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Lysozyme Transport to the Brain by Liposomes

Research Article

Mirjam M. Nordling-David#, Elior Rachamin#, Etty Grad and Gershon Golomb[1]*

Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

#Equally contributed to this work.

Submitted: July 12, 2018; Accepted: July 31, 2018; Posted August 14, 2018

Graphical Abstract

Abstract

Delivery of drugs into the brain is limited due to poor penetrability of many drugs via the blood-brain barrier. Previous studies have shown that the brain is kept under close surveillance by the immune system, implying that circulating phagocytic cells, such as neutrophils and monocytes, are crossing the blood-brain barrier. We hypothesized that charged liposomes could be transported to the brain following their phagocytosis by circulating monocytes. In this work, we investigated the capacity of circulating monocytes to be exploited as a drug delivery system following IV administration of nano-sized, positively fluorescently labeled liposomes containing the protein lysozyme. Negatively charged fluorescently labeled liposomes were used for comparison. By using a modified thin-film hydration technique, the desired properties of the liposomal formulations were achieved including: size, polydispersity index, high drug concentration and stability. In vitro results showed a significant time-dependent uptake of positively charged liposomes by RAW264.7 cells. In vivo results revealed that circulating white blood cells (mainly monocytes) contained high levels of fluorescently labeled liposomes. Screening of brain sections using confocal microscopy uncovered that a substantial amount of fluorescently labeled liposomes, in contrast to the fluorescent markers in solution, was transported into the brain. In addition, anti-CD68 immunofluorescent staining of brain sections, demonstrated co-localization of positively charged liposomes and macrophages in different brain sections. Furthermore, significantly higher levels of lysozyme were detected in brain lysates from rats treated with positively charged liposomes compared to rats treated with lysozyme solution. Taken together this confirms our hypothesis that the designed liposomes were transported to the brain following their phagocytosis by circulating monocytes.

Keywords

Blood-brain Barrier, Monocytes, Liposomes; Lysozyme, Brain delivery, Drug delivery system

License:

CC BY-NC-SA 4.0

Abbreviations

  • WBC white blood cells
  • MPS mononuclear phagocytic system
  • HP 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt
  • DSPC 1,2-distearoyl-sn-glycero-3-phospocholine
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DPPE-Rhod 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine Rhodamine B sulfonyl); DPPE-Rhodamine
  • DSPG 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol)
  • FBS fetal bovine serum
  • Lip(+)LYS Liposomal lysozyme (positively charged)

Rationale and Purpose

The blood-brain barrier (BBB) is a formidable permeability barrier, which excludes most drugs from entering the brain. Hence, developing drug delivery systems for brain disorders, through intact BBB, is of importance. The mononuclear phagocytic system (MPS), phagocytize particulate matter in the circulation. In addition, the brain is under immuno-surveillance. Consequently, we hypothesized that monocytes could be utilized to transport particulate delivery systems to the brain. We describe here the extent and mechanism of liposomal lysozyme (a 14 kDa protein) transport to the brain in rats.

Introduction

One of the major limiting factors in the development of new drugs for brain disease is the presence of an intact BBB. Only certain small and positively charged lipophilic molecules can passively diffuse across an undisrupted BBB, while certain nutrients and specific molecules can only enter the brain via transporters [1]. Understandably, developing an efficient and non-invasive drug delivery system for brain therapy, without affecting the BBB, is of high merit [2].

The MPS is a part of the immune system that consists of phagocytic cells, primarily monocytes and macrophages. Both circulating monocytes, and neutrophils [3] phagocyte foreign particles in the blood, including drugs delivered as nanoparticles (NP). Since the brain is under immunological surveillance, allowing monocytes and neutrophils to cross the BBB [4-7], these cells can be exploited to deliver particulate drugs across the BBB and into the brain. The propensity of monocytes for rapid recognition of particulate matter, the major clearance mechanism of IV administered particulate delivery systems [8, 9], has provided a rational approach to formulate specific “non-stealth” liposomes for increased uptake by circulating monocytes.

