Elastin-like polypeptide incorporated thermally sensitive liposome improve antibiotic therapy against musculoskeletal bacterial pathogens

Abstract

Musculoskeletal infections caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa in children and adults can lead to adverse outcomes including a need for extensive surgical debridement and limb amputation. To enable targeted antimicrobial release in infected tissues, the objective of this study was to design and investigate novel elastin-like polypeptide (ELP)-based thermally sensitive liposomes in vitro. ELP biopolymers can change their phase behaviour at higher temperatures. We hypothesised that ELP-TSL will improve therapeutic efficacy by releasing antimicrobial payloads locally at higher temperatures (≥39 °C). ELP-TSL library were formulated by varying cholesterol and phospholipid composition by the thin film and extrusion method. A broad-spectrum antimicrobial (Ciprofloxacin or Cipro) was encapsulated inside the liposomes by the ammonium sulphate gradient method. Cipro release from ELP-TSLs was assessed in physiological buffers containing ∼25% serum by fluorescence spectroscopy, and efficacy against Staphylococcus aureus and Pseudomonas aeruginosa was assessed by disc diffusion and planktonic assay. Active loading of Cipro achieved an encapsulation efficiency of 40–70% in the ELP-TSL depending upon composition. ELP-TSL Cipro release was near complete at ≥39 °C; however, the release rates could be delayed by cholesterol. Triggered release of Cipro from ELP-TSL at ∼42 °C induced significant killing of S. aureus and P. aeruginosa compared to 37 °C. Our in vitro data suggest that ELP-TSL may potentially improve bacterial wound therapy in patients.

Keywords:

• Musculoskeletal infections
• elastin-like polypeptide
• low-temperature sensitive liposomes
• non-oncologic applications of hyperthermia
• antimicrobial therapy

Introduction

Bacteria organisms such as Staphylococcus aureus and Pseudomonas aeruginosa cause chronic, non-healing infections of the musculoskeletal system in children and adults [1–3]. Musculoskeletal wounds are a serious and difficult-to-treat infection, affect an estimated 2.4–4.5 million people in the United States, and typically result from diabetes mellitus, peripheral vascular disease, open fracture, or surgery [4]. Clinically, musculoskeletal wounds are characterised by biofilm formation, and reduced bacterial susceptibility to treatment [5,6]. As a result, these wounds require extended-duration treatment (generally >4–6 weeks) with the combinations of antimicrobials, resection of tissues and amputation in some cases [7].

Prior approaches to improve antimicrobial delivery to wounds used antibiotic-impregnated polymeric beads and gels to selectively enhance drug accumulation in infected regions, and reduce exposure and toxicity to normal tissue [8,9]. Although superior to conventional therapy, these approaches can be limited by slow antibiotic elution rates of the available clinically applicable materials and may pose risks of systemic toxicity due to the biosynthesis of monomeric by-products and in some cases requires surgical removal of the delivery device. To overcome these limitations, we hypothesise that thermally controlled and enhanced delivery of antimicrobial agents using nanoparticles with real-time non-invasive hyperthermia control can provide physicians more precise dosing control and complete killing of wound-resident bacteria. Towards this goal, we recently developed and reported antimicrobial-loaded low-temperature sensitive liposomes (LTSLs), which contained a lysolecithin lipid that allowed for the rapid release of encapsulated antimicrobial (Ciprofloxacin or Cipro) upon being heated to mild hyperthermic temperatures (40–42 °C) [10]. However, the LTSL-based drug delivery system demonstrates high sensitivity to serum proteins and instability of the vesicles in the bloodstream [11,12].

More recently, Park et al. developed elastin-like polypeptide based thermally sensitive liposomes (ELP-TSL) encapsulating doxorubicin [13,14]. These ELP-TSLs are composed of DPPC, DSPE-PEG, cholesterol and a fatty acid–conjugated ELP [VPGXG]n pentapeptide repeat [15]. Compared to LTSLs that had a half-life of ∼1 h (0.92 ± 0.17 h), the researchers reported an improved pharmacokinetics (half-life = 2.03 ± 0.77 h) for ELP-TSL in mice model. We hypothesised that this formulation methodology may also provide precise control of antimicrobial release with temperature. Our in vitro data suggest that ELP-TSLs are indeed capable of targeted release of antimicrobials with heat, and antibiotic therapy against S. aureus and P. aeruginosa.

