Glucosylated pH-sensitive liposomes as potential drug delivery systems

Article abstract of DOI:10.1016/j.chemphyslip.2016.08.004

The inclusion of pH-sensitive components in liposome formulations can allow a more controlled and efficient release in response to low pH typical of some pathological tissues and/or subcellular compartments. On the other hand decorating the surface of liposomes with sugar moieties attributes to lipid vesicles specificity toward lectins, sugar-binding proteins overexpressed in many tumor tissues. A novel multifunctional pH-sensitive glucosylated amphiphile was synthesized and characterized as pure aggregate component and in mixtures with a natural phospholipid. The comparison of the properties of the new glucosylated amphiphile with respect to those of a previously described cationic structural analogue demonstrates that the pH-sensitivity can strongly affect drug release, lipid organization, as well as the exposure of the glucose residues on liposome surface and their ability to interact with Concanavalin A, a plant lectin used as model system.

Full text of DOI:10.1016/j.chemphyslip.2016.08.004

Accepted Manuscript
Title: Glucosylated pH-sensitive liposomes as potential drug
delivery systems
Author: Luisa Giansanti Alessandro Mauceri Luciano
Galantini Barbara Altieri Antonella Piozzi Giovanna Mancini
PII:
S0009-3084(16)30112-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.chemphyslip.2016.08.004
CPL 4490
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Chemistry and Physics of Lipids
Received date:
Revised date:
Accepted date:
9-5-2016
26-7-2016
31-8-2016
Please cite this article as: Giansanti, Luisa, Mauceri, Alessandro, Galantini, Luciano,
Altieri, Barbara, Piozzi, Antonella, Mancini, Giovanna, Glucosylated pH-sensitive
liposomes as potential drug delivery systems.Chemistry and Physics of Lipids
http://dx.doi.org/10.1016/j.chemphyslip.2016.08.004
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Glucosylated pH-sensitive liposomes as
potential drug delivery systems
Luisa Giansanti,a,b Alessandro Mauceri, b,c Luciano Galantini,c Barbara Altieri,a,b Antonella Piozzi,c
Giovanna Mancinib,*
aDipartimento di Scienze Fisiche e Chimiche, Università degli Studi dell’Aquila, Via Vetoio, 67010
Coppito (AQ) (Italy)
b CNR, Istituto di Metodologie Chimiche, Area della Ricerca di Roma 1, Via Salaria km 29,300,
00015 Monterotondo (RM), Italy
c Dipartimento di Chimica, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185
Roma (Italy)
Graphical abstract

Highlights
 A new glucosylated pH-sensitive amphiphile was prepared and characterized
 Glycosylated liposomes were investigated by DLS, DSC and fluorescence experiments
 pH-sensitive liposomes show different properties than their cationic analogues
 pH-sensitivity greatly influences lipid bilayer organization
 The interaction of glycosylated pH-sensitive liposomes with Concanavalin A
ABSTRACT
The inclusion of pH-sensitive components in liposome formulations can allow a more controlled
and efficient release in response to low pH typical of some pathological tissues and/or subcellular
compartments. On the other hand decorating the surface of liposomes with sugar moieties attributes
to lipid vesicles specificity toward lectins, sugar-binding proteins overexpressed in many tumor
tissues. A novel multifunctional pH-sensitive glucosylated amphiphile was synthesized and
characterized as pure aggregate component and in mixtures with a natural phospholipid. The
comparison of the properties of the new glucosylated amphiphile with respect to those of a
previously described cationic structural analogue demonstrates that the pH-sensitivity can strongly
affect drug release, lipid organization, as well as the exposure of the glucose residues on liposome
surface and their ability to interact with Concanavalin A, a plant lectin used as model system.
Keywords: glucosylated amphiphile, pH-sensitivity, liposomes, agglutination, Concanavalin A,
drug release
1. Introduction
Since ’70 liposomes have been widely investigated as drug delivery systems for their
biocompatibility, efficiency and ability to entrap either hydrophilic or hydrophobic drugs and to
improve their pharmacokinetic and pharmacodynamic profiles [1]. Liposomes mainly enter cells via
endocytosys and if the drug does not escape the endosome there is a high risk of degradation with a

