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org/molecularpharmaceutics Review

Relevant Physicochemical Methods to Functionalize, Purify, and

Characterize Surface-Decorated Lipid-Based Nanocarriers
Leń a Guyon, Anne-Claire Groo, and Aureĺ ie Malzert-Freó n*

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ABSTRACT: Surface functionalization of lipid-based nanocarriers (LBNCs) with targeting

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ligands has attracted huge interest in the field of nanomedicines for their ability to overcome
some physiological barriers and their potential to deliver an active molecule to a specific target
without causing damage to healthy tissues. The principal objective of this review is to
summarize the present knowledge on LBNC decoration used for biomedical applications, with
an emphasis on the ligands used, the functionalization approaches, and the purification
methods after ligand corona formation. The most potent experimental techniques for the
LBNC surface characterization are described. The potential of promising methods such as
nuclear magnetic resonance spectroscopy and isothermal titration calorimetry to characterize
ligand surface corona is also outlined.

KEYWORDS: lipid-based nanocarriers, surface functionalization, targeting ligands, characterization

1. INTRODUCTION
In recent years, development of various nanotechnology
platforms in the area of medical biology, including both
diagnostic applications and therapy, has gained importance.
This field of research have been developed notably to design
multifunctional nanoparticles as pharmaceutical drug carriers.
In fact, nanoparticles could help to pass through the poor
aqueous solubility and the lack of stability of newly discovery
drugs. Moreover, these nanocarriers present many advantages
linked to their size-related properties, potential for selective
targeting, and controlled drug release.1
Among these nanometric systems, liposomes, developed in
1965, are the first generation of lipid-based nanoparticle drug
carriers. They are formed by hydration of a thin lipid film that
leads to bilayers with the ability to mimic a cell membrane.2 Figure 1. Schematic presentation of some organic nanoparticles:
Thereafter, various organic structures and more particularly micelle, liposome, nanoemulsion, lipid nanocapsule (LNC), solid lipid
lipid-based nanocarriers (LBNCs) have been developed such nanoparticle (SLN), and nanostructured lipid carrier (NLC).
as micelles, liposomes, nanoemulsions, lipid nanocapsules
(LNCs), solid lipid nanoparticles (SLNs), and nanostructured choice of the nanocarrier type and the targeting ligand that
lipid carriers (NLCs) (Figure 1). These systems can be form the drug delivery system is important, and several
formulated from biocompatible and biodegradable materials elements have to be considered. The careful biological analysis
and demonstrated their ability to improve specificity and
efficacy of chemotherapeutic agents. According to the structure
of these nanosized systems, not only hydrophobic drugs but Received: August 20, 2020
also hydrophilic compounds can be encapsulated.3 Such Revised: November 16, 2020
colloidal carriers are very promising for organic drugs. Accepted: November 16, 2020
In order to decrease side effects and deliver the drug at a
target site, active and passive targeting with a combination of
other strategies such as stimuli sensitivity could be used.4 The

© XXXX American Chemical Society https://dx.doi.org/10.1021/acs.molpharmaceut.0c00857

A Mol. Pharmaceutics XXXX, XXX, XXX−XXX
 Table 1. Examples of Functionalized Lipid-Based Nanocarriers Developed for Biomedical Applications during the Last Four Yearsa
LBNC biomedical study
ligand type goal functionalization process drug/cargo application stage ref
proteins CD44 antibody (Fab’ of mAb) liposome targeting ligand for CD44 overexpressing cells: penetration into conjugation with preformed liposomes SATB1 siRNA gastric cancer vitro 32
solid tumors, reduction of immunogenicity and improvement of
pharmacokinetic profiles
trastuzumab (monoclonal antibod- liposome targeting ligand for HER2-overexpressing cancers conjugation of MAbs-SH with pre- paclitaxel & rapa- breast cancer vitro 33
ies) formed liposomes mycin
vivo
extracellular adherence protein liposome able to bind to several host cell extracellular matrix components conjugation of Eap with preformed colistin infections by vitro 34
Molecular Pharmaceutics

