Lipid Shell-Enveloped Polymeric Nanoparticles with High Integrity of

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Lipid shell-enveloped polymeric nanoparticles with high integrity of lipid shells improve mucus penetration and interaction with cystic fibrosis-related bacterial biofilms Feng Wan, Tommy Nylander, Sylvia Natalie Klodzinska, Camilla Foged, Mingshi Yang, Stefania G. Baldursdottir, and Hanne Mørck Nielsen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19762 • Publication Date (Web): 23 Feb 2018 Downloaded from http://pubs.acs.org on February 25, 2018

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ACS Applied Materials & Interfaces

Lipid shell-enveloped polymeric nanoparticles with high integrity of lipid shells improve mucus penetration and interaction with cystic fibrosis-related bacterial biofilms Feng Wan 1; Tommy Nylander 2; Sylvia Natalie Klodzinska 1; Camilla Foged 1; Mingshi Yang 1; Stefania G. Baldursdottir 1; Hanne M. Nielsen 1* 1

Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark; 2 Department of Physical Chemistry, Lund University, SE-221 00 Lund, Sweden *Corresponding author: [email protected]

Key words: lipid bilayer-enveloped polymeric NPs, nanomedicines, Pseudomonas aeruginosa, bacterial biofilm, FRET, QCM-D

1 ACS Paragon Plus Environment

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Abstract Nanoparticle (NP) mediated drug delivery into viscous biomatrices, e.g., mucus and bacterial biofilms, is challenging. Lipid shell-enveloped polymeric NPs (Lipid@NPs), composed of a polymeric NP core coated with a lipid shell, represent a promising alternative to the current delivery systems. Here, we describe facile methods to prepare Lipid@NPs with high integrity of lipid shells and demonstrate the potential of Lipid@NPs in effective mucus penetration and interaction with cystic fibrosis-related bacterial biofilms. Lipid shell-enveloped polystyrene NPs with high integrity of lipid shells (cLipid@PSNPs) were prepared by using an electrostatically mediated layer-by-layer approach, where the model polystyrene NPs (PSNPs) were first modified with positively charged poly-L-lysine (PLL) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), followed by subsequent fusion with zwitterionic, PEGylated small unilamellar vesicles (SUVs). The interaction of the PSNPs with SUVs was significantly enhanced by modifying the PSNPs with PLL and DOTAP, which eventually resulted in the formation of cLipid@PSNPs, i.e. Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs. Improved mucus-penetrating property of cLipid@PSNPs was demonstrated by quartz crystal microbalance with dissipation monitoring measurements. Furthermore, fluorescence resonance energy transfer measurements showed that the interaction of the cLipid@PSNPs with bacterial biofilms was significantly promoted. In conclusion, we prepare cLipid@PSNPs via an electrostatically mediated layer-by layer approach. Our results suggest that the integrity of the lipid envelopes is crucial for enabling the diffusion of Lipid@PSNPs into the mucus layer and promoting the interaction of Lipid@PSNPs with a bacterial biofilm.


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Introduction Chronic bacterial infection in lungs (e.g. Pseudomonas aeruginosa) constitutes the primary cause of morbidity and mortality in cystic fibrosis (CF) patients 1. The formation of recalcitrant and highly resistant biofilms in CF mucus challenges efficacious therapy with antibiotics. Thus, improving antibiotic treatment is a cornerstone for preventing chronic infections and reducing the bacterial load, exacerbation rates and loss of pulmonary function 2. Antibiotic treatment via inhalation represents a promising strategy, because it offers the opportunity for direct drug delivery to the site of infection and for achieving high local antibiotic concentrations, while limiting systemic side effects of the drug 2. However, efficient and targeted delivery of antibiotics into bacterial biofilms in CF patients by inhalation remains challenging. Nebulized aqueous solution of antibiotics is by far the most widely used dosage form. Inhaled antibiotics in solution are rapidly cleared from the lungs or inactivated by metabolic enzymes 3, resulting in short residence times and local drug concentrations below the effective minimum inhibitory concentration (MIC) in the vicinity of the bacteria. Consequently, these dosage forms require at least twice-daily administration, resulting in poor patience compliance. Importantly, sub-inhibitory concentrations of antibiotics may further induce the formation of bacterial biofilms

4-5

. Therefore,

nanomedicines are being explored to improve the treatment of CF-related lung infections by enhancing the penetration of antimicrobial agents into bacterial biofilms in a localized and controlled manner 6-8. Inhalable liposomal formulations have been shown to be promising for effective delivery of drugs into bacterial biofilms. It has been demonstrated that lipid bilayer vesicles with fluid phase membranes have a unique ability to fuse with cell membranes of bacteria, leading to significantly increased drug



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retention in the lungs and enhanced antimicrobial activity 9-11. Furthermore, certain types of liposomes are capable of penetrating the mucus layer and the biofilm due to the surfactant properties of lipids, thereby attaining access and close proximity to bacteria

