Liposome and protein based stealth nanoparticles

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Faraday Discussions Vol 161

Lipids & Membrane Biophysics

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[journal], [year], [vol], 00–00 | 1

This journal is © The Royal Society of Chemistry [year]

Liposome and protein based stealth

nanoparticles

Eugénia Nogueira,ab‡ Ana Loureiro,

ab‡ Patrícia Nogueira,

c Jaime

Freitas,c Catarina R. Almeida,

d Johan Härmark,

e Hans Hebert,

e

Alexandra Moreira,c Alexandre M. Carmo,

cf Ana Preto,

b Andreia C. 5

Gomes,b Artur Cavaco-Paulo

a*

DOI: 10.1039/b000000x

Liposomes and protein based nanoparticles were tuned with different

polymers and glycolipids to improve stealth and thus decrease their 10

clearance by macrophages. Liposomes were coated with polyethylene

glycol (PEG) and brain-tissue-derived monosialoganglioside (GM1).

Bovine serum albumin (BSA) nanoparticles were produced incorporating a

PEGylated surfactant (PEG-surfactant). All obtained nanoparticles were

monodisperse, with sizes ranging from 80 to 120 nm, with a zeta-potential 15

close to zero. The presented stealthing strategies lead to a decreased of

internalization levels by macrophages. These surface modified

nanoparticles could be used for production of new drug delivery

nanosystems for systemic administration (e.g. intravenous application).

20

1 Introduction

Nanotherapeutic systems have been extensively investigated to solve several

limitations of conventional drug delivery, such as high toxicity, high dosage, in vivo

degradation and short circulating half-lives1-3. When nanoparticles enter the cell via

specific receptor-mediated endocytosis, they bypass the recognition of P-25

glycoprotein, one of the main drug resistance mechanisms4. Applications of

nanoparticles are limited by their rapid recognition by macrophages of the

reticuloendothelial system (RES)5. The main sites of nanoparticle clearance are liver

a IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering,

University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal. E-mail: [email protected] b CBMA (Centre of Molecular and Environmental Biology), Department of Biology,

University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal c IBMC – Instituto de Biologia Molecular e Celular, Rua do Campo Alegre 823, 4150-180

Porto, Portugal d INEB - Instituto de Engenharia Biomédica, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal e Department of Biosciences and Nutrition, The Royal Institute of Technology, School of

Technology and Healt, Karolinska Institutet, Stockholm, Sweden f ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua Jorge

Viterbo Ferreira, 228, 4050-313 Porto, Portugal

‡ These authors contributed equally to this work.

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View Article OnlineDOI: 10.1039/C3FD00057E



2 | [journal], [year], [vol], 00–00


PEG

GM1

Fig. 1 Chemical structures of polyethyleneglycol (PEG) and glycolipid GM1 (a brain-tissue-derived

monosialoganglioside). 5

and spleen, where macrophages are in direct contact with the bloodstream6.

Nanoparticles are cleared from the bloodstream within minutes upon intravenous

injection, depending on their size and surface characteristics. The rapid uptake of

nanoparticles is problematic when the long-term circulation of nanoparticle-loaded 10

drug systems is necessary. Numerous interesting approaches for design and

engineering of long circulating vehicles have been described. Among them, surface

stabilization of nanoparticles and liposomes with a range of nonionic surfactants or

polymeric macromolecules has proved to be one of the most successful approaches

for keeping the particles in the blood for long periods of time7, 8. Surface enrichment 15

of nanocarriers with nonionic surfactants can be achieved by physical adsorption,

incorporation during the production of the carriers, or by covalent attachment to any

reactive surface groups. The presence of such surfactants on the particle surface

strongly reduces interparticle attractive Van der Waals forces while increasing the

repulsive barrier between two approaching particles. This steric mechanism of 20

stabilization involves an elastic as well as an osmotic contribution9.