consequent reduction of its efficacy [2]. Different strategies have been proposed to avoid this
phenomenon, and the use of pH-sensitive liposomes (i.e. liposomes containing a pH-sensitive
component) is one of the most promising strategies. In fact, pH-sensitive liposomes are stable at
physiological pH, whereas they can be destabilized and may show fusogenic properties under acidic
conditions, typical of endosomal compartment and of certain target tissue such as tumour [3]. As a
consequence of destabilization, the controlled release of the entrapped drug to the target tissue
occurs, thus avoiding lysosomal sequestration and degradation. Moreover, it is possible to modify
liposome surface to ascribe them certain features, such as increased circulation time in the
bloodstream and specificity toward certain targets, even at subcellular level, thus improving their
efficacy as drug delivery systems [4]. Over the last years many liposome formulations have been
developed providing improved therapeutic outcomes and reduced side effects. Therefore liposomes
with improved drug delivery potential for cancer therapy have been prepared, mainly based on the
active targeting to tumor tissues (mainly by folate and transferring) and on the release of the
chemotherapic agent triggered by pathological conditions typical of the microenvironment of tumor
cells [5]. One of approaches relies on the use of glycosylated amphiphile that, when included in
liposome formulations, increase their specificity to tumor tissue [6,7]. In fact, certain malignant
tissues overexpress lectins that are sugar-binding proteins located at cell surface and involved in
many recognition processes such as cell-cell adhesion, cell attachment to substratum and blood-
borne metastasis [8].
The combination of pH sensitivity and specificity for target tissue can hugely increase the potential
of liposomal formulations as delivery systems to cancer cells. Herein we report on the synthesis and
the physicochemical characterization of a novel pH-sensitive glucosylated amphiphile (GA) 1 and
on its employment as a component of liposome formulations. GA 1 is characterized by the presence
of a PEG spacer that links a glucose moiety to a tertiary ammine group bearing a methyl and a
hydrophobic alkyl chain. It is the pH-sensitive analogue of a previously reported cationic GA, 2,
that features a quaternary charged nitrogen as polar headgroup and that showed good ability to

interact with a plant lectin both as a monomer and as liposome component [9]. The aggregation
properties of GA 1 were investigated as a pure component and when included in liposomes of 1,2-
dimyristoyl-sn-glycero-phosphocholine, DMPC, (Chart 1) at different molar ratios. The effect of the
inclusion of GA 1 on DMPC bilayer properties was studied by differential scanning calorimetry
(DSC) measurements. The ability of the glucosylated liposomes to interact with lectins was
evaluated by optical density (OD) measurement based agglutination assay monitoring their ability
to bind Concanavalin A (ConA), a plant lectin usually used as model system in agglutination
experiments. Finally the sensitivity of the formulations to pH conditions was investigated.
2. Materials and Methods
2.1. Materials
DMPC was purchased from Avanti Polar Lipids (Alabaster, AL, USA). ConA, from Canavalina
ensiformis (Jack bean), phosphate-buffered saline (PBS; Aldrich; 0.01 M phosphate buffer; 0.0027
M KCl; 0.137 M NaCl; pH 7.4) Sephadex G-50, 1-pyrenemethanol (pyr-m) and all reagents
employed for the synthesis of 1 were purchased from Sigma-Aldrich. Compound 4 was prepared as
previously reported [9]. All solvents and chemicals were used as purchased without further
purification.
2.2. Instrumentation
1
A Bruker300 Avance spectrometer (operating at 300 MHz for H and 75 MHz for 13C) was used
1
to record NMR spectra. H resonances of deuterated solvents (with respect to TMS) were used as
internal standards.
A Fluoromax-4 Horiba-JobinYvon spectrofluorimeter was used to carry out steady-state
fluorescence experiments.
A Cary 300 UV–vis double beam spectrophotometer (Varian Australia PTY Ltd., Mulgrave, Vic.,
Australia) was used to carry out OD measurements.
A pHmeter Crison PH 25+ was used to measure the pH of the solutions.