We have shown that circulating monocytes could be exploited as transporters of non-PEGylated, negatively charged liposomes to the brain [10]. Brain uptake after IV administration of negatively charged serotonin liposomes in healthy rats was 2 times higher than the uptake after free drug administration. However, markedly higher brain uptake seems to be required to serve as a viable solution for brain drug delivery. It is also known that positively charged particles are more avidly internalized by the MPS in comparison to negatively charged particles [11, 12]. Accordingly, we hypothesized that positively charged liposomes could be exploited to effectively deliver high-molecular weight drugs to the brain following their phagocytosis by circulating monocytes.

The objective of this work was to develop and characterize positively charged and fluorescently labeled liposomal formulations containing the lysozyme protein (MW = 14 kDa). In addition, we examined the biodistribution of positively charged liposomes in comparison to negatively charged liposomes and to free drugs in solution, and further elucidated the mechanism of transport into the brain of intact rats.

Experimental design

A nanoparticulated delivery system of positively charged liposomes encapsulating lysozyme was developed. The liposomes contained fluorescent markers, the hydrophilic 8-Hydroxypyrene-1,3,6-trisulfonic acid tri-sodium salt (HP) in the aqueous core or the hydrophobic DPPE-Rhodamine (DPPE-Rhod) in the liposome membrane for monitoring uptake and biodistribution. Formulations studied were, (i) lysozyme encapsulated in positively charged liposomes, labelled with DPPE-Rhod; (ii) negatively charged liposomes containing HP (since lysozyme was not encapsulated efficiently in this liposomal formulation) for comparison purposes; and serving as controls, (iii) empty liposomes and iv) drugs in solution (lysozyme, DPPE-Rhod, and HP). First, we characterized the liposomal delivery systems, and evaluated the uptake and cytotoxicity in cell cultures. Next, we examined the biodistribution and brain transport in intact rats (animal care and procedures conformed to the standards for care and use of laboratory animals of the Hebrew University of Jerusalem, Israel and the National Institutes of Health, USA). Following treatment (IV or IP) with various formulations, phagocytosis by the MPS, biodistribution, toxicity, and brain uptake were determined. The co-localization of the liposomes with monocytes in the brain was assessed for validating the proposed mechanism of brain transport.

Materials and Methods

Liposomes preparation

Positively charged liposomes were prepared using a modified film thin hydration technique. Liposomes were composed of 1, 2-distearoyl-sn-glycero-3-phospocholine (DSPC, Lipoid, Ludwigshafen, Germany), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, Lipoid) and cholesterol (Sigma-Aldrich), at a molar ratio of 3:1:2, respectively. The lipophilic fluorescent marker, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissam-ine Rhodamine B sulfonyl), (DPPE-Rhod, Avanti Polar Lipids) was added to the film at a molar ratio of 0.05. The lipids were dissolved in tert-butanol (Arcos Organics) and lyophilized overnight. The obtained film was hydrated with 10 mg/mL of chicken egg-white lysozyme (Sigma-Aldrich) in phosphate-buffered saline, and rotated in a 60°C bath for 40 minutes at 90 rpm (Lip(+)LYS). The obtained liposomes were then homogenized using a thermo barrel extruder (Lipex Biomembranes). Non-encapsulated drug was removed by means of dialysis (300 kDa MW, Spectrum Laboratories, Inc.) in PBS overnight. Empty liposomes (empty-Lip(+)) were prepared by the same procedure without lysozyme in the hydration solution.

Despite numerous efforts, only minor encapsulation of lysozyme in negatively charged liposomes was obtained (most probably due the electrostatic interaction). Therefore, HP was encapsulated in negatively charged liposomes to serve as a comparison for the positively charged liposomes. Negatively charged liposomes were prepared by replacing DOTAP with the negatively charged lipid, 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG, Lipoid) at a molar ratio of 3:1:2. The lyophilized film was then hydrated with 100 mM of the hydrophilic fluorescent marker 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HP, Sigma-Aldrich) in PBS (Lip(-)HP). Un-encapsulated HP was removed by passing the liposomal suspension through a Sephadex G-50 column and eluted using PBS.