(Eap) and to rebind to the surface of S. aureus, (improve the invasion of liposomes (using DMTMM as a Salmonella
S. aureus into cells) cross-linker) OR by physical ad- enterica
sorption
anticarbonic anhydrase IX antibody liposome able to target carbonic anhydrase IX (CA IX, enzyme expressed on conjugation of anti CA-IX with DSPE- triptolide lung cancer vitro 20
the surface of lung cancer cells) PEG-Mal micelles and postinsertion
with preformed liposomes
vivo
transferrin & hydrazone SLN transferrin: targeting ligand for transferrin receptors incorporation in the formulation docetaxel & baica- lung cancer vitro 29
process lin
hydrazone: pH-sensitive moiety vivo
proteins and pep- transferrin & PFV (cell penetrating liposome transferrin: to target transferrin receptors (overexpressed on the incorporation in the formulation doxorubicin and glioblastoma vitro 35
tides peptide, sequence: PFVYLI) surface of glioblastoma cells U87 and brain endothelial cells process erlotinib
bEnd.3) PFV: to increase translocation of drugs across the BBB
into glioblastoma tumor cells (U87)
anticarbonic anhydrase IX liposome anti-CA IX: able to target CA IX CPP33: able to penetrate the cell conjugation of anti-CA IX antibody triptolide lung cancer vitro 30
antibody & CPP33 (tumor line- membrane of human nonsmall cell lung cancer (NSCLC) A549 and CPP33 with DSPE-PEG-Mal

B
age-homing cell-penetrating pep- cells micelles postinsertion with pre-
tide, RLWMRWYSPRTRAYG) formed liposomes
vivo
lactoferrin & RGD peptide NLC lactoferrin: to modulate the transcytosis across the BBB and incorporation in the formulation temozolomide glioblastoma vitro 22
permeation to GBM through receptor-mediated signaling process
pathways RGD: targeting ligand for integrins
vivo
peptides RLT peptide (low-density lipopro- liposome RLT: Targeting ligand for LDLRs and to assist endolysosome incorporation of DSPE-PEG-Mal in docetaxel lung cancer vitro 31
tein receptor (LDLR)-binding escape the formulation process and con-
peptide) jugation of RLT (RLT-SH) with
preformed liposomes
pubs.acs.org/molecularpharmaceutics

vivo
hexapeptide antagonist G liposome block the action of various neuropeptides through its ability to bind postinsertion of DSPE-PEG- doxorubicin lung cancer vitro 36
to several receptors on the surface of small cell lung cancer antagonist G micelles into lip-
osomes OR conjugation of antago-
nist G-SH with preformed lip-
osomes
peptides: RGD, cRGD (cRGDfK), liposome targeting ligand αv integrins Incorporation in the formulation plasmid DNA & gastric cancer vitro 37
RPARPAR, and iRGD (c process tumor homing
(CRGDKGPDC) peptides
vivo
angiopep-2 SLN targeting ligand for the lipoprotein receptor related protein 1 conjugation of angiopep-2 with car- docetaxel glioblastoma vitro 38
boxylic moieties of preformed SLN
vivo
NFL-TBS·40−63 peptide NLC targeting glioblastoma cells physical adsorption ferrocifen-type glioblastoma vitro 39
molecule
vivo
Review

Mol. Pharmaceutics XXXX, XXX, XXX−XXX

https://dx.doi.org/10.1021/acs.molpharmaceut.0c00857
 Table 1. continued
LBNC biomedical study
ligand type goal functionalization process drug/cargo application stage ref
aptamers RNA HB5 aptamer NLC to facilitate the receptor-mediated endocytosis that contribute to conjugation of HB5 aptamer with ATP aptamer-epi- lung cancer vitro 24
NLC internalization into SK-BR-3 tumor cells preformed amino-NLC gallocatechin
gallate-prot-
amine sulfate
vivo
microRNA (miR-542−3p) SLN targeting ligand for astrocyte-elevated gene-1 (oncogene associated physical adsorption all-trans retinoic gastric cancer vitro 40
with the tumor proliferation and growth) acid & Sorafenib
Molecular Pharmaceutics

vivo
small molecules FA liposome targeting folate receptors protein incorporation in the formulation 5-fluorouracil colorectal vitro 26
process cancer
vivo
FA & dextran SLN FA: targeting folate receptors protein incorporation in the formulation doxorubicin colorectal vitro 27
process cancer
dextran: to avoid biorecognization of FA residues on nanoparticles vivo
by FA transporters
glycyrrhetinic acid liposome targeting ligand for protein kinase C (overexpressed on the surface incorporation in the formulation curcumin liver cancer vitro 41
of hepatocellular carcinoma cells) process
vivo
FA liposome PEG: extend blood-circulation time incorporation in the formulation oleuropein prostate can- vitro 25
process cer
FA: targeting folate receptors protein (overexpressed in many vivo
cancer cells)