12

. To date, inhaled liposomal formulation of

ciprofloxacin (Pulmaquin®, Aradigm Corporation, California, USA) has been approved by the European Medicines Agency (EMA) for use in CF patients 2. In addition, results of Phase II clinical trials demonstrated good safety and efficacy of a liposomal formulation of amikacin (Arikace®, Insmed, New Jersey, USA) in CF patients with P. aeruginosa infection 13, and it is currently tested in a phase III clinical trial 6. Liposomal formulations possess a number of drawbacks, e.g., (i) instability of liposomes during storage, drying and inhalation, (ii) premature leakage of drug from liposomes before reaching the site of action, and (iii) poor sustained release properties 6. In this regard, hydrophobic polymeric nanoparticles (NPs) represents a promising alternative delivery system with sustained drug release properties, which may mediate extended contact time between the antibiotic and the bacterial biofilm, eventually increasing efficacy 11, 14. The interactions between hydrophobic polymeric NPs and mucin, which is composed of high molecular weight (200 kDa ~ 200 MDa) glycoproteins and constitutes the major component in mucus, seem to hinder the diffusion of NPs in mucus and bacterial biofilms

15-16

. Therefore, we hypothesize that lipid bilayer-enveloped polymeric NPs (Lipid@NPs),

composed of a polymeric NP core coated with a zwitterionic lipid bilayer, could be a promising alternative to the current delivery systems for effective delivery of antimicrobial agents into bacterial biofilms. The rationales for such a design are (i) adequate mucus-penetrating properties due to the presence of the lipid bilayer envelope; (ii) improved penetration into bacterial biofilms, (iii) lipid bilayer fusion with bacterial membranes; and (iv) enhanced colloidal stability and sustained drug release properties offered by the drug-loaded polymeric core 6, 17-18.


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In the present study, we demonstrate the mucus penetration and biofilm-interacting properties of Lipid@NPs. We firstly introduce an electrostatically mediated layer-by-layer approach, where model polystyrene NPs (PSNPs) are first surface-modified with positively charged poly-L-lysine (PLL) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), followed by fusion with zwitterionic, PEGylated small unilamellar vesicles (SUVs), to prepare lipid shell-enveloped PSNPs with high integrity of lipid shells (cLipid@PSNPs), i.e. Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs. The SUVs are mainly composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) due to its fusogenic property

with

bacterial

membrane,

and

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

methoxy(polyethylene glycol) 2000 (DSPE-PEG2000), which is used to improve the physicochemical stability and mucus penetrating property of the nanovehicles. Quartz crystal microbalance with dissipation monitoring (QCM-D) measurements displayed that both Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs possessed improved mucus-penetrating properties, as compared to nonenveloped PSNPs. In addition, fluorescence resonance energy transfer (FRET) measurements showed that interaction of cLipid@PSNPs with a Pseudomonas aeruginosa biofilm was significantly enhanced. Thus, Lipid@NPs may pose a promising delivery system for effective delivery of antimicrobial agents into bacterial biofilm.

Materials and methods Materials

Polystyrene nanoparticles (PSNPs in 2.5% w/v dispersion, 100 nm and 200 nm, surface plain) were purchased from Kisker-Biotech (Steinfurt, Germany). 1,2-dioleoyl-sn-glycero-3-phosphocholine



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(DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) 2000 (DSPE-PEG2000), cholesterol (chol), and DOTAP were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC18(3)] and 3,3'dioctadecyloxacarbocyanine perchlorate [DiOC18(3)] were purchased from Invitrogen (Carlsbad, CA, USA).

HEPES,

Tris,

PLL,

osmium

tetroxide

solution,

ammonium

molybdate,

N-(3-

dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanolamine hydrochloride (ETA-HCl), 11-mercaptoundecanoic acid (MUA), and porcine stomach mucin (Type III, bound sialic acid 0.5-1.5 %, partially purified powder), Mueller Hinton Broth (MHB), and deoxyribonucleic acid (DNA) from fish sperm were purchased from Sigma–Aldrich (Broendby, DK). Ultrapure water for sample preparation was obtained from a PURELAB® flex machine (ELGA LabWater, High Wycombe, UK).

Liposomes

The liposomes used in this study were prepared by using the thin lipid film method. Briefly, lipids (DOPC:DSPE-PEG2000, 96:4, molar ratio; or DOTAP:Chol, 55:45, molar ratio) were dissolved in chloroform:methanol (3:1, v/v). The organic solvent was evaporated under vacuum, and the lipid films were washed twice with 500 mL of ethanol (99.9%, v/v) followed by solvent evaporation overnight. Then, the lipid films were rehydrated using 5 mL HEPES buffer (10 mM, pH 7.4) at 50 °C with 2 min of vigorous vortexing every tenth min for 1 h resulting in a lipid concentration of 5 mg/mL (i.e. 5.79 mM for SUVs (DOPC:DSPE-PEG2000) and 8.90 mM for SUVs (DOTAP:Chol). SUVs were obtained from large multilamellar vesicle (LMVs) by 10 times extrusion at 50°C through polycarbonate filters with pore sizes of 50 nm (Whatman, UK) using an extruder (LIPEX Extruders, Northern Lipids, CA). The final lipid concentration (4.35±0.22 mg/mL) of SUVs dispersions were quantified by using a 6 ACS Paragon Plus Environment

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phospholipid assay kit (Wako Chemicals, Neuss, Germany) according to the manufacturer’s instructions.