Characterization of liposomal formulations

Size and zeta potential

Size, polydispersity index (PDI) and surface charge of drug-loaded and empty liposomes were determined at room temperature, following a 1:100 dilution with PBS, by means of a Zetasizer (Malvern Instruments, Malvern).

Cryogenic transmission electron microscopy (Cryo-TEM)

A drop (3 μl) of the liposomal suspension was applied to a glow discharged TEM grid (300-mesh Cu grid) coated with a holey carbon film (Lacey substrate, Ted Pella, Inc.). The excess liquid was blotted, and the specimen was vitrified by rapid plunging it into liquid ethane, pre-cooled with liquid nitrogen using Vitrobot Mark IV (FEI). The vitrified samples were examined at −177°C using a FEI Tecnai G2 12 TWIN TEM equipped with a Gatan 626 cold stage, and the images were recorded (4K × 4K FEI Eagle CCD camera) at 120 kV in a low-dose mode.

Determination of lipids concentration

Total phospholipid concentration was determined by means of HPLC (Alliance e2695, Waters) equipped with an ELS detector (Alltech 3300, Grace). Liposomes were dissolved with an IPA:chloroform solution (1:1), and were injected to a YMC-Pack PVA-Sil-NP column (250 × 4.6 mm, 5 μm, YMC America, Inc.), heated to 35°C. The mobile phases were composed of IPA:chloroform (1:1) (phase A), and IPA:chloroform:water (8:5:1) (phase B), both containing 0.02% v/v TEA and 0.005% v/v TFA, using a gradient starting at 80/20 to 100% with a flow rate of 1 mL/min.

Adsorption to serum proteins

Liposomes were diluted in fetal bovine serum (FBS, Biological Industries, Israel) at a ratio of 1:40 and incubated at 37°C for 24 and 48 hours. The dilution factor was chosen based on the maximal expected intravenous (IV) injection volume (0.5 mL) in rats, 0.3 kg body weight (BW) and blood volume of 20 mL. The affinity of serum proteins to positively and negatively charged liposomes was evaluated as a function of the liposomes size distribution pre- and post- incubation period, measured by means of a Zetasizer.

Drug loading and encapsulation yield

The concentration of encapsulated lysozyme was determined using a gradient reverse phase HPLC method. Drug containing liposomes were dissolved in 200 mM Octyl β-D-glucopyranoside (OGP, Sigma-Aldrich) and injected to an Xbridge BEH300 C18 column (100 × 4.6 mm, 3.5 μm, Waters). The mobile phase consisted of A, 0.1% TFA in water and B, 0.1% TFA in acetonitrile. Chromatographic separation was performed using a gradient of 95/5 to 5/95, at a flow rate of 1 mL/min, detected at 280 nm (PDA 2998 detector, Waters). Concentration of non-encapsulated lysozyme was determined using the filtrate from liposomes centrifuged with a 100 kDa centrifugal filter unit (Vivaspin, GE Healthcare). The concentration inside the vesicles was determined by subtracting the external concentration from total lysozyme concentration in the suspension.

Lysozyme bioactivity

The bioactivity of lysozyme was assessed using lyophilized Micrococcus lysodeikticus cells (Sigma-Aldrich). A Micrococcus lysodeikticus cell suspension was prepared using 66 mM potassium phosphate buffer (Merck), pH 6.2 where after 2.5 mL was pipetted into polystyrene cuvettes (Sarstedt). A 100 µl of a liposome suspension treated with or without OGP (test), and potassium phosphate buffer (blank) were added into the cuvette, mixed by inversion, and the decrease in absorption was recorded by means of a spectrophotometer (UV/VIS-Ultraspec 2100 pro) at λ=450 for 7 minutes. The slope of ΔA450 per minute was evaluated and lysozyme concentration was calculated using the following equation:

Where df is the dilution factor, 0.001 is the ΔA450 per unit of lysozyme, and 0.1 is the volume (mL) of liposomes or buffer added to the cell suspension.