C
N-acetyl-D-glucosamine NLC glucose receptor-targeting ligand incorporation in the formulation gemcitabine & pa- lung cancer vitro 42
process clitaxel
octanoyl galactose ester ME targeting ligand for asialoglycoprotein receptors (overexpressed on incorporation in the formulation Coix seed com- liver cancer vitro 43
the surface of hepatoma-cells) process pounds
vivo
galactose PE targeting ligand for asialoglycoprotein receptors (overexpressed on incorporation in the formulation doxorubicin & in- liver cancer vitro 44
the surface of HepG-2 or H22 cells) process docyanine green
vivo
attenuated bacteri- liposome targeting tumor conjugation with preformed liposomes doxorubicin colorectal vitro, 45
al microorgan- and physical adsorption cancer vivo
isms: Salmonella
pubs.acs.org/molecularpharmaceutics

a
Abbreviations used: NE, nanoemulsion; SLN, solid lipid nanoparticle; NLC, nanostructured lipid carriers; ME, microemulsion; PE, pickering emulsions; FA, folic acid.
Review

Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
of the target site is essential to choose ligands that can be
useful to target the specific area.
In fact, various biological barriers have to be considered
inside the human body (such as crossing cellular membrane,
tumor stroma, skin, intestinal or blood−brain barriers), and
mechanisms that are potentially available for crossing these
barriers are different from tissue to tissue.5 A wide variety of
ligands have been attempted to be incorporated onto
nanoparticle surfaces: peptides, antibody fragments, aptamers,
small molecules, etc. This functionalization was usually
achieved by using two different ways: ligands were attached
at the surface (1) by physical adsorption via electrostatic
complexation6 or (2) through a covalent conjugation of the
molecule with the nanoparticle surface.7
The characterization of such nanometric systems has been
shown to be a crucial step due to the complexity of the
nanostructure composition, the interactions with protein in
biological medium (formation of a biocorona), etc. It was
previously pointed out that complementary and more complex
methods should be used to provide reliable information even
for the determination of one parameter such as the size of
nanoparticles.8,9 Indeed, this characterization step may be
much more difficult after surface functionalization, but the
complete understanding of the entire structure is crucial before
preclinical studies.
Lipid-based nanoparticles have been the longest-studied
nanocarriers and present one of the promising drug-delivery
candidates. Some reviews are already focused on non-lipid-
based nanocarriers and more specifically on inorganic particles
or polymeric ones. Usually, articles are devoted to surface
functionalization and/or nanocarriers characterization.10−12
However, it is not the same approach of functionalization,
purification, and characterization function of the nanocarriers
used. For these reasons, the review will focus on lipid-based
nanocarriers and more particularly on the important character-
ization step after surface functionalization. Some examples of
lipid-based nanoparticles decoration used for biomedical
applications will be given with an emphasis on the ligands
used and the functionalization approaches. Then, purification
methods daily used in the laboratory will be summarized.
Finally, characterization techniques used in order to follow the
surface decoration will be described. Particular interest will be
given to nuclear magnetic resonance spectroscopy (NMR) and
isothermal titration calorimetry (ITC), and the potential of
these techniques will be outlined.
2. FUNCTIONALIZATION OF LIPID-BASED
NANOCARRIERS
2.1. Biomedical Application of Functionalized Lipid-
Based Nanocarriers. The development of functionalized
lipid-based nanocarriers (LBNCs) has been particularly
relevant especially in biomedical research. The lipid
components of these carriers are usually phospholipids,
cholesterol, and triglycerides, but also bile salts and free fatty
acids. These excipients, generally extracted from natural
sources, are known to be biocompatible (nontoxic and inert
to tissue) and biodegradable in vivo. One important advantage
of LBNCs compared to other carriers is that a number of these
lipids, such as phosphatidylcholine, stearic acid, cholesterol,
