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Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 136– 144

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fa

valuation of new amphiphilic PEG derivatives for preparing stealthipid nanoparticles

osario Pignatelloa,∗, Antonio Leonardia, Rosalia Pellitterib, Claudia Carbonea,ilvia Caggiac, Adriana Carol Eleonora Grazianoc, Venera Cardilec

Section of Pharmaceutical Technology, Department of Drug Sciences, University of Catania, viale A. Doria, 6, I-95125, Catania, ItalyInstitute of Neurological Sciences, National Research Council, Section of Catania, via P. Gaifami 18, I-95126, Catania, ItalySection of Physiology, Department of Bio-medical Sciences, University of Catania, viale A. Doria, 6, I-95125, Catania, Italy

i g h l i g h t s

PEG–lipoamino acid conjugatesshowed to make long circulatingnanocarriers.In this work we tested two of theabove conjugates to produce stealthSLN.Comparison was made with SLN pre-pared with DSPE–PEG and PEG 40monostearate.SLN showed a good stability profile inserum and at room temperature.PEG–LAA derivatives hinderedthe endocytic uptake of SLN bymacrophages.

g r a p h i c a l a b s t r a c t

Stealth solid lipid nanoparticles (SLN) were produced using two novel amphiphilic conjugates ofmPEG2000 and mPEG5000 carboxylic acids with a C-18 lipoamino acid moiety as a lipid anchor. The SLNshowed stealth properties similar to or greater than SLN produced using commercial PEG lipid derivatives.

a r t i c l e i n f o

rticle history:eceived 26 February 2013eceived in revised form 14 May 2013ccepted 16 May 2013vailable online 24 May 2013

eywords:EGylation

a b s t r a c t

Two novel amphiphilic conjugates of mPEG2000 and mPEG5000 carboxylic acids with a lipoamino acidas a lipid anchor (mPEG-C–LAA18), recently described as surface modifiers for drug nanocarriers, wereused to decorate solid lipid nanoparticles (SLN). The SLN were produced using a suitably adapted sol-vent injection method (the Quasi-emulsion solvent diffusion) and, for the sake of comparison, were alsoprepared using a commercial phospholipid PEG derivative (DSPE–PEG) and a lipid PEG (PEG 40 monostea-rate), commonly used to make stealth nanocarriers. The SLN were characterized in terms of technologicalproperties and stability in serum. An in vitro assay using murine macrophage cultures confirmed the abil-

tealth nanocarriersLNESDipoamino acidsmphiphilicityell uptake

ity of the PEG–LAA conjugates to hinder or retard the internalization of the nanoparticles by the endocyticcells.

© 2013 Elsevier B.V. All rights reserved.

acrophages

∗ Corresponding author at: Dipartimento di Scienze del Farmaco, Città Universi-aria, viale A. Doria, 6, 95125 Catania, Italy. Tel.: +39 0957384005.

E-mail addresses: r.pignatello@unict.it, r.pignatello@gmail.com (R. Pignatello).

927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfa.2013.05.059

1. Introduction

Nanosized carriers are innovative pharmaceutical forms able

to incorporate biologically active substances and to increaseand optimize their biopharmaceutical profile and/or therapeu-tic efficacy. Such systems are often designed in such a way toallow a slow release of the drug for prolonged times and/or to

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chieve a selective targeting of the cargo to its specific sites ofction.

In recent years, nanosized lipid carriers (LNC) have been pro-osed as new colloidal systems to improve the bioavailability andllow a controlled release of drugs. Compared to other colloidalelivery systems, the LNC can be easily produced on a large scale,nd containing physiologically biocompatible lipids reduces theisk of acute and chronic toxicity; in addition, their lipid core allowso modulate the drug release profile and protect labile drugs fromhemical or enzymatic degradation in vivo.

