Targeted PLGA nano- but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro.

Abstract

Vaccine efficacy is strongly enhanced by antibody-mediated targeting of vaccine components to dendritic cells (DCs), which are professional antigen presenting cells. However, the options to link antigens or immune modulators to a single antibody are limited. Here, we engineered versatile nano- and micrometer-sized slow-release vaccine delivery vehicles that specifically target human DCs to overcome this limitation. The nano- (NPs) and microparticles (MPs), with diameters of approximately 200nm and 2microm, consist of a PLGA core coated with a polyethylene glycol-lipid layer carrying the humanized targeting antibody hD1, which does not interact with complement or Fc receptors and recognizes the human C-type lectin receptor DC-SIGN on DCs. We studied how these particles interact with human DCs and blood cells, as well as the kinetics of PLGA-encapsulated antigen degradation within DCs. Encapsulation of antigen resulted in almost 38% degradation for both NPs and MPs 6days after particle ingestion by DCs, compared to 94% when nonencapsulated, soluble antigen was used. In contrast to the MPs, which were taken up rather nonspecifically, the NPs effectively targeted human DCs. Consequently, targeted delivery only improved antigen presentation of NPs and induced antigen-dependent T cell responses at 10-100 fold lower concentrations than nontargeted NPs.

Full text

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Targeted PLGA nano- but not microparticles specifically deliver antigen to human
dendritic cells via DC-SIGN in vitro
Luis J. Cruza,1, Paul J. Tackena,1, Remco Fokkinkb, Ben Joostena, Martien Cohen Stuartb, Fernando Albericioc,
Ruurd Torensmaa, Carl G. Figdora,⁎
aDepartment of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands
bLaboratory of Physical Chemistry and Colloid Science, Wageningen University, Wageningen, The Netherlands
cInstitute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, Barcelona, Spain
a b s t r a c ta r t i c l ei n f o
Article history:
Received 16 December 2009
Accepted 5 February 2010
Available online 13 February 2010
Keywords:
Delivery vehicle
Nanoparticle
Microparticle
Targeting
Immunogenicity
Biodegradation
PLGA
Vaccine efficacy is strongly enhanced by antibody-mediated targeting of vaccine components to dendritic
cells (DCs), which are professional antigen presenting cells. However, the options to link antigens or immune
modulators to a single antibody are limited. Here, we engineered versatile nano- and micrometer-sized
slow-release vaccine delivery vehicles that specifically target human DCs to overcome this limitation. The
nano- (NPs) and microparticles (MPs), with diameters of approximately 200 nm and 2 µm, consist of a PLGA
core coated with a polyethylene glycol-lipid layer carrying the humanized targeting antibody hD1, which
does not interact with complement or Fc receptors and recognizes the human C-type lectin receptor DC-SIGN
on DCs. We studied how these particles interact with human DCs and blood cells, as well as the kinetics of
PLGA-encapsulated antigen degradation within DCs. Encapsulation of antigen resulted in almost 38%
degradation for both NPs and MPs 6 days after particle ingestion by DCs, compared to 94% when
nonencapsulated, soluble antigen was used. In contrast to the MPs, which were taken up rather
nonspecifically, the NPs effectively targeted human DCs. Consequently, targeted delivery only improved
antigen presentation of NPs and induced antigen-dependent T cell responses at 10–100 fold lower
concentrations than nontargeted NPs.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Dendritic cells (DCs) are professional antigen presenting cells that
play a key role in regulating antigen-specific immunity. DCs roam our
bodies in a constant search for foreign intruders, such as bacteria and
viruses. Ingestion of these pathogens results in migration of DCs from
the periphery to the lymph nodes, where processed pathogen-derived
antigens are presented to T cells. This interaction between DCs and T
cellscontrolsthetypeandmagnitudeoftheresultingimmuneresponse
[1].Recently,preclinicalandclinical studiesexploitedDCsinanattempt
toimprovevaccineefficacy.Severalstudiesshowedthatvaccineefficacy
is strongly enhanced by antibody-mediated targeting of vaccine
components to specific surface receptors on DCs in vivo [2]. However,
the options to link a single antibody to multiple vaccine components,
such as (multiple) antigens and immune modulators, are limited.
Therefore, it was postulated that future targeted DC-based vaccination
strategies might benefit from encapsulation of vaccine components
withinnanoparticles(NPs)ormicroparticles(MPs)[2,3].Conjugationof
antibodies to slow-release particles allows targeting of relatively large
amounts of antigen to DCs and could provide long-lasting release of
antigens within the cell. Furthermore, slow-release systems allow for
co-encapsulation of immunomodulatory molecules that enhance the
efficacy of vaccination [3–6].
Previous studies have shown that delivery of particulate vaccine
carriers to DCs by passive or active targeting strategies enhances
immune responses in mouse models [7–9]. The aim of this study was
to translate these strategies to a human setting by generating targeted
NP and MP slow-release vaccines using components, or derivatives
thereof, that are currently being used in the clinic and to determine
the efficacy of these differentially-sized particles to specifically target
human DCs. The challenge faced is to create biocompatible and
biodegradable slow-release vaccine delivery vehicles with a size and
composition that allow effective targeting of DCs. MPs are likely to be
handled differently from NPs by human DCs since they enter the cell
via distinct endocytic mechanisms.
Journal of Controlled Release 144 (2010) 118–126
Abbreviations: CF, carboxyfluorescein; DC, dendritic cell; DIEA, N,N-diisopropy-
lethylamine; DLS, dynamic light scattering; DMF, N,N-dimethylformamide; FDA, food
and drug administration; HOBt, 1-hydroxybenzotriazole; MP, microparticle; NP,
nanoparticle; PBL, peripheral blood lymphocyte; PBMC, peripheral blood mononuclear
cell; PEG, polyethylene glycol; PVA, polyvinyl alcohol; TT, tetanus toxoid.
