs. (B) Body weight of the mice as a function of time post-infection and antimicrobial injection. Data are presented as means ± SD over 5 mice per group. (C-H) The number of CFUs retrieved from 1 g of homogenized organ tissue for different organs, excised 4 days after intra-vascular antimicrobial injection. Data are presented as means ± SD over 5 mice per group. Asterisks above the data points indicate statistical significance at p < 0.01 (**) and p < 0.0001 (****, Students’ T-test).
5
killing than PBS, with encapsulated ACNs performing better than bare ones (Figures 4B and 4C). m-LLNs performed similarly against both S. aureus WHGFP
(Figure 4B) and S. aureus Xen36 internalized in murine macrophages.
Staphylococcal Killing in Murine Infection Models. Acute peritoneal infection is a life-threatening condition invoking rapid action of macrophages to clear infection.51,52
Therefore, we first evaluated the bacterial killing efficacy of our m-LLNs in a murine, peritoneal infection model53
(see schematic in Figure 5A). Moreover, use of a peritoneal infection model also allows a relatively easy way to isolate macrophages with intra-cellular bacteria.
Plating of homogenized intra-peritoneal fluid extract (Figure 5B) yielded most CFUs after injection of saline. Injection with ciprofloxacin yielded 2 log-units less CFUs than PBS injection, while injection with ACNs was significantly (p < 0.05) more effective in clearing staphylococci from peritoneal fluid than ciprofloxacin. However, the targeting ability of m-LLNs towards staphylococcal surfaces (see Figure 3) proofed its value by reducing the number of peritoneal CFUs with respect to PBS by 4 log-units, which is significantly more than achieved by ACNs with leukocyte membrane encapsulation or ciprofloxacin (Figure 5B).
In addition, similar experiments were conducted in which peritoneal fluid extract was taken 1 day after staphylococcal injection to harvest macrophages with intra-cellular staphylococci for use in an intra-vascular infection model. Possible extra-cellular staphylococci were removed from macrophage surfaces by washing with lysostaphin. Immuno-cyto staining and fluorescence microscopy demonstrated that on average 1 mL of peritoneal fluid extract contained 5 x 107
macrophages, each possessing 8 staphylococci per macrophage (see also Figure S8).
Since macrophages with intra-cellular S. aureus can spread via the blood circulation to infect various organs,9
we evaluated the antimicrobial efficacy of our LLNs also in an established murine organ infection model9
after intra-vascular injection of macrophages with intra-cellular staphylococci. 2 h after intra-vascular infection of macrophages with intra-cellular S. aureus WHGFP
, mice were injected with a single dose of PBS, ciprofloxacin, ACNs or m-LLNs and sacrificed at day four post-treatment after which various organs were removed, homogenized and plated (see Figure 6A). Body weight of the mice showed little variation over the course of the infection period till sacrifice (Figure 6B). Staphylococcal CFUs retrieved per gram homogenized organ tissue are summarized in Figures 6C-6H for blood, heart, liver, spleen, lung and kidney tissue, respectively. m-LLNs were most efficacious in eradicating infecting staphylococci from all organs examined, with ACNs performing slightly better than ciprofloxacin especially in the blood and kidneys. In livers, spleens and lungs, ciprofloxacin demonstrated no antimicrobial efficacy compared with PBS treatment, while m-LLNs showed up to 3 log-unit reduction in these organs.
CONCLUSIONS
Intracellular pathogens are extremely recalcitrant to conventional antibiotic treatment and can spread infections via the intravenous pathway. Here, we developed antimicrobial conjugated nanoparticles loaded into LLNs which can efficiently eradicate intracellular pathogens both in vitro and in vivo. Our LLNs are fabricated using cell membrane abstracted from lab-cultured macrophages. The nano-sized LLNs possess hollow structures and can stabilize ACNs for more than 4 weeks. LLNs, inheriting Toll-like receptors on the surfaces from their mother cells, can target extracellular pathogens and subsequently, initiate the internalization of LLNs. The antimicrobial conjugation significantly lowered the minimal bactericidal concentration of the antimicrobials owing to their synergistic effect. Notably, peritoneal and intravenous infections with MDR intracellular staphylococci in murine models were cured significantly faster using LLNs than with similarly un-loaded ACN nanoparticles or systemic ciprofloxacin treatment. Herewith, ACN-un-loaded LLNs may provide a powerful alternative to current antibiotic treatment.
