Screening and optimization of ligand conjugates for lysosomal targeting

Article abstract of DOI:10.1021/bc200336j

The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal immunofluorescent microscopy, subcellular fractionation, and flow cytometry. Confocal microscopy demonstrated that liposomes modified with derivatives of rhodamine B provide a good rate of colocalization with the specific lysosomal markers. The comparison of fluorescence of FD in lysosomes isolated by subcellular fractionation also showed that the efficiency of lysosomal delivery of the liposomal load by liposomes modified with some of synthesized ligands was significantly higher compared to that with plain liposomes. These results were additionally confirmed by flow cytometry of the intact cells treated with liposomes loaded with 5-dodecanoylaminofluorescein di-β-d-galactopyranoside, a specific substrate for the intralysosomal β-galactosidase, using a number of cell lines, including macrophages with induced phenotype of lysosomal enzyme deficiency; two of the synthesized ligands-rhodamine B DSPE-PEG_2k-amide and 6-(3-(DSPE-PEG_2k)-thioureido) rhodamine B-demonstrated enhanced lysosomal delivery, in some cases, higher than that for commercially available rhodamine B octadecyl ester, with the best results (the enhancement of the lysosomal delivery up to 75% greater in comparison to plain liposomes) shown for the cells with induced lysosomal enzyme deficiency phenotype. Use of liposomes modified with rhodamine B derivatives may be advantageous for the development of drug delivery systems for the treatment of lysosome-associated disorders.

Full text of DOI:10.1021/bc200336j

ARTICLE
pubs.acs.org/bc
Screening and Optimization of Ligand Conjugates for Lysosomal
Targeting
Igor Meerovich, Alexander Koshkaryev, Ritesh Thekkedath, and Vladimir P. Torchilin*
Center for Pharmaceutical Biotechnology & Nanomedicine, Northeastern University, 312 Mugar Hall, 360 Huntington Avenue, Boston,
Massachusetts 02115, United States
ABSTRACT:
The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into
lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially
lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery
of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these
liposomes and their content to lysosomes in HeLa cells was investigated by confocal immunofluorescent microscopy, subcellular
fractionation, and flow cytometry. Confocal microscopy demonstrated that liposomes modified with derivatives of rhodamine B
provide a good rate of colocalization with the specific lysosomal markers. The comparison of fluorescence of FD in lysosomes
isolated by subcellular fractionation also showed that the efficiency of lysosomal delivery of the liposomal load by liposomes
modified with some of synthesized ligands was significantly higher compared to that with plain liposomes. These results were
additionally confirmed by flow cytometry of the intact cells treated with liposomes loaded with 5-dodecanoylaminofluorescein di-β-
D-galactopyranoside, a specific substrate for the intralysosomal β-galactosidase, using a number of cell lines, including macrophages
with induced phenotype of lysosomal enzyme deficiency; two of the synthesized ligands—rhodamine B DSPE-PEG2k-amide and
6-(3-(DSPE-PEG2k)-thioureido) rhodamine B—demonstrated enhanced lysosomal delivery, in some cases, higher than that for
commercially available rhodamine B octadecyl ester, with the best results (the enhancement of the lysosomal delivery up to 75%
greater in comparison to plain liposomes) shown for the cells with induced lysosomal enzyme deficiency phenotype. Use of
liposomes modified with rhodamine B derivatives may be advantageous for the development of drug delivery systems for the
treatment of lysosome-associated disorders.
’ INTRODUCTION
LSD clinical manifestations typically include deterioration of
neurological function (50% of LSDs are associated with central
nervous system (CNS) disorders), as well as pulmonary, hepatic,
splenic, cardiovascular, and renal dysfunction. Other tissues and
functions also often affected in LSDs include the immune system,
bone, connective tissue, skeletal and cardiac muscle, dermis, and
ocular function.5,6 The clinical course and the severity of individual
LSDs usually correlate with levels of residual enzyme activity.
The most extensively used strategy for treatment of LSDs is
enzyme replacement therapy (ERT), based on the exogenous
Many pharmaceutical agents, including various large (enzymes,
antibodies, other polypeptides) and small molecules, must be de-
livered specifically to particular cell organelles in order to effi-
cientlyexerttheir therapeuticaction. Such deliveryisstillmainly an
unresolved problem,1 although some attempts have been made to
target mitochondria and nuclei using liposomes modified with
mitochondriotropic2 or nucleotropic agents.3
Lysosomes represent one of the important intracellular targets.
In a number of inherited human metabolic disorders, the lysoso-
mal storage disorders (LSDs), defects in the lysosomal enzymes,
lead to a progressive accumulation of unmetabolized substrates.4
LSD symptoms may vary widely depending on the particular
mutations inherited and the specific metabolic pathways affected;
Received: June 27, 2011
Revised:
August 24, 2011
Published: September 13, 2011
r
2011 American Chemical Society
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|

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Figure 1. Synthesis of derivatives of Neutral Red and rhodamine B.
administration of active enzymes.7 Also, among the potential
advances in LSD treatment is the so-called “chaperone therapy”,
also called “enzyme enhancement therapy”, aimed at the im-
properly folded mutant proteins, that are stabilized by the
exogenously administered small molecules, resulting in an in-
crease in the levels of functional enzyme. It was shown to be a
promising treatment strategy for several LSDs including Gaucher
disease, the GM1 and GM2 gangliosidoses, and Fabry disease.8
However, this procedure remains limited and expensive because
of the instability, poor delivery, and rapid degradation of the
administered enzymes.
The use of liposome-immobilized enzymes introduced more
than 30 years ago new opportunities for enzyme therapy,
especially in the treatment of diseases localized to liver cells
which are natural targets for liposomes.9 Still, these liposome-
based preparations are not in general clinical use for ERT, and a
clear need to sharply increase the efficiency of the delivery of
the liposomal enzymes to lysosomes inside cells still exists.
Improvement in liposome-based enzyme delivery can be achieved
by using liposomes specifically targeted to lysosomes.
Another important aspect related to the lysosomal targeting
for drug delivery is the involvement of lysosomes in the treat-
ment of cancer via the activation of the lysosome-dependent cell
death pathway.10 It has been proven that the moderate permea-
bilization of lysosomal membranes can result in cell apoptosis.
On the other hand, secreted lysosomal cathepsins degrade
protein components of the extracellular matrix and thus con-
tribute actively to tumor angiogenesis.11 Thus, it may be useful to
develop a delivery system with specific lysosomal targeting that
can provide inhibition of lysosomal enzymatic activity in cancer
cells or deliver lysosome-destabilizing agents that will cause
cancer cell apoptosis.
A large variety of small molecules have been identified which
specifically target and accumulate in lysosomes. Among them,
Neutral Red and rhodamine B are routinely used for the
visualization of lysosomes and other acidic organelles in live
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ARTICLE
cells.12,13 The commercially available rhodamine B octadecyl
ester is used widely to monitor membrane fusion14 and for the
study of lysosomal metabolism.15 As shown by Vult von Steyern
et al.,16 rhodamine B accumulates specifically in the lysosomes of
denervated skeletal muscle. Huth et al. have shown a preferential
build-up of the liposomes within lysosomes, when the liposomal
membrane was labeled with rhodamine B-phosphoethano-
lamine.17 With this in mind, we originally proved the general
concept of the possibility to prepare the lysosome-targeted
liposomes based on the use of commercially available Rhodamine
B octadecyl ester.18
The aim of the current work was to synthesize different ligands
based on Neutral Red (NR) and rhodamine B (RhB) with short
and long poly(ethylene glycol) spacers suitable for introduction
into the lipid bilayer and compare their ability to enhance the
lysosomal delivery of these ligand-modified liposomes loaded
with the model compound FITC-dextran.
for rhodamine B octadecyl ester (R18, as ligand III), which also
was commercially available.