Materials and methods

Cipro hydrochloride (Cipro) was purchased from LC laboratory (St Louis, MO). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MSPC) and N-(Carbonyl-methoxypolyethyleneglylcol-2000)-1,2-distearoyl-sn-glycerol-3-phosphoethanolamine sodium salt (DSPE-MPEG 2000) were obtained from Corden Pharma (Boulder, CO). Cholesterol was acquired from Calbiochem (San Diego, CA). The 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) was obtained from Avanti Polar Lipid (Alabaster, AL). ELP-lipid conjugate (Stearoyl[C18]-VPGVGVPGVGVPGVG-NH2; 95% purity, MW: 1512 Da) were obtained from Peptron Inc. (Germantown, Maryland). Trypticase soy agar and broth were obtained from Becton Dickinson (Sparks, MD). Staphylococcus aureus ATCC 29213 buffer was obtained from the Oklahoma Animal Disease Diagnostic Laboratory (Stillwater, OK). Methicillin- resistant Staphylococcus aureus, a clone of USA300, was obtained from the Centre for Biologics Evaluation and Research, Office of Vaccines Research and Review, U.S. Food and Drug Administration (FDA). The Pseudomonas aeruginosa was a community human isolate from the Stillwater Medical Center, Stillwater, OK.

Synthesis of cipro-loaded liposome

ELP-TSLs were designed with different ratios of phospholipid (DPPC, DSPC) and cholesterol (Table 1) as previously described by Park et al. [13]. In the formulations, DPPC served as the main lipid and the molar ratio of cholesterol was varied from 15–25%. For liposome synthesis, phospholipids, cholesterol and ELP-steoryl were dissolved in chloroform (1 ml chloroform/10 mg total lipid). The organic solvent was evaporated to dryness in a rotary evaporator to yield a lipid film. The thin film of lipids for ELP-TSL synthesis (25–50 mg total lipid) was hydrated with 2 ml of 250 mM ammonium sulphate (pH:4) solution by vortexing in 55 °C water bath for 10–15 min. The liposome suspension was then extruded five times through two stacked polycarbonate filters of 200 nm pore size to yield bland homogeneous liposome. Next, the exterior buffer of liposome suspension was exchanged with 25 mM (1/6 of 1X PBS) phosphate-buffered saline by size exclusion chromatography (SEC) using PD-10 column (GE Healthcare Bio-Sciences, Pittsburgh, PA). Encapsulation of cipro (2 mg cipro/100 mg total lipid) into liposomes was carried out actively using a pH gradient method described by Mayer et al. [16]. Unencapsulated cipro was removed with a PD-10 column using 1X PBS (pH 7.4) as eluent. Encapsulation efficiency was determined by computing the difference between cipro recovered from the total amount of cipro added using a prior developed calibration curve.

Table 1. Design and composition of ELP-TSL library.

	ELP-TSL (Mole ratio)
Components	ELP-TSL (100:0):15	ELP-TSL (75:25):15	ELP-TSL (75:25):25
DPPC	55	41.25	41.25
DSPC	0	13.75	13.75
DSPE-PEG2000	2	2	2
Cholesterol	15	15	25
MSPC	0	0	0
ELP	0.41	0.41	0.41

Characterization of ELP-TSL

ELP-TSL library were characterised for size by measuring hydrodynamic diameter using a dynamic light scattering (DLS) instrument (Brookhaven Instruments, Holtsville, NY). The liposomes were also characterised for zeta potential using a Brookhaven ZetaPALS (Brookhaven Instrument, Holtsville, NY) in PBS and ddH₂O. Briefly, samples were diluted by adding 10 μL of synthesised ELP-TSL or LTSL to 1.5 ml of 25% FBS. An average of three measurements was taken to determine the mean hydrodynamic diameter (± SD) and zeta potential of liposomes. In addition, stability of ELP-TSL was assessed by periodically measuring size over 48 h.