In vitro evaluation

Quantitative uptake evaluation in vitro (FACS)

RAW264.7 cells (murine monocyte/ macrophage) were seeded in 12-well plates, 1 х 105 cells per well, containing Dulbecco’s modified eagle medium (DMEM, BI) enriched with 10% FBS, 2 mM L-glutamine and 100 Units/mL penicillin and 100 mg/mL streptomycin. The next day, cells were treated with 0.05, 0.25, 0.5, and 1.2 mg/mL Lip(+)LYS diluted in PBS, for 1, 4, and 24 hours. After treatment the cells were washed, trypsinized, and collected by centrifugation. Cells were thereafter re-suspended with PBS and analyzed by means of iCyt eclipse flow cytometer (Sony Biotechnology, Inc.). A total of 50,000 cells were counted for each measurement in a rate of 20 μl/min. Untreated cells served as a control group, and fluorescence of the gated cells was measured using the FCS Express software (De Novo Software).

Qualitative uptake evaluation (confocal microscopy)

RAW264.7 cells were seeded on coverslips and treated as described above. After treatment the cells were washed and fixed for 10 minutes using a 4% paraformaldehyde solution (J.T. Baker chemicals). Cell’s nucleus was thereafter stained with 10 μg/mL Hoechst solution (Sigma-Aldrich) for 10 minutes. Each coverslip was mounted on a microscope slide with mounting medium (Sigma-Aldrich), and the slides were examined by means of an Olympus FV10i confocal laser scanning microscope 1 x 60 (Olympus America, Inc.).

Cell viability assay (MTT assay)

The RAW264.7 cells were seeded in 24-well plates (40,000 cells/well) containing complete growth medium. The cells were treated on the following day with 0.5 and 1.2 mg/mL Lip(+)LYS, or lysozyme solution (20 and 40 µg/mL). Each experiment was performed in duplicate and 10% v/v dimethyl sulfoxide (DMSO, Sigma-Aldrich) served as a positive control. After 24 hours or 48 hours incubation, 100 μl of 5 mg/mL thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich) was added to each well, containing 1 mL of growth medium, and incubated for 60 minutes. The unreacted dye was thereafter removed, and the purple formazan product was dissolved in 100 µl/well DMSO for 30 minutes at 37°C. Cell viability was determined by means of a plate reader (Cytation 3, BioTrek) at λ=540 nm and the number of viable cells were normalized to untreated cells.

In vivo evaluation

Animal care and procedures conformed to the standards for care and use of laboratory animals of the Hebrew University of Jerusalem, Israel, and the National Institutes of Health (NIH, USA). Animals were fed with standard laboratory chow and tap water ad libitum. A total of 34 naïve male Sabra rats (BW 250–320 g BW; Harlan, Jerusalem, Israel) were used in this study. In all experiments, the animals were randomly divided into subgroups. In the treatment groups, rats were injected IV (jugular vein) with Lip(+)LYS, Lip(-)HP, or IP with the free fluorescent marker in solution (DPPE-Rhod dissolved in corn oil or HP in PBS). Untreated rats served as control.

Toxicity

Lip(+)LYS (4 mg/kg lysozyme, 1.2 mg/kg DPPE-Rhod) and DPPE-Rhod (1.2 mg/kg) were injected IV and IP, respectively, (n=5 each group). Untreated rats served as control. Heparinized blood was drawn 24 h after treatment by cardiac puncture under general anesthesia. Blood specimens were centrifuged (4,000 rpm, 10 min, 4°C), and enzyme levels of aspartate transaminase (AST) and alanine transaminase (ALT), enzymes typically used as biomarkers for liver toxicity assessment, were analyzed according to the routine protocol of the Department of Clinical Biochemistry, Hadassah Hospital.