Synthetic Procedures. Synthesis of Octadecanoic Acid
(8-(Dimethylamino)-3-methylphenazin-2-yl)amide (I, NRstear). I was
made starting with the Neutral Red acidÀbase indicator (NR),
according to procedure developed by Suzuki et al.21 Neutral Red
(120 mg) was dissolved in distilled water, and 0.1 M aqueous
NaOH was added. The desalted Neutral Red was extracted with
chloroform. The organic fraction was dried over Na2SO4 and
evaporated, resulting in 68.6 mg of free-base form of Neutral Red.
The intermediate product and 75 mg of triethylamine (TEA,
7.4 mmol) were dissolved together in 22.5 mL of tetrahydrofuran
(THF) and the mixture was stirred for 1 h at room temperature.
Stearoyl chloride (128 mg, 0.42 μmol) of was dissolved with
0.75 mL of THF and added to the reaction mixture, which was
stirred for 24 h at room temperature. The THF was evaporated
and the residue obtained was dissolved in 20 mL of chloroform.
The chloroform phase was washed twice with 20 mL of 1 N HCl
and 1 N NaOH, and once with the same deionized water, dried
over Na2SO4, and evaporated. The residue was purified by silica
gel column chromatography with n-hexaneÀethyl acetate (1:3,
Rf = 0.83) as the eluent to yield the final product (18.4 mg
’ MATERIALS AND METHODS
Materials. Egg phosphatidylcholine (ePC), cholesterol (Chol),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly-
ethylene glycol)-2000] (DSPE-PEG2k-amine) and 1,2-dioleoyl-
sn-glycero-3-phosphoethanolamine (DOPE) were purchased
from Avanti Polar Lipids (Alabaster AL, USA). Fluorescein
isothiocyanate-dextran (FD) with molecular weight (MW) of
4400, Neutral Red (NR), rhodamine B (RhB), rhodamine B
octadecyl ester (R18), rhodamine B isothiocyanate (RhB-ITC),
polyoxyethylene(MW 3400)-bis(p-nitrophenyl carbonate)
(PEG3.4k-(pNP)2), triethylamine (TEA), β-galactosidase (product
number G0413), protease inhibitor cocktail (PIC), phorbol
myristate acetate (PMA), and conduritol B epoxide (CBE) were
purchased from Sigma-Aldrich (St. Louis MO, USA). p-Nitro-
phenylcarbonyl-(polyethylene glycol-3400)-dioleylphosphatidyl-
ethanolamine (pNP-PEG3.4k-DOPE) was prepared and purified
according to the method in ref 19. Bio-Gel A-1.5 m sorbent
was purchased from Bio-Rad (Hercules CA, USA). Lysosome
Enrichment Kit, Coomassie-based protein assay kit, and
DyLight 350-conjugated goat antimouse IgG were obtained
from Pierce Biotechnology (Rockford IL, USA). Fluorescein
di-β-D-galactopyranoside (FDG), dodecanoylamino fluores-
cein di-β-D-galactopyranoside (C12FDG), and 5-(pentafluoro-
benzoylamino) fluorescein di-β-D-glucopyranoside (PFB-FDGlu)
were purchased from Invitrogen/Molecular Probes, Inc. (Eugene
OR, USA). Mouse monoclonal (H4B4) anti-lysosome-asso-
ciated membrane protein antibody (anti-Lamp2) was purchased
from Abcam (Cambridge MA, USA). Fluoromount-G mounting
medium was purchased from SouthernBiotech (Birmingham AL,
USA). Cell cultures of human epithelial cervical cancer CCL-2
(HeLa), mouse melanoma CRL-6475 (B16(F10)), mouse Lewis
lung carcinoma CRL-1642 (LLC), mouse embryo fibroblasts
CRL-1658 (NIH/3T3), rat myocardium myoblasts CRL-1446
(H9c2), and human monocytes (U-93720) were purchased from
the American Type Culture Collection (ATCC, Manassas VA,
USA). Cell culture media and supplements were from CellGro
(Kansas City MO, USA). All other chemicals and buffer compo-
nents were analytical-grade preparations. Distilled and deionized
water was used in all experiments.
1
of purified product, yield 13%). H NMR (CDCl3, 400 MHz)
δ 0.83À0.91 (m, 3H, ÀCH3), 1.22À1.43 (m, 30H, ÀCH2À),
1.53À1.61 (m, 2H, ÀCH2CH2COÀ), 1.75À1.84 (m, 3H,
ArÀCH3), 2.17 (s, 1H, ArÀNHÀAr), 3.19 (s, 6H, ArdN+-
(CH3)2), 3.96À4.00 (m, 1H, ArÀH), 4.19À4.24 (t, J = 6.05 Hz),
4.64 (s, 1H, Ar-H), 4.72 (s, 1H, ArÀH), 5.04 (s, 1H, ArÀH),
7.06 (d, J = 7.06 Hz, 1H, ArÀH), 7.39 (s, 1H, ArÀH), 7.53
(dd, J = 9.64 Hz, 1H, ArÀH), 7.70, 7.90 (s, 1H, ArÀH), 7.98
(d, J = 9.61 Hz, 1H, ArÀH).
Synthesis of DOPE-PEG3.4K-carbonyl 8-(Dimethylamino)-3-
methylphenazin-2-yl)amide (II, NRPEG). II was made from NR
by reaction with p-nitrophenylcarbonyl-(polyethylene glycol-
3400)-dioleylphosphatidylethanolamine (pNP-PEG3.4k-DOPE),
according to ref 19 using less basic conditions (pH 7.5) to avoid
precipitation of NR that occurs at pH above 8.0. pNP-PEG3.4k
-
DOPE (6 mg, 1.37 μmol) of was dissolved in 2 mL of phosphate
buffered saline with pH adjusted to 7.5 containing a 3-fold
excess of NR (1.2 mg; 4.15 μmol). After fully dissolving the
pNP-PEG3.4k-DOPE, the resultant mixture was sparged with
nitrogen and stirred for 24 h at room temperature. The excess of
NR was separated by dialysis against deionized water using
Spectra/Por cellulose ester dialysis membranes (Spectrum La-
boratories, Rancho Domingues, CA, USA) with a molecular
weight cutoff size of 2000 Da. Product was purified from the
nonconjugated DOPE-PEG3.4k-COOH by silica gel column
chromatography with chloroformÀmethanol (4:1, Rf = 0.65)
as the eluent to yield the final product (yield of purified product
1
2.3 mg, 38%). H NMR (CDCl3, 400 MHz): δ 0.87À0.89
(m, 6H, ÀCH3 of fatty acid chains), 1.00À1.50 (m, 44H,
ÀCH2À of fatty acid chains), 1.5À1.8 (s, 4H, ÀCOOÀ
CH2ÀCH2À), 1.90À2.14 (m, 8H, ÀCH2ÀCH2ÀCHd),
2.20À2.50 (m, 4H, CH2ÀCH2ÀCOOÀ of fatty acid chains),
3.18 (s, 6H, ArdN+(CH3)2), 3.20À3.50 (m, 2H, ÀCH2À
CH2ÀNH2CO), 3.53À3.78 (m, PEG chains), 3.78À3.85 (m,
2H, CH2ÀCH2ÀOÀ), 4.20À4.48 (m, 2H, ÀOÀCH2ÀCH-
(CH2À/OÀ), 5.07À5.18 (m, 1H, ÀOÀCH(CH2À)2), 5.19À
5.41 (m, 4H, ÀCHdCHÀ), 6.90À8.00 (m, 5H, ArÀH), 8.06À
8.32 (m, 1H, ArÀNHÀCO).