Due to its unique physical properties, PEG is commonly used to improve the

stability and biological performance of colloidal drug carriers (Fig. 1). The grafting

of PEG to the surface of a colloidal carrier has been clearly shown to extend the

circulation lifetime of the vehicle5. The ability of PEG to fulfil this role has been 25

attributed mostly to its physical properties such as unlimited water solubility, large

excluded volume and high degree of conformational entropy10. The unique physical

properties of the polymer and the extended circulation lifetime have been largely

attributed to the reduction or prevention of protein adsorption11. However, there is

little evidence that the presence of PEG at the surface of a vehicle actually reduces 30

total serum protein binding12. Others have shown that the steric barrier that PEG

provides prevents aggregation of colloidal carriers and thus enhances their stability

in vivo13. More recently, some groups have suggested a ‘‘dysopsonization’’

phenomenon where PEG actually promotes binding of certain proteins that then act

to mask the vehicle14. 35

It has been demonstrated that surface-modified liposomes with gangliosides have

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a prolonged circulation time in the blood stream compared to non-modified

liposomes15. Several glycolipids have been tested in studies of RES uptake of

liposomes after intravenous injection: the glycolipid GM1 (a brain-tissue-derived

monosialoganglioside; Fig. 1) significantly decreased RES uptake when incorporated

on the liposome surface and the formulation remained in blood circulation for 5

several hours16. The stability of the bilayer reduces vesicle susceptibility to lysis by

mouse plasma components (e.g. lipoproteins) and to perturbation or penetration by

plasma or cell surface proteins. Furthermore, due to the inhibition of complement

activation, the assembly of the membrane attack complex at the vesicle bilayer is

prevented. It has been postulated that the negative charge in GM1 is “shielded” by a 10

bulky, neutral, hydrophilic sugar moiety that contributes to macrophage avoidance

by decreasing or preventing protein adsorption or opsonization 15, 16. Subsequently,

it was suggested that a balance between the opsonic molecule and these suppressive

proteins (dysopsonins) could regulate the quantity and the rate of clearance of

liposomes from the blood by hepatic macrophages17. Dysopsonins could modulate 15

the rate of liposome uptake by reducing the amount of liposome bound opsonin and,

hence, protect the phagocytic cells from being destroyed by excessive binding and

ingestion of liposomes, particularly those vesicles which are more resistant to

lysosomal esterases9,17.

Herein we report the surface modification of nanoparticles with different 20

polymers and glycolipids to improve stealth. Liposomes incorporating PEG and

GM1, and BSA nanoparticles incorporating a PEGylated surfactant were extensively

characterized in order to study morphology alterations promoted by stealth agents.

Additionally, the stealth degree of nanoparticles incorporating these molecules was

evaluated by analysing the internalization of these nanoparticles by human 25

macrophages.

2 Materials and methods

2.1 Materials

EPC (Phosphatidyl choline from egg lecithin) and DSPE-MPEG ([N-(carbonyl-30

methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-

phosphoethanolamine] were obtained from Lipoid GmbH (Germany). Cholesterol

(CH), BSA, Fluorescein isothiocyanate (FITC), BSA-FITC conjugate and PEGylated

surfactant were obtained from Sigma (USA). GM1 was obtained from Avanti Polar

Lipids (USA). 35

2.2 Liposome preparation

Liposomes composed of EPC/CH/DSPE-MPEG or GM1 were prepared by thin film

hydration method. Briefly, a known amount of EPC, CH and DSPE-MPEG or GM1

was dissolved in chloroform in a 50 mL round bottom flask. To macrophages 40

internalization studies, FITC was incorporated in the lipidic film at 1:10 ratio (v/v)

relative to final volume. The organic solvent was evaporated by rotary evaporator

followed by additional evaporation under reduced pressure by high vacuum system

to remove traces of chloroform. The resulting dried lipid film was dispersed in

phosphate buffered saline (PBS). The mixture was vortex mixed above the phase-45


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transition temperature (room temperature) to yield multilamellar vesicles, which