A similar procedure was used to prepare DiOC18(3)-labeled SUVs (DOTAP:Chol), DiIC18(3)-labeled SUVs (DOPC:DSPE-PEG2000), and FRET dyes (DiOC18(3)/DiIC18(3))-labeled SUVs (DOPC:DSPEPEG2000). For DiOC18(3)-labeled SUVs (DOTAP:Chol) and DiIC18(3)-labeled SUVs (DOPC:DSPEPEG2000), 1.5% (w/w) of DiOC18(3) and DiIC18(3) was added to the specific lipid mixture before film formation, respectively. For the FRET dyes (DiOC18(3)/DiIC18(3))-labeled SUVs, 0.75% (w/w) DiOC18(3) and 0.75% (w/w) DiIC18(3) were mixed with the specific lipid mixture.

Nanoparticle modification with PLL and DOTAP

Prior to surface modification, the PSNPs suspensions were dialyzed against ultrapure water for 24 h using dialysis cassettes (molecular weight cutoffs 10K, Fisher Scientific, DK). The PSNPs were collected into a volumetric flask with final concentration of 10 mg/mL (in 10 mM HEPES buffer, pH 7.4). To modify PSNPs with PLL and DOTAP, PSNPs suspensions were diluted to a concentration of approximately 1 mg/mL using HEPES buffer (10 mM, pH 7.4) (1.05±0.08 mg/mL determined by optical density at 600 nm, OD600), followed by addition of PLL solution at a concentration of 1 mg/mL and DOTAP SUVs (5 mg/mL, total lipid concentration), respectively (See the composition in detail in supporting information, Table S-1and Table S-2). For both types of coatings, the mixtures were kept at room temperature (rt) for 20 min with 10 s vortexing every 5 min. Non-adsorbed PLL and DOTAP SUVs were removed by centrifugation (18,000 g, 20 min, rt). The resulting PLL and DOTAP modified PSNPs (i.e. PLL-PSNPs and DOTAP-PSNPs) were washed once using ultrapure water and re-dispersed in HEPES buffer (10 mM, pH 7.4) using bath sonication.



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Lipid shell-enveloped PSNPs (Lipid@PSNPs)

Lipid shell-enveloped PSNPs were prepared by co-incubating the zwitterionic, PEGylated liposomes (DOPC:DSPE-PEG2000) with PSNPs, or surface-modified PSNPs (PLL-PSNPs or DOTAP-PSNPs) by a method previously reported 19. Briefly, PSNPs, PLL-PSNPs or DOTAP-PSNPs (1 mg/mL in HEPES buffer,10 mM, pH 7.4) were co-incubated with DOPC:DSPE-PEG2000 SUVs (See the composition in detail in supporting information, Table S-3) at rt for 20 min with 10 s vortexing every 5 min. Excess lipids/liposomes was removed by centrifugation (18,000 g, 20 min, rt). The resulting lipid shellenveloped PSNPs (i.e. dLipid@PSNPs, Lipid@PLL-PSNPs, and Lipid@DOTAP-PSNPs) were washed once using ultrapure water and re-dispersed in HEPES buffer (10 mM, pH 7.4) using bath sonication.

Z-average and zeta potential

The z-average and zeta potential of the liposomes and NPs dispersions were determined by using dynamic light scattering and laser Doppler electrophoresis, respectively. The samples were diluted using HEPES buffer (10 mM, pH 7.4) to a concentration of 0.1 mg/mL. The measurements were carried out at 25 °C by using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser and 173° detection optics. Data acquisition and analysis were performed by using the Malvern DTS v6.30 software (Malvern Instruments).

Lipid molecules on Lipid@PSNPs

The theoretical number of lipid molecules in a 220 nm unilamellar liposome was calculated according to Equation 1:


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=


[ ( ) ( ) ]

(Equation 1)

where d is the average diameter of the liposomes, h is the thickness of the bilayer, and A is the lipid headgroup area. The headgroup area and the bilayer thickness of phosphatidylcholine is approximately 0.64 nm2 and 4 nm, respectively 20. The theoretical number of lipid molecules in a 220 nm liposome is approximately 460,000.

To quantify the amount of lipid molecules on Lipid@PSNPs, fluorescently labeled SUVs (as described above) were used to prepare fluorescently labeled Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs. A volume of 20 µL of fluorescently labeled Lipid@PLL-PSNPs or Lipid@DOTAP-PSNPs (1 mg/mL) was mixed with 180 µL DMSO to dissolve the NPs. Then, 150 µL diluent of Lipid@PLL-PSNPs or Lipid@DOTAP-PSNPs suspensions was added to the wells in a 96-well plate with transparent bottom (Thermo Fisher Scientific, Hvidovre, Denmark). The fluorescence intensity was measured by using a FLUOstar OPTIMA plate-reader (BMG LABTECH, Offenburg, Germany). For quantification of DiOC18(3) representing DOTAP on Lipid@DOTAP-PSNPs, samples were measured by excitation at 485 nm and emission at 535 nm. For quantification of DiIC18(3) representing DOPC:DSPE-PEG2000 on Lipid@ PSNPs, the samples were measured by excitation at 545 nm and emission at 590 nm. Linearity was simultaneously determined in the range of 0.00125~0.05 mg/mL for both DOTAP and DOPC:DSPE-PEG2000 (See Figure S-2 in supporting information).