A Malvern Nano-ZetaSizer spectrometer, equipped with a 5 mW HeNe laser (λecc= 632.8 nm) and
a digital logarithmic correlator was used to perform DLS measurements. The normalized intensity
autocorrelation functions were measured at an angle of 173° at 25.0 ± 0.1 °C and analyzed by using
the cumulant fit.
A METTLER TA 3000 calorimeter provided with a TC 10 A processor was used to perform DSC
measurements by keeping the cell (DSC30) under N2 flow.
2.3. Synthesis of GA 1
GA 1 was synthesized following the procedure reported in Scheme 1.
2.3.1 Preparation of N-methylhexadecanamide 2.
5 mL of aqueous CH3NH2 (40%) were added to 10 mL of CH2Cl2 and, after cooling at 0°C, the
solution was magnetically stirred in a round bottom flask. 3 mL (10 mmol) of palmitoyl chloride
were added dropwise and the mixture was left to react at room temperature for 24 h. The organic
layer was extracted with aqueous NaOH 2 M, then with aqueous HCl 1 M and finally with a
saturated aqueous solution of NaCl. The organic layer was dried over anhydrous MgSO4 and, after
filtration, the solvent was removed under reduced pressure to give 2.48 g of 2 (yield 92%) as a
white solid. 1H-NMR (δ CDCl3, 300 MHz) ppm: 5.79 (s, 1H, NH); 2.74-2.83 (os, 3H, NCH3); 2.17
3
(t, JHH = 7.6 Hz, 2H, COCH2); 1.54-1.72 (m, 2H, COCH2CH2); 1.15-1.33 (m, 26H,
3
13
NCH2CH2[CH2]13CH3); 0.87 (t, JHH = 6.5Hz, 3H, CH2CH3). C-NMR (δ CDCl3, 75 MHz) ppm:
175.16; 38.12; 33.27; 31.04; 31.00; 30.96; 30.84; 30.71; 27.61; 27.15; 24.04; 15.47.
2.3.2 Preparation of N-methylhexadecanamine 3.
2.48 g (9.2 mmol) of N-methylhexadecanamide 2 were added in small portions to an ice-bath
cooled suspension of 0.91 g (24.0 mmol) of LiAlH4 in 100 mL of dry THF. The reaction was kept
at reflux for 24 h. The mixture was then cooled at room temperature and 7 mL of 10% aqueous
solution of NaOH, 5 mL of H2O and 80 mL of THF were added. The mixture was filtered and the
solvent removed under reduced pressure. The oily residue was purified by chromatography on silica
gel (eluent from CHCl3 100% to CHCl3/MeOH = 80:20) to give 1.54 g of 3 (yield 65%) as a white

solid. 1H-NMR (δ CDCl3, 300 MHz) ppm: 2.53 (t, 3JHH = 7.2 Hz, 2H, NCH2); 2.40 (s, 3H, NCH3);
2.12 (s, 1H, NH); 1.35-1.46 (m, 2H, NCH2CH2); 1.09-1.31 (m, 26H, NCH2CH2[CH2]13CH3); 0.85
3
13
(t, JHH = 6.6 Hz, 3H, CH2CH3). C-NMR (δ CDCl3, 75 MHz) ppm: 53.32; 37.56; 33.27; 31.04;
30.97; 30.91; 30.71; 28.66; 24.03; 15.45.
2.3.2 Preparation of GA 1
0.34 g (1.3 mmol) of N-methylhexadecanamine 3 and 0.45 g (0.9 mmol) of 4 were suspended in 8
mL of dry CH3CN. The mixture was heated to reflux for two weeks. The solvent was removed
under reduced pressure and the residue purified by chromatography on silica gel (eluent from
CHCl3 100% to CHCl3/MeOH = 80:20) to give 0.29 g of 1 (yield 48%) as a brown oil. 1H-NMR (δ
DMSO-d6, 300 MHz) ppm: 8.09 (s, 1H, C=CH); 4.85-4.59 (m, 2H, OCH2C=C); 4.50 (t, 3JHH = 5.1
Hz, 2H, NNCH2CH2O); 4.24 (d, 3JHH = 7.8 Hz, 1H, H-1); 4.05 (s, 4H, OH); 3.80 (t, 3JHH = 5.1 Hz,
2H, NNCH2CH2O); 3.75-3.65 (m, 3H, OCH2CH2NCH3, H-6[1H]); 3.55-3.42 (m, 9H, OCH2CH2O,
H-6[1H]); 3.19-2.89 (m, 8H, H-2, H-3, H-4, H-5, OCH2CH2NCH2); 2.66 (s, 3H, NCH3); 1.67-1.52
3
(m, 2H, CH3NCH2CH2); 1.31-1.15 (m, 26H, NCH2CH2[CH2]13CH3); 0.83 (t, JHH = 6.6 Hz, 3H,
CH2CH3). 13C-NMR (δ DMSO-d6, 75 MHz) ppm: 145.40; 126.38; 103.91; 78.76; 78.50; 75.17;
71.88; 71.54; 71.44; 71.36; 70.54; 70.27; 63.27; 59.21; 58.09; 51.13; 43.98; 33.12; 30.84; 30.76;
30.52; 28.61; 28.29; 23.91; 15.76. Elemental analysis for C, H, N was within the theoretic value.
2.4. Determination of critical micellar concentration (cmc) of GA 1
Aqueous solutions (3 mL) of amphiphile 1 at concentrations between 0.1 M and 1 mM were added to a
defined amount of pyrene in order to obtain ~ 0.5 M final concentration of pyrene (prepared from 7 L of a
160 M stock solution of pyrene in absolute ethanol dried by a nitrogen flux). The solutions were kept at
40°C under stirring for 12 h. Emission spectra of the solutions were acquired in the range 350-450 nm
(exc=335 nm) at 25°C. The cmc was defined as the concentration where the plot of intensity ratio of the third
(I3, 380 nm) and first (I1, 370 nm) vibronic peaks of pyrene I3/I1 versus GA 1 concentration begins to raise.
2.5 Determination of pKa value of amphiphile 1 in aggregative conditions