were then extruded (extruder supplied by Lipex Biomembranes Inc., Vancouver,

Canada) through polycarbonate filters of 200 nm pore size (Nucleopore), and then

several times through polycarbonate filters of 100 nm pore (Nucleopore) to form

large unilamellar vesicles. 5

2.3 BSA nanoparticle preparation

The preparation of BSA nanoparticles was achieved by emulsion using a high-

pressure homogenizer (APV-2000, Denmark). BSA previously dissolved in PBS at

concentration of 10 mg/mL was emulsified with the organic solvent (vegetable oil) 10

by subjecting the mixture to different numbers of homogenization cycles at high

pressure. PEGylated surfactant was dissolved in aqueous phase of initial formulation

for nanoparticles production. To macrophages internalization studies, BSA-FITC

conjugate was incorporated in the initial formulation at 1:20 ratio (m/m) relative to

BSA protein. 15

2.4 Determination of zeta-potential and size

The zeta-potential (ζ-potential) values and size distribution of liposomes and BSA

nanoparticles were determined at pH 7.4 (PBS) and at 25.0°C, using a Malvern 20

zetasizer NS (Malvern Instruments) by electrophoretic laser Doppler anemometry

and photon correlation spectroscopy (PCS), respectively. Lipid concentration was

kept constant at 400 µM and protein concentration was kept constant at 10 mg/mL.

The values for viscosity and refractive index were taken as 0.890 cP and 1.330,

respectively. 25

2.5 Microscopy imaging analysis

For scanning transmission electron microscopy (STEM) analysis, nanoparticles

suspensions were dropped in Copper grids with carbon film 400 meshes, 3 mm

diameter. The shape and morphology of nanoparticles were observed using a NOVA 30

Nano SEM 200 FEI. For transmission electron microscopy (TEM) analysis, the

nanoparticles samples were applied to glow discharged carbon-coated copper grids

followed by negative staining with a solution of 1% (w/v) uranyl acetate. Imaging

was performed using a JEOL JEM2100F transmission electron microscope operating

at an acceleration voltage of 200 kV. Images were recorded with a Tietz (Tietz 35

Video and Image Processing Systems GmbH, Gauting, Germany) 4k*4k CCD

camera at a magnification of 100,000 x.

2.6 Macrophage internalization

Human peripheral blood mononuclear cells were isolated from buffy coats of healthy 40

volunteers from the Immuno-haemotherapy Department of Hospital de São João

(Porto, Portugal) by centrifugation over Lymphoprep (Axis-Shield). The ethics

committee granted approval for the study and all volunteers gave their informed

consent. Monocytes were further purified by magnetic cell sorting using CD14-

recognizing microbeads (Miltenyi Biotec). Monocyte-derived macrophages were 45


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generated in RPMI 1640 (supplemented with 10% FCS, 100 U/mL penicillin and

100 µg/mL streptomycin) in the presence of 50 ng/mL M-CSF (PeproTech) for 7

days followed by a 24h activation with 20 ng/mL IL-4 (PeproTech). Binding and

internalization of liposomes by macrophages was assessed with an ImageStream

multi-spectral imaging flow cytometer (Amnis). Briefly, macrophages were 5

incubated for 1 h with 100 µg/mL liposomes in complete RPMI at 37ºC. After

incubation, cells were washed twice with PBS (pH 7.4). Images of cells were

acquired on a 6-Channel ImageStreamX Imaging Flow Cytometer (Amnis, EMD

Millipore) at the Bioimaging Center for Biomaterials and Regenerative Therapies

(b.IMAGE, INEB, Porto, Portugal) equipped with 488 nm and 785 nm excitation 10

lasers, with 40 x magnification and controlled by INSPIRE software. Approximately

20.000 events with a brightfield (BF) area lower limit of 20 µm2 were collected per

sample. Analysis was performed with IDEAS 5.0 (Amnis, EMD Millipore). The

percentage of cells containing internalized liposomes was determined with the

Internalization Wizard. Briefly, after excluding images out of focus and selecting 15

single cells, a gate was drawn on the liposome positive cells and a mask

corresponding to the interior of the cell was created on the basis of the BF image.