Fluorescence resonance energy transfer (FRET)

A FRET pair of hydrophobic dyes (DiOC18(3) as the donor and DiIC18(3) as the acceptor) was incorporated in the membrane of DOPC:DSPE-PEG2000 liposomes. To measure FRET phenomena, the



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ﬂuorescence spectra of DiOC18(3) and DiIC18(3) in SUVs or dissolved in acetone were recorded using a Tecan Safire 2 Multimode Microplate Reader (Tecan, Männedorf, Switzerland) with an excitation wavelength of 450 nm and emission scans from 495–650 nm. The signal was quantified using Equation 2:

=

!18(3) %C18(3) +

(Equation 2)

C18(3)

where ()*+,(-) and (.*+,(-) represent the fluorescence intensities at 510 and 570 nm, respectively. To investigate the interaction of NPs with SUVs, 200 µL of the mixture of fluorescence dye-labeled SUVs and NPs (i.e. PSNPs, PLL-PSNPs, DOTAP-PSNPs) prepared as described above were added to 96well plates to measure the FRET ratio.

Transmission electron microscopy (TEM)

Morphological analysis of the NPs was carried out by using a Tecnai G2 20 TWIN transmission electron microscope (FEI, Hillsboro, OR, USA). Samples of 100 µL NPs were mixed with 100 µL of 2% (w/v) osmium tetroxide in water to fix the lipid and kept at rt for 20 min. Excess osmium was removed by centrifugation (12,000 g, 10 min, rt). The samples were redispersed in 100 µL ultrapure water and mounted onto a TEM grid, followed by staining with 1% (w/v) ammonium molybdate for 1 min. The grid was dried by removing the liquid using a filter paper. All observations were made in bright field mode at an acceleration voltage of 10 kV. Digital images were recorded with a Gatan Imaging Filter 100 CCD camera (Gatan, Pleasanton, CA, USA).

Quartz crystal microbalance with dissipation monitoring (QCM-D)


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QCM-D measurements were performed with an E4 system from Q-Sense (Gothenburg, Sweden) using carboxylic acid-functionalized, gold-coated quartz crystals with a fundamental frequency of 4.95 Hz (QSX301, Q-Sense). To deposit a stable mucin layer on the crystals, mucin was immobilized on the crystals by using the amine-coupling method

21

. Briefly, the crystals were first functionalized with a

monolayer of carboxylic-acid (SAM-COOH) by immersion in a 1 mM ethanolic solution of MUA for at least 12 h at rt. The resulting functionalized gold-coated quartz crystals were subsequently rinsed with ethanol, dried and mounted in the QCM-D chamber. While mounted in the chamber, the crystals were exposed to an aqueous solution of 200 mM EDC and 50 mM NHS for 10 min, followed by rinsing with ultrapure water and citrate buffer (10 mM, pH 4.0). Afterwards, 0.5 mg/mL mucin in citrate buffer was applied for 30 min, followed by rinsing with the citrate buffer for 10 min. Unreacted NHS-ester was deactivated by rinsing with 1 M ETA-HCl (in Tris buffer, 10 mM, pH 8.5) for 15 min. Following another rinsing with citrate buffer (10 mM, pH 6.0) for 10 min, NP suspensions (in citrate buffer, 10 mM, pH 6.0) were applied in the QCM-D chamber, and changes in frequency (∆f) and energy dissipation factor (∆D) were simultaneously recorded. All experiments were conducted at 37°C and at a constant flow rate of 50 µL/min. QCM-D measurements were performed in at least triplicate, and representative measurements are presented.

Bacterial biofilms

To prepare a medium mimicking cystic fibrosis sputum, 200 mg of DNA from fish sperm was added to 1 L of MHB and the medium was sterilized at 121 °C for 15 min. Prior to use, P. aeruginosa (PA01) from cryostock was grown overnight (15-18 hours) in MHB at 37°C. The overnight inoculum was transferred to fresh MHB and incubated for 2-3 hours to reach exponential growth phase. Immediately before use, the bacterial suspension was adjusted to 0.5 McFarland standard (1 × 108 CFU/mL, OD600 11 ACS Paragon Plus Environment


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approximately 0.08-0.1) and further diluted 1:20. A culture sample was plated for contamination check every time bacteria were grown from cryostocks. For preparation of the biofilm, 100 µL of MHB-DNA medium was placed in each well of a 96-well flat-bottomed polystyrene tissue-culture-treated cell culture plates (Costar Corning®, Corning, NY, USA). The medium was inoculated with 10 µL of adjusted P. aeruginosa (PA01) to yield a final concentration of 5×105 CFU/mL and incubated statically at 37 °C in ambient air. The biofilm was used after 24 h for the 1-day biofilms or the MHBDNA medium was replaced with fresh medium every 24 h for 72 h for the 3-day biofilms.

To investigate the interaction between the bacterial biofilms and the NPs, the biofilms were rinsed with HEPES buffer (10 mM, pH 7.4) twice, and 200 µl of NP suspensions (0.1 mg/mL) was added to each well. FRET ratios were determined as described above at 0, 15, 30, 45, 60, 90 and 120 min.

Statistics

All measurements were performed at least in triplicate, unless otherwise stated. Excel (Microsoft, USA) was applied for data analysis and statistical analysis. Data are presented as means ± standard deviation (SD). The student’s t test is used to assess the statistically significant differences between two findings. The significant statistical difference is defined according to p < 0.05.