and glycerol monooleate have been used in FDA-approved
pharmaceutical applications. Thus, they have well-defined
safety and toxicological profiles. These nanocarriers have a
core−shell structure including a core that could act as a
D https://dx.doi.org/10.1021/acs.molpharmaceut.0c00857
Review
reservoir for active drugs and a shell which is usually a
protective membrane. However, in some cases the shell has
both properties: it acts as a protective membrane that could
also be used to entrap lipophilic drugs. Thus, LBNCs have
been developed for their special characteristics related to their
structural properties and composition: a high drug-loading
ability, high biocompatibility, and a tailorable surface to
improve the targeting, biodistribution, and pharmacokinetic
profile.13
Table 1 summarizes the main functionalized LBNCs
developed for biomedical applications during the last four
years. Each example is given with particular emphasis on ligand
type and goal, the functionalization method, and the drug and/
or cargo used function of the biomedical application. As shown
in this table, the research area is mainly focused on the
treatment of various cancers. Among various LBNCs, the most
studied examples are liposomes, which primarily consist of a
lipid phospholipid bilayer, i.e., the major components of cell
membranes.14 The powerful drug carriers liposomes can
encapsulate both hydrophobic and hydrophilic drugs, improv-
ing not only their solubility but also their stability,
bioavailability, and activity.15 Several liposomal chemother-
apeutic formulations have been approved for the treatment of
cancers by the European Medicines Agency (EMA) and the
U.S. Food and Drug Administration (FDA). In particular,
Doxil16 was the first FDA approved formulation in 1995. The
growing number of liposomal formulations that are undergoing
different stages of clinical trials show the potential for the
application of LBNCs. For example, doxorubicin-loaded
liposomes functionalized with the cetuximab Fab fragment
have been studied as first-line therapy in patients with
advanced triple-negative, EGFR-positive breast cancer.17
Upon intravenous administration, blood proteins such as
opsonins adsorb at the surface of the nanocarriers, leading to
their quick recognition by cells of the mononuclear phagocyte
system (MPS) and their elimination from the bloodstream.
This process leads to low accumulation of drugs at the target
site with many side effects. To target the desired diseased site
and obtain the optimal therapeutic response, passive and active
targeting strategies were developed.18
Passive targeting consists of using the disease anatomy to
accumulate the drug at a desired site and prevent nonspecific
distribution by optimizing the delivery system characteristics.
Nanocarriers with a 10−100 nm diameter range, and with a
hydrophilic and neutral surface charge, have a greater chance
of reaching the targeted tumor or inflammatory tissues, and
selectively accumulate in the tumor interstitium upon
enhanced permeability and retention effect. To render “stealth”
the drug delivery systems, i.e., able to escape to the MPS
through increased repulsion forces, hydrophilic and neutral
compounds are used for surface modification, in particular,
polysaccharides, and poly(ethylene glycol) (PEG). In this last
case, the scientific community speaks about “PEGylated
nanoparticles”.
Active targeting is a promising strategy in which a
recognizable cell-specific ligand is linked to the nanocarrier
surface to be more selective toward targeted tissues and even
cells to treat. In the case of cancers, many receptors are
overexpressed in diseased cells compared to normal cells.
Targeting these receptors by a specific ligand is an interesting
approach to increase drug internalization in cancer cells.
Several extracellular targets for active drug targeting have been
studied, such as epidermal growth factor receptor (EGFR),
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Figure 2. Schematic presentation of different strategies used to functionalize LBNCs. (A) Direct ligand conjugation with preformed LBNC, (B)
postinsertion of lipid−PEG or lipid−PEG−ligand micelles, (C) incorporation of lipid−PEG−ligand within the LBNC formulation process, or (D)
noncovalent adsorption of the ligand.