Internalization was estimated by comparing the liposomal FITC fluorescence

intensity inside the cell with the fluorescence intensity in the entire cell, and the

percentage of cells with internalized liposomes was quantified. 20

In the case of BSA nanoparticles, the binding and internalization by macrophages

was assessed by flow cytometer analysis, using FACScalibur from BD Biosciences.

Briefly, macrophages were incubated for 1 h with 100 µg/mL of BSA nanoparticles,

labelled with FITC, in complete RPMI at 37ºC (allowing binding and

internalization). As negative control, macrophages were incubated with complete 25

RPMI. After incubation, cells were washed twice with PBS (pH 7.4) and then

resuspended in PBS for FACS analysis. The internalization was determined as the

geometrical mean fluorescence intensity of BSA nanoparticles corrected for the

background staining of cells incubated with no nanoparticles (negative control).

Mean fluorescence intensity equal to or below 1.0 means no internalization. 30

2.7 Statistical analysis

Statistical analyses were performed with GraphPad Prism software (version 5.0).

Differences were tested for statistical significance at p≤0.05 by one-way ANOVA.

35

3 Results and discussion

3.1 Stealth liposomes

Liposomes have gained extensive attention as carriers for a wide range of drugs due

to being both nontoxic and biodegradable as they are composed of substances

naturally occurring in biological membranes18. Biologically active materials 40

encapsulated within liposomes are protected to a varying extent from immediate

dilution or degradation, which makes them good drug delivery systems for the

transport of bioactive compounds to pathologically affected organs19, 20. Despite

certain technical advantages, at least one obstacle currently limits the widespread


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implementation of liposomes as drug carriers in vivo: unmodified liposomes do not Table 1 Physicochemical characterization of liposomes and BSA nanoparticles in the presence of

stealth agents (DSPE-MPEG, GM1 and PEGylated surfactant).

Nanoparticles

type Stealth agent

Z-average

(d.nm)

Polydispersity

index (PdI) ζ-potential

(mV)

Stability

along time*

Liposomes

- 141.12 (±1.08) 0.147 (±0.014) 0.614 (±1.16) < 1 week

5% DSPE-

MPEG 118.50 (±2.09) 0.043 (±0.010) -0.593 (±0.713) > 16 weeks

10% DSPE-

MPEG 113.30 (±2.55) 0.027 (±0.012) -1.24 (±1.34) > 16 weeks

10% GM1 123.00 (±2.32) 0.041 (±0.016) -6.67 (±1.62) > 16 weeks

BSA

nanoparticles

- 223.23 (±2.08) 0.183 (±0.017) -12.28 (±1.17) > 20 weeks

2.5mg/mL PEG-

surfactant 134.68 (±4.77) 0.111 (±0.019) -0.519 (±0.255) > 20 weeks

5 mg/mL PEG-

surfactant 77.24 (±10.01) 0.184 (±0.004) -1.59 (±0.18) > 20 weeks

* Particle size (nm), polydispersity index, ζ-potential (mV) and stability of liposomes along time, liposomes with or without 5%, 10% of DSPE-MPEG and 10% GM1, BSA nanoparticles

with or without 2.5mg/mL and 5mg/mL of PEGylated surfactant (PEG-surfactant). Values represent the mean ± SD of 3 experiments. 5

survive long in circulation, but instead are removed by macrophages of the RES

within few minutes of administration6. Recently, the inclusion of PEG in liposomal

bilayers has extended blood circulation time of conventional liposomes and

considered as a major breakthrough in liposome-mediated drug delivery5. PEG can 10

be incorporated on the liposome surface by several ways, the most used method

being the binding of polymer at membrane surface by crosslinking with a lipid (for

example, PEG - distearoylphosphatidylethanolamine [DSPE])9.