Results and discussion

Co-incubation of non-modified PSNPs with liposome suspension results in incomplete lipid envelopes


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The dLipid@PSNPs were prepared via direct co-incubation of the zwitterionic, PEGylated SUVs with non-modified PSNPs. There were no significant differences (p > 0.05) in z-average between nonmodified PSNPs and dLipid@PSNPs, irrespective of the investigated surface area ratio (SA ratio, lipid bilayer:PSNPs) (Table I and Figure S-1 in the supporting information). Although the zeta potential of the resulting dLipid@PSNPs was less negative than the non-modified PSNPs, it was still significantly different from that of the zwitterionic, PEGylated SUVs (p < 0.05), irrespective of the investigated surface area ratio (SA ratio, lipid bilayer:PSNPs) (Table I and Figure S-1 in the supporting information). This is likely due to the fact that the negatively charged groups on PSNPs were not completely shielded by a lipid bilayer. In addition, the measured number of lipid molecules per PSNP (Figure 1) was only about 30% of that of 220 nm liposome. These results imply that the conventional co-incubation of nonmodified PSNPs with liposome suspension leads to a low NP coating efficiency, which can seemingly be explained by the formation of bilayer patches covering incompletely the PSNPs 22.This could be the result of the weak interactions between PSNPs and zwitterionic, PEGylated SUVs.

Therefore, FRET was thus applied to assess the interaction of the zwitterionic, PEGylated SUVs with the PSNPs and the changes in structure of lipid bilayer after the fusion with PSNPs. FRET is a physical phenomenon, which relies on the distance-dependent energy transfer from a donor molecule to an acceptor molecule

23

. FRET measurements have been widely used in biomedical research due to the

sensitivity to changes in the distance between the donor molecule and the acceptor molecule

24-27

. In

this study, DiOC18(3) and DiIC18(3) were selected as donor and acceptor molecules, respectively, and incorporated into SUVs. In ultrapure water, a strong DiIC18(3) signal was observed (Figure 2a) due to the close proximity to DiOC18(3) in the SUV lipid bilayer, resulting in a FRET ratio of 0.75. The FRET



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signal disappeared after disintegration of the SUVs by acetone, (Figure 2a) because DiOC18(3) and DiIC18(3) are not in close proximity. Simple addition of the non-modified PSNPs to the FRET dyes-labeled SUVs dispersion led to a slight increase in fluorescence intensity at 510 nm and decrease in fluorescence intensity at 570 nm (Figure 2b), indicating that some of the SUVs interacted with PSNPs and that the structure of the lipid bilayer was distorted. However, the FRET ratio of the dLipid@PSNPs collected by centrifugation showed a significant decline compared to that of the FRET dyes-labeled SUVs (Figure 2c). This suggests that the distance between DiOC18(3) and DiIC18(3) increases after coating onto the PSNPs and thus results in a loss of the FRET effect. This can be explained by a comparable mechanism to that of the supported lipid bilayers formed onto the silica nanoparticles, in which SUVs deform and rupture to form lipid bilayer patches onto the silica nanoparticles after adhesion to the silica nanoparticles. The successive adsorption of SUVs to the silica nanoparticles leads to the formation of complete lipid bilayer with the coalescence of neighboring bilayer patches

22

. There is seemingly no sufficient SUVs adsorbed onto

PSNPs due to the weak interaction between the zwitterionic, PEGylated SUVs and the PSNPs, eventually resulting in the incomplete lipid bilayer patches on the PSNPs. Furthermore, the spreading of the lipids on the solid supports seems to result in the increased distance between DiOC18(3) and DiIC18(3) and the loss of the FRET effect 22, 28.

Surface-modified PSNPs with PLL and DOTAP improves the integrity of lipid envelopes

To improve the integrity of the lipid envelopes on PSNPs, an electronically mediated layer-by-layer approach was applied, where PSNPs were first modified by using positively charged PLL or DOTAP,


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followed by the co-incubation of the surface-modified PSNPs (PLL-PSNPs or DOTAP-PSNPs) with zwitterionic, PEGylated SUVs (Figure 3). After surface modification with PLL, the z-average of the PSNPs increased significantly from 220 nm to 240 nm (p < 0.05), whereas the zeta potential of the PLL- PSNPs turned to be positive (approximately +15 mV ~ +35 mV) and increased with higher PLLto-PSNP mass ratios (Table I and Figure S-1 in the supporting information). A mass ratio of 5:1 was selected for further studies due to the more narrow size distribution (Table I and Figure S-1 in the supporting information). Overall, similar results were obtained when introducing surface modifications with DOTAP (Table I and Figure S-1 in the supporting information), yet with DOTAP- PSNPs, a SA ratio of 2:1 was considered optimal as a further increase in the SA of the DOTAP bilayer to PSNPs did not result in an additional increase in the zeta potential of the resulting DOTAP-PSNPs (Table I and Figure S-1 in the supporting information).