As the other colloidal carriers, however, LNC must remainong enough in the general circulation to reach the targetites or to release for the expected times the carried actives.ne of the major strategy to obtain a long plasmatic circula-

ion is to modify the surface of these vectors by hydrophilicacromolecules, which will physically mask them to the com-

lement system, producing those known as ‘sterically stabilized’r ‘stealth’ systems [1,2]. Polyethylene glycol (PEG) is proba-ly the most used hydrophilic polymer to this aim, and several

ipid derivatives of PEGs have been incorporated in liposomes,anoparticles and LNC in general to overcome a rapid bloodlearance of such colloidal nanocarriers [3–5]. For instance, to pro-uce stealth liposomes, amphiphilic PEGs have been produced byerivatizing the polymer chains with lipophilic moieties, such as,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE–PEG) or,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE–PEG),o enhance their incorporation within the phospholipid bilayers6]. Cholesterol, phosphatidic acid, fatty acids and diglycerides,eramides, as well as synthetic lipids are further cases of lipophilicodifiers that have been linked to PEGs to facilitate their anchoring

o the surface of lipid-based nanocarriers [7–11].In recent researches, we have described some novel series of

mphiphilic derivatives of poly(ethylene glycol) (PEG), by cova-ently linked to either mono- and dicarboxy-, or mono-amino- andiaminoPEGs with �-lipoamino acids (LAA), thus obtaining differ-nt mono- or homo-disubstituted PEG conjugates [12–14].

LAA are synthetic compounds, which combine the structuralharacteristics of lipids (for the presence of the hydrophobic sidehain), with those of amino acids [15–18]. LAA have been exploiteds promoieties able to modify and/or modulate the amphiphilicityf drug molecules, thus affecting their interaction with biomem-ranes and penetration through biological barriers [19–22].

Because of the peculiar lengths of the side alkyl chain ofhe used LAA (16–20 carbon atoms), that well fit the struc-ure of the lipid moiety in phospholipids and fatty acids, theseEG–LAA conjugates can be suitably proposed as surface modi-ers for liposomes, lipid nanocapsules and polymeric nanoparticles23–25].

In this further note, we describe the use of two of these PEG–LAAonjugates for the surface modification of solid lipid nanoparticlesSLN), with the aim of obtaining carriers able to escape opsoniza-ion and rapid cell uptake. We thus produced pegylated SLN bysing two conjugates of �-methoxy–PEG carboxylic acid (mPEGC),

aving a molecular weight of 2000 or 5000 Da, with a LAA bear-

ng a C-15 side chain (LAA18) (Fig. 1). For the sake of comparison,tealth SLN were also produced using the commercially available

ig. 1. General structure of mPEG2000C–LAA18 (m = 44) and mPEG5000C–LAA18m = 96).

ochem. Eng. Aspects 434 (2013) 136– 144 137

DSPE–PEG and PEG 40 monostearate, two of the more commonlyused surface modifiers for liposomes and nanoparticles.

The produced SLN systems were characterized for their physico-chemical properties and stability in serum. Incubation withmacrophage cultures of fluorescent-labeled SLN was used to evalu-ate the ability of the different pegylation means to hinder or retardthe macrophage uptake of these carriers.

2. Materials and methods

2.1. Materials

Lipoid S 100 was gifted by Lipoid GmbH (Ludwigshafen,Germany); according to the Lipoid catalog, it contains 100%soybean 1-palmitoyl-2-oleoyl-phosphatidylcholine. Stearic acidwas purchased from Carlo Erba (Milan, Italy); PEG 40 mono-stearate was purchased from Sigma–Aldrich Chimica Srl (Milan,Italy); DSPE–mPEG was purchased from Genzyme (Postfach,Switzerland). Rhodamine-DHPE (Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylam-monium salt) [26] was an InVitrogen product (Life Tech-nologies Italia, Monza, Italy). The used PEG–LAA conjugates(mPEG2000C–LAA18 and mPEG5000C–LAA18) were synthetized aspreviously published [13,14], by an EDAC-assisted conjugation ofthe LAA18 methyl ester [27] with the corresponding mPEG car-boxylic acid (Iris Biotech, Germany, for PEG2000 and Sigma-Aldrichfor PEG5000, respectively).