⁎ Corresponding author. Department of Tumor Immunology, Nijmegen Centre for
Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Postbox 9101,
6500 HB Nijmegen, The Netherlands. Tel.: +31 24 3617600; fax: +31 24 3540339.
E-mail address: c.figdor@ncmls.ru.nl (C.G. Figdor).
1Authors contributed equally to this paper.
0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2010.02.013
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel

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In this study, PLGA is used as a biodegradable slow-release
polymer that allows for effective encapsulation of all kinds of drugs
and antigens [5,6,10]. PLGA is used in a host of Food and Drug
Administration (FDA)-approved therapeutic devices and has a long
safety record [3]. A wide variety of biodegradable PLGA particles
generated in various ways are utilized for sustained delivery of
bioactive molecules as an injectable depot formulation. For vaccina-
tion purposes, antigen is mostly formulated in PLGA MPs that are
ingested by phagocytic cells following administration, resulting in
particle degradation and antigen presentation. However, PLGA NPs
are also used as carriers of pathogen- or tumor-derived antigens and
induce antigen-specific immune responses in mouse models [11–13].
PLGA particles release their contents within days, weeks or even
months, depending on particle composition and the solvent, which is
usually water or phosphate buffered saline [10]. However, surpris-
ingly little is known about PLGA release kinetics within the living cell.
In addition to slow-release kinetics, the targeted particles should
display‘stealth’-like propertiestoavoidrapidnonspecific clearanceby
cells other than DCs to reach their target site. To date, the most
effective mitigation of rapid particle clearance is accomplished by
surface grafting polyethylene glycol (PEG) to build a sterically
repulsive shield that protects the particle from nonspecific interac-
tions. PEG-coated particles display a prolonged circulation half-life in
blood and reduced rate of uptake by the liver when compared to non-
coated particles [14,15]. Here, specific targeting of particles towards
DCs is accomplished by grafting of a DC-specific antibody directed
against the C-type lectin DC-SIGN onto a lipid-PEG layer on the
particle surface. Receptors of the C-type lectin family represent
promising targets for in vivo DC vaccination strategies. DC-SIGN is one
of the most DC-restricted C-type lectin receptors in humans and
mediates antigen presentation, two key features that are required for
specific delivery of antigens to DCs [2]. We have previously generated
a mouse antibody against human DC-SIGN that effectively targets DCs
in a primate model [16]. A humanized form of this antibody (hD1)
effectively targets conjugated antigen to DC-SIGN in vitro and in vivo,
thereby enhancing antigen presentation [17,18]. The hD1 antibody is
low-immunogenic and its Fc tail does not react with complement or
Fc receptors. Similar antibodies, such as the Eculizumab antibody,
which is used as a negative control for hD1 in our studies, are already
approved by the FDA for treatment of patients and are currently in use
in the clinic [19,20].
In this study, we determined the kinetics and subcellular location
of PLGA-encapsulated antigen degradation by DCs. In addition, we
engineered versatile PLGA-based, DC-SIGN-targeted and PEG-coated
MP and NP vaccines and compared these differentially-sized particles
with respect to specific uptake and processing by human DCs and
nonspecific interactions with human blood cells. As far as we know,
this is the first study assessing antibody-mediated delivery of PLGA
NPs and MPs to human DCs.
2. Materials and methods
2.1. Materials
PLGA (Resomer RG 502 H, lactide:glycolide molar ratio 48:52 to
52:48)waspurchasedfromBoehringerIngelheim,Germany.Solventsfor
peptide synthesis and PLGA preparation (dichloromethane, 2-propanol,
N,N′-dimethylformamide and ethyl acetate) were obtained from Merck
(Germany). SATP (N-hydroxysuccinimide (NHS) esters of S-acetylthioa-
cetic and propionic acid) reagent was obtained from Pierce (USA).
Polyvinyl alcohol (PVA) was purchased from Sigma (USA). Lipids
purchased from Avanti Polar Lipids (USA) include 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[maleimide(PEG)2000] (ammoni-
um salt), DSPE-PEG(2000)maleimide and 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[methoxy(PEG)-2000] (ammonium salt)
(mPEG 2000 PE). The phospholipid 2-(4,4-difluoro-5-(4-phenyl-1, 3-
butadienyl)-4-bora 3a, 4a-diaza-s-indacene-3-pentanoyl)-1-hexadeca-
noyl-sn-glycero-3-phosphocholine and DQ Green BSA (DQ-BSA) were
from Molecular Probes (The Netherlands).
The followingantibodieswere used:anti-humanHLA-DR/DPclone
Q5/13 [21], Alexa Fluor 647-labeled goat anti-human and goat anti-
mouse IgG (Molecular Probes). Humanized anti-DC-SIGN (hD1) and
its isotype control (h5G1.1) were described before [18,19] and were
kindly provided by Alexion Pharmaceuticals (USA).
2.2. Peptide synthesis
The TT epitope comprises the 830–844 region of tetanus toxoid. 5
(6)-carboxyfluorescein (CF) and TT epitopes were linked by a Lys-Lys
cathepsin-like cleavage site. The peptide antigen (FITC-KKQYIKANSK-
FIGITEL-NH2) with the extreme N-terminal modified FITC was
synthesized manually according to standard protocols of solid-phase
peptide synthesis, using the Fmoc/tert-butyl strategy. Side chain
protecting groups used were tert-butyl for Glu, Thr, Ser and Tyr; trityl
for Gln and Asn and Boc for Lys. Nα-Fmoc-protected amino acids were
incorporated using 1-hydroxybenzotriazole (HOBt) and N,N′-diiso-
propylcarbodiimide in N,N-dimethylformamide (DMF) for 1.5 h and
monitoredbyKaiserninhydrintests.Whenrequired,recouplingswere
performed with 1H-benzotriazolium,1-[bis(dimethylamino)methy-
lene]-,tetrafluoroborate(1-),3-oxide, HOBt and N,N-diisopropylethy-
lamine (DIEA) in DMF. The Fmoc-protecting group was cleaved with a
20% piperidine solution in DMF (2×10 min). After completion of the
sequence, the peptide-resin was labeled with FITC. CF (5 equiv.), 7-
azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluoro-
phosphate (5 equiv.), HOBt (5 equiv.), and DIEA (10 equiv.) were
dissolved in a 9:1 mixture of DMF:dichloromethane, pre-activated for
10 min and then added to the peptide-resin and stirred for 12 h. The
FITC-TT peptide was cleaved in trifluoroacetic acid, triisopropylsilane
and water (95:2.5:2.5) for 1 h.