Differential scanning calorimetry (DSC)

Lipid films were synthesised as mentioned earlier. The films (ELP-TSL, and LTSL: DPPC:DSPE-PEG: MSPC: 86.54:3.85:9.62 molar ratio) were hydrated with 1X PBS to create multilamellar vesicles. The liposomal suspensions were transferred into aluminium pans hermetically sealed with lids (TA instruments, New Castle, DE) and scanned in DSC instrument (TA instruments, New Castle, DE) from 35 °C to 50 °C at an average heating rate of 1 °C/min.

Thermal scan of cipro release from ELP-TSL

Release of cipro from the liposomes as a function of temperature (25–42 °C) was assessed by measuring the dequenching of the drug in PBS containing 25% fetal bovine serum (FBS). Briefly, samples (50 mg lipids containing ∼1 mg cipro) were diluted 50-fold in 3 ml of 10% FBS and placed in a quartz cuvette equipped with a stopper and magnetic stirrer. A fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies, Santa Clara, CA) equipped with an external temperature controller was used to measure cipro release from the liposomes by excitation at 320 nm and fluorescence emission monitored at 420 nm. The data were collected at every 1 °C over temperatures ranging from 25° to 45 °C. Each temperature point was held for 2 min to ensure full equilibration. From 39–42 °C fluorescence readings were taken every 0.2 °C and each point was equilibrated for 1 min.

The drug release efficiency was calculated using following equation: where Ii – initial fluorescence intensity, It – fluorescence intensity of sample at a temperature, (If) – fluorescent intensity after adding TritonX-100.

Cipro release kinetics of ELP-TSL

The release of cipro from ELP-TSL in 25% FBS as a function of time was measured using Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA). For fluorescence measurement, at the same volumes, as described in 2.4, the samples were equilibrated to the desired temperature (25, 37, 38, 39, 40, 41 and 42 °C) for 30 min. Baseline fluorescence measurements for each sample were taken at 25 °C and complete release of cipro was achieved by introducing 1% Triton X-100 for 5 min at 45 °C. Data were obtained as percentage release of encapsulated cipro at any given temperature. Based on the data obtained from 2.1 to 2.4, ELP-TSL with the composition (DPPC: DSPC: DSPE-PEG: cholesterol: ELP-steoryl; molar ratio: 41.25:13.75: 2: 0.41) that showed maximal encapsulation efficiency and complete release at >40 °C was selected for further evaluations in 2.5 and 2.6.

Transmission electron microscopy (TEM) imaging of ELP-TSL and LTSL

ELP-TSLs were imaged by the negative staining technique for TEM. LTSLs was hydrated and synthesised as previously published and served as the positive control [10]. The liposomes were diluted 100x in PBS. A 10 μL drop of diluted liposomes was pipetted onto a carbon grid (Lacey or Holey grid) and left for 1 min so that the liposomes adsorbed to the grid, then the liquid was wicked away with a piece of filter paper. The grid was allowed to dry for 30 s. For negative staining a 9 μL drop of 2% phosphotungstic acid (PTA) was pipetted onto the grid and left for 30 s, then it was wicked away with a piece of filter paper. Again, the grid was briefly dried before imaging. The imaging was conducted at 200 kV at 60 000 X using a JEOL JEM-2100 TEM (JEOL, Peabody, MA).