5
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SUPPORTING INFORMATION
Materials and methods
The Synthesis of Dual-Antimicrobial Conjugate
A mixture of triclosan (2.89g, 10 mmol, 1.0 equivalent), triethylamine (1.11g, 11 mmol, 1.1 equivalent) in anhydrous dichloromethane (100 mL) was cooled to 0 to -5 °C. To this reaction mixture, chloroacetyl chloride (1.13g, 10 mmol, 1.0 equivalent) in 20 mL dry dichloromethane was added drop wise with constant stirring over a period of 1 h maintaining the temperature constant. The reaction mixture was then stirred at room temperature overnight, diluted with 100 mL dichloromethane, washed with 100 mL 5% HCl (1×), and 100 mL 5% sodium hydroxide solution (1×). The organic layer was washed with saturated aqueous NaCl, dried over anhydrous magnesium sulfate, filtered and solvent was removed under reduced pressure. The crude product was purified by silica gel column to afford the corresponding triclosan chloroacetyl derivative as colorless oil (yield: 82%). 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.37 – 7.23 (m, 3H), 6.99 (d, J =
7.0 Hz, 1H), 6.89 (d, J = 7.0 Hz, 1H), 4.34 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 165.02, 150.59, 146.73, 141.01, 130.61, 130.14, 129.22, 128.38, 127.65, 126.40, 124.05, 120.99, 119.79, 40.35.
To a solution of ciprofloxacin (331 mg, 1.0 mmol) in DMF (5 mL) was added the triclosan chloroacetyl derivative (336 mg, 1.0 mmol), trimethylamine (0.416 mL, 3.0 mmol) and potassium iodide (249 mg, 1.5 mmol) under an argon atmosphere. The mixture was stirred at room temperature overnight. After that, the resulting reaction mixture was poured into water (50 mL). The resulting precipitate was filtered off, washed with water and recrystallized from methanol. Ciprofloxacin-triclosan conjugate was obtained as a yellow powder (560 mg, 85%). 1H NMR (400 MHz, DMSO) δ 15.20 (s, 1H), 8.66 (s, 1H), 7.89 (d, J = 13.1 Hz, 1H), 7.76 (d, J = 2.5 Hz, 1H), 7.55 (t, J = 7.1 Hz, 2H), 7.40 (dd, J = 8.8, 2.5 Hz, 2H), 7.16 (d, J = 8.8 Hz, 1H), 6.96 (d, J = 8.9 Hz, 1H), 3.81 (s, 1H), 3.55 (s, 2H), 2.73 (s, 4H), 1.31 (d, J = 6.7 Hz, 2H), 1.17 (s, 2H), 13C NMR (100 MHz, DMSO) δ 176.80, 168.18, 166.38, 151.23, 148.38, 146.51, 145.56, 141.87, 139.59, 130.56, 129.26, 128.94, 128.87, 127.97, 125.11, 124.93, 121.92, 120.67, 111.51, 111.28, 106.81, 58.15, 51.78, 49.82, 49.77, 36.29, 8.05. MALDI-TOF MS (m/Z, [M+H]+, calculated: 660.0866, 662.0836, 661.0889, 663.0870, 664.0807; found: 660.0869, 662.0841, 661.0933, 663.0888, 664.0795)
Isolation of Cell Membrane. J774 murine macrophages and THP-1 human momnocytes were purchased from American Type Culture Collection. J774 cells were cultured in Dulbecco’s Modified Eagle’s Medium (high glucose, Gibco, Thermo Fisher Scientific, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 0.1% L-Ascorbic acid 2-phosphate sesquimagnesium salt (AA2P, Sigma), while THP-1 cells in RPMI medium 1640 (Gibco) containing 10% FBS and 1% L-Glutamax (Gibco).
In order to harvest the cell membrane, cells were harvested and resuspended at a concentration of 2.5 × 107 cell mL–1 in ice-cold Tris-magnesium buffer (TM buffer, pH 7.4, 0.01 M Tris and 0.001 M MgCl) and 1 EDTA-free mini protease inhibitor tablet (Pierce, ThermoFisher Scientific) per 10 mL of solution. Cells were enucleated using a sonicator (Vibra cell model 375, Sonics and Material, Inc., Danbury, CT) for 4× 10 s while
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cooling in an ice/water bath. The cell homogenate was mixed with 1 M sucrose to a final concentration of 0.25 M sucrose, and then centrifuged at 2000g and 4 °C for 10 min. The supernatant was collected and further centrifuged at 20,000g and 4 °C for 30 min to collect the cell membrane. The cell membrane was washed with ice-cold TM-buffer with 0.25 M of sucrose and collected by centrifugation at 20,000g and 4 °C for 30 min. The cell membrane was stored at -20°C for further use.