2.2. Preparation of naked SLN

‘Non stealth’ SLN were prepared by an appropriate modificationof a solvent injection technique known as Quasi-emulsion solventdiffusion (QESD) [28,29], using the same formulation that, in previ-ous studies, showed good technological characteristics and stability[30,31].

The method involves the dissolution of the lipid, in this casestearic acid (20 mg) and the surfactant (300 mg of Lipoid S 100,equivalent to 1%, w/v) in 5 ml of ethanol at room temperature. Theorganic solution is then injected through a thin syringe in 30 ml ofHPLC-grade water under constant agitation using an Ultra-TurraxT25 at a speed of 24,000 rpm. After 15 min the suspension was leftunder slow magnetic stirring at room temperature for 24 h to allowthe complete evaporation of ethanol.

2.3. Preparation of ‘stealth’ SLN

Using the above method, pegylated SLN were produced byadding either the commercial PEG derivatives (PEG 40 monostea-rate or DSPE–mPEG) or the PEG–LAA conjugates (PEG2000C–LAA18or PEG5000C–LAA18) in the organic phase, at three different con-centrations (0.5%, 1% or 3%, w/v) (Table 1).

To study the cell uptake, other series of both naked or pegylatedSLN were obtained by adding the tracer dye rhodamine-DHPE, arhodamine-labeled glycerophosphoethanolamine lipid. SLN wereobtained by adding 5 �g of rhodamine-DHPE to the organic phase.

2.4. Size measurement

The average particle size and polydispersity index (PDI) weredetermined by dynamic light scattering with a Nanosizer ZS90(Malvern Instruments, UK). The experiments were carried out usinga 5 mW He–Ne laser diode operating at 633 nm as a light source;

size measurements were taken at a fixed scattering angle of 90◦.To obtain the mean diameter and PDI of the samples, a third-order cumulant fitting correlation function was performed by theanalyzer. The real and imaginary refractive indexes were set at

138 R. Pignatello et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 136– 144

Table 1Outline of stealth SLN produced using different PEG derivatives.

% of PEG derivative (w/w) mPEGC2000–LAA18 mPEGC5000–LAA18 DSPE–PEG PEG 40 monostearate

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sis consisted of ten measurements in triplicate for each sample,nd results were expressed as mean size ± S.D.

.5. Zeta potential

The electrophoretic mobility and Zeta potential were deter-ined by the technique of laser Doppler anemometer with the

bove Nanosizer ZS90 apparatus. An appropriate amount of eachample (80 �l) was diluted with 20 ml of HPLC-grade water andubmitted to the analysis. Zeta limits ranged from −120 to 120 mV.trobing parameters were set as follows: strobe delay −1.00, onime 200.00 ms, off time ms. A Smoluchowsky constant F (Ka) of.5 was used to achieve zeta potential values from electrophoreticobility. Each reported value (Fig. 4) is the mean ± S.D. of up to

00 measurements, automatically defined by the instrument as aunction of the internal quality control.

.6. Evaluation of physical stabilty

To assess the physical stability of the prepared SLN, samplesere stored for 9 months at room temperature and away fromirect light, in closed glass vials. At predetermined time intervals,ize and zeta potential values were reevaluated.

.7. Evaluation of SLN stability in bovine serum

The stability of SLN in bovine serum was assessed by modifying method described in literature [32]. Each sample was diluted five-old with bovine serum, closed in a glass vial and was placed in anncubator at 37 ◦C. Changes in mean size and PDI were evaluated asbove described, every 30 min for 4 h.

.8. Incubation with macrophage cultures

The experiments were carried out using J774 cells, a murine celline of monocyte-macrophages, which were purchased from ATCCRockville, MD, USA). Cells were cultured in Dulbecco’s modifiedagle Medium (DMEM) containing 10% fetal bovine serum, 4.5 g/llucose, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 �g/mltreptomycin (Invitrogen, Milan, Italy), and incubated at 37 ◦C in a% CO2 atmosphere. The medium was replaced every 3 days andhe subcultures were performed every 8–10 days.