Evaluation of cipro activity by Kirby–Bauer disc diffusion assay

Disc diffusion evaluation was performed against Staphylococcus aureus and Pseudomonas aeruginosa biofilm. Briefly, blank and cipro-loaded ELP-TSL were heated to 37, 39 and 42 °C in a water bath in 25% FBS for 15 min. The samples were then centrifuged at 4000 × g in a filter tube with a cut-off of 10 kDa to extract the filtrate containing the released cipro. The concentration of cipro in the filtrate was measures spectrophotometrically. Bacteria were grown overnight in tryptic soy broth (TSB) at 37° C in a shaking incubator at 200 RPM, centrifuged at 4000 RPM for 10 min and the media were replaced with sterile PBS. The culture was then matched to a 0.5 McFarland latex turbidity standard (1.5 × 10⁸ CFU/mL). For disc diffusion, a lawn of the bacteria was generated onto a tryptic soy agar (TSA) plate using a sterile swab. Next, 5 mm diameter sterile paper discs inoculated with 20 μL of diluted filtrate containing ∼5 μg ciprofloxacin was placed in each quadrant of the bacterial lawn, and the plates were incubated overnight at 37° C. The antimicrobial activity of cipro-loaded ELP-TSL and blank was determined by measuring in millimetres the zone of growth inhibition around the disc. Each temperature point was tested in triplicate and the mean and standard error were determined.

Efficacy of ELP-TSL against Methicillin Resistant S. aureus ( MRSA)

A single colony of methicillin-resistant S. aureus removed from a TSA plate was placed in TSB, and cultured overnight at 200 rpm for 18 h at 37 °C. The culture was centrifuged, the supernatant was removed and replaced with PBS to match a 0.5 McFarland latex turbidity standard. Treatment groups (n = 6/group) included the following: MRSA (± Heat), MRSA (Cipro ± Heat) and MRSA (ELP-TSL ± Heat). Each group was treated with 300 μl of sample containing 14 μg cipro/ml. Hyperthermia was performed by placing the sample in a water bath for 30 min at the corresponding water temperature for the group. After hyperthermia treatment, the serially diluted samples were plated and incubated at 37° C for 48 h to calculate Log₁₀ CFU/mL.

Statistical analysis

Treatment groups were compared for differences in mean bacterial CFU using analysis of variance (ANOVA) followed by Tukey’s multiple comparisons post hoc test. All analyses were performed using Sigma Plot (Systat Software Inc. San Jose, CA). All p values were two-sided, and a p values less than 0.05 indicated statistical significance. Values are reported as mean ± SD.

Results

Physicochemical characterisation of liposome

The hydrodynamic diameter, zeta-potential and encapsulation efficiency of Cipro loaded ELP-TSL library are given in Table 2. The encapsulation efficiency of ELP-TSL formulation ranged from 40–70%. The diameter of the ELP-TSLs ranged from ∼150–170 nm with a polydispersity index (PDI) of less than 0.137, and the zeta potential from −35 to −43 mV. In contrast, the zeta potential in ddH₂O was close to neutral for all the formulations. The stability of ELP-TSL was excellent with hydrodynamic diameter and zeta potential close to baseline measurements at 48 h (data not shown).

Table 2. Particle size and zeta potential of cipro loaded ELP-TSL library.

		Zeta-potential (mV)
Liposomes	Hydrodynamic Diameter (nm)	PBS	ddH2O	Encapsulation efficiency (%)
ELP-TSL (100:0):15	154 ± 2.6	−35 ± 1.9	−1.0 ± 2.5	29.2
ELP-TSL (75:25):15	165 ± 3.3	−38 ± 1.5	−0.7 ± 2.4	68.5
ELP-TSL (75:25):25	169 ± 1.1	−43 ± 1.4	−0.8 ± 1.7	46.5

Thermal scan from ELP-TSLs and LTSL

The release of cipro from DSPC/cholesterol incorporated ELP-TSL was <5–10% from 37–38 °C and was more gradual and complete at ≥39 °C was noted. Also, ELP-TSL formulation with the higher molar level of cholesterol (25% vs. 15%) delayed cipro release by 30–40% at 40–42 °C (Figure 1).

Figure 1. Thermoscan assay in physiological buffer containing 25% serum for cipro loaded ELP-TSL prepared with a different molar ratio of DPPC: DSPC ratio (100:0 and 75:25) and cholesterol (15 and 25 molar ratio). ELP-TSL were tuneable and shifted transitional temperature and temperature of maximum drug release depending on lipid composition.