Antimicrobial Conjugated Nanoparticle Preparation. For the preparation of antimicrobial conjugated nanoparticles (ACNs), 200 μL dual antimicrobial conjugate stock solution in dimethyl sulfoxide (10 mg mL–1
) was added dropwise to 5 mL ultrapure water under magnetic stirring (2500 rpm) to form a nanoparticle suspension. After 30 min further stirring, the nanoparticle suspension was collected and further centrifuged at 20,000g (centrifuge 5417R, Eppendorf, Germany) and 4 °C for 30 min to collect the nanoparticles. Then, nanoaparticles were rinsed once with ultrapure water (5 mL) to remove the residue organic solvent. Finally, the nanoparticles were collected and re-suspended in 2 mL ultrapure water to form the ACNs suspension with a final concentration of 1 mg mL−1
and stored in a refrigerator at 4 °C.
For the preparation of the ACN-loaded leukocyte-like nanocarriers (LLNs), the binary drug nanoparticle suspension (2 mL), was mixed with leukocyte membranes collected from 5 × 106
cells. The resulting mixture was homogenized using a sonicator (Vibra cell model 375, Sonics and Material, Inc., Danbury, CT) for 4 × 10 s while cooling in an ice/water bath. The encapsulated ACNs were centrifuged at 20,000g and 4°C for 30 min and washed once with cold ultrapure water. Finally, CAN-LLNs were collected and re-suspended in 2 mL ultrapure water to a final concentration of 1 mg mL−1
and stored at 4°C for further use.
Nanoparticle Characterizations. Zeta potentials of the nanoparticles were measured at 25°C using a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK) in 10 mM potassium phosphate as a function of storage time up to four weeks. In addition, hydrodynamic diameters of the nanoparticles were measured using the same instrument. UV-VIS absorption spectra were measured using an UV−VIS spectrophotometer (Shimadzu, Japan). All experiments were done at a nanoparticle concentration of 0.1 mg mL–1
. Transmission electron microscopy (TEM) was performed using a Glacios Cryo-TEM (Thermo Scientific, Massachusetts, United States) at an acceleration voltage of 200 kV. TEM samples were prepared by applying a drop let of a nanoparticle suspension onto a carbon coated copper grid and drying it at room temperature.
Dual Antimicrobial Conjugate Release In Vitro. To determine the release of binary drug from both cell membrane encapsulated and non-encapsulated ACNs, 2 mL of freshly prepared ACN suspensions (1.0 mg mL−1
) was transferred into a dialysis bag (molecular weight cut off: 12−14 kDa) and subsequently immersed in 20 mL of a 10 mM phosphate buffer (pH 7.4 and pH 5.0) at 37°C. Aliquots (1 mL) of the dialysis solution were collected every 30 min up to 72 h, and the absorbance of the solutions at 281 nm was recorded on a UV− VIS spectrophotometer (Shimadzu, Japan). The volume of the stock dialysis solution was kept constant by adding 1 mL of fresh buffer, after each aliquot was taken.
Proteomics Analysis. In order to identify the proteins on the separated cell membranes, proteomics analysis was employed. Cell membrane proteins were precipitated using the ProteoExtract Kit (Merck, Darmstadt, Germany), as described in the user guide. Precipitated proteins were solubilized in 25 mM ammonium bicarbonate containing 0.1% RapiGest (Waters, Eschborn, Germany) (80°C, 15 min). Proteins were reduced by adding 5 mM DTT (45 min, 56°C), and free cysteines alkylated with iodoacetamide (Sigma, Taufkirchen, Germany) (15 mM, 25°C, 1 h in the dark). 0.2 μg porcine sequencing grade trypsin (Promega, Mannheim, Germany) were added and the samples were incubated overnight at 37°C. After digestion, RapiGest was hydrolysed by adding 10 mM HCl (37°C, 10 min). The resulting precipitate was removed by centrifugation (13,000 g, 15 min, 4°C), and the supernatant was transferred into an autosampler vial for peptide analysis via LC-MS, as described previous.S1
Interaction of Nile Red-Loaded LLNs with Planktonic Staphylococci and Intracellular Staphylococci. Loading the LLNs with Nile red was essentially done as described above for binary drug. Briefly, a Nile red stock solution in dimethylformamide (40 μL, 1 mg mL−1
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suspension (collected from 5 × 106
cells) in phosphate-buffered saline (PBS) to a total volume of 2 mL. The resulting mixture was homogenized using a sonicator (Vibra cell model 375, Sonics and Material, Inc., Danbury, CT) for 4 ×10 s while cooling in an ice/water bath. The Nile red-loaded nanoparticles were centrifuged at 20,000g and 4°C for 30 min and washed once with cold ultrapure water. Finally, the LLNs were collected and re-suspended in 2 mL ultrapure water to form the Nile red-loaded nanoparticles suspension with a Nile red concentration of 20 μg mL−1
and stored in a refrigerator at 4°C.