fibroblast growth factor receptors (FGFR), folate receptors, or
transferrin receptors. Consideration might also be given to the
delivery of drugs directly to a desired organelle with
mitochondrial or lysosomal targeting, using intracellular
targets. Another approach is to consider receptors that are
overexpressed in the tumor microenvironments and tumor
vasculature. These targets include integrins (glycoproteins
overexpressed in neovascular epithelial tumor cells) or cluster-
of-differentiation (CD44, overexpressed in several tumors).
Different natures of targeting ligands are nowadays used:
antibodies (or their fragments), peptides, aptamers, and small
molecules.
Proteins. Antibodies or their fragments, i.e., fragment
antigen-binding (Fab’)/single-chain fragment variable (scFv),
are commonly used to realize active targeting. The main
advantage of using a fragment instead of the whole antibody is
to prevent the potential inactivation of antibody during surface
functionalization. One example of antibody is anti-carbonic
anhydrase (CA) IX that could specifically target to CA IX
overexpressed, due to hypoxia, in many solid tumors such as
lung tumors. This anti-CA IX targeting ligand was used to form
docetaxel-loaded immunoliposomes for lung cancer treatment.
Results showed a 1.7-fold higher binding activity in CA-IX
positive A549 lung cancer cells compared with nontargeted
E https://dx.doi.org/10.1021/acs.molpharmaceut.0c00857
liposomes.19 Similarly, anti-CA IX functionalized triptolide-
encapsulated liposomes have shown a higher efficacy in vivo
for lung cancer therapy. In fact, pulmonary delivery of targeted
triptolide-liposomes exhibited the strongest tumor growth
inhibition after eight treatment doses compared to nontargeted
liposomes, free triptolide, and the saline groups. Moreover, the
lifespan of targeted triptolide-liposomes treated mice was
significantly prolonged with a median survival time of up to 90
days, twice longer than in the control group (compared with
nontargeted triptolide-liposomes that could prolong the
median survival time to 71 days).20
Peptides. The LBNC surface is functionalized with peptides
by covalent coupling or physical adsorption. Cell-penetrating
peptides (CPP) with nonspecific internalization properties or
cell-targeting peptide (CTP) with receptor-specific binding are
used.21 The tripeptide RGD was used in several studies for its
capacity to target integrins, which are transmembrane
receptors overexpressed on many solid tumors and in tumor
neovasculature.22 Chen et al. formulated doxorubicin-loaded
liposomes with RGD surface functionalization and showed a
2.5-fold higher cellular uptake of the drug in human glioma cell
line (U87MG) compared with the nontargeted doxorubicin-
loaded liposomes.23
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Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
Table 2. Advantages and Drawbacks of Different Strategies Used to Functionalize LBNCs
advantages
Direct ligand conjugation with preformed • No modification of the LBNC
LBNC composition before conjugation
postinsertion of lipid-PEG-ligand micelles • Relatively simple protocol
• Ligand/LBNCs ratio can be easily
adjusted
incorporation of lipid−PEG-ligand within • Characterization of lipid−PEG−
the LBNC formulation process ligand before the incorporation
• No release of the entrapped drugs
Noncovalent adsorption of the ligand • Simple method
• Less time-consuming than the other
strategies
• Not requiring ligand or nanoparticle
modification
Aptamers. These molecules are small single-stranded DNA
or RNA able to target specific receptors. HB5 aptamers were
used by Liang et al. to functionalize nanostructured lipid
carriers (NLC). These HB5-functionalized nanocarriers,
loaded with ATP aptamer bound to epigallocatechin gallate
and protamine sulfate, showed to enhance NLC internalization
into a human breast cell line (SK-BR-3, HER2 receptor
overexpressed) by promoting the receptor-mediated endocy-
tosis.24
Small molecules. Many small molecules are used as
targeting ligands such as folate25−27 or affibody molecules
which are small proteins imitating monoclonal antibodies.28
Among the reported folate-conjugated LBNCs studies, Nassir
et al. developed oleuropein-loaded liposomes functionalized
with folate for prostate cancer treatment. In vivo results
showed an improved efficacy of the functionalized nanocarriers
compared with plain oleuropein solution in terms of tumor
volume reduction, body weight maintenance, and survival
probability. As usual, these authors combined passive targeting
thanks to PEGylation and active targeting through overex-
pressed folate receptor binding.25 It can be noted that the use
of a stealth nanocarriers surface functionalized with a targeted
ligand is a strongly developed approach to reach the desired
site.
To optimize the therapeutic response, LBNCs may be
functionalized by using both a specific ligand and a pH
sensitive lipid-conjugate.29 Li et al. functionalized solid lipid
nanoparticles (SLN) with both transferrin (Tf) and hydrazine
(pH sensitive moiety) for lung cancer therapy.29 In this
strategy, Tf-PEG-hydrazone (hz)-glyceryl monostearate
(GMS) is used to allow the drug release upon specific
stimulus, i.e., the acidic environment of tumors in the present