2.3. Microparticle preparation
Microparticles (MPs) with entrapped FITC-TT peptide or DQ-BSA
were prepared using the w/o/w emulsion solvent evaporation–
extraction method. In brief, 50 mg of PLGA was emulsified under
sonification in 1 mL of methylene chloride containing FITC-TT peptide
(2 mgin100 µLinwater)orDQ-BSA(2 mgin100 µLinwater)for60 s.
This first emulsion was rapidly added to 1 mL of 1% polyvinyl alcohol/
7% ethyl acetate in distillated water. In some preparations 1% mPEG
2000 PE and 0.5% DSPE-PEG(2000)maleimide or the phospholipid 2-
(4,4-difluoro-5-(4-phenyl-1, 3-butadienyl)-4-bora 3a, 4a-diaza-s-
indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocho-
line were added and vortexed vigorously for 30 s. This solution was
added to 200 mL of 0.3% PVA/7% of ethyl acetate in distilled water and
stirred overnight to evaporate ethyl acetate. Next, the MPs are rinsed
three times with distillated water through centrifugation at 3000g at
4 °C. Finally, the MPs were lyophilized.
2.4. Nanoparticle preparation
NPs with entrapped FITC-TT peptide or DQ-BSA were prepared
using an o/w emulsion and solvent evaporation–extraction method.
In brief, 50 mg of PLGA in 3 mL of methylene chloride containing FITC-
TT peptide (2 mg in 100 µL in water) or DQ-BSA (2 mg in 100 µL in
water) was added dropwise to 25 mL of aqueous 2% PVA and
emulsified for 90 s using a sonicator (Branson, sonofier 250). A
combination of 6 mg DSPE-PEG(2000)maleimide and 6 mg mPEG
2000 PE were dissolved in methylene chloride and added to the vial.
The methylene chloride was removed by a stream of nitrogen gas.
Subsequently, the emulsion was rapidly added to the vial containing
the lipids and the solution was homogenized for 30 s using a
sonicator. Following overnight evaporation of the solvent at 4 °C,
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the NPs were collected by ultracentrifugation at 60,000g for 30 min,
washed three times with distillated water and lyophilized.
2.5. Quantifying antigen content of MPs and NPs
Antigenentrapmentefficiency was determined by digesting10 mg
NPs or MPs in 3 mL 0.8 N NaOH overnight at 37 °C. FITC-TT content
was determined by measuring fluorescence relative to a standard
curve (485 nm excitation and 530 nm emission) using the CytoFluorII
(Applied Biosystems, USA). Entrapment efficiency for DQ-BSA was
determined by measuring protein content of digested particles using
Coomassie Plus Protein Assay Reagent (Pierce) according to the
manufacturer's protocol. Antigen entrapment efficiency was deter-
mined by dividing the amount of the antigen encapsulated by the
theoretical amount assuming all was encapsulated.
2.6. Scanning electron microscopy (SEM)
NP and MP morphology was studied by SEM. Particles were
transferred to metallic stubs with double-sided conductive tape.
Subsequently, samples were ion-coated with gold with a sputter
coater (EDWARDS, Scan coat, United Kingdom) for 180 s in a vacuum
at a current intensity of 40 mA. Samples were analyzed on a Jeol JSM-
6310 scanning electron microscope.
2.7. Dynamic light scattering and zeta potential
Dynamic light scattering (DLS) measurements on MPs and NPs
were performed on an ALV light-scattering instrument equipped with
an ALV5000/60X0 Multiple Tau Correlator and an Oxxius SLIM-532
150 mW DPSS laser operated at a wavelength of 532 nm. A refractive
index matching bath of filtered cis-decalin surrounded the cylindrical
scattering cell, and the temperature was controlled at 21.5±0.3 °C
using a Haake F3-K thermostat. For each sample the auto-correlation
function, g2(τ), was recorded ten times at a detection angle of 90°. For
each measurement the diffusion coefficient, D, was determined using
the 2nd order cumulant and the corresponding particle diameter was
calculated assuming that the particles were spherical in shape. Zeta
potential measurements were performed on NPs and MPs diluted in
distilled deionized water using a Malvern ZetaSizer 2000 (UK).
2.8. Conjugating antibodies to MPs and NPs
Protected sulfhydryl groups were introduced to hD1 and h5G1.1
antibodies with SATP and were reduced with hydroxylamine
hydrochloride (Pierce) using the manufacturer's protocol. Subse-
quently, antibodies (200 μg/mL) were conjugated to DSPE-PEG(2000)
maleimide-containing NP and MP preparations in PBS. Next, particles
were washed with PBS to remove unbound antibodies. The presence
of hD1 and h5G1.1 on the particle surface was confirmed by staining
NPs and MPs with goat anti-human secondary antibodies, followed by
analysis on a FacsCalibur flow cytometer using CellQuest software (BD
Biosciences, USA).
2.9. Cells
Granulocytes and peripheral blood mononuclear cells (PBMCs)
were obtained from buffy coats of healthy individuals and were
purified using Ficoll density centrifugation. Peripheral blood lympho-
cytes (PBLs) and DCs were obtained from PBMCs as reported
elsewhere [18]. In brief, PBMCs were allowed to adhere for 1 h at
37 °C. Non-adherent cells (PBLs) were gently removed, washed and
cryopreserved. Adherent monocytes were cultured in the presence of
IL-4 and GM-CSF (500 and 800 U/mL, respectively; Schering-Plough
International, USA) for 6 days to obtain immature DCs. DCs were
cryopreserved until use. Cells were cultured in X-VIVO 15 medium
(Cambrex, Belgium) supplemented with 2% human serum.