To evaluate the macrophage uptake of the different carriers, × 104 of J774 cells were previously seeded for 2 h on each cov-rslip placed into sterile 12-wells plates and after incubated for upo 4 h at 37 ◦C with 100 �l of the rhodamine-labeled different SLNormulations, except for the controls. In each experiment, two wellsere used a ‘blank’ (unloaded SLN) and two as a control (no SLN

dded) to monitor the autofluorescence of J774 macrophages with

r without the SLN, respectively.

After incubation for 4 h with the SLN formulations, the viabilityf the cells, checked out by the MTT test, was greater than 95% (dataot shown).

3a 4a3b 4b3c 4c

For fluorescence evaluation, coverslips were washed andmounted in PBS/glycerol (1:1, v/v) and placed on glass microscopeslides. Coverslips were analyzed on a Reichert Jung fluorescentmicroscope (Leica, Milan, Italy) and positive cells were countedover the entire coverslips.

3. Results and discussion

Similarly to other colloidal drug carriers, the problem of a rapidrecognition by the immune system and clearance from the sys-temic blood flow has been observed after injection of lipid-basednanoparticles. To circumvent such phenomenon and increase thehalf-life of LNC in plasma, sterically stabilized (stealth) systemshave been developed [33,34].

Apart a former paper by Muller et al. who used poloxamer 407and poloxamine 908 to modify the surface of SLN [35], and works byGarcia-Fuentes and Alonso who compared the performance of PEGwith chitosan [36], PEG polymers have become the ‘gold standard’in recent years for this aim. In general, analysis of recent literatureindicates that a common strategy is followed to prepare stericallystabilized SLN or NLC: due to the peculiar nature of the SLN matrix,PEG derivatives (and typically, PEG2000) are pre-conjugated to alipophilic moiety to reinforce and prolong the attachment of thehydrophilic polymer onto the nanoparticle surface. One approachis using a stearic acid–PEG conjugate; this surface modifier has beengenerally used by professor Gasco’s group to modify the surface ofstearic acid based SLN. Such stealth SLN have shown to prolongthe permanence time of doxorubicin in blood in rats and rabbits[37,38]. Even more importantly, this strategy of pegylation affectedthe distribution pattern of the SLN and of the anticancer drug inthe various organs, and especially in brain, hearth and liver. Anin situ model to evaluate the stealth properties of nanocarriers hasbeen also proposed by Wand and Wu [39]. SLN, both plain or dec-orated with PEG stearate, were injected intraperitoneally to miceand their phagocytosis by resident macrophages was measured forsome hours. Pegylated SLN confirmed to be uptaken less efficientlythan uncovered particles [39].

Other reported amphiphilic compounds are phospholipid–PEGconjugates. The same above research group had previouslyobserved that the in vitro phagocytosis by murine macrophagesof stearic acid SLN was reduced when the carriers were stericallymodified by either stearic-acid PEG2000 or DPPE–PEG2000 [40]. Inanother report, DSPE–PEG was used to produce long circulatingstearic acid SLN, which showed a longer permanence in blood (witht1/2� between 5 and 10 h) than the unpegylated carrier [41].

A different strategy to strongly attach the PEG corona tonanoparticle surface has been proposed by Acar et al. [42], whoprepared very small iron oxide nanoparticles coated with interdig-itated bilayers composed of an inner layer of 10-undecenoic acid(UD) and outer layers of a PEG ester of UD. These nanoparticlesshowed to be resistant to aggregation, after dilution and even after�-irradiation.

The research presented here is part of a wider study, aimedat evaluating novel amphiphilic PEG derivatives, in which a LAA

moiety is chosen to anchor the hydrophilic polymer to the sur-face of lipid- and polymer-based nanocarriers. With respect to thestearic acid residue [37–39], LAA, because of their amphiphilicstructure [17], should ensure a more complete and harmonic

Physicochem. Eng. Aspects 434 (2013) 136– 144 139

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nteraction with lipid and phospholipid nanomatrices, allowing suitable retention of the PEG corona on their surface [23–25].oreover, compared to the various commercial phospholipid–PEG

erivatives, like DSPE–PEG, that are structurally well appropriateo decorate a liposome surface, but can disclose some steric hin-rance within the lipid matrix of SLN/NLC, the linear acyl chain ofhe LAA moiety can also permit to efficiently bind the PEG portion,roducing stable nanoparticles.