case. Transferrin functionalized SLN were prepared by an
emulsification method for the loading of both docetaxel and
baicalin in order to have a combined chemotherapy. In this
strategy, transferrin is used to target receptors overexpressed
on the surface of glioblastoma cells. Then, the PEG layer is
able to shed from the SLN in the acidic tumor environment
allowing drug release. These SLNs were studied in vivo: results
revealed a higher antitumor efficiency with functionalized SLN
compared to nondecorated nanocarriers, showing the interest-
ing potential of this targeting approach. Moreover, incorpo-
ration of two ligands on the surface of LBNCs was studied by
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drawbacks ref
• Reactive moieties need to be present and available at the 34, 38, 45,
surface of LBNCs. 46
• Stability of LBNCs under coupling conditions with ligands
• Postinsertion approach may be challenging and have to be 19, 36,
optimized for each LBNC/ligand type. 47−51
• Leakage of the LBNC membrane resulting in release of the
entrapped drug
• Stability of LBNCs under incubation conditions
• Modification of the initial LBNC structure 22, 25, 37,
51
• Conformation of the lipid-PEG-ligand in the LBNC: ligand
could be oriented outside or inside the LBNC.
• Less stable over time and in physiological conditions 29, 34, 45,
52, 53
Lin et al. to target the same cells for lung cancer therapy.
Anticarbonic anhydrase IX (CA IX) antibody and CPP33 dual-
ligand liposomes for efficiently delivering triptolide (TPL) to
NSCLC by pulmonary administration were investigated.
Interestingly, results revealed that dual-ligand liposomes had
a much better penetrating ability in 3D tumor spheroids than
either nonmodified liposomes or single-ligand-modified lip-
osomes.30
The LBNC surface is functionalized with various targeting
ligands according to the intended biomedical application, but
the functionalization process itself may impact the targeting
ligand efficiency. The most common strategies used to perform
this functionalization are detailed in the next section.
2.2. Functionalization Process. As stealth nanocarriers
usually contain PEG at their surface, this moiety is commonly
considered in the coupling strategy between the LBNC and a
selected ligand. Four strategies are observed in LBNC
functionalization (Figure 2 and Table 2): (A) Direct ligand
conjugation with preformed LBNC, (B) postinsertion of lipid-
PEG-ligand micelles, (C) incorporation of lipid-PEG-ligand
within the formulation process, or (D) noncovalent adsorption
of the ligand at the surface of LBNC.
2.2.1. Strategy A: Direct Ligand Conjugation with
Preformed LBNC. In this strategy, ligands are conjugated to
lipids or PEG chains used as excipients in the formulation of
the LBNCs, or may be covalently linked to the surface of
preformed LBNCs (Figure 2A).34,38,45 A variety of classical
reactions such as amide bond ligation, thioesters bond
formation by the maleimide−thiol addition reaction, disulfide
bridge, hydrazone bond formation, or the use of biorthogonal
chemistry have been used. In the context of lung cancer, Lee et
al. developed cationic liposomes encapsulating small interfering
RNA (siRNA), which could effectively target epidermal growth
factor receptors (EGFR, surface receptors which are overex-
pressed in many solid tumors such as breast or nonsmall cell
lung cancer).46 A thiolated anti EGFR antibody was
conjugated with the maleimide groups of the distal termini
of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-malei-
mide (polyethylene glycol) (DSPE-PEG-MAL) chains of
preformed liposomes by overnight incubation at 4 °C. This
common approach can be performed if reactive moieties are
present and available at the surface of LBNCs.
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Figure 3. Scheme of different parameters that have to be adjusted all along the formulation and functionalization processes.
2.2.2. Strategy B: Postinsertion of Lipid-PEG-Ligand
Micelles. Liposomes, thanks to their lipid bilayer structure,
were frequently functionalized using this strategy (Figure 2B).
Lipid-PEG, which are amphiphilic compounds, form micelles
above their critical micellar concentration.19,36,47 Preformed
liposome formulations are incubated with these micelles, in
controlled experimental conditions (agitation duration and
speed, temperature), allowing a transfer of the lipid-PEG
conjugate within the phospholipidic bilayer of liposomes.48
After this lipid-PEG postinsertion, a chemical reaction
(strategy 1) is used to covalently link ligands with the
nanoparticle surface (Figure 2B). Alternatively, the ligand can
be first linked to lipid-PEG before the incorporation of micelles
into liposomes by postinsertion. This surface engineering
technique was used with doxorubicin-encapsulating liposomal
formulation with the aim to target vascular endothelial growth
factor (VEGF) receptor.49 First, FabV antigen-binding frag-
ments were conjugated with the end of DSPE-PEG-MAL
chains in order to form DSPE-PEG-MAL-FabV by the
maleimide−thiol addition reaction. After this conjugation
step, incorporation of DSPE-PEG-MAL-FabV was performed
by coincubation with preformed liposomes at 55 °C for 30
min. The efficiency of this postinsertion approach depends on