2.10. Antigen release kinetics
DCs were cultured at 105cells/well in 100 µL X-VIVO 15 medium
without phenol red (Cambrex) supplemented with 2% human serum,
either in the presence or absence of 0.5 µg soluble or PLGA-
encapsulated DQ-BSA. After 2 h at 37 °C, the medium containing
DQ-BSA or DQ-BSA NPs or MPs that had not been taken up was
collected and fluorescence of the cells was measured in a CytoFluor II
(Applied Biosystems, USA) at an excitation wavelength of 485 nm and
an emission wavelength of 530 nm. The cells were cultured at 37 °C
for 6 days and fluorescence released within DCs is followed on the
CytoFluor II. Background fluorescence was determined in wells
containing only DCs cultured without DQ-BSA, DCs cultured with
empty MPs or NPs not carrying DQ-BSA or wells with soluble or
encapsulated DQ-BSA without cells. The total amount of fluorescence
that could be reached was estimated by measuring fluorescence
generated upon digestion of NPs or MPs in 0.8N NaOH to release
entrapped DQ-BSA and trypsin digestion. Trypsin degrades the self-
quenched BSA into peptide fragments with an average length of less
than 8 amino acids, thus relieving quenching. Maximum fluorescence
released from MPs or NPs that were not taken up by the cells was
determined the same way. Maximum fluorescence released from DQ-
BSA that was not taken up by the cells was determined by trypsin
digestion. The maximum amount of fluorescence that could theoret-
ically be generated in the cells was calculated by subtracting the
maximum fluorescence released from DQ-BSA, NPs or MPs that were
not taken up from the total amount of fluorescence that could be
reached. The degree of antigen degradation was expressed as the
percentage of fluorescence measured at various time points (minus
background) relative to the maximum amount of fluorescence that
could theoretically be generated in the cells.
2.11. Live imaging of antigen degradation
DCs were cultured in RPMI 1640 without phenol red (Gibco, Life
Technologies, the Netherlands) supplemented with 10% FCS and
1:10,000 (v/v) LysoTracker Red (Molecular Probes). Labeled cells
were analyzed at 37 °C with a Zeiss LSM 510 microscope equipped
with a type S heated stage CO2controller and PlanApochromatic
63×1.4 oil immersion DIC lens (Carl Zeiss, Germany). DQ-BSA MPs
(3 µg/mL) were added to the culture medium and DCs were analyzed
during MP uptake for 1 h. Subsequently, DCs were washed to remove
MPs that had not been taken up and cells were analyzed for another
9 h. Cells were imaged using Zeiss LSM Image Browser version 3.2
(Carl Zeiss) and processed with Image J version 1.32j software
(National Institutes of Health, http://rsb.info.nih.gov/ij).
2.12. MP internalization assay
InternalizationofMPscontainingFITC-TTwasconfirmedbyconfocal
microscopy. Cells were fixed on poly-L-lysine coated glass slides and
stained with anti-human MHC class II antibody (clone Q5/13) or IgG2a
isotype control, followed by a secondary mAb goat anti-mouse Alexa
647 antibody. Cells were imaged with a Bio-Rad MRC 1024 confocal
system operating on a Nikon Optiphot microscope and a Nikon 60×
planApo 1.4 oil immersion lens. Pictures were analyzed with Bio-Rad
Lasersharp 2000 and Adobe Photoshop 7.0 (Adobe Systems, USA)
software.
2.13. Particle binding and uptake
Particle binding was studied by incubating cells with 100 μg/mL
PLGA particles for 1 h at 4 °C in culture medium. Subsequently, cells
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were washed and analyzed by flow cytometry. Uptake was studied by
incubating cells with 100 μg/mL PLGA particles at 37 °C and
determining changes in cell-associated fluorescence over time. At
the indicated time points, a fraction of the cells were washed and
analyzed by flow cytometry on a FacsCalibur (Becton Dickinson, USA).
2.14. Targeting MPs and NPs to DCs within a mixed blood cell population
DCs were stained with a biotinylated mannose receptor-specific
antibody(clone15-2,BioLegend)andstreptavidinAlexa647(Invitrogen).
Leukocytesisolatedfrombuffycoatsweremixedwithpre-stainedDCsina
20:1 ratio in culture medium. Cells were incubated with NPs and MPs
(100 μg/mL) at 37 °C for 3 h. Subsequently, cells were analyzed by flow
cytometry on a FacsCalibur. The relative particle-derived fluorescence for
the various cell fractions incubated with the indicated particles was
calculated relative to the mean cell fluorescence of DCs incubated with
hD1-NPs or hD1-MPs, which was set at 100%.
2.15. Antigen presentation assay
DCs and PBLs of donors that respond to TT-antigen were obtained
from buffy coats after informed consent. PBLs were restimulated once
for 1 week with TT peptide to increase the amount of TT-responsive T
cells. DCs (105) were incubated with various amounts of targeted or
control MPs and NPs for 2 days at 37 °C. Following washing, the
restimulated PBLs were added to the DCs. Four days after addition of
the PBLs, proliferative responses were determined by adding tritiated
thymidine (1 µCi [0.037 MBq]/well; MP Biomedicals, the
Netherlands) to the cell cultures. Tritiated thymidine incorporation
was measured after 16h in a scintillation counter.
3. Results
3.1. Incorporation of FITC-labeled antigen in PLGA
In our studies, tetanus toxoid (TT) 830–844 peptide was used as a
clinically relevant model antigen to study presentation of PLGA-
encapsulated antigens. This TT peptide is used as a nonspecific vaccine
helper epitope in clinical trials to increase immune responses by
increasing the helper T cell response [22,23]. The TT peptide was
covalently linked to FITC (FITC-TT) by continuous solid-phase
synthesis [24], which should allow visualization of incorporated
peptide and uptake of PLGA containing FITC-TT peptide by DCs. The
FITC label was separated from the TT peptide by a cleavage site that is
recognized by the phagosomal/endosomal protease cathepsin, allow-
ing peptides to be independently processed for presentation. The
entrapmentefficiency ofFITC-TTpeptidewithin PLGA(PLGA-FITC-TT)
MPs was 91%. The MPs were spherical and 2.1 µm in diameter
(Table 1).