The conjugated phospholipid DSPE-MPEG was incorporated in a molar ratio of

5% into the lipidic film of these new formulations. The particle size and 15

polydispersity index (PdI) of liposomes were determined by dynamic light scattering

(DLS). The measurement gives the average hydrodynamic diameter of the particles,

the peak values in the hydrodynamic diameter distribution and the PdI that describes

the width of the particle size distribution. The results indicated that the size

distribution pattern of EPC/CH liposomes was also influenced by DSPE-MPEG, as 20

in its presence, the average size of liposomes tended to be smaller (Table 1). The

electrostatic repulsion among liposomes with DSPE-MPEG might reduce the

feasible aggregation of liposomes after extrusion, resulting in smaller average size21.

Liposomes without DSPE-MPEG had a mean particle size of 141.12 ± 1.08 nm and a

precipitate formed within a few days of preparation, which prevents its further use. 25

In contrast, liposomes with 5% DSPE-MPEG had a mean particle size of 118.50 ±

2.09 nm and remained in a clear solution during the 16 weeks of analysis. PdI is a

parameter that defines the particle size distribution of nanoparticles22. PdI values are

given in brackets and range from 0 to 1, with a higher value indicating a less


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homogeneous size distribution. Liposomes with DSPE-MPEG exhibited a narrow

Fig. 2 Inclusion of DSPE-MPEG or PEGylated surfactant yields spherical vesicles with narrower

size distribution. Representative (A) STEM and (B) TEM images of liposomes containing 5%

DSPE-MPEG; (C) STEM and (D) TEM images of liposomes containing 10% DSPE-MPEG; (E) 5

STEM and (F) TEM images of BSA nanoparticles with 2.5 mg/mL of PEGylated surfactant; (G)

STEM and (H) TEM images of BSA nanoparticles with 5 mg/mL of PEGylated surfactant.

size distribution (PdI <0.1), which indicated monodispersed solutions, in which all

of the liposomes had similar physicochemical characteristics (Table 1). The higher 10

PdI value showed by liposomes without DSPE-MPEG revealed a broad particle size

distribution (Table 1).

Apart from size determination, ζ-potential was also evaluated to obtain an

indication of liposome surface potential. The surface potential of the particles cannot

be measured directly, thus the ζ-potential (electrical potential at the surface of 15

hydrodynamic shear around the colloidal particle) is usually determined as a

characteristic parameter for the particle charge23. Both liposomal formulations

possess a ζ-potential close to zero (Table 1), suggesting that inclusion of DSPE-

MPEG does not alter the formation of neutral liposomes.

Using STEM (Fig. 2A) and TEM (Fig. 2B) imaging we showed that liposomal 20

formulations are clearly composed of spherical homogeneous particles with smooth

surfaces. This morphology would offer the highest potential for controlled release

and protection of incorporated drugs, as they provide minimum contact with the

aqueous environment, as well as favour the longest diffusion pathway. Comparing

particles with any other shape, spherical particles also require the smallest amount of 25

surface-active agent for stabilization, because of their small specific surface area23.

These results are in agreement with DLS results showing that a very similar particle

size distribution.

It is well established that modification of the liposome surface with PEG greatly

reduces the opsonization of liposomes and their subsequent clearance by the RES 30

(mononuclear- phagocyte)5, 22. Therefore, the stealth degree of liposomes containing

PEG was evaluated by macrophage internalization. Liposomes without stealth agent

were not included due to their physical instability (see data of Table 1), as they

precipitate within few days of preparation. Thus, this formulation was not used for

biological assays, where serum proteins from complete medium promote even more 35

A E C

B D F

G

H


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Fig. 3 Increased stealth degree of liposomes incorporating DSPE-MPEG and GM1. Bars represent

the percentage of macrophages with internalized liposomes (100 µg/mL) after 1 h of incubation at

37 ºC, as determined by ImageStream analysis. Values are the mean + SD of 2 experiments. *P

<0.05%, **P<0.01. 5

instability of this liposomal formulation. Despite the presence of 5% DSPE-MPEG,

liposomes were recognized and internalized by macrophages even after a short

incubation period (1 h; Fig. 3). Therefore, to improve their stealth degree, the

concentration of DSPE-MPEG on liposomes was increased from 5% to 10%. The 10

physicochemical characterization of these liposomes showed that no significant

differences were observed comparing these two DSPE-MPEG concentrations (Table

1; Fig. 2). However, the degree of macrophage uptake depends on the concentration

of DSPE-MPEG in liposomes: a concentration of 10% DSPE-MPEG decreased

uptake by macrophages to 13%, unlike the 90% observed for liposomes with 5% 15

DSPE-MPEG (Fig. 3). We showed that increasing DSPE-MPEG concentration

clearly improved the stealth degree of liposomes, as the internalization of liposomes

by macrophages is greatly reduced.