Conjugates of Neutral Red and Rhodamine B. The studied
cnojugates and paths of their synthesis are shown in Figure 1.
Substances were synthesized from commercially available
Neutral Red, rhodamine B, and rhodamine B isothiocyanate, except
Synthesis of Rhodamine B DSPE-PEG2k-amide (IV, RamPEG).
IV was made from rhodamine B by amidification with
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ARTICLE
DSPE-PEG2k-amide. Rhodamine B (7.7 mg, 16.1 μmol) was dis-
solved in 2 mL of dimethylformamide (DMF). Thionylchloride
(2.0 mg, 16.2 μmol; used as catalyst) was added to the rhodamine B
and the mixture was stirred at 55 °C for 40 min. DSPE-PEG2k-amine
(15 mg, 5.37 μmol) was added dropwise to the stirred solution of
activated rhodamine B, and the resulting mixture was stirred for
additional 7 h at 70 °C under a reverse condenser. The resulting
mixture was diluted with deionized water 1:5, and the nonreacted
rhodamine B was separated by dialysis against deionized water using
Spectra/Por cellulose ester dialysis membranes with cutoff size of
2000 Da. Product was purified from the nonconjugated DSPE-
PEG2k-amine by silica gel column chromatography with chloro-
formÀmethanol (4:1, Rf = 0.62) as the eluent to yield the final
product (yield of purified product 2.0 mg, 11%). 1H NMR (CDCl3,
400 MHz): δ 0.87À0.89 (t, 6H, ÀCH3 of fatty acid chains),
1.15À1.70 (m, 60H, ÀCH2À of fatty acid chains, + 6H, ArÀN-
(CH2CH3)2), 2.22À2.44 (q, 4H, CH2ÀCH2ÀCOOÀ of fatty acid
chains), 3.18À3.42 (m, 2H, OPO3ÀCH2ÀCH2ÀNH2COÀArd
N+(CH3)2), 3.20À3.50 (m, 2H, ÀCH2ÀCH2ÀNH2COÀ),
3.43À4.50 (m, ÀCH2ÀCH2ÀOÀ of PEG chains), 5.07À5.18
(m, 1H, ÀOÀCH(CH2À)2), 6.78 (m, 2H, ArÀH), 7.00 (m, 2H,
ArÀCONH2ÀCH2), 7.53 (td, 1H, ArÀH), 8.02 (m, 1H, ArÀH).
Synthesis of Rhodamine B 2-(DOPE-PEG3.4k-carbonyl)-ami-
noethyl Ester (V, RestPEG). V was synthesized starting with the
rhodamine B 2-aminoethyl ester, synthesized from rhodamine B
as described by Derkacheva et al.22 and generously provided by
the authors. Rhodamine B 2-aminoethyl ester (2.6 mg, 4.6 μmol)
was dissolved in 0.5 mL of methanol and activated by adding
0.47 mg (4.6 μmol) of TEA. pNP-PEG3.4k-DOPE (6 mg, 1.37 μmol)
was dissolved in an activated rhodamine B 2-aminoethyl ester
solution. The resultant mixture was sparged with nitrogen and
stirred for 24 h at room temperature. The excess of rhodamine B
2-aminoethyl ester was separated by dialysis against deionized
water using Spectra/Por cellulose ester dialysis membranes with
a cutoff size of 2000 Da. Product was purified from the non-
conjugated DOPE-PEG3.4k-COOH by silica gel column chro-
matography with chloroformÀmethanol (4:1, Rf = 0.52) as
the eluent to yield the final product (yield of purified product
cellulose ester dialysis membranes with a cutoff size of 2000 Da.
Product was purified from the nonconjugated DSPE-PEG2k-
amine by silica gel column chromatography with chloroformÀ
methanol (4:1, Rf = 0.56) as the eluent to yield the final product
(yield of purified product 5.8 mg, 33%). 1H NMR (CDCl3, 400
MHz): δ 0.77À0.99 (m, 6H, ÀCH3 of fatty acids), 1.03À1.51
(m, 6H, ArÀN(CH2CH3)2), 1.57À1.72 (m, 4H, ÀCH2COOÀ
of fatty acids), 2.01, 2.28À2.39 (m, 4H, ÀCH2COOÀ of fatty
acids), 3.21À3.35 (m, 2H, ÀCH2ÀCH2ÀNHCOOÀ) 3.41À
3.45 (m, 4H, ArÀN(CH2CH3)2), 3.50À4.25 (m, PEG chains),
5.00À5.18 (m,1H, ArÀH), 6.23À6.26 (dd, J = 6.26 Hz, 1H,
ArÀH), 6.60, 6.80À6.94 (m, 1H, ArÀH), 7.26 (m, 1H, ArÀH),
7.51À7.56 (s, 1H, ÀCH2ÀNHÀCSÀNHÀ), 7.69À7.74 (m,
1H, ArÀH), 7.81À7.87 (d, J = 7.83 Hz, 1H, ArÀH).
Preparation of Liposomal Formulations. Plain and ligand-
modified FD-loaded liposomes were obtained from lipid films
prepared by evaporating the solvent from mixtures of chloroform
solutions of egg phosphatidylcholine (ePC) and cholesterol
(7:3 molar ratio) in chloroform,19 optionally supplemented with
respective ligands (1 mol %) in ethanol or chloroform. After
removing the solvent on a rotary evaporator followed by freezeÀ
drying on a Freeze-Dry System Freezone 4.5 (Labconco, Kansas
City, MO), films were redissolved in chloroform and re-dried.
The films were hydrated by vigorous vortexing with phosphate-
buffered saline (PBS: 137 mM NaCl, 8 mM Na2HPO4, 2.7 mM
KCl, 1.5 mM KH2PO4, pH 7.4) or PBS supplemented with FD
(molecular weight 4400, 45 mg/mL) to produce a total lipid
content of 10 mg/mL. The hydrated lipid films were extruded
21 times through Nuclepore polycarbonate membranes with
200 nm pore size (Whatman, Clifton, NJ) using an Avanti Mini-
Extruder device (Avanti Polar Lipids, Alabaster, AL). Liposomes
were separated from non-incorporated FD by gel-filtration on a
BioGel 1.5 M column (0.7 Â 24 cm). Final lipid content in the
liposomal fraction was calculated from the ratio of volumes of
loaded formulation and the eluted fraction. Effective inclusion of
FD into liposomes was evaluated using the BioTek Synergy HT
microplate reader (BioTek, Winooski, VT) by measuring fluor-
escence (ex/em: 485/528 nm) of liposomes diluted 1/100 with
PBS (pH 7.4) supplied with 0.2% Triton X-100 (to avoid a
possible FRET effect), and calculating FD concentration accord-
ing to a preliminary calibration made under the same conditions,
with subsequent normalization to total lipid content.