Analysis of PLGA-FITC-TT particles by flow cytometry revealed that
virtually all particles were fluorescent (Fig. 1A). Confocal microscopy
revealedPLGAmicrospheres with theFITC-labeledpeptideapparently
located at the particle surface, but this was likely due to quenching or
the fact that the light source was unable to penetrate the dense PLGA
particles and reach FITC-TT peptide within the particle (Fig. 1B). To
verify that the bulk of FITC-TT peptide encapsulated within the PLGA
particle was indeed shielded from detection, the fluorescence
associated with PLGA-FITC-TT particles was compared before and
after complete hydrolysis of the PLGA polymer. As expected, release of
FITC-TT peptides by PLGA degradation resulted in a strong, 150-fold,
increase in fluorescence (Fig. 1C).
3.2. Encapsulated antigen is presented by human DCs
Human monocyte-derived DCs were incubated with PLGA-FITC-TT
MPs to establish that the vaccine delivery vehicles are taken up and
the TT peptide is presented. Confocal laser scanning microscopy
revealed that particles enter the cell within 1h (Fig. 2A). Incubation of
PLGA-FITC-TT with DCs from donors that respond to TT-antigen
induced proliferation of autologous T cells to the same level as DCs
thatwerepulsedwithTTpeptideexogenously.This Tcell proliferation
was peptide specific and was not induced by DCs incubated with PLGA
MPs without TT peptide, showing that the PLGA-encapsulated TT
peptide was effectively presented by human DCs (Fig. 2B).
3.3. Kinetics and subcellular location of antigen degradation within DCs
Antigen presentation by DCs requires release of the antigen from
PLGA within endosomal or phagosomal compartments, followed by
degradation of the antigen into peptide fragments that are presented
on the cell surface. Since it was not possible to quantify the release of
our FITC-TT peptide from PLGA particles within living cells, we used a
model protein antigen to determine the kinetics of PLGA-encapsulat-
ed antigen degradation within the DC. The protein is labeled to such a
high degree with fluorescent BODIPY dye that the fluorescence is self-
quenched. Quenching is relieved by release of the antigen from PLGA
within the cell and subsequent hydrolyses of the antigen to
fluorescent peptides by cellular proteases [25–27]. This allowed us
to perform pulse-chase experiments. As expected, almost no antigen
degradation was detected when PLGA particles were incubated in
culture medium without cells. Uptake of particles by DCs resulted in
approximately 13% of antigen degradation within one day, increasing
to almost 38% after 6 days. No significant differences in antigen
degradation kinetics were observed between NPs and MPs (Fig. 3).
Soluble antigen was degraded much faster than encapsulated antigen.
Uptake of soluble antigen by DCs resulted in 27% antigen degradation
within 2 days, increasing to 94% after 6 days.
Antigen degradation could not be monitored after 6 days due to
the limited lifespan of cultured human DCs. Live cell imaging
experiments revealed that fluorescent peptides were generated
within the lysosomal compartments of DCs already 1h after PLGA
particles were added to the cells (Supplementary movies 1 and 2 and
supplementary Fig. 1). Together, these data show that degradation of
encapsulated antigen starts rapidly after particle uptake, despite the
fact that PLGA particles retain slow-release characteristics within DCs.
3.4. Surface grafting of PLGA particles with a PEG-lipid layer
Recently, Duncanson et al. [28] reported a novel polymeric particle
comprised of poly(lactic acid) (PLA) with incorporated PEG-lipids.
Here, we explore whether a similar strategy can be used to formulate
PLGA particles coated with a combination of PEG-lipids and
Table 1
PLGA NP and MP characterization. PLGA MPs with FITC-TT peptide (PLGA-FITC-TT MP),
PLGA MPs and NPs with DQ-BSA (PLGA-DQ-BSA) and lipid-PEG-coated PLGA MPs and
NPs (PLGA-FITC-TT-PEG) are characterized by DLS and zeta potential measurements.
Particle diameter data represent the mean value±SD of DLS data. Zeta potential data
represent the mean value±SD of 5 readings. The adsorption of the lipid-PEG layer to
PLGA shields particle surface charge, which is evident from the change in zeta potential.
FITC-TT-antigen content of PLGA particles was determined by particle digestion and
measuring fluorescence relative to standard controls. DQ-BSA antigen content of PLGA
particles was determined by particle digestion and measuring protein content. Data
represent % of encapsulated antigen relative to the amount of antigen added during
particle formation±SD of experiments performed in triplicate. The amount of antibody
introduced onto PLGA particles was determined by Coomassie dye protein assay and is
depicted as the mean±SD of two experiments.
Sample Diameter
(nm)
Zeta potential
(mV)
Antigen
(% w/w)
Antibodies
(µg/mg PLGA)
PLGA-FITC-TT MP
PLGA-DQ-BSA MP
PLGA-DQ-BSA NP
PLGA-FITC-TT-PEG MP
PLGA-FITC-TT-PEG NP
2099±144
2002±121
239±14
1809±218
202±4
−44.7±1.6
−44.9±1.8
−43.6±2.2
−32.7±1.6
−28.6±0.4
90.8±1.2
75.7±2.6
55.7±2.6
89.8±1.9
78.2±1.8
30.6±2.1
20.0±1.2
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functionalized PEG-lipids for the introduction of targeting antibodies
on the particle surface (see Fig. 4 for a diagram).