Small aliquots of a HCl 1 mM aqueous solution were added to a 0.1 mM aqueous solution of
amphiphile 1 (above its cmc) in saline buffer NaBr 0.390 mM. The pKa value of 1 was estimated by
using the Gran’s plot (after equivalence point):
10pHVa=Ka-1(Ve-Va)
where Va is the total added volume of acid and Ve is the equivalence volume.
2.6. Liposome preparation
Aqueous dispersions of DMPC/1 liposomes were prepared according to a reported procedure
[10]. Lipid films were prepared on the inside wall of a round-bottom flask by evaporation of
solutions containing the proper amount of DMPC and 1 (dissolved in CHCl3) to obtain the desired
molar percentage mixture. In the case of experiments relative to determination of the surface
potential value (Ψ°) of DMPC/1 liposomes, heptadecyl-7-hydroxycoumarin (HC) (dissolved in THF)
was added to the lipid solution to obtain a final 15 μM concentration. The obtained lipid films were
kept overnight under reduced pressure (0.4 mbar); then a proper amount of PBS was added to
obtain a lipid dispersion of the desired concentration. The aqueous suspensions were vortex-mixed
and the obtained multilamellar vesicles (MLV) were subjected to DSC measurements at a 0.1
mg/μL (14.8 mM total lipids) concentration. For all the other experiments the MLV were freeze-
thawed six times from liquid nitrogen temperature to 50 °C and were then extruded (10 times)
through a 100 nm polycarbonate membrane.
2.7 Determination of aggregate size by DLS
DLS measurements were carried out at 25°C on aqueous solutions of amphiphile 1 (80 mM) and
suspensions (1.25 mM total lipids in PBS) of DMPC/1 liposomes at 95:5, 80:20, 70:30 molar ratio.
2.8 Determination of thermotropic properties of liposomes
DSC measurements were carried out on 30 μL of MLV aqueous dispersions at 0.1 mg/μL (148
mM total lipids) using aluminium pans. Two heating scans were recorded at the rate of 5°C min-1
and two subsequent heating scans were recorded at the rate 1°C min-1. Under the experimental