This is in good agreement with the current scaling models for polymers at

interfaces, which predict a mushroom-brush transition in PEG conformation at 5% 20

of PEG-lipid, when PEG coils start to repel each other and extend out from the

surface on which they are grafted12. If the polymer density is low it is said to be in

the mushroom regime, when the graft density is high the polymers are said to be in

the brush regime (Fig. 4)24. Nanoparticle populations bearing a predominant surface

of PEG molecules as high density mushroom- brush intermediate and/or brush 25

configuration are most resistant to phagocytosis and poorly activated the human

complement system. Conversely, those populations with a predominant surface PEG

in a mushroom regime are potent activators of the complement system and are prone

to phagocytosis9. Therefore, surface heterogeneity explains why liposomes with 5%

PEG are rapidly internalized by macrophages, while the presence of 10% PEG 30

reduces significantly their internalization.

Apart from addition of PEG, the glycolipid GM1 has been described as significantly

decreasing uptake by RES macrophages25. In order to evaluate the stealth degree of

these liposomes, GM1 was incorporated in a molar ratio of 10% in the lipidic film of

these new formulations. Liposomes incorporating 10% GM1 showed a reduced 35


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Mushroom regime Brush regime

Fig. 4 A schematic diagram of a PEG-grafted bilayer at low grafting concentration (mushroom

regime) and a PEG-grafted bilayer at high grafting concentration (brush regime) [Adaptated from

Tirosh O. et al., 199812]. 5

macrophage uptake, just 35% of macrophages internalized liposomes after 1 h of

contact (Fig. 3). However, the stealth degree of liposomes incorporating GM1 is

lower that of liposomes incorporating the same concentration of PEG. This disparity

can be associated with the different thickness of steric barrier of PEG and GM1. The 10

basic concept is that a hydrophilic polymer or a glycolipid, such as PEG or GM1,

possessing a flexible chain, occupy the space immediately adjacent to the liposome

surface (“periliposomal layer”), forming a steric barrier that tends to exclude other

macromolecules from this space9. Consequently, access and binding of blood plasma

opsonins to the liposome surface are hindered, and thus interactions of RES 15

macrophages with such liposomes are inhibited. In this way, the steric barrier

provided by the amphipathic PEG was estimated to be 6 nm thick, compared to 2.5

nm for GM1, so GM1 is a weak steric barrier compared with PEG26, what could be

of increased value for intravenous application.

20

3.2 Stealth BSA nanoparticles

Proteins are a class of natural molecules that have potential use in both biological

and material applications. Albumin nanoparticles are easy to prepare in defined

sizes, reproducible and carry reactive groups (thiol, amino, and carboxylic groups)

on their surfaces that can be used for ligand binding and/or other surface 25

modifications. Conjugation of surface modifying ligands to the surface can easily be

performed by covalent linkage. In the albumin–ligand combinations, the protein acts

as a biodegradable carrier for drug delivery whereas the ligand is used for modifying

the pharmacokinetic parameters (e.g. surfactants), enhancing the nanosystem

stability (e.g. poly-L-lysine), prolonging its circulation half-life (e.g. PEG), slowing 30

the drug release (e.g. cationic polymers) or as a targeting agent (e.g. folate,

thermosensitive polymers, transferrin, apolipoproteins and monoclonal antibodies)27-

29. Protein nanoparticles have shown efficacy as biodegradable carriers, which can

incorporate a variety of drugs in a relatively non-specific fashion. They have been

extensively studied as suitable for drug delivery because they present advantages 35

over the other drug delivery systems such as biodegradability, greater stability

during storage, stability in vivo, non-toxicity, non-antigen and ease to scale up

manufacturing. Different methods for protein nanoparticle preparation are available,

with main techniques being desolvation, emulsification, thermal gelation and,

recently, nano spray drying, nab-technology and self-assembly techniques have been 40


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10 | [journal], [year], [vol], 00–00


described29.