Alternatively, for the evaluation of lysosomal uptake of the
liposomal load by flow cytometry, both plain and ligand-modified
liposomes were produced with a load of C12FDG, a 12-carbon
lipophilic variant of fluorescein di-β-D-galactopyranoside (FDG).23
C12FDG solubilized in DMSO was added to a mixture of ePC,
Chol, and ligand dissolved in chloroform, at 1.5% molar to total
lipids. After evaporation of the solvents, the lipid film was
redissolved in chloroform. After a second chloroform evapora-
tion and freezeÀdrying of the film, C12FDG-loaded liposomes
were prepared by hydration of the lipid film in PBS with
subsequent extrusion and separation from non-incorporated
ligands and C12FDG as described above. To estimate C12FDG
loading, C12FDG-liposomes were resuspended in PBS at 150 μg/mL
and incubated with or without recombinant β-galactosidase
(0.635 μg/mL) for 24 h at 37 °C. After the liposome dissolution
with 0.2% Triton X-100 (to avoid a possible FRET effect), the
fluorescent intensity of the C12-fluorescein produced by enzy-
matic hydrolysis of C12FDG was measured on a BioTek Synergy
HT microplate reader (ex/em: 485/528 nm) and normalized for
lipid content.
1
4.0 mg, 62%). H NMR (CDCl3, 400 MHz) δ 0.84À0.91
(m, 6H, ÀCH3 of fatty acids), 1.09À1.20 (m, 6H, ArÀ
N(CH2CH3)2), 1.23À1.37 (m, 32H, ÀCH2À of fatty acids),
1.40À1.43 (m, 6H, ArdN+(CH2CH3)2), 1.54À1.62, 1.66À
1.84 (m, 4H, ÀCH2ÀCH2ÀCOOÀ), 1.97À2.04 (m, 8H,
ÀCH2ÀCHdCHÀ), 2.24À2.31 (m, 6H, ÀCH2ÀCOÀ),
3.30À3.38 (m, 2H, ÀCH2ÀNHÀCOOÀ), 3.44À3.48 (m,
4H, ArÀN(CH2ÀCH3)2), 3.54 (s, 2H, ÀCH2ÀNHÀCOÀ),
3.58À3.84 (m, PEG chains), 3.92À4.00, 4.10À4.17 (m, 4H,
ÀPO4ÀCH2À), 4.18À4.22 (m, 2H, ÀCH2ÀOÀCOÀ),
4.36À4.42 (m, 4H, ÀOÀCH(CH2ÀOÀ)2), 5.18À5.23 (m,
2H, ArÀH and ÀOÀCH(CH2ÀOÀ)2), 5.29À5.38 (m, 4H,
ÀCHdCHÀ of fatty acids), 5.84À5.90 (s, 1H, ArÀH),
6.80À6.93 (m, 1H, ArÀH), 7.04À7.20 (m, 2H, ArÀH),
7.27À7.34 (m, 1H, ArÀH), 8.33 (dd, J = 27.89 Hz, 1H, ArÀH).
Synthesis of 6-(3-(DSPE-PEG2k)-thioureido) Rhodamine B
(VI, RtuPEG). VI was made starting with a rhodamine B
isothiocyanate (mix of isomers, from Sigma). Rhodamine B
isothiocyanate (7.7 mg, 16.1 μmol) was dissolved in 1 mL
of methanol, and the solution was used to dissolve 15 mg
(5.38 μmol) of DSPE-PEG2k-amine; the resultant mixture was
sparged with nitrogen and stirred for 48 h at room temperature.
After the reaction, the excess of Rhodamine B isothiocyanate was
separated by dialysis against deionized water using Spectra/Por
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ARTICLE
Characterization of Liposomes. Liposome size distribution
was determined by dynamic light scattering using a Coulter
N4MD Submicrometer Particle Size Analyzer (Beckman-
Coulter, Fullerton, CA). Zeta-potential of liposomal preparations
was measured at 25 °C in 0.1 mM KCl solution (diluted to a lipid
content of 0.2À0.3 μg/mL) using the Zeta-Plus Instrument
(Brookhaven Instruments, Holtsville, NY).
Cell Cultures. The fluorescent microscopy and subcellular
fractionation studies were done on HeLa cells, used for experi-
ments up to passage 16. For the cell experiments, HeLa cells were
grown at 37 °C at 5% CO2 and 95% humidity in Dulbecco
Modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL
streptomycin, and 2 mM glutamine. To reseed the cell cultures or
harvest them for subcellular fractionation or flow cytometry
studies, cells were detached by trypsinization with 0.5% trypsin
in PBS containing 0.025% EDTA. B16(F10), LLC, NIH/3T3,
and H9c2 cells were grown and treated according to the same
general protocol.
The U-937 monocytes, that were used for modeling the
phenotype of lysosomal enzyme deficiency disorder, were grown
in flasks suspended in RPMI-1640 medium supplemented with
10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and
2 mM glutamine. To induce the needed phenotype, the cells
were first seeded in 6-well plates 5 Â 106 cells per well in
complete RPMI-1640 and cultured for 48 h in the presence of
10nM phorbol myristate acetate (PMA; Sigma-Aldrich).24 The
mature attached macrophages were washed twice with the sterile
PBS and cultured for an additional 72 h in complete RPMI-1640,
supplied with 200 μM of conduritol B epoxide (CBE; Sigma-
Aldrich).25,26 To harvest the macrophages for flow cytometry
studies after the incubation with liposomes, the cells were detached
using 4 mg/mL lidocaine in PBS containing 0.025% EDTA.
Lysosomal β-Glucocerebrosidase Enzymatic Activity As-
say. The efficiency and stability of the induction of the lysosomal
enzyme deficiency phenotype in U-937 cells were evaluated by
the residual enzymatic activity of the lysosomal β-glucocrebro-
sidase after the treatment of the cells with PMA and CBE
immediately, as well as after different recovery times after the
removal of the CBE-containing medium replaced by the fresh
complete RPMI-1640 medium. For this evaluation, the cells were
seeded and subsequently treated with PMA and CBE as de-
scribed above. At specific time points after removal of the CBE,
the cells were detached using lidocaine and EDTA, dispersed
in 300 μL of PBS (pH 7.4), supplemented with 50 μM of specific
β-glucocerebrosidase substrate, 5-(pentafluorobenzoylamino)
fluorescein di-β-D-glucopyranoside (PFB-FDGlu), and incu-
bated for 1 h at 37 °C. Residual enzymatic activity, resulting
in the formation of the fluorescent fluorescein derivative, was
evaluated from the intensity of the green fluorescence deter-
mined at the emission wavelength of 520 nm (channel FL-1) by
flow cytometry, with macrophages not treated with the CBE as
a control.
complete liposome-free medium (denoted further in the text as
4 + 20 h incubation).