conditions, reproducible thermal recordings were obtained. Uncertainty on temperatures was
determined to be 0.1°C.
2.9 Determination of surface potential (Ψ°) of liposomes
An indirect method, described in literature and based on the pH-sensitive fluorophore HC [811],
was exploited to determine Ψ° of DMPC/1 liposomes at molar ratios 95:5, 80:20 and 70:30 (total
lipid concentration [DMPC]+[1]=5 mM) adding HC dissolved in THF to the lipid film to obtain a
final concentration of 15 μM. Fluorescence measurements of liposome suspensions in PBS were
performed by scanning the excitation wavelength between 300 and 400 nm, at the emission
wavelength of 450 nm, varying the pH of solution between 2 and 12 by addition of concentrated
aqueous NaOH or HCl. The extent of dissociation of liposomes including HC was monitored by the
ratio of the excitation fluorescence intensities at 380 and 330 nm (pH-independent isosbestic point).
pKa of HC associated with the cationic liposome bilayer (pKa charged) corresponds to the inflection
point of the plot of I380/I330 ratio as a function of pH. The surface potential (Ψ°) was obtained by a
conversion and rearrangement of the Boltzmann equation:
ψ° = -e-1(pKa charged- pKa neutral)kBTln10
where kB is the Boltzmann constant, T is the absolute temperature, e is the electron charge and
neutral
pKa
is the pKa of HC associated with DMPC bilayer chosen as neutral reference.
2.10 Agglutination of glucosylated liposomes by ConA monitored by OD measurements
The variation of specific turbidity at 25°C of 1 mL samples of 0.83 mM DMPC/1 (95:5, 80:20
and 70:30) liposomes in PBS upon the addition of ConA (0.33 mg/mL) was recorded over 500
minutes. Scans were carried out in a 1-cm quartz cell upon addition of ConA in PBS at 525 nm
immediately after addition of ConA and every minute.
2.11 Evaluation of release from DMPC/1 liposomes as a function of pH
Lipid films of DMPC and DMPC/1 at 95:5, 80:20, 70:30 molar ratio were hydrated with 2 mL of a
solution of pyr-m 0.1 mM in PBS to obtain 1 mM total lipid concentration. 150 μL of the extruded

suspension were added to 3 mL of a 5∙10-5 M solution of FeCl3 in PBS at pH=7.4 or at pH 4.7 (the
pH was adjusted by addition of small volumes of concentrated HCl before the beginning of the
release test). The release of pyr-m was monitored at 30°C by following the decrease of the intensity
of the first vibronic peak of pyr-m at 375 nm (λex= 335 nm) as a function of time.
Rate release of pyr-m at pH 4.7 was evaluated by plotting the percentage of drug released from
liposomes as a function of the square root of time according to Higuchi model. Release rate
constant (Kd) were evaluated as the slope of the linear plot obtained.
3. Results
3.1 Characterization of GA 1
Aqueous solutions of GA 1 at 10 mM and 4 °C are completely transparent and stable, thus
indicating that GA 1 is characterized by low (<4 °C) Krafft point and Krafft temperature. The cmc
of GA 1 was determined by a fluorescence method reported in the literature and resulted (5.1 ±
0.3)∙10-6 M [12]. Above its cmc GA 1 forms micelles characterized by a hydrodynamic diameter
(dh) = 6 ± 0.8 nm as determined by DLS measurements. The application of the Gran’s plot to the
data obtained by the acid-base titration of GA 1 gave an apparent pKa = 3.7 ± 0.2.
3.2 Characterization of DMPC/1 liposomes
DLS measurements of DMPC/1 liposomes at 95:5, 80:20 and 70:30 molar ratio showed that all the
investigated formulations feature a stable monomodal distribution with a dh centered at ~100 nm
(Table 1).
The thermograms relative to DMPC/1 liposomes are reported in Fig. 1. The main transition
temperature (Tm) is shifted to lower values with respect to the Tm of DMPC (Fig. 1) and the extent
of the decrease depends on the amount of GA 1 in the formulation. Also ΔH values are lower if
compared to ΔH of liposome composed of pure DMPC (Table 1). It can be observed that when the
amount of GA 1 in the formulation is low the profile of the peak relative to the main transition is

very similar to that of pure DMPC liposomes. On the other hand, when the amount of GA 1
increases the peak in the thermograms are sensibly broader.
Finally, as reported in Table 1 similar positive values of Ψ° were estimated for all the DMPC/1
formulations by means of the spectrofluorimetric titration.
3.3 Interaction of DMPC/1 liposomes with ConA
DMPC/1 liposomes at molar ratio 80:20 and 70:30 showed a neat increase of OD upon the addition
of ConA, whereas no significant OD changes were detected in the case of the 95:5 formulation (Fig.
2). At molar ratio 80:20 vesicle agglutination occurs faster and to a higher extent with respect to the
70:30 formulation. Moreover, 80:20 liposome suspension showed a decrease of OD ~ 400 minutes
after the addition of ConA clearly indicating precipitation of particles. DMPC liposomes (in the
absence of glucosylated lipids) agglutinate in the presence of ConA over a longer range of time
with respect to DMPC/1 liposomes, and give rise to precipitation after 24 hours due to aspecific
interactions.
3.4 Evaluation of release from DMPC/1 liposomes as a function of pH
The capability of DMPC and DMPC/1 liposomes to release an entrapped drug was evaluated at
physiological pH (7.4) and acidic pH (4.7) by monitoring the decrease of the intensity of
fluorescence of pyr-m, a fluorescent hydrophilic molecule used as drug model, upon dilution in PBS
containing FeCl3 as quencher. The intensity of the fluorescent signal is proportional only to pyr-m
retained in the internal compartment of liposomes, because when the dye is released from the
internal aqueous compartment of liposomes, its fluorescence is quenched by FeCl3 dissolved in the
bulk. At pH 7.4 DMPC and 95:5 DMPC/1 liposomes release less than 10% of the entrapped dye
over 3 h of incubation upon dilution, whereas in the case of 80:20 and 70:30 DMPC/1 liposomes
the amount of released dye reaches ~20% (data not shown). At pH=4.7 all the formulations show a
higher release with respect to neutral conditions (Figure 3). However, it is evident that liposomes
devoid of the glucosylated component release the lowest amount of dye (25% after 3 h), whereas all