Here we report the production of protein nanoparticles by emulsification using a

high pressure homogenizer. After several steps of optimization, small and stable

nanoparticles were obtained. The BSA concentration as well as the ratio between

aqueous phase and organic phase (vegetable oil) are important parameters for 5

nanoparticles formation process, as previously described for microspheres formation

using ultrasound30. We observed that BSA nanoparticles prepared using lower

percentage of vegetable oil (0.5% (v/v)) and higher BSA concentration (10 mg/mL)

results in smaller and more stable nanoparticles (data not shown). Based on this, we

introduced the PEGylated surfactant in aqueous phase to produce PEGylated BSA 10

nanoparticles. As mentioned above, the conjugation of surface modifying ligands to

nanoparticles can easily be performed by covalent linkage. Chemical coupling of

PEG to protein nanoparticles (PEGylation) is the most frequent way to improve in

vivo longevity of drug carriers, extending their circulation half-life by more than 50-

fold31. PEGylated BSA nanoparticles were prepared without chemical surface 15

modification. PEGylated surfactant was introduced in the aqueous phase of the

initial formulation for nanoparticle production and small PEGylated nanoparticles

were obtained by subjecting of the mixture (aqueous phase/vegetable oil) to high

pressure homogenization. The nanoparticle size was determined by DLS and results

demonstrated that the size distribution of BSA nanoparticles was also influenced by 20

presence of PEG-surfactant. The average size of nanoparticles tended to be smaller

in the presence of PEG-surfactant (Table 1). BSA nanoparticles prepared without

PEGylated surfactant had an average size of 223.23 ± 2.08 nm and, in contrast,

nanoparticles containing 5 mg/mL of PEG-surfactant had an average size of 77.24 ±

10.01 nm. Smaller particles lead to a reduced adsorption of proteins at the surface 25

and in turn to a reduced uptake by phagocytes32. Smaller particles (<100 nm) with a

hydrophilic surface are essential in achieving the reduction of opsonization reactions

and subsequent clearance by macrophages33. Apart from size, features of the

nanoparticle surface also influence their uptake by phagocytes33, 34 by directly

affecting particle size, because they represent the major determinant for protein 30

adsorption. The obtained PEGylated BSA nanoparticles present characteristics that

can promote the reduction of proteins adsorption and opsonization.

The results on ζ-potential of BSA nanoparticles demonstrated that formulations

containing PEG-surfactant present ζ-potential value close to zero (Table 1). The

addition of PEGylated surfactant in the initial solution for nanoparticle formation 35

allowed the adjustment of surface properties, and the negative values of ζ-potential

(approximately -13mV) obtained for nanoparticles prepared without PEG-surfactant,

reach approximately neutral values when this surfactant is present in the

formulation.

Using STEM and TEM imaging, PEGylated BSA nanoparticles prepared using 40

high pressure homogenization were shown to present a spherical form and smooth

surfaces (Fig. 2C and 2D).

In order to determine the stealth degree of these PEGylated BSA nanoparticles,

the internalization of PEGylated BSA nanoparticles (containing 2.5 or 5 mg/mL

PEG-surfactant) by activated macrophages was tested. The PEGylated surfactant has 45

the dual function of being a stealth agent and yielding small BSA nanoparticles.