Immunofluorescence Microscopy. Intracellular trafficking
and localization of FD-loaded ligand-modified liposomes were
preliminarily tested using a fluorescent microscope Nikon
Eclipse E400 (Japan) equipped with a 6 V-20W halogen lamp,
filter blocks for blue (EX/DM/BA 330À380/400/435 nm), green
(465À495/505/515À565), and red (528À553/565/600À600)
spectral channels and 100Â oil-immersion objective. After the
initial evaluation, the samples obtained by incubation of cells with
liposomes that provided better colocalization with lysosomes
were re-evaluated using a Zeiss LSM 700 upright confocal micro-
scope (Thornwood, NY) equipped with a 63Â, 1.4-numerical
aperture plan-apochromat oil-immersion objective. HeLa cells
were grown on microscope coverslips in 6-well plates seeded in
complete DMEM medium at 104 cells per well. When cells reached
40À50% confluence, plain and ligand-modified liposomes were
added to cells in amounts providing an equal FD load for all formula-
tions with an average concentration of total lipids 50 μg/mL and
incubated as described. After incubation, cells were washed with
PBS and fixed with 4% paraformaldehyde in PBS (pH 7.4) for
15 min at room temperature (RT), followed by a PBS wash,
quenching with NaBH4 in PBS for 5 min, and another PBS wash.
The cells were then permeabilized by incubation with 0.2%
saponin and 1% BSA in PBS for 10 min at RT, washed three
times with a blocking solution (1% BSA in PBS, pH 7.4), and
kept for 30 min in the same buffer. The cells were stained with the
mouse antihuman Lamp2 mAb diluted with blocking solution
(1:50) for 60 min at RT, and washed five times with the blocking
solution. Visualization was achieved by cell incubation with
DyLight 350-conjugated goat antimouse IgG (1:100 dilution)
for 60 min at RT followed with five washes with the blocking
solution. Individual coverslips were mounted cell-side down onto
fresh glass slides with fluorescence-free glycerol-based mounting
medium (Fluoromount-G; Southern Biotechnology Associates,
Inc.) and studied under bright light and under epifluorescence
with UV, Rhodamine/TRITC, and Fluorescein/FITC filters.
Co-localization of liposomes and lysosomal markers was char-
acterized for individual cells (N = 8 to 20 for different liposomal
formulations) by Pearson’s correlation coefficient (PCC) and
Manders’ overlap coefficient (MOC),27 calculated using ImageJ 1.42
software (National Institutes of Health, Bethesda, MD) with
MBF bundle of plug-ins (McMaster University, Hamilton, ON,
Canada).
Evaluation of Lysosomal Delivery by Subcellular Fractio-
nation. Hela cells were grown to 90% confluence under
the above-described conditions and incubated with lipo-
somes added at amounts calculated to provide the same FD
load on cells for all formulations in complete DMEM for
4 + 20 h.
In order to isolate the lysosome-enriched fractions, the cells
((∼0.7À1.0) Â 108 cells per sample) were collected by trypsi-
nization and washed with ice-cold PBS. The cell pellet was
resuspended in 1 mL of the reagent A of the Lysosome Enrich-
ment Kit (Pierce Biotechnology, Rockford IL, USA) comple-
mented with 1% (v/v) of a protease inhibitor cocktail. After
2 min incubation on ice, the cells were lysed by sonication
(20 bursts, 3 s each, at 6 W). The cell lysate was treated with 1 mL
of the reagent B of the same kit. The mixture was gently shaken
several times and centrifuged at 500 g for 10 min at 4 °C to
pellet nuclei and any remaining intact cells. The postnuclear
supernatant was then adjusted with OptiPrep gradient medium
Interaction of Liposomes with Cells in Vitro. Cells grown to
the needed extent of confluence were incubated with liposomes
added at amounts calculated to provide the same FD or C12FDG
load to cells for every liposomal formulation, in their respective
serum-free media for 4 h, then washed twice with medium to
remove nonbound liposomes and used for studies. When re-
quired, cells treated with liposomes for 4 h were washed twice
with fresh medium to remove nonbound liposomes and incu-
bated for an additional chase period of 20 h at 37 °C in 5% CO2 in
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Table 1. Composition and Properties of Plain and Ligand-Modified Liposomal Formulations
composition (mol %)
C12FDG loading
liposomal formulation
ePC
Chol
ligand
C
12FDG
size ( SD (nm)
zeta ( SD (mV)
FD/lipids (mg/mg)
(% to Lip-C12FDG)
Lip-FD
70
70
70
70
70
70
70
70
70
70
70
70
70
70
30
30
30
30
30
30
30
30
30
30
30
30
30
30
—
—
1
—
1.5
—
—
—
—
—
1.5
—
—
1.5
—
—
1.5
200 ( 70
205 ( 85
210 ( 72
169 ( 47
153 ( 27
232 ( 51
240 ( 75
201 ( 40
208 ( 30
179 ( 68
180 ( 49
198 ( 42
179 ( 44
184 ( 65
À13.7 ( 6.5
À10.1 ( 1.2
À8.1 ( 4.9
À20.1 ( 4.6
À31.0 ( 8.2
11.5 ( 2.5
0.31 ( 0.16
—
—
100
Lip-C12FDG
Lip-NRstear-FD
Lip-NRPEG-FD
Lip-RestPEG-FD
Lip-R18
0.06 ( 0.01
0.17 ( 0.01
0.25 ( 0.02
—
—
1
—
1
—
1
—
Lip-R18-FD
1
À6.4 ( 4.6
À10.2 ( 1.0
À0.6 ( 3.1
À5.2 ( 0.5
À33.3 ( 4.4
À0.3 ( 1.3
À11.4 ( 7.3
À28.1 ( 3.5
0.28 ( 0.15
—
—
Lip-R18-C12FDG
Lip-RamPEG
1
112 ( 30
—
1
—
Lip-RamPEG-FD
Lip-RamPEG-C12FDG
Lip-RtuPEG
1
0.39 ( 0.12
—
—
1
145 ( 37
—
1
—
Lip-RtuPEG-FD
Lip-RtuPEG-C12FDG
1
0.26 ( 0.07
—
—
1
119 ( 29
(Pierce Biotechnology; 60% v/v solution of Iodixanol) to 15% of
Iodixanol, loaded onto the top of discontinuous density gradient
with the following steps from top to bottom: 17%, 20%, 23%,
27%, and 30% of Iodixanol (by respective dilutions of OptiPrep),
and subjected to ultracentrifugation at 145 000 g for 2 h at 4 °C
using Beckman Coulter Optima XL ultracentrifuge equipped
with SW41Ti swinging bucket rotor (Beckman Coulter, Krefeld,
Germany). A total of 5 resultant fractions were collected from the
top of the tube, diluted with PBS to the equal total volume of
6 mL, pelleted at 30 000 g for 30 min at 4 °C using Beckman
Coulter Optima TLX Tabletop equipped with TLA-100.3 fixed
angle rotor (Beckman Coulter, Krefeld, Germany), and then
resuspended in equal volumes of cold PBS. All collected fractions
were evaluated for lysosomal β-galactosidase activity and ana-
lyzed for protein, FD, and ligand content.
and trypsinized. The harvested cells were washed twice with PBS,
resuspended in 1 mL of ice-cold PBS, and their fluorescence
determined using a fluorescence-activated cell sorter (FACS).
Data acquisition was performed on a Becton Dickinson FACScan
(Becton Dickinson, San Jose, CA), and the data were ana-
lyzed using CellQuest software (Becton Dickinson). The
green and red fluorescence were determined respectively at
the emission wavelengths of 520 and 580 nm (channels FL-1 and
FL-2). To eliminate possible overlap of rhodamine fluorescence
with channel FL-1, the compensation between FL-1 and FL-2
channels was applied using the cells incubated with nonloaded
ligand-modified liposomes as an additional control.