DSC of liposomes

DSC did not show a sharp peak of phase transition throughout the temperature range from 39 °C up to 49 °C for the ELP vesicles; however, a difference in the progressive pattern of endothermic heat flow in each sample was noted. Notably, at 40 °C or greater, 100:0:15 and 75:25:15 showed a broad peak of transition suggesting some changes in the phase behaviour. In contrast, LTSL showed a sharp peak at ∼41.6 °C (Figure 2).

Figure 2. Thermogram of ELP-TSLs and LTSLs. There was no sharp peak of transition in ELP-TSLs, however, a progressive increase in heat flow was noted for 75:25:25:0.4.

TEM of liposomes

TEM studies showed that like lysolecithin-based LTSLs, ELP-TSLs were spherical at room temperature with the aqueous core showing the likely presence of crystalised cipro (Figure 3(a,b)).

Figure 3. TEM images of LTSL and ELP-TSL (DPPC:DSPC: Cholesterol: 75:25:15 molar ratio) loaded with cipro. Crystallized cipro is seen within the core of the liposomes (arrows).

Release kinetics of cipro from ELP-TSL in 25% FBS containing medium

Selected ELP-TSL from 2.4 reached maximum drug-release (> 95%) within 1 min at ∼40 °C. At body temperature, ELP-TSL released 20% of the encapsulated cipro in 15 min. of incubation, and ∼40% cipro was released at 30 min (Figure 4).

Figure 4. Release kinetics of ELP-TSL (DPPC:DSPC: Cholesterol:75:25:15 molar ratio) in PBS containing 25% FBS. ELP-TSL were incubated in 25% FBS for 30 min. At 37 °C, within 5 min. of incubation in 25% serum, ELP-TSL maintained stability and retained >90% of cipro. After 15–30 min. of incubation, ELP-TSL retained greater than 50–60% of the content at body temperature and achieve near complete release at hyperthermia.

Disc diffusion antimicrobial sensitivity test for drug-loaded LTSL and ELP-TSL

Against S. aureus, ELP-TSL caused no inhibition at 37 °C (Figure 5(a,b)). At 39 °C, ELP-TSL produced a significantly higher zone of inhibition against S. aureus (17 mm ± 1 mm) and P. aeruginosa (15.5 mm ± 2 mm), respectively. At 42 °C, ELP-TSL showed a similar trend and produced a zone of inhibition of 21 mm ± 1 mm and 20 mm (± 1 mm) for S. aureus and P. aeruginosa respectively. Bland liposomes were not toxic against the bacteria (Figure 5(c)).

Figure 5. (a) Evaluation of thermosensitive release and killing of S. aureus and P. aeruginosa treated with ELP-TSL (DPPC:DSPC: Cholesterol:75:25:15 molar ratio) in 25% FBS, by disc diffusion method. The amount of drug on the disc ranges from 0 to 5 μg (b) Zones of inhibition for S. aureus and P. aeruginosa respectively. Bacterial killing was observed only at 39 °C and 42 °C. (c) Evaluation of toxicity of bland liposomes. No zone of inhibition was observed for S. aureus or P. aeruginosa, respectively, treated with TSL (without ELP) and bland ELP-TSL liposomes.

Efficacy of ELP-TSL against MRSA

Compared to body temperature, mild hyperthermia alone killed 6% of MRSA. The combination of ELP-TSL and hyperthemia resulted in ∼30% killing at mild hyperthermia compared to 37 °C (p < 0.001). Cipro alone demonstrated the same rate of killing at the body and hyperthermic temperatures since the drug was freely available to MRSA.