Subsequently, the co-incubation of PLL-PSNPs and DOTAP-PSNPs with the zwitterionic, PEGylated SUVs resulted in a significant increase in z-average for both Lipid@PLL-PSNPs and Lipid@DOTAPPSNPs (Table I and Figure S-1 in the supporting information). In addition, the zeta potentials of Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs were -18.9 mV and -13.3 mV, respectively. It is worthy to note that zeta potential of Lipid@PLL-PSNPs approximates to that of zwitterionic SUVs applied in this study (no significant difference, p > 0.05, Table I and Figure S-1 in the supporting information). The less negatively charge of Lipid@DOTAP-PSNPs could be the result of the coexistence of DOTAP with DOPC and DSPE-PEG2000 in the lipid shell (Figure 1). Furthermore, the number of lipid molecules on each Lipid@PLL-PSNP and Lipid@DOTAP-PSNPs significantly increased compared to that of dLipid@PSNPs (p < 0.01) and was comparable to the number of lipid molecules in a 220 nm liposome (Figure 1). These results suggest that electronically mediated layer-by-



Page 16 of 31

layer approach improves the integrity of the lipid shell on PSNPs. Meanwhile, the results also suggest that the lipid envelopes are likely organized as lipid bilayers, which is supported by the presence of a seemingly continuous lipid bilayer on the surface of Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs (Figure 4).

The improved integrity of the envelopes on PSNPs may be a result of enhanced interaction of PLLPSNPs and DOTAP-PSNPs with SUVs. Exposure of PLL-PSNPs and DOTAP-PSNPs to the FRET dyes-labeled SUVs led to an increased DiOC18(3) signal and a simultaneous decrease in the DiIC18(3) signal as compared to that observed when adding non-modified PSNPs (Figure 2b). The difference in the FRET signal indicates a stronger interaction of SUVs with PLL-PSNPs and DOTAP-PSNPs. Further, the FRET ratios of the purified Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs were also significantly higher than that of dLipid@PSNPs and approached that of FRET dyes-labeled SUVs (Figure 2c), suggesting an improved integrity of the lipid envelopes. Interestingly, it was observed that the FRET ratio for Lipid@DOTAP-PSNPs was lower than that of Lipid@PLL-PSNPs. This may be attributed to different mechanisms of lipid shell formation on the PLL-PSNPs and DOTAP-PSNPs, respectively.

In the case of PLL-PSNPs, electrostatic interactions promoted the adsorption of SUVs onto PLLPSNPs and increased the number of SUVs on the surface of the PLL-PSNPs. Subsequently, deformation and rupture of the SUVs occur due to enhanced adhesion forces, which seemingly results in the formation of complete lipid bilayer covering PLL-PSNPs 22, 29. For the DOTAP-PSNPs, however, it is more likely that an electrostatically mediated lipids fusion and exchange process between DOTAP bilayers of DOTAP-PSNPs and the zwitterionic SUVs influence the formation of the lipid shell 30. This


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lipid exchange process leads to a mixture of DOTAP and DOPC present in the lipid envelopes, as displayed that approximately 50% of DOTAP was present in the lipid coat of Lipid@DOTAP-PSNPs (Figure 1). As a result, the distance between DiOC18(3) and DiIC18(3) increased, eventually resulting in the loss of the FRET signal.

cLipid@PSNPs display improved mucus penetrating properties

Immobilization of mucin on the QCM-D crystal was effectively achieved by covalent grafting of the mucin onto NHS ester-functionalized surfaces. After surface functionalization of gold-coated crystals, the introduction of mucin to the chamber led to a fast decrease in frequency, suggesting mucin adsorption onto the surface of crystals (Figure 5a). In addition, the increase in the dissipation factor suggests viscoelastic properties of the adsorbed mucin layer (Figure 5a). After rinsing with buffer, no significant changes in frequency was observed, suggesting that the adsorbed mucin layer is highly stable, as expected, due to the formation of covalent bonds 21.

Upon introducing the NPs suspensions to the immobilized mucin layer in the chamber, the changes in frequency and dissipation factor were simultaneously monitored. The addition of 0.2 mg/mL of PSNPs resulted in a large decrease in frequency (∆f ≈ 900 Hz) indicating strong interactions between nonmodified PSNPs and the mucin layer (Figure 5b). Correspondingly, the dissipation factor reached a level of ∆D ≈ 120×10-6, which indicates the presence of a soft, thick adsorbed layer (Figure 5c) 31. In comparison, exposure of both types of lipid bilayer-enveloped PSNPs to the mucin layer resulted in reduced changes in frequencies and dissipation factors (∆f ≈ 250 Hz, ∆D ≈ 70×10-6 and ∆f ≈ 400 Hz, ∆D ≈ 100×10-6 for Lipid@DOTAP-PSNPs and Lipid@PLL-PSNPs, respectively). This implies that the lipid envelope may potentially improve the mucus-penetrating properties of NPs by reducing the



interaction of the NPs with the mucin layer

32

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. In line with this, information on the stiffness of the

interaction between the adsorbed NPs and the mucin layer was obtained through ∆D versus ∆f plots. A less steep slope indicates NP adsorption without a significant increase in the dissipation, which is characteristic of a strong interaction between adsorbed NPs and the mucin layer. In contrast, a steeper slope suggests a relatively weak interaction

33-34

. Relatively steep slopes were observed for

Lipid@DOTAP-PSNPs and Lipid@PLL-PSNPs (Figure 5d), indicating that the lipid coating also decreased the stiffness of the interaction between the adsorbed NPs and the mucin layer.