the formulation containing GA 1 release a much higher amount (~50% after 3 h) of the entrapped
dye.
The obtained Kd values are 2.26, 3.80, 4.42 and 5.13 % min-1/2 for DMPC, DMPC/1 95:5, DMPC/1
80:20, DMPC/1 70:30 liposomes, respectively (r2 were greater than 0.955, the graph from which Kd
were evaluated are reported in Figure SI 2). It can be clearly noticed that, differently from what
observed evaluating the amount of released pyr-m, Kd values increase as a function of the amount
of GA present in the formulation.
4. Discussion
GA 1 is the pH-sensitive analogue of GA 2, a quaternary ammonium surfactant that was shown able
to interact with Con A but that can not be included in liposome formulations above 5 molar
percentage because of its detergent effect [9]. Thus the tertiary ammine GA 1 was synthesized to
have the possibility of increasing the amount of glucosylated component in the formulation.
Further, because the protonation of the new lipid attributes to it a detergent effect, its presence in
the formulation allows controlling drug release in acidic compartments such as lisosomes and/or
tumour tissues. Moreover, its pH-sensitivity can facilitate the release of the entrapped drug to the
target tissue.
As expected, GA 1 features a cmc (5.4∙10-6 M) lower than the cmc of GA 2 (1∙10-3 M) [9]. In fact,
GA 1 is only partially protonated in micellar aggregates, therefore it has a behavior that is in
between those of ionic and non-ionic surfactants that usually feature lower cmc values with respect
to ionic surfactants. The low apparent pKa value of 3.7 obtained in the case of GA 1 micelles
indicates that within the aggregates the amphiphile has less basic properties than expected for a
tertiary ammine, i.e. in the micelle the protonation equilibrium shifts toward the deprotonated form,
in agreement with data reported in the literature for other surfactants with tertiary amine head group

[14]. Literature reports show that the extent of the shift of pKa mainly depends on the dissociation
extent of the surfactant in the aggregate [15], on the electrostatic interaction between the polar
headgroups [16] and on a charge effect due to a dielectric discontinuity between the water
environment and the interior of the micelle (aspect that influences transfer of charged and
uncharged species from bulk water to the surface of a micelle) [17].
The absence of a net cationic charge on the headgroup of GA 1 reduces both detergent and
disturbing effects on the lipid bilayer of liposomes with respect to its cationic analogue GA 2 [9]. In
fact, DLS measurements show that phospholipid liposomes containing up to 30 molar % of GA 1
are stable and feature a hydrodynamic radius of ≈ 100 nm, in good agreement with the dimension
imposed by the extrusion protocol, whereas 10 molar % of GA 2 partially solubilizes the lipid
bilayer of phospholipid vesicles. This is an interesting result because it demonstrates that it is
possible to decorate the surface of liposomes with a higher amount of targeting residues.
DSC measurements allowed investigating the influences of the presence of GA 1 on the lipid
organization of DMPC liposome. Both the shift of Tm to lower values and the decrease of ΔH
associated with the transition from gel to liquid-crystalline phase with respect DMPC liposomes
(Fig. 1, Table 1) suggest that the packing of lipid in the bilayer is loosened by the presence of the
synthetic amphiphile [18]. In the case of DMPC/1 liposomes at 95:5 molar ratio a very low decrease
of Tm and ΔH values are observed, indicating that the interaction of the alkyl chains are slightly
reduced with respect to the mere DMPC whereas the interaction between polar headgroups is not
affected by the presence of the GA 1 [19]. On the other hand, when the same amount of GA 2 was
included in DMPC liposomes, though the decrease of Tm was the same, the effect on lipid packing
was significantly different. In fact GA 2, being scarcely miscible with DMPC, had an evident
disturbing effect on the phospholipid bilayer indicated by the large range of temperature at which
the transition occurs [9]. The increase of the amount of GA 1 in the formulation causes a significant
reduction of lipid packing, interaction between polar headgroups and cooperativity of the transition.
This behavior is rather different compared to that observed in other mixed systems containing a pH-