Discussion

Staphylococcus aureus and Pseudomonas aeruginosa are typically community-acquired and cause nosocomial infections all over the world. A critical challenge to their treatment regardless of location (e.g. cystic fibrosis, superficial wounds, etc.) is their ability to establish biofilms. These biofilms typically require 500- to 5000-fold higher dose of antibiotic than needed to kill non-biofilm bacteria [17]. This occurs because biofilms have increased cellular density [18], demonstrate drug efflux properties [19] and has modulated levels of β-1,3-glucan with overexpression of exopolysaccharide glycocalyx modulation that sequester the drug molecules and impedes access to bacteria [20,21]. These combine to require long-duration treatment (generally >6 weeks) with combinations of antimicrobials, resection of tissues, amputation, and the emergence of drug resistance [7,22,23].

One approach to enhancing antimicrobial targeting of biofilms can be through the development of nanoparticle-encapsulated drug delivery system. A variety of antibiotic-laden polymeric and liposome nanoparticles for wound therapy to improve efficacy, ease of administration, and safety over free antibiotics has been reported [24,25]. In particular, liposomes are especially attractive since they are biocompatible and demonstrate a high degree of membrane stability (reduced leakage) [26]. Preclinical studies have shown that small and cationic liposomes are capable of permeating biofilms, reinforcing their role as effective permeabilising agents [27]. Most of the liposome-based antimicrobial system release the drug slowly (<5–10% over several hours to days), and thus this approach may not be applicable for biofilm therapy that requires bolus and high-dose antimicrobial delivery for bacterial clearance. Thus, to capture the full therapeutic advantage of nanotechnology, it is important to improve our ability to control and selectively release liposome-borne drug payloads rapidly within biofilms at sufficient concentration. In prior research, we showed that low temperature-sensitive liposomes (LTSL) sensitive to mild, non-destructive elevations above normal body temperature achieves precise, localised delivery of liposomal antimicrobial payloads in biofilms [10]. However, lysolecithin-based LTSL have short half-life and systemic stability [11,28]. Recently, TSLs doped with ELP with enhanced systemic stability for doxorubicin delivery were reported [13]. ELPs are composed of the pentapeptide VPGXG and some equivalent variations, where X represents any amino acid except l-proline. Many studies have established that the choice of guest amino acid in the pentapeptide sequence affects the physicochemical properties and their sensitivity to stimuli such as heat [29]. Importantly, Park et al. showed that a fatty acid conjugated ELP insertion in LTSL imparts high thermosensitivity and biocompatibility [13]. This was achieved by conjugating a single hydrocarbon tail (steoryl group) at the N-terminus of ELP for incorporation into the liposome bilayer. In contrast to LTSL that demonstrates a half-life of less than an hour, ELP-TSL formulated using this methodology achieved a plasma half-lives of ∼5 h with EPR like accumulation of doxorubicin in solid tumour [30].

To test whether the high systemic stability of ELP-TSL can be leveraged for antimicrobial treatment, we loaded ciprofloxacin (cipro). Cipro is a second-generation fluoroquinolone antimicrobial drug with known bactericidal action against Gram-positive and negative bacteria such as Escherichia coli, Haemophilus influenza, Klebsiella pneumonia, Legionella pneumonia, Moraxella catarrhal, Staphylococcus aureus, Streptococcus pneumonia and Staphylococcus epidermis [31]. Chemically, cipro is a water-soluble drug, and thus, an excellent candidate for loading into the aqueous core of LTSLs. To impart membrane stability, cholesterol lipid was incorporated into the ELP-TSL membrane at a molar ratio of 15–25%, and this methodology appeared to delay cipro release in the hyperthermia range (Figure 1). Compared to bland liposomes, the zeta-potential study of liposomes loaded with cipro was significantly negative (Supplementary Tables 2 and 3). Cipro is a highly charged molecule, and during loading, they may complex with the liposome membrane by electrostatic interaction to affect encapsulation efficiencies and zeta potential. Oh et al. noted extensive and irreversible aggregation of liposome during cipro loading when the liposomes were composed of more than 50 mol% negative lipids. In contrast, the incorporation of cholesterol in the lipid membrane improved encapsulation by modifying membrane potential [32]. Our size and TEM data suggest that ELP-TSL composition provide the crucially needed properties of stable cipro encapsulation, without causing charge-induced irreversible aggregation of drug and lipids in 25% FBS under the relevant conditions and temperatures over 24–48 h (Table 2 and Figure 3). However, unlike LTSLs that demonstrate sharp Tm, DSC data for ELP-TSL Tm in our study was not conclusive. This is in line with data from Park et al. study where the mechanism of drug release without Cipro appears to be driven by the destabilisation of the liposome membrane and enhanced permeation induced by the conformational change of ELP from random coil to β-turn at the mild hyperthermic temperatures rather than melting of the lipids themselves [14]. We also adopted the Park et al. ELP library scheme for our in vitro investigations [14]. In our experience, ELP-TSLs with (75:25):25: 0.41) molar compositions were most efficient and reproducible in their encapsulation and release of cipro. For the other formulation, we experienced significant differences in the encapsulation of cipro between various batches, and this discrepancy require further investigation. In terms of serum stability, ELP-TSL appeared to have low liposomal permeation at 37 °C (Figure 2). This also reduced bacterial killing at body temperatures and high zone of inhibition at 42 °C for the ELP-LTSL (Figure 5 and 6). Additional data and proof that the ELP-TSL formulation is more effective in inducing bacterial killing in the mild hyperthermia ranges in vivo should be obtained to prove this hypothesis.