For control, Nile red-loaded PC-liposomes were prepared according to the procedure that was essentially the same, as described above for the LLNs, the only difference being that a Nile red stock solution in dimethylformamide (40 μL, 1 mg mL−1
) was mixed with phosphocholine-based phospholipids (DPPC, DSPC and DOPC) and cholesterol (Avanti Polar Lipids) in a chloroform:methanol mixture (200 μL, 3:1 v/v, 5 mg mL−1
) to a total volume of 2 mL in PBS. The resulting mixture was sonicated and centrifuged to remove the organic solvents.
Fluorescence-activated cell sorting (FACS). To study the interaction of the LLNs with planktonic staphylococci,
bacteria were cultured and harvested according to our previous protocol.S2
After harvesting, 50 μL of Nile red-loaded m-LLN, h-LLN and PC-liposome suspensions (0.5 mg mL−1
) were mixed with 1.2 mL of a S.
aureus WHGFP
(1 × 108
bacteria mL−1
) suspension in sterile 2 mL Eppendorf tubes. After 1 h incubation at 37°C, suspensions were centrifuged twice at 6500 rpm for 5 min (centrifuge 5417R, Eppendorf, Germany) and resuspended in 5 mL of PBS. Finally, flow cytometry was performed using a BD LSR-II flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, U.S.) and dada analyses were performed using FlowJo analysis software (FlowJo LLC, Ashland, Oregon, U.S.).
Confocal laser scanning microscopy (CLSM). In order to study the interaction of Nile red- loaded LLNs with
staphylococci, a 500 μL droplet of a S. aureus WHGFP
suspension (109
bacteria mL–1
) was pipetted into a 6-well-plate to allow adhesion for 1 h at 37°C. Next the suspension was discarded and the wells were washed twice with 500 μL PBS to remove planktonic bacteria and 2 mL Nile red-loaded nanoparticles (0.1 mg mL−1
) was added to each well. The interaction of the LLNs with fluorescent staphylococci was subsequently studied using CLSM (Leica TCS SP2 Leica, Wetzlar, Germany) with a HCX APO L40×/0.80 W U-V-1 objective. An argon ion laser at 488 nm and a green HeNe laser were used to excite the GFP and Nile red, respectively and fluorescence was collected at 500 – 535 nm (GFP) and 583 – 688 nm (Nile red). All data were acquired and analyzed using Leica software, version 2.0 and ImageJ software.
Bacteria-Induced LLNs Internalization. J774 macrophages were seeded at a density of 4 × 105
cells mL−1
into a 6-well-plate to allow culture for 12 h at 37°C in a CO2 incubator. Then, macrophages were infected
with S. aureus WHGFP
at a ratio of 20 bacteria per macrophage. Macrophage cultures were maintained in growth media supplemented with 100 μg mL−1
of gentamycin to inhibit the growth of extracellular bacteria. After culture for 24 h at 37°C in a CO2 incubator, the suspension was discarded and the wells were washed once
with 1 mL PBS to remove any planktonic bacteria. After removal of the growth medium, 2 mL freshly prepared Nile red-loaded LLNs (0.1 mg mL–1
) were added to each well and cultured at 37°C for 2 h, after which the suspension was removed and macrophages were rinsed with PBS (1 mL well−1
) and subsequently fixed with 3.7% paraformaldehyde solution for 15 min at ambient temperature and permeabilized with 0.1% Triton X-100 (1 mL well−1
) in PBS. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) solution in PBS (4 μg mL−1
) for 1 h at room temperature. Macrophages with internalized red-fluorescent LLNs were observed using a confocal laser scanning microscopy (TCS SP2, Leica, Wetzlar, Germany), equipped with an argon ion laser at 488 nm to excite green fluorescent protein, Nile red and a violet (405 nm) laser to excite DAPI. Fluorescence was detected at 430−500 nm (blue), 500 – 535 nm (GFP) and 583−688 nm (red), respectively.