sensitive synthetic amphiphile [20]. Such a difference could be ascribed to the hygroscopic effect of
the PEG spacer that, causing the removal of water from the hydration shell of lipid headgroups,
affects the Tm value [21]. The weaker interactions of the alkyl chains of lipid membrane induce a
reduction in the cooperativity of the transition as reflected by the width of the DSC transition peak.
As a matter of fact, it has been previously observed that, in the same condition, a longer
polyoxyethilenic spacer determines a decrease of cooperativity of the main transition [13].
Ψo of DMPC/1 liposomes was determined by the spetrofluorimetric titration of HC, a pH-sensitive
probe that, when embedded in a lipid bilayer, locates in proximity of the headgroups. The fact that
all the investigated formulations, independently from their composition, feature similar values of Ψo
indicates that GA 1, as also observed in the micelles it forms, is not protonated even when included
in phospholipid liposomes. Despite composed of uncharged and zwitterionic lipids, all the
formulations show positive Ψo values: this result can be ascribed to a different localization of the
probe in DMPC/1 liposomes with respect to liposomes of pure DMPC (chosen as neutral reference),
as also observed in other systems [22]. The shift of HC in the bilayer is probably correlated with the
loosening of bilayer packing induced by the addition of GA 1 as inferred from DSC experiments.
We also investigated the targeting properties of DMPC/1 liposomes by agglutination assay
evaluating their ability to interact with ConA, a tetrameric protein that, thanks to the presence of
multiple binding sites for glucose, binds to more than one glycosylated liposome leading to
aggregation of vesicles. The absence of interaction with ConA observed in the case of DMPC/1
liposomes 95:5 cannot be ascribed to the low density of glucose moieties on vesicles surface
because liposomes containing the same percentage of GA 2 in the same experimental conditions
showed a great ability to interact with ConA [9]. The different behaviour of DMPC/1 and DMPC/2
liposomes is probably related to a different exposure of the glucose residue to the bulk due to the
different organization of the lipid bilayer, as suggested by DSC experiments. Agglutination was
observed upon the addition of ConA to 80:20 and 70:30 DMPC/1 liposome suspensions. The minor
and slower OD increase observed in the case of 70:30 with respect to 80:20 molar ratio indicates

that in the presence of a higher amount of GA 1 agglutination yields smaller aggregates. Evidently,
the increase of the sugar moieties concentration on the liposome surface suppresses liposome
crosslinking upon addition of ConA, due probably to a preferential intraliposome partial saturation
of the protein binding sites. Literature reports confirm the dependence of the binding efficiency of
carbohydrates with lectins on surface density of sugar moieties [23]. It must be stressed that under
agglutination conditions the fraction of sugar moieties accessible to ConA is always larger than the
protein binding sites ([GL1]/[ConA] binding sites being ≈ 2 and 3.5 for DMPC/1 80:20 and 70:30,
respectively). This means that the binding sites of ConA are probably saturated in all the
investigated samples and an increase of GA1 fraction although determining an increase of sugar
moieties does not promote a sensitive increment of the ConA sugar binding degree.
Finally, the pH-sensitivity of the investigated glucosylated formulations was assessed by
fluorescence experiments comparing the release of a hydrophilic molecule, pyr-m, at neutral (7.4)
and acidic pH (4.7), the latter being typical of endosomal compartment and of certain tumour cells
[3]. At pH=7.4 DMPC/1 liposomes at 95:5 molar ratio show the same modest capability of DMPC
liposomes to release pyr-m, confirming that the presence of the neutral GA at low percentage does
not affect packing. When the percentage of 1 in the formulation raises the release of pyr-m from the
internal aqueous compartment is twofold higher indicating a loose packing of lipid molecules, in
good agreement with results obtained by DSC experiments. At acidic pH, the release of the drug
from all formulations containing the GA, independently of its amount, is significantly enhanced and
is sensibly higher with respect to the release observed from DMPC liposomes. The absence of
significant differences by increasing the GA content was observed also in the case of other pH-
sensitive pegylated liposomes [24] and can probably be ascribed to the increased sterical stability
conferred to liposomes by high amount of PEG-lipids. Drug release rate is an important parameter
that can greatly affect the therapeutic efficacy of liposome formulation [25]. The fact that the plot
from which the Kd values were evaluated are linear indicates that Higuchi’s model was a good
fitting model and that the release of pyr-m from the investigated liposomes was driven mainly by a