The data were tested for statistical significance using the
Student’s paired t test. P-values were considered significant at
p e 0.05.
Protein concentration in each fraction was determined using a
Coomassie protein assay in triplicate by measuring absorption at
595 nm using BioTek Synergy HT microplate reader (BioTek
Instruments, Winooski, VT, USA). FD and ligand content in
fractions were determined by measuring the fluorescence inten-
sity of equal volumes of each fraction in triplicate, using the same
microplate reader at 485/528 nm and 530À590 nm (ex/em),
respectively. The quantity of FD in each subcellular fraction was
calculated using a calibration by standard FD solutions in PBS.
The lysosomal β-galactosidase activity was evaluated by dis-
persing 50 μL of each fraction in 150 μL of PBS supplemented
with 15 μM of fluorescein di-β-D-galactopyranoside (FDG) and
incubating the mix for 18 h at 37 °C. The intensity of fluores-
cence of the resultant product was measured using a microplate
reader at 485/528 nm.
Measured and calculated values for each ligand, FD, and
relative β-galactosidase activity were normalized to the protein
content, and their fractional distribution was calculated as a
percent of the normalized value in each fraction to the total sum
of all fractions.
Flow Cytometry Analysis. The binding of plain and
rhodamine-based ligand-modified liposomes to cultured cells was
evaluated by flow cytometry. Control (untreated) HeLa cells
or cells treated with liposomes were washed twice with DMEM
’ RESULTS
All liposomal formulations were characterized for size, zeta-
potential, and FD or relative C12FDG content (see Table 1).
In all cellular studies in vitro, liposomes were added for
incubation with cells at dilutions providing the same FD or
C12FDG load to cells for all liposomal formulations.
Intracellular Localization of Ligand-Modified Liposomes.
The lysosomotropic ability of ligands was initially tested by
fluorescent microscopy of HeLa cells incubated with plain or
ligand-modified FD-loaded liposomes, fixed and treated as
described in Methods.
Both derivatives of Neutral Red showed low or no colocaliza-
tion of liposomal FD with antibody-marked lysosomes (PCC/
MOC for Lip-NRstear-FD and Lip-NRPEG-FD liposomes were,
respectively, À0.032/0.007 and 0.403/0.567) and were excluded
from further study, while samples of cells incubated with
rhodamine B derivatives showed elevated colocalization of
liposomal load in comparison to plain Lip-FD liposomes under
the same conditions and were reanalyzed using the more accurate
method of confocal microscopy.
Figures 2 and 3 show representative confocal fluorescence
micrographs of HeLa cells incubated with plain or ligand-modified
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Figure 2. Confocal microscopy images of the intracellular distribution of FD-loaded liposomes modified with rhodamine B derivatives (incubation 4 h).
Rows: “Plain” — cells incubated with plain Lip-FD liposomes (as control), rows III to VI — cells incubated, respectively, with Lip-R18-FD, Lip-
RamPEG-FD, Lip-RestPEG-FD, and Lip-RtuPEG-FD liposomes. Columns: A (blue channel) — fluorescence of anti-Lamp2 antibody-stained
lysosomes; B (red channel) — fluorescence of RhB-derivative modified liposomes; cells incubated with plain liposomes were not observed under this
regime, respective position Plain-B is filled with a blank panel; C (green channel) — fluorescence of FITC-dextran; D — Overlay of A, B, and C images
with their respective DIC image. Scale bar = 20 μm.
FD-loaded liposomes for 4 h, without and with an additional
chase period of 20 h.
lipid load of 50 μg/mL and then kept at the same level
(9.8 μg/mL).
Estimation of colocalization of ligand-modified liposomes
with lysosome-specific marker (Table 2) after 4 h of incubation
showed that liposomes with rhodamine B derivatives provided a
much higher rate of lysosomal localization in comparison to such
for plain FD-loaded liposomes (MOP = 0.410; PCC = 0.619).
An additional chase period of 20 h after the 4 h incubation of
cells with liposomes generally decreased the colocalization of
FITC-dextran with anti-Lamp2 mAb-labeled lysosomes for both
plain and ligand-modified liposomes. However, lysosomal colo-
calization of FD delivered by rhodamine-conjugated liposomes
remained significantly higher than for plain Lip-FD liposomes
(Figure 3, Table 2).
Evaluation of Intracellular Distribution of FD-Loaded
Ligand-Modified Liposomes Incubated with Cultured HeLa
Cells, by Subcellular Fractionation. To study the organelle
distribution of ligand-modified liposomes and their FD load by
subcellular fractionation, the cultured HeLa cells were incu-
bated for 4 + 20 h with liposomes diluted to provide the same
FD load on cells, originally chosen to keep the average liposomal
As shown by β-galactosidase activity assay, about 60% of the
lysosomal β-galactosidase was concentrated in fraction 1 with a
minor part (about 20%) in fraction 2 and negligible amounts in
other fractions.
The fractional distribution of each ligand was calculated as a
percent of the fluorescence measured at 530/590 nm (ex/em)
for each fraction and normalized to the protein content to the
total sum of normalized fluorescent intensities of fractions 1 to 5.
Results shown in Figure 4A demonstrate that most of all
rhodamine B-based liposome-attached ligands accumulated in
the lysosome-enriched fractions (about 50% in fraction 1 or
60À70% in fractions 1 + 2).
The fractional distribution of absolute protein-normalized
content of FD is demonstrated in Figure 4B. The absolute
accumulation of FD in the first fraction compared with the value
for the same fraction from fractionation of cells incubated
with plain FD-loaded liposomes showed statistical significant
difference for Lip-R18-FD (p = 0.05) and Lip-RtuPEG-FD
(p = 0.005), with more moderate enhancement for Lip-RamPEG-FD
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Figure 3. Confocal microscopy images of the intracellular distribution of FD-loaded liposomes modified with rhodamine B derivatives (incubation 4 +
20 h). Rows: “Plain” — cells incubated with plain Lip-FD liposomes (as control), rows III to VI — cells incubated respectively with Lip-R18-FD, Lip-
RamPEG-FD, Lip-RestPEG-FD, and Lip-RtuPEG-FD liposomes. Columns: A (blue channel) — fluorescence of anti-Lamp2 antibody-stained
lysosomes; B (red channel) — fluorescence of RhB-derivative modified liposomes; cells incubated with plain liposomes were not observed under this
regime, respective position Plain-B is filled with blank panel; C (green channel) — fluorescence of FITC-dextran; D — Overlay of A, B, and C images
with their respective DIC image. Scale bar = 20 μm.