Killing of Planktonic Staphylococci In Vitro. To determine the MIC of LLNs and their composing antimicrobials, 100 μL of each antimicrobial in PBS (Triclosan, ciprofloxacin, 1 : 1 mixture of Triclosan and ciprofloxacin, ACN) or LLNs (both with an equivalent amount of antimicrobial concentration between 0 and 80 μg mL−1
) was applied to 100 μL of a S. aureus WHGFP
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bacteria mL−1
). The MIC values were taken as the lowest drug concentration at which bacterial growth was absent. Subsequently, the MBC values were determined by plating aliquots of suspensions with concentrations yielding no visible growth of bacteria on TSB agar plates after being incubated for 24 h at 37°C, and the lowest concentration at which colony formation remained absent was taken as the MBC.
Killing of Intracellular Staphylococci by Triclosan, Ciprofloxacin, ACNs and LLNs In Vitro. J774 murine macrophages were plated at a density of 4 × 105
cells mL−1
and infected with staphylococci (S. aureus WHGFP
and S. aureus Xen36) at a ratio of 20 bacteria per macrophage. Macrophage cultures were maintained in growth media supplemented with 100 μg mL−1
of gentamycin to inhibit the growth of extracellular bacteria and LLNs or their composing antimicrobials were added to the growth media 1 day after infection.
The survival of intracellular staphylococci was assessed 24 h after addition of the antibiotics. Macrophages were lysed with Hanks buffered saline solution supplemented with 0.1% bovine serum albumin (BSA) and 0.1% Triton-X, and serial dilutions of the lysate were made in PBS containing 0.05% Tween-20. The number of surviving intracellular bacteria was determined by plating on tryptic soy agar plates with 5% defibrinated sheep blood.
Intraperitoneal Infection Model for Testing Efficacy of the m-LLNs. Eight-week-old female mice, ICR (CD-1) (35 g to 40 g each) were obtained from Vital River Laboratory Animal Technology Co. (Beijing, China). All animals were housed in the on-site animal facility of Nankai University and experimental procedures were approved by the Institutional Animal Care and Use Committee of Nankai University, Tianjin, China. For intraperitoneal infection, each mouse was subjected to a peritoneal injection of a dose of 2 × 108
multidrug resistant S. aureus WHGFP
. At day 1 after bacterial injection, infected animals were randomly assigned into four groups of five animals each, receiving intraperitoneal injection of (i) 200 μL 154 mM saline (untreated control), (ii) 200 μL ciprofloxacin in PBS (1 mg mL–1
), (iii) 200 μL ACN in PBS (1 mg mL–1
) and (iv) 200
μL mLLN in PBS (1 mg mL–1
). Treatment was initiated 1 day post-infection and continued for 2 consecutive days. All mice were killed on day 3 after infection, peritoneal macrophages were harvested by washing the peritoneal cavity with 5 mL cold PBS. Macrophages were lysed by homogenization on a JY98-IIIDN sonicator (Scientz, Ningbo, China), and serial dilutions of the lysate were made in PBS solution containing 0.05% Tween-20. The number of surviving intracellular bacteria was determined by plating on tryptic soy agar plates supplemented with 5% defibrinated sheep blood.
Intravenous Infection Model for Evaluating the Killing Efficacy of the ACNs and m-LLN. Eight-week old female mice, ICR (CD-1), were obtained from Vital River Laboratory Animal Technology Co. (Beijing, China). Each mouse was subjected to a peritoneal injection of a dose of 2 × 108
multidrug resistant
S. aureus WHGFP
. At day 1 after bacterial injection, infected animals were killed and the peritoneum was flushed with 5mL of cold PBS. Peritoneal washes were centrifuged for 5 min at 1,500 r.p.m. at 4 °C in a table-top centrifuge (5424 R, Eppendorf, Hamburg, Germany). The pellet containing peritoneal macrophages, was collected and cells were treated with 50 μg mL−1
of lysostaphin (Sigma Aldrich, from Staphylococcus
staphylolyticus) for 20 min at 37°C to eradicate any extracellular staphylococci. Peritoneal macrophages were
washed three times with ice-cold PBS to remove the lysostaphin. Next, peritoneal macrophages from different pairs of donor mice were pooled, and injected in recipient mice by intravenous injection into the tail vein, two hours after staphylococcal injection. Infected animals were randomly assigned into four groups of five animals each, receiving intravenous injection of (i) 200 μL 154 mM saline (untreated control), (ii) 200 μL ciprofloxacin in PBS (1 mg mL–1
), (iii) 200 μL ACNs in PBS (1 mg mL–1
) and (iv) 200 μL m-LLNs in PBS (1 mg mL–1
). All mice were killed on day 4 after infection, blood, hearts, livers, spleens, lungs and kidneys were harvested in 5 mL of sterilized PBS. The organs were homogenized using a JY98-IIIDN sonicator (Scientz, Ningbo, China). The number of surviving bacteria per gram homogenized organ tissue was determined by plating serial dilutions of the tissue homogenate in PBS 0.05% Tween on tryptic soy agar with 5% defibrinated sheep blood.