.1. SLN characterization and short-term stability study

In this study, in comparison to two commercially avail-ble PEG polymers, DSPE–PEG and PEG 40 monostearate, twoonjugates of LAA18 with carboxy–mPEG, having a meanolecular weight of 2000 and 5000 Da, respectively, were

ested (namely, mPEGC2000–LAA18 and mPEGC5000–LAA18)Table 1).

A recent research by our group has already demonstrated thepplicability of the QESD method to obtain stable SLN systems30,31]. Therefore, for this study a similar base formulation washosen to produce pegylated lipid nanoparticles. The techniqueonsists of slowly introducing a solution of the lipid matrix in aater-miscible solvent, in a water phase containing a surfactant

nd under high-speed agitation. The resulting ‘quasi-emulsion’ ofhe organic solution in water is followed by a counter-diffusionf the aqueous phase inside the organic solvent droplets and aradual evaporation of the solvent itself, finally leading to theanoprecipitation of the lipid matrix [43]. This preparative proce-ure is mainly recommendable for the low quantity of emulsifiers

nd pharmaceutically compatible solvents required, that is ofaramount importance when specific applications, such as par-nteral or ophthalmic ones are intended. Moreover, the possibilityf avoiding high temperatures makes the method also suitable for

ig. 3. Stability (mean particle change) of stealth SLN prepared with: (a) DSPE–mPEG, (b) Pere stored at room temperature along the experiment.

SLN. DLS measurements were performed using a Nanosizer ZS90; samples werepreviously diluted tenfold with double distilled water. Error bars refer to three cyclesof ten measurements for each sample.

thermosensitive drugs. Finally, technically complex equipment isnot necessary, even during the scale-up step.

Naked SLN showed a mean diameter of 216 nm (Fig. 2). After 9months of storage at room temperature, this formulation did notsignificant changes neither in terms of mean size and size homo-geneity (PDI value) (Fig. 2). For the SLN prepared using DSPE–PEG,mean particle size decreased as increasing the percentage of thePEG derivative (Fig. 3a). All the samples however showed a rel-

ative stability up to 1 month of storage at room temperature, interms of mean particle size (Fig. 3a) and PDI values, that remainedin a 0.08–0.30 range (not shown). Also using PEG 40 monostea-rate, a mean size comprised between 190 and 254 nm was obtained

EG 40 monostearate, (c) mPEG2000C–LAA18, and (d) mPEG5000C–LAA18. All samples

140 R. Pignatello et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 136– 144

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ig. 4. Zeta potential ± S.D. values (up to 10 sets of 10 measurements) of naked andtealth SLN.

ith a very high size homogeneity (PDI 0.16–0.20; not shown); alsohese SLN were relatively stable for 1 month at room temperatureFig. 3b).

The incorporation of both the tested PEG–LAA conjugates alsoroduced uniformly dispersed SLN systems, with a mean sizeround 250–270 nm for the lower PEG–LAA concentrations, and

further reduction of particle size using the highest concentration3%) (Fig. 3c and d). PDI values remained in a very narrow range

0.09–0.21, not shown) also upon storage at room temperature.

Literature gives contrasting data as concerns the effect of addi-ion of lipid PEG derivatives on SLN size. In some instances, a strong

ig. 6. Changes in mean particle size after incubation with bovine serum at 37 ◦C for thonostearate, (c) mPEG2000C–LAA18, and (d) mPEG5000C–LAA-18.