diffusion-controlled mechanism [26]. The obtained Kd values are in good agreement with those
reported in literature investigating drug release profile from pegylated liposomes in vitro [27].
Differently from what observed evaluating the amount of drug released, the release rate seems to be
strictly dependent on the amount of GA present in the formulation, thus suggesting that their
efficacy as drug delivery system could be greatly affected by the presence of the glucosylated pH
sensitive component.
As a whole, these results indicate that the new pH-sensitive liposomes have a good potential as
delivery systems in which the release of the drug is triggered by pH sensitivity.
4 Conclusions
Recent efforts in liposome research are aimed at developing multifunctional formulations to
increase their specificity to target tissue while controlling drug release thus improving their efficacy
as drug delivery systems. We synthesized and characterized a novel pH-sensitive GA 1 and
included it in DMPC liposomes at different molar ratios. The results of our investigations indicate
that at physiological pH the new amphiphile is present in its deprotonated form both when self
assembled, as pure component, in micelles and when included in DMPC liposomes. The
comparison of the properties of DMPC/GA 1 liposomes with those containing its cationic analogue
GA 2, previously described, points out that the presence of the neutral amphiphile has a modest
detergent effect on the lipid bilayer of phospholipid liposomes thus allowing to obtain stable
formulations containing up to 30% of 1. This represents a good result for targeting. Moreover,
acidic pH induced a significant increase of drug release from all DMPC/1 liposomes investigated.
These features together with the targeting ability, make these multifunctional pH-sensitive
glucosylated particles good candidates as drug delivery systems.

Corresponding Author
* E-mail: giovanna.mancini@uniroma1.it
Notes
The authors declare no competing financial interest.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to
the final version of the manuscript
Acknowledgements
We acknowledge technical and administrative support by Mrs. Stefania Tarquini and Mr. Marco
Pastore and the financial support of PRIN project Multinanoita (project number 2010JMAZML)
and of the FIRB project 2012 RBFR12BGHO.

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Fig 1. Thermograms relative to DMPC/1 MLVs (at different molar ratios) organized according to
increasing percentages of GA 1, the first thermogram from the bottom being relative to vesicles
formulated only with DMPC. Scan rate is 1 °C/min.
Fig 2. Agglutination of DMPC/1 vesicles at 95/5 (red trace), 80/20 (blue trace) and 70/30 (green
trace) molar ratio in PBS upon the addition of ConA monitored by optical density. ConA
concentration 0.33 mg/mL; total lipids concentration 0.83 mM.

Fig 3. Percentage of 1-pyrenemethanol retained by DMPC/1 vesicles at 100/0 (black circles), 95/5 (red
circles), 80/20 (green down triangle) and 70/30 (yellow up triangle) molar ratio in PBS at pH 4.7 as a
function of time. The reported data represent the average values of at least three different and independent
experiments of each sample. Error in determination is within 5% (see Figure SI 1).
Chart 1. Liposome components.

Scheme 1. Synthetic scheme for the synthesis of GA 1

Table 1. Thermodynamic parameters of DMPC/1 MLV and SUV. Temperature and ΔH values
were determined with an accuracy of ±0.1°C and ±0.5 kJ/mol, respectively.
DMPC/1
Tm (°C)
ΔH (kJ/mol)
dh (nm)
Ψo (mV)
95/5
80/20
70/30
23.8
22.0
21.9
20.8
25.8
23.2
96 ± 8
84 ± 5
26 ± 6
29 ± 5
34 ± 5
108 ± 16
Tm of DMPC liposomes: 24.1°C; ΔH of DMPC liposomes: 28.9 kJ/mol (data from ref [130]).