Fully formulated nanoparticules were developed for intravenous application with

sizes smaller than 150 nm. It is known that bigger particles promote themselves

opsonization with serum proteins present in the cell culture medium and subsequent


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[journal], [year], [vol], 00–00 | 11


Fig. 5 Increased stealth degree of BSA nanoparticles containing PEGylated surfactant. Mean

fluorescence intensity of macrophages (MØ) (negative control), macrophages incubated with 100

µg/mL of NP containing 2.5 or 5 mg/mL of PEG-surfactant for 1 h. Values are the mean + SD of 2

experiments. *P <0.05%. 5

clearance by macrophages32, 33. Larger BSA particles without the stealth agent were

therefore not subjected to macrophage internalization.

Macrophages incubated with nanoparticles containing 2.5 mg/mL of PEG-

surfactant showed a higher mean fluorescence intensity value than those incubated 10

with 5 mg/mL. These results indicate that nanoparticles containing 2.5 mg/mL PEG-

surfactant are more readily recognized and internalized by macrophages after 1 h of

incubation (Fig. 5). As in the case of liposomes containing 10% PEG, PEGylated

BSA nanoparticles population containing 5 mg/mL of PEG-surfactant showed more

resistance to phagocytosis. These results allowed us to infer that the surface of these 15

nanoparticles population present a predominant number of PEG molecules and this

neutral and hydrophilic surface can generate stealth nanoparticles, which present a

significant reduction in internalization by macrophages. PEG molecules forms a

dynamic molecular “layer” over the nanoparticle surface, due to the chain flexibility

and electrical neutrality of the PEG backbone35-37. This neutral and hydrophilic 20

surface can generate stealth particles, decreasing the phagocytic uptake and thus

leading to a longer half-life in blood circulation, which is of great value for

intravenous application.

4 Conclusions 25

We report the relevance of surface modification of nanoparticles with different

polymers and glycolipids to improve stealth and decrease their clearance by

macrophages. Liposomes incorporating PEG and GM1, and BSA nanoparticles

incorporating PEG-surfactant were produced in order to study morphological

alterations promoted by these stealth agents. The results reveal that inclusion of 5% 30

PEG decreased liposome size and led to the formation of a monodispersed solution.

In the case of BSA nanoparticles, a reduction in particle size was also observed and


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12 | [journal], [year], [vol], 00–00


their ζ-potential was close to zero in the presence of PEG-surfactant. The presence

of 5% of PEG was insufficient to reduce the internalization of liposomes by

macrophages, although the increase to 10% greatly improved the stealth degree of

liposomes. This observation is in accordance with the current scaling models for

polymers at interfaces, which predict that liposomes incorporating a higher 5

concentration of PEG (brush regime) were more resistant to phagocytosis and poorly

activated the human complement system. We observed that liposomes incorporating

the glycolipid GM1 stealth agent clearly reduced macrophage uptake, but not to the

same extent as when using a similar concentration of PEG. This difference can be

related with the higher thickness of steric barrier of liposomes with PEG that tends 10

to exclude plasma opsonins to the liposomal surface. Our data also showed that the

presence of PEGylated surfactant at concentration of 5 mg/mL improves the stealth

degree of BSA nanoparticles. This concentration of PEGylated surfactant appears to

promote PEG chains sufficiently extended into aqueous solution to shield the surface

of nanoparticles and improve resistance to macrophage uptake. 15

Summing up, we produced nanoparticles liposome and BSA derived using stealth

agents surface modifiers in order to decrease size and internalization by activated

RES macrophages, which could be used for the fabrication of drug delivery systems

with improved qualities for systemic administration like intravenous application.

20

Acknowledgements

Eugénia Nogueira (SFRH/BD/81269/2011) and Ana Loureiro

(SFRH/BD/81479/2011) hold a scholarship from Fundação para a Ciência e a

Tecnologia (FCT). We thank the Immuno-haemotherapy department of Hospital de

São João (Porto, Portugal) for providing buffy coats of healthy volunteers. This 25

work has received funding from the European Union Seventh Framework

Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827

NANOFOL. This work was also supported by FEDER through POFC – COMPETE

and by national funds from FCT through the project PEst-C/BIA/UI4050/2011.

30

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Liposome and protein based stealth nanoparticles

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