Table 2. Averaged Coefficients of Colocalization of Ligand-Modified Liposomes and Liposomal FD Load with Anti-Lamp2 mAb
Labeled Lysosomes, Determined from Micrographs Obtained by Confocal Immunofluorescent Microscopya
incubation 4 h
Ligand to anti-Lamp2 mAb FD to anti-Lamp2 mAb
incubation 4 + 20 h
Ligand to anti-Lamp2 mAb FD to anti-Lamp2 mAb
liposomal formulation
PCC
MOC
PCC
MOC
PCC
MOC
PCC
0.363
MOC
Lip-FD (Plain)
—
—
0.410
0.619
—
—
0.508
Lip-R18-FD (III)
0.769
0.637
0.726
0.816
0.864
0.790
0.816
0.877
0.756***
0.656**
0.604**
0.862***
0.857***
0.769**
0.745**
0.902***
0.708
0.523
0.544
0.695
0.791
0.693
0.711
0.805
0.726**
0.588*
0.613**
0.632**
0.808**
0.735**
0.760**
0.767**
Lip-RamPEG-FD (IV)
Lip-RestPEG-FD (V)
Lip-RtuPEG-FD (VI)
a PCC À Pearson’s correlation coefficient, MOC À Mander’s overlap coefficient. Values are presented as mean for N cells (N = 8 to 20 for different
liposomal formulations). * p < 0.05. ** p < 0.01. *** p < 0.001.
and absence of FD delivery enhancement for Lip-RestPEG-FD
liposomes (Table 3). The observed increase of FD fluorescence
was not determined by a possible overlap of RhB and FITC
spectral channels as was shown previously for liposomes mod-
ified with rhodamine B octadecyl ester18 by comparing the green
channel fluorescence of fractions obtained from nontreated cells
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or cells incubated with ligand-modified liposomes without a
FD load.
Thus, liposomes modified with R18, RtuPEG, and RamPEG
which demonstrated enhancement of lysosomal delivery of their
FD load were chosen for further studies.
Flow Cytometric Evaluation of General Uptake of Ligand-
Modified Liposomes by Cell. The general interaction of cells
with FD-loaded liposomes modified with rhodamine-based
ligands (R18, RtuPEG, and RamPEG) was evaluated with tumor
(HeLa and B16(F10)) and normal (NIH/3T3 and H9c2) cell
lines using the flow cytometry method.
Liposomes were added to cells in amounts calculated to
provide the same total FD content in cell medium (originally
chosen to have average lipid load on cells 100 μg/mL, then kept
the same) of 36.44 μg/mL. After incubation with liposomes for
4 h, cells were harvested as described in Methods and used for
measurements. Since used cell lines had different autofluores-
cence, results were presented as relative FL-1 fluorescence values
normalized to the value of FL1 for nontreated cells.
Results are shown in Table 4. In most cases, cells incubated
with FD-loaded liposomes surface-modified with rhodamine B
derivative ligands showed a significant increase of FL-1 channel
fluorescence in comparison to plain FD-loaded liposomes.
Evaluation of Lysosomal Targeting by Flow Cytometry
Using Liposomes Loaded with C12FDG. To additionally con-
firm and compare the ability of liposomes modified with rhoda-
mine B derivatives to specifically target lysosomes, we evaluated
their specific lysosomal targeting by flow cytometric analysis of
live cells using liposomes loaded with C12FDG, a lipophilic
substrate for the lysosomal β-galactosidase, as was proposed
in ref 18, assuming FL1 fluorescence will be emitted only
by product of its hydrolysis by lysosomal β-galactosidase
(C12-fluorescein).
The plain and ligand-modified liposomal formulations were
prepared, loaded with C12FDG and characterized as described
in Methods. Despite the introduction of C12FDG into the
lipid bilayer of the liposomes, its effective load (evaluated by
β-galactosidase cleavage of C12FDG localized on the surface of
the intact liposomes) varied between plain and ligand-modified
liposomes. Because of this, we studied the lysosomal uptake of
ligand-modified liposomes with intact cultured cells, adding the
liposomal formulations to cells in amounts that provided the
same total load of C12FDG as plain C12FDG-loaded liposomes at
concentration of 200 μg/mL by total lipids.
The lysosomal uptake was evaluated on a number of cultured
tumor and normal cell lines including HeLa, B16(F10) melanoma,
LLC carcinoma, NIH/3T3 fibroblasts, and H9c2 myoblasts.
Considering the general purpose of the study, the enhance-
ment of the lysosomal load was also specially evaluated on U-937
monocytes matured to macrophages with induced phenotype of
lysosomal enzymatic deficiency disorder.24À26 The acquisition of
the lysosomal enzyme deficiency phenotype by the cells was
confirmed by evaluation of the residual lysosomal β-glucocer-
ebrosidase enzymatic activity (see Methods) immediately as well
as 24, 48, and 72 h after the removal of the CBE-containing
medium. It was observed that, immediately after the removal of
the CBE, the cells retained about 35% of the β-glucocrebrosidase
activity of the control (macrophages without CBE treatment).
Figure 4. Relative fractional distribution of protein-normalized fluores-
cence of rhodamine B derivatives (A) and fractional distribution of
protein-normalized FD content (B) in fractionated HeLa cells incubated
with different ligand-modified liposomes for 4 + 20 h (number of
experiments n = 6).
Table 3. Characterization or Intracellular Distribution of Liposomal FD Delivered by Different Ligand-Modified Liposomes
paired t test of FD content
relative content
of FD in fraction
1 (% from total
sum of fractions
1 to 5)
in fraction 1 against the
fraction 1 of sample
incubated with plain
FD-loaded liposomes
(P-value)
content of FD
in fraction 1
(ng/mg
ratio of FD
contents in fraction 1
and loaded
content of FD
in loaded lysate,
(ng/mg proteins)
liposomal formulation
proteins)
nuclei-free lysate
Lip-FD
27.8 ( 8.5
43.1 ( 20.2
35.9 ( 18.3
40.1 ( 23.8
38.7 ( 12.9
73.2 ( 22.5
106.7 ( 50.0
91.6 ( 46.5
72.3 ( 42.8
132.8 ( 44.3
35.9 ( 9.7
45.2 ( 11.5
36.3 ( 12.9
26.8 ( 18.7
47.6 ( 16.8
—
2.04
2.36
2.52
2.70
2.79
Lip-R18-FD
0.05
0.19
0.94
0.005
Lip-RamPEG-FD
Lip-RestPEG-FD
Lip-RtuPEG-FD
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Table 4. General FD Delivery to Cells by Ligand-Modified Liposomes
cell line
liposomal formulation
HeLa
B16(F10)
NIH/3T3
H9c2
Ratios of Averaged Normalized General Uptake of FD by Different Cells (By Values of Fluorescence Intensity in FL-1 Channel) Provided by
Ligand-Modified Liposomes to the Value for Plain Liposomesa
Lip-R18-FD
1.73
1.65
1.67
1.55
1.38
1.97
1.25
0.90
1.16
1.40
1.24
1.43
Lip-RamPEG-FD
Lip-RtuPEG-FD
Paired t test of Averaged Normalized General Uptake of FD Provided by Ligand-Modified Liposomes in Comparison with the Value for Plain Liposomes (P-value)
Lip-R18-FD
0.0001
0.0001
0.0003
0.013
0.002
0.002
0.036
0.237
0.006
0.03
0.02
0.05
Lip-RamPEG-FD
Lip-RtuPEG-FD
a Due to different autofluorscence of cell lines, values of FL-1 fluorescence of cells incubated with liposomes were used normalized to the respective
values for nontreated cells. Number of experiments n = 3 to 6.