Addition of fluorescently tagged phospholipids to the second
emulsion step during PLGA particle formation resulted in adsorption
of a lipid layer surrounding the particles (Fig. 5A). This fluorescent
lipid layer was detected on virtually all particles analyzed by flow
cytometry (Fig. 5B). Subsequently, the fluorescently-labeled lipids
were substituted by PEG-lipids and functionalized PEG-lipids to
generate PLGA-PEG vaccine delivery vehicles.
Fig. 2. Uptake of PLGA MPs with FITC-TT peptide by DCs results in antigen presentation.
DCs were incubated with FITC-TT containing MPs (green) for 1 h to confirm uptake by
human DCs. Cells were analyzed by confocal laser scanning microscopy. Cell surface was
visualized by MHC class II staining (blue). The image represents the middle focal plane of
the DCs, with iris set at 2 nm(A). Presentation of PLGA-encapsulated FITC-TT peptide was
studiedbyculturingDCsfor18hinculturemediumalone,ormediumsupplementedwith
empty PLGA MPs (PLGA) or 0.1 µg of FITC-TT peptide encapsulated within PLGA MPs
(PLGA TT). In addition, DCs were pulsed with 1 µM TT peptide as a positive control for
antigen presentation (TT). Subsequently, autologous TT-responsive peripheral blood
lymphocytes were added. After 3 days, cellular responses were assessed in a proliferation
assay. Data are mean proliferation indices ± SD relative to medium control for
experiments performed in triplicate. Significant difference from medium control
according to ANOVA and Dunnett’s test: ⁎P b .01.
Fig. 3. PLGA-encapsulated protein antigen is slowly processed by human DCs. The
degradation of PLGA-encapsulated or nonencapsulated protein antigen was deter-
mined following uptake by human DCs. PLGA NPs (circles) and MPs (triangles) carrying
the self-quenched model protein antigen DQ-BSA or soluble DQ-BSA (squares) were
incubated in culture medium in the presence (closed symbols) or absence (open
symbols) of human DCs. Two hours after addition of antigen, DCs were washed and
fluorescence generated by antigen degradation was followed for 6 days. Antigen
degradation is depicted as the percentage of fluorescence measured at various time
points relative to the maximum amount of fluorescence that could theoretically be
generated by the cells upon complete degradation of the amount of DQ-BSA taken up
during the 2 hour pulse. Experiments were performed in sixplo and one representative
experiment out of three is shown. Data are mean values ± SD. The curves representing
the degradation of NPs and MPs in the presence of DCs (closed circles and triangles)
showed a high degree of overlap and were plotted at positions x − 0.1 and x + 0.1
days, respectively, for reasons of clarity.
Fig. 1. FITC-TT peptide antigen is entrapped within PLGA particles. The presence of
FITC-TT in PLGA MPs was confirmed by measuring particles with (black line) or without
(grey, filled) encapsulated FITC-TT peptide by flow cytometry (A). PLGA FITC-TT MPs
were mounted on glass slides and analyzed by confocal laser scanning microscopy.
FITC-TT peptide was detected as a green fluorescent ring surrounding the PLGA
particles (B). The apparent surface localization of FITC-TT peptide was likely due to
quenching or the fact that the light source was unable to penetrate the dense PLGA
particles and reach FITC-TT-peptide within the particle. Therefore, fluorescence of
intact and degraded PLGA particles was measured in a fluorimeter to confirm that the
major part of fluorescent peptides is shielded from detection and located within the
particles. Data are mean values of 3 experiments ± SD performed in triplicate (C).
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3.5. Engineering targeted NPs and MPs
To compare DC-specific delivery of targeted MPs to that of NPs, we
generated PLGA-PEG MPs and NPs. Scanning electron microscopy and
DLS both indicated the diameter of the PEG-coated MPs to be around
2 μm, while the NPs were approximately 200 nm in diameter (Fig. 6A
and Table 1). The PEG-lipid layer was successfully introduced on the
PLGA particlesurface, as was reflected by the reduced zeta potential of
the particles compared to PLGA without PEG (Table 1). This reduction
in zeta potential is due to the fact that the PEG layer shields the
negative charges of the carboxylic acid groups present on the PLGA
surface [29]. The DC-specific antibody hD1 was introduced on the
PLGA particles by adsorption of a PEG-lipid layer of which part of the
PEG-lipids contained a maleimide group. Sulfhydryl groups were
introduced into the antibodies and conjugated to the functionalized
PEG-lipid molecules. Analysis by flow cytometry and confocal
microscopy confirmed that antibodies were present on the PLGA
surface (Fig. 6B and C). The PLGA-PEG NPs and MPs used in this study
contained approximately 20–30 µg antibody per mg PLGA (Table 1).
3.6. Specific uptake of NPs, but not MPs, is effectively enhanced by
targeted delivery
NPs and MPs with hD1 antibody did bind to human DCs, while NPs
and MPs carrying the isotype control or no antibody did not. This
confirms that targeted MPs and NPs bind DCs via the specific binding
region of the hD1 antibody and not through interactions between
antibodyFcdomainsandFcreceptors(Fig.7A).DespitethefactthatMPs
and NPs showed similar binding characteristics when incubated with
DCs at 4 °C, there were large differences in particle uptake kinetics at
37 °C. DCsrapidly accumulatedhD1-coatedNPs when compared to NPs
carrying no or isotype control antibodies, which were taken up more
gradually over time. In contrast, specifically targeting MPs to DC-SIGN
increased uptake only marginally when compared to nontargeted MPs
(Fig. 7B). Off note, uptake of NPs and MPs by DCs did not result in
enhanced expression of CD80, CD83 and CD86, indicating particle
uptake had no effect on DC maturation status (data not shown). DCs
represent a relatively rare cell population and vaccine delivery vehicles
will encounter numerous cells before reaching a DC. Intravenous
injectionoftargetedvaccinesmightproveeffectiveforDCtherapy,since
DC-SIGN-specific antibodies havebeenshown to reach DCsin all lymph
nodes tested in nonhuman primates[16]. To mimic a situation in which
vaccine delivery vehicles are injected into the human bloodstream, we
determined the effectiveness of targeting NPs and MPs to DCs within a
mixed blood cell population. Therefore, monocyte-derived human DCs
werecoculturedwithleukocytesisolatedfrom wholebloodinaratioof
1:20. DCs took up the majority of targeted, but also nontargeted, NPs
and MPs, confirming they are cells specialized in sampling their
Fig. 4. Diagram of targeted PLGA vaccine particle. The FITC-TT peptide antigen is
encapsulated within PLGA particles. The particles are shielded by a lipid-PEG layer.