Fig. 5. Mean size changes of naked SLN upon incubation with bovine serum at 37 ◦C.

size increase was reported, e.g., [38,40], while other studies, evenwith analogous systems, indicated no significant size change, e.g.,[37,44]. These aspects have not been studied in details in the pub-

Our hypothesis to explain the variability on particle size observedfor stealth SLN, in comparison with naked SLN, was that the dif-ferent PEG derivatives, and in particular the PEG–LAA conjugates

e SLN prepared with different molar percentages of (a) DSPE–mPEG, (b) PEG-40

R. Pignatello et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 136– 144 141

Fig. 7. Fluorescence microscopy pictures of J-774 macrophage cultures untreated (a) or incubated with different nanocarriers: (b) 1 h with naked SLN; 2 h with SLN preparedusing 0.5% DSPE–mPEG (3a) (c) or 0.5% PEG 40 monostearate (4a) (d); 2 h (e) or 4 h (f) with SLN prepared using 0.5% mPEG2000C–LAA18 (1a); 2 h (g) or 4 h (h) with SLNprepared using 3% mPEG2000C–LAA18 (1c): 2 h (i) or 4 h (l) with SLN prepared using 3% mPEG5000C–LAA18 (2c).

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sed in this study, behaved as surfactants, partially rearrangingithin the lipid matrix of the colloidal systems instead of preciselyositioning onto their surface.

.2. Zeta potential determination

In contrast with what observed for particle size, the effect ofhe presence of surface modifiers, such as PEG derivatives, on SLNurface charge has been reported with uniform findings. Gener-lly a reduction of the Zeta potential value has been observed fortealth systems compared to naked ones, due to the presence ofhe hydrophilic polymer chains that, amassing on nanoparticlesurface, mask their real charge [36,38,40,45].

The results obtained with our systems would require furthertudies for a more complete interpretation. However, preliminaryeasurements showed that, according to the literature, the Zeta

otential values of stealth SLN decreased slightly compared to thenpegylated nanoparticles. This effect was even more evident withhe PEG2000C–LAA18 conjugate at the concentrations of 0.5% and% (samples 1a and 1c). In all the other cases, a slight increase ofhe surface charge was measured. A likely explanation could be theact that these systems underwent a rearrangement, in which theydrophilic chains of the PEG polymer were positioned in such aay to lead the stearic acid residues outside, with a consequent

ncrease of the net negative surface charge (Fig. 4). An indirect con-rmation of the difficulty in identifying the distribution of PEG–LAAetween the nanoparticles and the external medium, as well as itsxact positioning in the SLN (e.g., within the lipid matrix or ontoheir surface) and its surface organization, has been given us by theelative variability obtained when a quantitative assessment of PEGas attempted, using a published method [46] (data not shown).ecause of the intrinsic significance and scientific interest of this

nformation, we have planned to examine in a more systematicay such aspects in a future study.

Interestingly, the Zeta potential values of the different SLNystems changed within a narrow interval (around ±12%) duringtorage at room temperature (data not shown). This piece of infor-ation further supports the conclusion that the produced lipid

ystems, whatever the positioning of the PEG–LAA derivatives, areelatively stable in the tested storage interval and conditions.

.3. Stability studies in serum

The stability of the produced stealth and naked SLN was eval-ated in the presence of bovine serum, in terms of changes inarticle mean size after an incubation of 4 h [32]. In general, imme-iately after contact with serum a decrease of the initial particleize was registered. This effect can probably be attributed to theact that, while in water the PEG chains, being strongly hydrated,re arranged in a more voluminous shape on the carrier surface,n the presence of serum the volume of hydration of the polymer

as reduced, with the consequent fall of the measured ‘apparent’iameter.

Accordingly to this hypothesis, along the incubation time nakedLN did not modify their mean size (Fig. 5), suggesting that theydrophilic polymer layer did not undergo further rearrangementsr detachment. In general, an analogous behavior was showny the SLN produced with the different PEG derivatives (Fig. 6).n few cases, and especially using lower concentration of theEG2000C–LAA18 conjugate (1), a reduction of particle size waseasured after the 3rd–4th h of incubation, probably due to the

act that the polymer begins to separate from the carrier matrix

Fig. 6c). The problem of the gradual loss or detachment (shedding)f PEG molecules from the nanoparticle surface has been studiednd discussed by several Authors [2]. This phenomenon is linked to

various extent to the properties and composition of the carrier and

cochem. Eng. Aspects 434 (2013) 136– 144

the PEG derivative used, and would finally influence the circulationtime and therapeutic efficiency of the stealth carrier [2,45,47–49].