Interestingly, it was observed that Lipid@DOTAP-PSNPs possessed weaker interaction with mucin than Lipid@PLL-PSNPs, i.e. improved mucus-penetrating properties. This might be attributed to the more virus-like surface of Lipid@DOTAP-PSNPs. The coexistence of positively charged DOTAP molecules on the surface of Lipid@DOTAP-PSNPs leads to the less negative charge (-13.3 mV of zeta potential, close to neutral), thus seemingly close to the virus-like surface (i.e. hydrophilic and with a high charge density, however neutral due to the high concentration of cationic and anionic groups). The previous studies have shown that the virus-mimicking nanoparticles can prompt the mucus penetrating 35-36

, though the mechanism is not yet fully understood. In contrast, Lipid@PLL-PSNPs present more

negatively charges (-18.9 mV of zeta potential) due to lacking of positively charged DOTAP molecules, as a consequence, the weaker ‘slippery’ properties.

Higher integrity of lipid shell improves the interaction with bacterial biofilm

The interaction between bacterial biofilms and liposomes has been assessed by using FRET, and the change in the FRET signal after incubation of liposomes with bacterial biofilms is indicative of a fusion process 9. Here, FRET dye-labeled SUVs were incubated with a P. aeruginosa biofilm, and the


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dynamic changes in the normalized FRET ratio during a 2 h incubation period were measured. The normalized FRET ratio decreased sharply and instantaneously from 1 to 0.6 (Figure 6a), which indicates a strong interaction/fusion of the SUVs with the bacterial biofilm. In contrast, incubation of dLipid@PSNPs with the bacterial biofilm did not result in any obvious decline in the FRET ratio over 2 h, suggesting that the incomplete lipid envelope does not promote an interaction between the dLipid@PSNPs and the P. aeruginosa biofilm. In comparison, both Lipid@DOTAP-PSNPs and Lipid@PLL-PSNPs showed enhanced interaction with the P. aeruginosa biofilm, suggesting that the integrity of the lipid shell is crucial for promoting the fusion of Lipid@PSNPs with the bacterial biofilm. Both the Lipid@DOTAP-PSNPs and the Lipid@PLL-PSNPs showed weaker interactions with one-day bacterial biofilms, as compared to those of control SUVs, which may be a result of (i) significantly larger particle sizes of the Lipid@DOTAP-PSNPs and Lipid@PLL-PSNPs decreased fluidity/flexibility of lipid shell after adsorption onto the surface of PSNPs

38

37

and (ii) the

. It should be

noted that Lipid@DOTAP-PSNPs showed stronger interactions with one-day bacterial biofilms than those displayed by Lipid@PLL-PSNPs. This could be attributed to the fact that DOTAP and DOPC molecules coexist in the lipid bilayer of Lipid@DOTAP-PSNPs, which could lead to a more flexible lipid bilayer. In addition, the positive charge of DOTAP may also benefit the interaction with the bacterial biofilm.

Interestingly, it was observed that both Lipid@DOTAP-PSNPs and Lipid@PLL-PSNPs also interacted with a stronger three-day bacterial biofilm, however especially for Lipid@DOTAP-PSNPs the interaction was decreased (Figure 6b). This could be attributed to the fact that more extracellular polymeric substances (EPS) were present in the biofilm at increased age of the bacterial biofilm, which hampers the interaction of the Lipid@DOTAP-PSNPs and Lipid@PLL-PSNPs with the bacterial



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biofilm 39. To overcome a denser EPS matrix barrier, it is thus necessary to further reduce the particle size and optimize the composition of the lipid shell.

Conclusion Here, we present facile methods to prepare lipid shell-enveloped polymeric NPs via an electrostatically mediated layer-by-layer process. Compared to the conventional method, the electrostatically mediated layer-by-layer approach improves the integrity of lipid envelopes formed on PSNPs. As a consequence, the improved integrity of the lipid envelopes enables the diffusion of Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs into the mucus layer, and promotes the interaction of Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs with a bacterial biofilm. It is worthy to note that Lipid@DOTAP-PSNPs presents a more intensive interaction with the bacterial one-day biofilm as compared to that of Lipid@PLL-PSNPs, whereas similar, but reduced interactions are observed with the three-day bacterial biofilm. Overall, lipid shell-enveloped polymeric NPs may represent a promising approach to improve the delivery of antimicrobial agents-loaded polymeric NPs into CF-related respiratory bacterial biofilm infection.

Supporting Information. Calculation of the amount of PSNPs and lipids used according to the different surface area ratio, the composition of the mixture used for preparation of PLL-PSNPs, DOTAP-PSNPs and Lipid@PSNPs (dLipid@ PSNPs, Lipid@PLL-PSNPs and Lipid@DOTAPPSNPs), size distribution by intensity and zeta potential distribution of PSNPs, SUVs (DOPC/DSPEPEG), SUVs (DOTAP/Chol,), PLL-PSNPs, DOTAP-PSNPs, dLipid@PSNPs, Lipid@PLL-PSNPs, and


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Lipid@DOTAP-PSNPs, and linearity between fluorescent intensity and lipids concentration in the range of 0.00125~0.05 mg/mL.

Acknowledgements

This work was funded by the Independent Research Fund Denmark, Technology and Production Sciences (DFF–4093-00062) (FW) and the University of Copenhagen 2016 Programme of Excellence Research Centre for Control of Antibiotic Resistance (UC-CARE) (SNK). We also acknowledge the Alfred Benzon Foundation (DK) and Drug Research Academy (University of Copenhagen, DK) for funding the FLUOstar OPTIMA plate reader. The authors would also like to thank the Core Facility for Integrated Microscopy (CFIM), Faculty of Health and Medical Sciences, University of Copenhagen for technical assistance with TEM analyses.