Some of the lipid-PEG molecules contain a maleimide group, allowing introduction of
antibodies on the particle surface.
Fig. 5. Introduction of lipids to the particle surface. Fluorescently labeled lipids were
added during particle formation. Particles were transferred to slides and analyzed by
confocal scanning laser microscopy. The fluorescent lipid layer surrounding the
particles is shown in red (A). Fluorescence of PLGA particles generated with (solid
grey) or without (black line, no fill) fluorescent lipids present during particle formation
was determined by flow cytometry. The presence of the fluorescent lipid layer could be
confirmed on 98% of the lipid-treated particles (B).
Fig. 6. Antibodies are introduced on the surface of PLGA NPs and MPs. Morphology of
NPs (A, left panel) and MPs (A, right panel) with PEG-lipids was analyzed by scanning
electron microscopy. Presence of the antibodies on the PLGA particle surface was
confirmed by flow cytometry. NPs and MPs were stained with fluorescently-labeled
secondary antibodies and analyzed on a flow cytometer (B). PLGA MPs were mounted
on glass slides and analyzed by confocal laser scanning microscopy to visualize
antibodies present on the particle surface. FITC-TT peptide was detected as a green
fluorescent ring surrounding the PLGA particles (see also Fig. 2A). Antibodies on the
particle surface were detected by secondary antibody staining with Alexa 647-labeled
anti-human IgG. The images represent the middle focal plane of particles and show split
channels of the FITC signal (FITC-TT), the Alexa 647 signal (antibody) and a merged
picture showing the antibody in red and the FITC-TT peptide in green (C).
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environment (Fig. 7C). DCs internalized NPs and MPs targeted to DC-
SIGN more effectively than nontargeted controls. Similar to the results
shown in Fig. 7B, targeting NPs was much more effective than targeting
MPs, as was demonstrated by the relatively high nonspecific uptake of
MPsbyDCs.Furthermore,targetedMPswerescavengedrelativelymore
efficiently by other phagocytes in the leukocyte population than
targeted NPs. On average, the relative amount of MPs acquired by a
leukocyte was 11% of that acquired by DCs upon targeting DC-SIGN,
while this was only 5% for NPs (Fig. 7C).
3.7. Targeted delivery increases antigen presentation induced by NPs, but
not by MPs
DCs from donors that respond to TT-antigen were exposed to the
vaccine delivery vehicles and incubated with autologous T cells to
determine the efficiency of antigen presentation following uptake of
targeted and nontargeted NPs and MPs. Specific targeting of MPs to
DCs did not significantly enhance antigen presentation (Fig. 8). This
was likely due to the fact that targeting only marginally affected the
efficiency by which DCs take up MPs (Fig. 7B). In contrast to MPs,
targeting NPs to DCs effectively increased antigen presentation.
Targeted NPs induced similar cellular responses to nontargeted NPs
at 10–100 fold lower concentrations (Fig. 8). Interestingly, targeted
and nontargeted MPs induced the same level of T cell proliferation as
targeted NPs at the highest antigen concentrations tested. This indi-
cates that the level of nonspecific antigen uptake via MPs is relatively
high and could only be matched by NPs upon engraftment of the
DC-SIGN antibody.
4. Discussion
The PLGA NPs and MPs in this study are developed using
components, or derivatives thereof, that are used in the clinic, to
carry vaccine components to human antigen presenting cells. Our DC-
specific antibodies effectively targeted PLGA-based vaccine NPs, but
notMPs,to humanDCs. Nonspecific uptakeof NPs,which likelyoccurs
via the endocytic mechanism of macropinocytosis, was inefficient
since uptake of NPs was efficiently boosted by the anti-DC-SIGN
antibody. In contrast, there appeared to be substantial nonspecific
phagocytic uptake of MPs by DCs, as well as by other blood cells,
despite the PEG layer being present on the MP surface. A previous
study by Kwon et al. [8] reports on targeted delivery of MPs to mouse
DCs using DEC-205 antibodies. Uptake of these MPs by mouse bone-
marrow derived DCs in vitro is twofold enhanced by the antibody
when compared to isotype control-coated particles, which is similar
to the effect we observed upon targeting of MPs to human DCs in the
mixed blood cell populationexperiments. Despite therelativelyminor
effect of their DEC-205 antibody on MP uptake in vitro, it does
enhance MP vaccine efficacy in vivo [8].
The antigen degradation studies showed that antigen encapsulat-
ed in the PLGA particles was slowly released and processed by human
Fig. 8. SpecifictargetingofNPstohumanDCsenhancesantigenpresentation.Presentation
ofTTpeptide wasstudiedbyculturingDCsfor2daysinculturemediumalone,ormedium
supplemented with various concentrations of NPs and MPs carrying the anti-DC-SIGN
antibody hD1 (αDC-SIGN, closed circles) or its isotype control 5G1.1 (isotype, open
circles). Subsequently, autologous TT-responsive PBLs were added. After 4 days, cellular
responses were assessed ina proliferation assay.Data are meanproliferation indices ± SD
relative to medium control for 3 experiments.