the potential formation of lipid-PEG-ligand micelles even if it is
not systematically detailed in the studies.48 Indeed, Wicki et al.
postinserted DSPE-PEG-MAL-FabV into liposomes without
any preliminary studies about the self-assembling properties of
such conjugates.50 Moreover, although being largely used, the
postinsertion approach may be challenging as it depends on
the incubation conditions (see section 2.3), and the
incorporation of the lipid moiety may be done without leakage
of the liposomal membrane resulting in release of the
entrapped drug. Lastly, as for strategy A, unsuitable incubation
conditions, especially high temperature, could also degrade
encapsulated labile molecules (such as peptide or RNA).51
2.2.3. Strategy C: Incorporation of Lipid-PEG-Ligand
within the LBNC Formulation Process. In this approach
(Figure 2C), the lipid-PEG-ligand conjugate is synthesized
prior to the formulation process and added with the other
excipients at the first step of the formulation protocol.22,25,37
Biswas et al. used this strategy to develop mitochondria-
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targeted liposomes.51 Phosphatidylethanolamine-PEG (PE−
PEG) was first conjugated to the targeted ligand: triphenyl-
phosphonium (TPP). Then, PE−PEG−TPP was added in the
formulation process, allowing the incorporation into the
liposomal lipid bilayer. Results of in vitro and in vivo studies
in a breast cancer model showed mitochondrial targeting with
an increased cytotoxicity of paclitaxel liposomes compared to
unmodified liposomes. The preparation of the lipid−PEG−
ligand conjugate before the incorporation in liposomes allowed
us to fully characterize the final lipid. However, with this
strategy the ligand could be oriented outside the LBNC (as
illustrated in Figure 2C) or inside the LBNC. If the last
situation occurred, the targeting will not be efficient.
2.2.4. Strategy D: Noncovalent Adsorption of the Ligand.
Noncovalent adsorption was also used to functionalize LBNCs
by either physical adsorption or ionic binding (Figure
2D).29,34,45 In physical adsorption, ligand is linked to the
surface through weak interactions such as electrostatic,
hydrogen binding, hydrophobic, and van der Waals attractive
forces. This strategy was used for example with a bacterial
derived protein such as extracellular adherence protein (Eap),
derived from Staphylococcus aureus. Menina et al. investigated
functionalization of colistin-loaded liposomes by both covalent
coupling of Eap and physical adsorption.34 For surface
adsorption, liposomes were incubated with Eap under stirring
for 1 h at room temperature and the excess of protein was
removed by centrifugal ultrafiltration before quantification.
Results revealed that the high functionalization efficiency was
obtained by physical adsorption thanks to the adhesive
properties of Eap via electrostatic interactions.52 Moreover,
the Eap adsorption to liposomal surfaces was shown to
enhance the internalization of the drug into epithelial cells,
resulting in a decrease of intracellular bacteria compared with
the treatment with unfunctionalized liposomes. This simple
method is less time-consuming than the other strategies and
does not require any ligand or nanoparticle modification.53
However, the adsorption method was demonstrated to be less
stable over time and in physiological conditions than the
conjugation techniques.1
2.3. Optimization of the Functionalization Process.
2.3.1. Impact of Process Parameters. The functionalization
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Table 3. Summary of the Main Advantages and Drawbacks of Nanoparticle Purification Approaches Described in This
Sectiona
purification methods advantages
centrifugation and • simple and easy separation process
ultracentrifugation
diafiltration centrifugal device • simple and easy separation process
(DCD)
• purification of small volumes (limited
volume-handling capacity)
dialysis • gentle purification method
• change the dispersant to achieve the desired
properties (physiological pH and
osmolality)
gel filtration chromatography and • high resolution
size exclusion chromatography
(SEC)
• fast method
tangential flow filtration (TFF) • high reproducibility
• automated process
• easy to scale-up
a
NP, nanoparticles.
efficiency depends on the nanocarrier composition, its
membrane fluidity, the anchor type, and the nanocarrier
stability. Higher membrane packing, in particular, below the
transition temperature of phospholipids forming liposomes,
and slower DSPE−PEG−ligands and LBNCs interactions
kinetics are observed at low temperatures. In consequence, it is
important to define the best process parameters to promote
the ligands insertion in terms of incubation temperature (from
4 to 65 °C generally) and duration (from few minutes to 24 h)
(Figure 3). The stirring mode (lateral agitation and/or vortex)
and rate must be also optimized to promote the ligand
incorporation without inducing detrimental destabilization of
the nanocarriers or denaturation of the targeting ligands during
the postinsertion process.54
2.3.2. Effect of Excipients. In some cases, the introduction
of defined excipients in the formulation of nanocarriers may
prevent their detrimental destabilization during the post-
insertion process and favor the insertion of the targeting ligand.