Biodistribution

Uptake evaluation

Animals (n=22) were randomly divided into 5 groups and injected IV with Lip(+)LYS (2 mg/kg lysozyme, 1.2 mg/kg DPPE-Rhod), Lip(-)HP (6 mg/kg HP), or IP with the free fluorescent marker in solution (1.2 mg/kg DPPE-Rhod or 6 mg/kg HP). Intact rats served as control. Four and 24 hours after treatment, the rats were anesthetized by isoflurane inhalation and subjected to intracardiac perfusion with PBS via the left ventricle. Following perfusion, liver, spleen, kidneys, and the brain were harvested and washed with PBS. Organs were scanned by means of a typhoon scanner at λex 560 nm; λem 580 nm (Lip(+)LYS) or λex 488 nm; λem 519 nm (Lip(-)HP) followed by ImageJ analysis. The relative mean fluorescence intensity of the tested organ was obtained by subtracting the measured mean fluorescence intensity from corresponding untreated organ. Brains were incubated in 30% sucrose solution (Sigma-Aldrich) overnight, embedded in optimal cutting temperature compound and thereafter snap frozen. The frozen tissue was sectioned (10–30 μm thick sections) with a cryostat (Sakura Finetek). Two or 3 sections were mounted on each slide and stained with DAPI (DAPI Fluoromount-G; SouthernBiotech) after fixation with 4% PFA and scanned by means of a Zeiss LSM 710 laser scanning confocal microscope (Carl Zeiss Microscopy) followed by ImageJ analysis.

Quantification of uptake by WBC

Heparinized blood was drawn 4 and 24 h after treatment by cardiac puncture under anesthesia. Red blood cells were lysed (Erythrolyse, 1:20 dilution, AbD, Serotec), and the pellet was washed twice with FACS buffer (1% BSA in PBS). Data were acquired on a BD FACScan (BD Bioscience) and analyzed with FCS Express software (De Novo Software). The population of white blood cells (WBC) was gated according to forward and side scattering, monocyte and granulocyte populations were gated according to their typical forward and side scattering, and the fluorescence of the gated cells was measured.

Macrophages co-localization with liposomes in the brain

Slides containing brain sections were fixed with 4% PFA in PBS for 15 minutes, followed by washing with 0.1% polyoxyethylene 20 sorbitanmonolaurate (J.T.Baker) in PBS (PBS-T). Sections were blocked by 3% Fraction V BSA in PBS for one hour and then incubated over night with a primary CD68 antibody (Bio-Rad), diluted 1:25 with CAS block (Life Technologies). One day after, sections were washed with PBS-T and incubated with secondary Cy2 conjugated antibody (Jackson ImmunoResearch) diluted 1:50 in CAS block. Each slide was mounted, stained with DAPI, and scanned by laser scanning confocal microscope (Olympus).

Quantification of lysozyme in the brain

Protein extraction from brain tissue

Animals were divided into 3 groups (n=7 each) and injected IV with Lip(+)LYS (3 mg/kg lysozyme) or lysozyme solution (3 mg/kg). Intact rats served as control. Four hours after treatment, the rats were anesthetized by isoflurane inhalation and subjected to intracardiac perfusion with PBS via the left ventricle. Following perfusion brains were harvested and washed with ice-cold PBS and then weighed and minced in a petri dish, followed by the addition of a protease inhibitor cocktail (Sigma-Aldrich), diluted 1:100 in PBS. Brains were then homogenized in a plastic tube on ice for 10 seconds, followed by sonication for 10 minutes using a tip-sonicator (Qsonica, LLC). Homogenates were then centrifuged for 10 minutes at 14,000× g, and the supernatant was removed and stored at −20°C pending analysis.