Fig. 7. Interactions of targeted NPs and MPs with human DCs and blood cells. NPs and
MPs carrying the anti-DC-SIGN antibody hD1 (αDC-SIGN NP and MP), its isotype
control 5G1.1 (isotype NP and MP) or no antibodies (NP and MP) were incubated with
human DCs for 1 h at 4°C to study binding. DCs cultured in medium without particles
were included as a negative control (medium). Next, cells were washed and analyzed
by flow cytometry to detect bound FITC-TT containing particles. Significant difference
from medium control according to ANOVA and Dunnett’s test: ⁎P b 0.01 (A). To study
uptake of targeted and nontargeted particles by human DCs, the experiment in panel A
was repeated at 37°Cfor 0, 1 or 18h. Cellswere washed and analyzed by flow cytometry
to detect FITC-TT containing particles associated with the DCs (B). DCs were stained for
mannose receptor expression, added to leukocytes (ratio 1:20) and cultured for 3 h at
37°C in the presence or absence of FITC-TT containing NPs and MPs carrying the anti-
DC-SIGN antibody hD1 (αDC-SIGN, white bars), its isotype control 5G1.1 (isotype, grey
bars) or no antibody (black bars) to study whether DCs are specifically targeted within
a mixed blood cell population. The relative fluorescence of the mannose receptor
positive (DC) and negative (leukocytes) cells was calculated relative to the mean cell
fluorescence of DCs incubated with αDC-SIGN NPs or αDC-SIGN MPs, which were set at
100% (C). Data are mean values ± SD of experiments performed in triplicate.
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DCs over a period of days within the lysosomal compartment of the
cell. Antigen degradation kinetics are strikingly similar for NPs and
MPs. Particle size determines the diffusion path length and affects
autocatalysis, two important parameters that influence the release
rate of entrapped molecules. Increasing particle size increases
diffusion path length, which negatively influences the release rate.
This is counteracted by the increase in autocatalysis of larger particles,
which might explain the marginal differences in release rate that are
sometimes found between differently-sized particle formulations
[30]. Although there were no differences between NPs and MPs,
comparison of encapsulated and soluble antigen clearly showed that
encapsulation in PLGA protects the antigen from rapid degradation.
Studies in mice have revealed that targeted delivery of particulate
vaccine carriers to DCs enhances immune responses. Delivery of
particles to DCs is achieved by passive or active targeting. Small NPs
are passively transported by the lymphatics to the lymph nodes,
where the DCs reside [7,9]. Active targeting of particulate vaccine
carriers is accomplished by grafting DC-specific antibodies to the
particle surface, which allows delivery of vaccine carriers to specific
DC subsets. This is advantageous since various DC subsets display
differential antigen processing capabilities. For example, targeting a
soluble chimeric protein consisting of a DC-specific antibody and an
antigen mainly induces antigen presentation to CD8+T cells whenthe
chimeric protein targets DEC-205-expressing DCs whereas it induces
presentation to CD4+T cells upon targeting DCIR2-expressing DCs
[31]. There are only a few reports on antibody-mediated targeting of
particulatevaccinecarriersto DCsin vivo.One studyinvolves particles
generated by an acid-degradable cross-linker and an acid-degradable
primary amine monomer carrying antigen and DEC-205 antibodies.
Upon subcutaneous injection, the DEC-205 targeted MPs are taken up
by DCs that migrate to draining lymph nodes resulting in antigen-
specific CD8+T cell activation [8]. In contrast to earlier reports on
DEC-205 targeted strategies using soluble proteins, these particles do
not require additional adjuvants to induce potent T cell responses,
suggesting theypossessan inherentcapacityto induceDC maturation.
A second study on active targeting of particulates in vivo uses
liposomes carrying tumor antigens and single chain antibodies
directed against mouse CD11c or DEC-205 grafted onto the liposomes
by a metal-chelating strategy. Part of the liposomal preparations also
carry immunostimulatory molecules to mature or activate DCs. Only
the targeted liposomes carrying immunostimulatory molecules show
protective immunity against tumors in mice following intravenous
injection, emphasizing that both antigen and DC activation are
required to induce immunity [32]. The use of single chain antibodies
for liposome targeting reduces nonspecific interactions between the
antibodyand cell-surface receptorsthat can significantlycontributeto
particle uptake, a problem often encountered upon targeting particles
carrying whole antibodies [33]. Here, we use a composite IgG2/IgG4
humanized antibody to circumvent nonspecific interactions. Uptake
of particles carrying the isotype control antibody by DCs and
leukocytes was comparable to that of particles without antibody,
showing IgG2/IgG4 composite antibodies provide a valuable tool for
targeted particle delivery. Coating PLGA particles with avidin to yield
a bridge by which the particle surface binds biotinylated ligands is a
strategy that is often used for cell-targeting [34]. However, avidin
itself binds to lectins and negatively-charged cell-surface moieties
because it is heavily glycosylated and positively charged [35].
Moreover, upon targeting particulate vaccines to DCs it seems likely
that the induced immune responses will also be directed against the
avidinitself,whichlimitsvaccineeffectiveness.Directconjugation of a
humanized antibody to the particle surface, in our case the PEG-lipid
layer, avoids unwanted immune responses directed against the
targeting moiety of the nanocarrier itself. In contrast to mouse studies
on targeted antigen delivery, which usually target DEC-205, our
antibody recognizes DC-SIGN. DEC-205 represents an excellent target
to study DC targeting in mice, but in humans it is expressed by many
differentcell types[36]. DC-SIGNisa morelikelytarget in humansdue
to its relatively DC-specific expression pattern [2].
Taken together, the proof of principle studies in mice and our
findings using human DCs hold great promise for targeted NP-based
vaccine therapeutics, since multiple vaccine components can be
delivered to DCs by a single targeted particle. Together with the
rapidly progressing understanding of DC biology, antigen processing
and presentation, it opens up novel approaches in nano-medicine for
intelligent design of vaccines that might ultimately guide antigens
into the appropriate cellular compartment, while at the same time
properly activating the DC, prompting the optimal immune response.
Acknowledgments
The authors wish to thank the Microscopic Imaging Center of the
NCMLS for use of their facilities and the technicians of the NCMLS
Tumor Immunology Department Clinical DC group for assistance. This
work was supported by the Marie Curie Research Training Network
Immunanomap.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi: 10.1016/j.jconrel.2010.02.013.
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