Table 5. Lysosomal Uptake of C12FDG Delivered to Cells by Ligand-Modified Liposomes
cell line
liposomal formulation
HeLa
B16(F10)
LLC
NIH/3T3
H9c2
U-937
Ratios of Averaged Normalized Lysosomal Uptake of C12FDG by Different Cells (By Values of Fluorescence Intensity in
FL-1 Channel) Provided by Ligand-Modified Liposomes to the Value for Plain Liposomes a
Lip-R18-C12FDG
1.17
1.27
1.12
1.09
1.02
1.26
1.47
1.15
1.29
1.04
1.22
1.13
1.15
1.12
1.16
1.35
1.75
1.55
Lip-RamPEG-C12FDG
Lip-RtuPEG-C12FDG
Paired t test of Averaged Normalized Lysosomal Uptake of C12FDG Provided by Ligand-Modified Liposomes in Comparison
with the Value for Plain Liposomes (P-value)
Lip-R18-C12FDG
0.013
0.017
0.035
0.024
0.38
0.00002
0.001
0.073
0.001
0.005
0.014
0.020
0.007
0.149
Lip-RamPEG-C12FDG
0.0074
Lip-RtuPEG-C12FDG
0.003
0.021
0.0004
a Due to different autofluorscence of cell lines, values of FL1 fluorescence of cells incubated with liposomes were used normalized to the respective values
for nontreated cells. Number of experiments n = 3 to 6.
The enzymatic activity recovered to 47À49% of control by
24À48 h post-CBE removal and to 62% after 72 h. All experi-
ments with flow cytometric evaluation of the lysosomal delivery
of liposomal load with activated U-937 cells were performed
within a short time range after induction of the lysosomal
deficiency by adding the liposome-containing medium immedi-
ately after removal of CBE.
The results presented in Table 5 show that, for all tested cell
lines, the cells incubated with C12FDG-loaded liposomes mod-
ified with rhodamine B derivatives used demonstrated signifi-
cantly increased C12FITC fluorescence compared to the cells
treated with the plain liposomes, for at least two out of three
ligands. The extent of the enhancement of the lysosomal target-
ing of the model substance (and the ligand providing the best
enhancement) differed among cell lines, achieving the maximum
values for cells with the induced phenotype of lysosomal
disorder.
delivery of a liposome-entrapped model substance (FITC-dex-
tran with a relatively high molecular mass of 4400 Da) to these
organelles.
In the current study, we attempted to develop and study other
potential lysotropic ligands based on commercially available
rhodamine B and Neutral Red, routinely used for the visualiza-
tion of lysosomes in live cells.12,13 A number of liposomal
formulations with a ligand-modified surface were prepared using
synthesized RhB and NR derivatives and loaded with FD. Their
interaction with cultured cells and the ability to deliver FD to
lysosomes were investigated using fluorescent microscopy, sub-
cellular fractionation, and flow cytometry in comparison to plain
FD-loaded liposomes and previously studied liposomes modified
with rhodamine B octadecyl ester.
The fluorescent microscopy studies have demonstrated that
liposomes modified with RhB derivatives generally show an
elevated colocalization of both the liposome-attached ligand
and loaded FD with a specific lysosomal marker Lamp2
(visualized using anti-Lamp2 monoclonal antibodies), while
liposomes modified with derivatives of NR show much less effect.
Additional study by subcellular fractionation of cells incubated with
FD-loaded liposomes modified with rhodamine B derivatives and
comparison of the fluorescence of lysosome-enriched fractions
’ DISCUSSION
In our earlier paper,18 we demonstrated that liposomes
modified with rhodamine B octadecyl ester acquire the ability
to specifically target lysosomes and allow for an increased
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with those for the cells treated with control nonmodified liposomes
confirmed the lysosome-targeting ability of three rhodamine B de-
rivatives.
Since methods such as cell staining for microscopy or sub-
cellular fractionation of cell lysates are considered disruptive, this
tendency was checked on intact live cells of several cell lines by
flow cytometry, proving both an increased general accumulation
of rhodamine-modified liposomes by cells and their increased
delivery into the lysosomes, thus confirming the data obtained by
microscopy and subcellular fractionation methods.
The evaluation of the specific lysosomal delivery of a model
drug substance by liposomes modified with rhodamine B deri-
vatives showed the best results for the cells with an induced pheno-
type of a lysosomal enzyme deficiencydisorder(theenhancement of
the lysosomal uptake of C12FDG by different rhodamine B-based
conjugates in the range 35À75% in comparison to nonmodified
liposomes). Still, it should be noted that the development of drug
delivery systems for specific LSDs, such as Gaucher disease, GM1
and GM2 gangliosidoses, and Fabry disease, requires evaluation
of the efficiency of lysosomal delivery by those systems using the
respective specific cell lines and animal models.
TEA, triethylamine; PIC, protease inhibitor cocktail; PMA, phor-
bol myristate acetate; CBE, conduritol B epoxide;pNP-PEG3.4k-
DOPE, p-nitrophenylcarbonyl-(polyethylene glycol-3400)-dioleyl-
phosphatidylethanolamine; mAb, monoclonal antibody; IgG,
immunoglobulin G; FDG, fluorescein di-β-D-galactopyranoside;
C12FDG, dodecanoylamino fluorescein di-β-D-galactopyrano-
side; PFB-FDGlu, 5-(pentafluorobenzoylamino) fluorescein di-
β-D-glucopyranoside; anti-Lamp2, mouse monoclonal (H4B4)
antilysosome-associated membrane protein antibody; THF, tet-
rahydrofuran; NRstear, octadecanoic acid (8-(dimethylamino)-3-
methylphenazin-2-yl)amide; NRPEG, DOPE-PEG3.4k-carbonyl
8-(dimethylamino)-3-methylphenazin-2-yl)amide; RamPEG,
rhodamine B DSPE-PEG2k-amide; RestPEG, rhodamine B 2-
(DOPE-PEG3.4k-carbonyl)-aminoethyl ester; RtuPEG, 6-(3-(DSPE-
PEG2k)-thioureido) rhodamine B; FBS, fetal bovine serum; PBS,
phosphate buffered saline; RT, room temperature; BSA, bovine
serum albumin; DIC, differential interference contrast; PCC,
Pearson’s correlation coefficient; MOC, Mander’s overlap coeffi-
cient; FACS, fluorescence-activated cell sorter; Lip, liposomes;
EDTA, ethylenediamintetraacetic acid; SD, standard deviation
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’ CONCLUSION
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The study has demonstrated that modification of the liposo-
mal surface with Rhodamine B-based ligands increases the
delivery of liposomal FITC-dextran to lysosomes. Liposomes
modified with two of the synthesized ligands—rhodamine B
DSPE-PEG2k-amide and 6-(3-(DSPE-PEG2k)-thioureido) rho-
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Corresponding Author
*Vladimir P. Torchilin, Ph.D., D.Sc., Professor and Director,
Center for Pharmaceutical Biotechnology and Nanomedicine,
Northeastern University, 312 Mugar Hall, 360 Huntington Ave.,
Boston, MA 02115. E-mail: v.torchilin@neu.edu. Phone: 617-
373-3206. Fax: 617-373-4201.
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’ ACKNOWLEDGMENT
The research was funded by NIH grant RO1 CA 128486 to
Vladimir P. Torchilin.
’ ABBREVIATIONS
ePC, egg phosphatidylcholine; Chol, cholesterol; DSPE-PEG2k-
amine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino-
(polyethylene glycol)-2000]; DOPE, 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine; FD, fluorescein isothiocyanate-dextran;
NR, Neutral Red; RhB, rhodamine B; R18, rhodamine B
octadecyl ester; RhB-ITC, rhodamine B isothiocyanate; PEG3.4k
(pNP)2, polyoxyethylene(MW 3400)-bis(p-nitrophenyl carbonate;
-
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Bioconjugate Chemistry
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