3.4. Macrophage uptake studies

To assess whether the steric stabilization induced on the SLNby the PEG derivatives was effective in contrasting the uptakeby endocytic cells, fluorescent SLN were incubated with murinemacrophages (J-774 cells). Control (untreated) cells showed a weakdiffuse fluorescence (Fig. 7a). Cells incubated with naked (nonpegylated) fluorescent SLN displayed instead a large amount ofinternalized nanoparticles (Fig. 7b).

Incubation of macrophages with the different pegylated SLNshowed a common trend: by increasing the initial PEG derivativeconcentration, a greater ability to reduce cell uptake (expressedas intracellular fluorescence) was registered. Such behavior wasobserved for all the systems, prepared with either the commercialPEGs or PEG–LAA conjugates. Until the second hour of incubation,all the nanoparticles containing the highest PEG molar concentra-tion (3%) were evidently able to circumvent the cellular uptakewhile, after 4 h, all systems began to be recognized and phagocy-tized.

At lower concentrations (0.5% and 1%) the ‘stealth’ effect wasless effective. Fluorescent spots were in fact clearly visible insidethe cells incubated with such nanoparticles. In particular, commer-cial PEGs were only able to partially mask the surface of SLN and,after 4 h of incubation, the uptake of these nanoparticles was almostcomplete (Fig. 7c and d).

In the case of PEG2000C–LAA18 conjugate further differenceswere noted. At a 0.5% concentration, this conjugate did not effec-tively mask the SLN surface, and after two hours the fluorescentparticles were easily phagocytized. At 1%, after 2 or 4 h of incuba-tion a moderate quantity of nanoparticles was incorporated (Fig. 7eand f).

At 3% concentration (batch 1c), a strong stealth effect wasobserved up to 2 h: after 4 h the system began to be recognizedand nanoparticles were clearly visible inside and on the edge ofcells (Fig. 7g and h).

Finally, the SLN produced using PEG5000C–LAA18 at the lowerconcentrations (0.5 and 1%; batches 2a and 2b) did not showstrong differences. In both cases, up to 2 h a partial uptake wasobserved, which increased progressively after 4 h. At 3% concen-tration (batch 2c), the behavior was similar to that offered bycommercial DSPE–mPEG (SLN batch 3c). Until the second hourthere was an evident circumvention of phagocytosis, while after4 h the nanoparticles were recognized and appeared clearly insidethe cells (Fig. 7i and l). Further studies are however planned to take aquantitative measurement of the behavior of the different carriers.

4. Conclusions

In this paper we have described the behavior of sterically sta-bilized SLN, obtained by using two new amphiphilic conjugates ofcarboxy–mPEG2000 or –mPEG5000 with one LAA having a C-18 alkylchain, compared to commercial DSPE–mPEG and PEG 40 monostea-rate polymers.

Findings of this research showed a good stability of the pre-pared nanoparticle suspensions and a decrease of their mean sizewhile increasing the percentage of the pegylating agent used, aneffect that was more evident in the presence of the two PEG–LAAconjugates at the highest tested molar concentration (3%).

In vitro studies on murine macrophage cultures showed aremarkable capacity of the surface modification of hindering thecell internalization of the SLN, and in particular using a 3% molarconcentration of all the tested PEG derivatives. This behavior was

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lso confirmed by the stability profile of the SLN upon incubationith bovine serum, where a reduction of particle size, due to a prob-

ble leakage of the lipid PEG derivatives from the carrier surface,as observed only after 3–4 h of incubation.

Further studies are in progress to evaluate if using otherEG–LAA conjugates, in which the LAA moiety has a differ-nt alkyl chain length (16 or 20 carbon atom) and PEG andAA are bound in a different manner (e.g., conjugates of LAAith amino–PEG) [12,14], the steric stabilization of nanoparticles

ould be changed or optimized further, in view of their in vivovaluation.

eferences

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