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**

6 Number of lipids/PSNP (X105)

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5 4 3 2 1

TA PPS N Ps Li pi d@ D O

Ps Li pi d@ PL LPS N

PS N Ps dL ip id @

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Figure 1. Average number of lipid molecules (dense pattern: DOTAP; sparse pattern: DOPC) on each PSNP. Data represents mean values ± SD (n = 3). ** means p < 0.01.



15

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Figure 2. Representative fluorescence spectra in water (full line) and ACE (dashed line) of FRET dyeslabeled SUVs prepared by using 0.75% (w/w) DiOC18(3) and 0.75% (w/w) DiIC18(3) (a); Changes in fluorescence spectra of FRET dyes-labeled SUVs in ultrapure water (black) and after addition of PSNPs (red), PLL-PSNPs (green) and DOTAP-PSNPs (blue) (b); FRET ratios of FRET dyes-labeled SUVs, dLipid@PSNPs, Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs (c). Data represents mean values ± SD (n ≥3). ** means p < 0.01.



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Figure 3. Schematic representation of the preparation of Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs by using the electrostatically mediated layer-by-layer approach.


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Figure 4. Representative transmission electron micrographs of PSNPs, scale bar = 100 nm (a); Lipid@PLL-PSNPs, scale bar = 200 nm (b); Lipid@DOTAP-PSNPs, scale bars = 200 nm (c).



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Figure 5. Representative QCM-D measurements of changes in frequency (∆f, red) and dissipation (∆D, blue) as a function of time during adsorption of mucin onto the crystal (a); the change in frequency (b) and dissipation factor (c) due to the introduction of 0.2 mg/mL PSNPs (black squares), 0.2 mg/mL Lipid@PLL-PSNPs (red triangles), 0.2 mg/mL Lipid@DOTAP-PSNPs (blue circles) to the mucin layer; ∆D vs ∆f plots of the NPs adsorption measurements illustrated in b and c (d). The measurements were conducted at 37°C with a flow rate of 50 µL/min and the presented data was measured at the 5th overtone. The first and the second arrows indicate the addition of mucin (a) or NPs (b, c) and rinsing with citrate buffer (10 mM, pH6.0), respectively.


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Figure 6. Time-resolved FRET ratios of FRET dyes-labeled SUVs (black squares), dLipid@PSNPs (olive triangles), Lipid@PLL-PSNPs (red circles), and Lipid@DOTAP-PSNPs (blue triangles) after incubation with one-day biofilms (a); three-day biofilm (b). Data represents mean values ± SD (n = 3).


ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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Table I Z-average, PDI, and zeta potential of PSNPs (non-modified), SUVs (DOPC/DSPE-PEG2000), SUVs (DOTAP/Chol), surfacemodified PSNPs (PLL-PSNPs and DOTAP-PSNPS), dLipid@PSNPs, Lpid@PLL-PSNPs and Lipid@DOTAP-PSNPs. Results denote mean values ±SD (n≥3). Samples PSNPs DOPC/DSPE-PEG2000 SUVs DOTAP/Chol SUVs

Size (nm) 212.9±4.7 74.9±10.8 76.3±3.4

PDI 0.03±0.02 0.05±0.01 0.13±0.02

Zeta potential (mV) -55.5±1.5 -17.9±2.9 48.4±7.7

2.5:1a 5:1a 10:1a 20:1a

227.2±3.0** 223.4±5.1** 240.8±11.6** 254.5±6.6**

0.10±0.02 0.06±0.04 0.11±0.08 0.20±0.02

33.6±2.1 24.4±4.5 17.5±3.6 12.3±1.0

DOTAP-PSNPs

8:1b 4:1b 2:1b 1:1b

239±6.7** 227.9±14.1** 222.7±6.8* 211.8±6.7

0.04±0.02 0.04±0.03 0.06±0.05 0.02±0.01

39.7±1.5 36.9±2.3 34.7±3.9 28.1±2.1

dLipid@PSNPs

8:1c 4:1c 2:1c

213.4±11.6 220.7±16.4 219.7±17.8

0.05±0.02 0.04±0.01 0.07±0.01

-44.6±5.5## -43.9±6.2## -43.9±2.1##

PLL-PSNPs

Description Non-modified

Lpid@PLL-PSNPs 2:1c 238.0±32.4** 0.03±0.01 -18.9±3.5 Lipid@DOTAP-PSNPs 2:1c 220.9±6.5** 0.03±0.02 -13.3±5.8## * and ** means p < 0.05 and 0.01 respectively referred to z-average of PSNPs (non-modified); # and ## means p < 0.05 and 0.01 respectively referred to zeta potential of SUVs (DOPC/DSPE-PEG2000). ‘a’ represents the mass ratio of PSNPs to PLL used for preparation of PLL-PSNPs; ‘b’ represents the SA ratio of DOTAP lipid bilayer to PSNPs; ‘c’ represents the SA ratio of DOPC lipid bilayer to PSNPs.


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Lipid Shell-Enveloped Polymeric Nanoparticles with High Integrity of

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