Figure 6. Percentage killing of methicillin-resistant S. aureus (MRSA) treated with Cipro loaded ELP-TSL at 37 °C and 42 °C. ELP-TSL (DPPC:DSPC:Cholesterol: 75:25:15 molar ratio); in combination with mild hyperthermia significantly improved killing of MRSA compared to 37 °C. Cipro alone demonstrated similar efficacy at 37 °C and 42 °C since the free drug was freely available to the bacteria.

Our in vitro investigation has several limitations. The main question is whether ELP-TSL will similarly, or even further, enhance drug delivery and efficacy compared to LTSL in vivo. Prior research in cancer models has shown that this is indeed achievable [13]. Studies are currently underway in our laboratory to further investigate ELP-TSL pharmacokinetics in mice and rat models. A significant limitation is to rely only on the performance of cipro to assess treatment efficacy and that this encapsulation methodology may not be readily adaptable to carry virtually any compatible antibiotic (e.g. gentamicin, daptomycin). We believe that we will need to adapt and modify our choice of drug and route of administration in such cases to ensure broad applicability and eventual translatability of our proposed therapeutic system. Finally, ELP-TSL may enhance drug delivery and improve therapeutic efficacy compared to systemic antimicrobial therapy, yet they may still require follow-up surgeries for the eradication of drug-resistant pathogens. Previously, we have shown that hyperthermia modulates the tissue microenvironment to improve tumour drug penetration relative to liposomes alone [33]. An additional benefit of heat is its ability to improve drug uptake in cell likely by an increase in the membrane permeability [34]. An increased permeability can hypothetically disrupt the inner electrostatic equilibrium of bacterial membranes, and impact is viability. Thus, we believe that heat combined ELP-TSL may be effective against drug-resistant pathogens. This is evidenced by our initial studies with MRSA where heat alone, and its combination with ELP-TSL achieved relatively higher bacterial killing rate at hyperthermic temperatures following single treatment (Figure 6).

In conclusion, building on prior success combining LTSLs and ELP-TSL with hyperthermia for targeted drug delivery to the solid tumour and infectious study [35–38], in this study, we reported for the first time a new method of antimicrobial loading in TSLs with ELP. Incorporation of ELP in the lipid bilayer confers temperature controlled drug permeation of antimicrobials in vitro, and at higher temperatures (42 °C) caused significant bacterial killing, suggesting that the proposed heat-targeted antimicrobial therapy may have clinical utility against musculoskeletal infections.

Acknowledgements

Research reported in this publication were supported by the Center for Veterinary Health Sciences seed support, the Oklahoma Center for Advancement of Science and Technology (HR17–060-1), and OSU Kerr (to A.R.) and McCasland Foundation (to J.M.) Chairs. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Oklahoma State University.

Disclosure statement

No potential conflict of interest was reported by the authors.