Quantification of lysozyme (Western blot)

Semi-quantitative analysis of lysozyme levels in the brains was conducted by means of Western blot. Total protein concentration was measured using the Bradford protein assay, and protein lysates were diluted to achieve similar amounts of loaded protein in each well. SDS-PAGE was performed under reducing conditions on a 12% polyacrylamide gel for an hour, followed by protein transfer to a nitrocellulose membrane overnight. The membrane was probed with primary lysozyme antibody (anti-lysozyme; hen egg white, rabbit antibody), diluted 1:1000 with 5% BSA in TBS-T for 2 h and then with goat anti-rabbit HRP-conjugated secondary antibody, diluted 1:10,000 in TBS-T for 30 minutes. Finally, the membrane was shaken in peroxide-enhanced chemiluminescent (ECL) mix for one minute and signal intensity was measured with a ChemiDoc Imager and analyzed using the Image Lab software (Bio-Rad).

Statistical analysis

Data are expressed as the mean ± standard error of mean (SEM). Statistical analysis of liposomes uptake in vitro, cytotoxicity in vitro, uptake by WBC and WB quantification was conducted using one or two-way analysis of variance (ANOVA). For statistical analysis of biodistribution and brain uptake in vivo, student’s t-test for independent means was used. Differences were termed statistically significant at p<0.05.

Results

Liposome physicochemical properties

Liposomal formulations obtained by the modified thin-film hydration technique are described in Table 1. The mean vesicle size was ~175 nm, with a positive (+19 mV) or negative (−38 mV) zeta potential, a low PDI (<0.1), and an encapsulation yield (EY%) of 19% and 4%, for lysozyme and HP, respectively.

Table 1. Composition and physicochemical properties of the tested liposomal formulations.

Name

Formulation

Composition

Physicochemical properties

Molar ratio

Lipids (mg/mL)

Drug*

(mg/mL)

Size

(nm)

PDI

ζ potential

(mV)

EY

(%)

Lip(+)LYS

DSPC:Chol:DOTAP

3:2:1

49 ± 4

Lysozyme 2 ± 0.4

179±7

0.09 ± 0.01

+19±0.5

19 ±4

Lip(-)HP

DSPC:Chol:DSPG

3:2:1

46 ± 2

HP

2 ± 0.2

169±0.8

0.08 ± 0.02

-38 ± 0.2

4±0.3

Mean ± SEM. *After removal of free drug. EY: Encapsulation yield

Figure 1. Representative cryo-TEM micrograph of Lip(+)LYS, scale bar = 200 nm.

In order to evaluate the stability of Lip(+)LYS over time, the formulation was stored in PBS for one year at 4°C, and examined periodically for size, PDI, and zeta potential changes. After a period of one, six and 12 months the observed changes in liposomes size, PDI, and zeta potential were found insignificant (less than: 10 nm, 0.005 and 3 mV, respectively; Fig. 2A-C).

Fig. 2. The effect of storage time and incubation in serum on liposome stability. Lip (+)LYS stability over time at 4°C was verified by size (A), PDI (B) and zeta potential (C) (mean ± SEM). Adsorption of serum proteins to Lip (+)LYS was measured before and 24 hours after incubation in FBS, red and green lines, respectively, (D).

Adsorption to serum proteins could change the biodistribution and stability of the liposomes, therefore we examined size changes following incubation in serum (Fig. 2D and Fig S1). A significant change in size and PDI of Lip(+)LYS following 24 hours of incubation with serum proteins was noted. The liposomes exhibited a higher PDI (0.276) with an increase of 25% from their initial mean size (Fig. 2D). It should be noted that no leakage of lysozyme was observed.

Bioactivity of lysozyme

Lysozyme bioactivity was determined by calculating the rate of decreased optical density (of lysozyme) following reaction with the substrate, Micrococcus lysodeikticus normalized to lysozyme solution (Fig. S2B). Intact Lip(+)LYS demonstrated partial enzymatic bioactivity of 45%, whereas when broken, following addition of OGP (clear solution), the enzymatic bioactivity was found to be 90%. Empty liposomes (empty-Lip(+)) served as a negative control as they did not show any bioactivity (Fig. 3).

Fig. 3. Bioactivity of lysozyme encapsulated in liposomes. Data normalized to lysozyme solution (200 Units/mL). Empty liposomes served as negative control (mean ± SEM).