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Figure S1. NMR spectra of chloroacetylated triclosan in d-CHCl3 at 0 °C. (A) 1
H NMR and (B) 13
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Figure S2. 1H NMR spectra of antimicrobial conjugate in d6-DMSO at 0°C. (A) 1H NMR and (B) 13C NMR.
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0 10 20 30 40 50 60 70 80 90 100 655 660 665 670 Mass/Charge Scale: 146.4109 Date: 28-NOV-2017 Time: 15:24:45 Mode: Positive Scans: 1 Varian QFT-ESI File: Tri-Cip(2)_ESI.trans 660.0869 662.0841 661.0933 663.0888 664.0795 655.3794Figure S3. Electrospray ionization (ESI) mass spectrum of antimicrobial conjugate measured in
DMSO. :DYHOHQJWK QP $&1 PXULQHPDFURSKDJHV P//1 KXPDQPDFURSKDJHV K//1 $EVRU E D Q F H D X $ % &
Figure S4. (A) UV-vis absorption spectra of ACNs at different concentration as a function of wavelength. (B) Linear regulation between UV-vis absorption and ACN concentration in a 10 mM phosphate buffer at pH 7.4. (C) UV-VIS absorption as a function of wavelength of ACN, m-LLN, h-LLN, murine macrophages and human monocytes in 10 mM PBS at 37°C. The 282 nm absorption peak represents ACNs.
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17 34 43 55 72 95 170 130 kDaFigure S5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showing
the protein profiles in m-LLNs and h-LLNs in the presence or absence of ACN loading.
Figure S6. (A) Membrane proteins found in significantly different abundance in m-LLNs in the presence or absence of ACN loading. Data obtained using LC-MS. (B) Same as panel A, now for the membrane proteins in h-LLNs.
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Figure S7. Staphylococci targeting PC-liposomes and LLNs. (A) Exemplary CLSM micrographs
of captured S. aureus WHGFP after 1 h incubation with Nile red-loaded PC-liposome (control) and LLNs (0.1 mg mL–1). Scale bars represent 25 μm. (B) Quantified fluorescent intensity from red channels of panel B. Error bar represents the standard deviations over 15 images from 3 experiments with separately prepared nanoparticles. Asterisks above the data points indicate statistical significance at p < 0.001 (***, Students’ T-test) between PC-liposomes and LLNs.
Figure S8. Staphylococci resident inside peritoneal macrophages. (A) Exemplary CLSM
micrographs of intra-macrophageal S. aureus WHGFP
after intraperitoneal injection of stahylococci and peritoneal fluid collection and culture in well-plate. Scale bar equals 25 μm. (B) Number of staphylococci per macrophage quantified from 75 cells in 5 different samples, showing around 8 staphylcocci resident in one macrophage.
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Supplementary References
(S1) Molinaro, R.; Corbo, C.; Martinez, J. O.; Taraballi, F.; Evangelopoulos, M.; Minardi, S.; Yazdi, I. K.; Zhao, P.; De Rosa, E.; Sherman, M. B.; et al. Biomimetic Proteolipid Vesicles for Targeting Inflamed Tissues. Nat. Mater. 2016, 15 (9), 1037–1046.
(S2) Liu, Y.; van der Mei, H. C.; Zhao, B.; Zhai, Y.; Cheng, T.; Li, Y.; Zhang, Z.; Busscher, H. J.; Ren, Y.; Shi, L. Eradication of Multidrug-Resistant Staphylococcal Infections by Light-Activatable Micellar Nanocarriers in a Murine Model. Adv. Funct. Mater. 2017, 27 (44), 1701974.