For example, Tomasina et al. showed that an incubation
temperature comprised at 50 or 60 °C during 90 min induced
a reorganization of the nanoparticles as observed from PdI
becoming higher than 0.2. A macrogolglyceride (Labrasol) is
used to bring flexibility to the structure and allows the
solubilization and the incorporation of DSPE−PEG−FA in the
shell. Thanks to this excipient, detrimental structural
reorganization was avoided by maintaining the incubation
temperature at 30 °C. Thus, the introduction of DSPE−PEG−
FA into nanoparticles was performed at 30 °C through a 5 min
pipetting.55
2.3.3. Ligand/LBNCs Ratio. Studies of ligand-functionalized
LBNCs have been reported especially for the development of
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drawbacks ref
• caking 65
• difficulties in redispersing NP
• loss of NP if insufficient centrifugation force is applied
• sheer forces can affect the NP characteristics
• concentration polarization 66
• membrane fouling
• caking
• loss of NP if insufficient centrifugation force is applied
• sheer forces can affect the NP characteristics
• time consuming process 73
• risk of microbial contamination
• inadequate removal of relatively large molecule impurities
• premature release of NP payload
• small volume of sample can be processed at a time 19, 67,
68
• irreversible adsorption onto the column stationary phase
• poor resolution between large impurities and small NP
• potential interactions with membrane 70−73
• need to optimize TFF parameters (trans-membrane pressure,
flow rate and flux) that have an impact on the NP
characteristics
• cost of the equipment
anticancer nanovectors. In this case, the uptake by cancer cells
must be maximized, while the surface functionalized LBNCs
detection by the immune-system must be as low as possible.56
In this goal, the ligand/LBNCs ratio must be varied to define
the optimal coverage.
2.3.4. Lipid−PEG−Ligand. The postinsertion of lipid−
PEG−ligand micelles into nanocarriers is the most widely used
functionalization strategy. The hydrophilic spacer PEG is
generally covalently linked to 1,2-distearoyl-sn-glycero-3-
phosphorylethanolamine (DSPE) used as a buoy intended to
be entrapped into the nanocarrier wall. DSPE−PEG could be
incorporated directly at the beginning of the formulation
process or through a postinsertion procedure. Thus, the
terminal groups of PEG can be functionalized to carboxylic
acids, amine, or sulfhydryl reactive groups. The covalent
coupling with targeting moieties is then allowed via the
formation of amide or disulfide. The use of a coupling agent
like N,N′-dicyclohexylcarbodiimide may be required to
accelerate the reaction, or the conjugation may be optimized
by the use of a linker such as cyanuric chloride.
2.3.5. PEG Spacer Length. As studied by Kubas et al., the
PEG spacer length may have a significant influence on receptor
binding ability of the coupled ligand.51 From competition
assays with 125I-labeled echistatin, these authors showed that
the binding capacities of c(RGDyK) ligands conjugated to
oligo(ethylene glycol) spacers (EGn) were weakened on
U87MG integrin-expressing human glioma cells when the
EG length increased, in correlation with a decrease of the RGD
concentration.58
2.3.6. PEG Density. Besides, it is well-known that 2000 Da
and higher molecular weight PEG, covalently attached to the
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lipid buoy, offers steric stabilization of the colloidal entity. The
PEG density on the surface of LBNCs can be changed not only
by adjusting the molecular weight (chain length) of PEG but
also the molar ratio (grafting efficiency) of PEG incorporation.
Typically, a significant reduction of plasma protein adsorption
onto biodegradable PEG-coated poly(lactic acid) (PLA),
poly(lactic-co-glycolic acid) (PLGA), or poly(ε-caprolactone)
(PCL) nanoparticles is obtained with a PEG content between
2 and 5 wt %.59
Above a low threshold PEG density, DSPE−PEG moieties
reorganize by passing from a “mushroom” to a “brush”
conformation. This configuration not only conveys greater
protein repulsion and enhanced LBNC lifetime in the
bloodstream, but it is also helpful to the ligand presentation
and facilitates its interactions with receptors present at the
surface of cells to treat. Moreover, the structure of the PEG
layer is strongly influenced by their molecular weight. It has
been pointed out that longer PEG chains (such as 5000 Da)
interact via van der Waals interactions and interchain hydrogen
bonding, resulting in a condensation of the outer layer size.57
2.3.7. Ligand Density. Nevertheless, above a high
concentration, DSPE−PEG micelles could induce a destabili-
zation by solubilization of the delivery system.60 Cao et al.
revealed also that the cellular uptake does not necessarily
linearly increase with ligand density. Indeed, competitive
interactions between adjacent folate moieties and folate−folate
receptors may occur at high grafting density, leading to limited
flexibility of multivalent ligands and consequently to decreased
targeting capacity.61 By using a mathematical model, Ghaghada
et. al showed that above a relative density of 500 folate ligands
per liposomes, the uptake of targeted liposomes by C6 glioma
cells could increase no more, probably due to a down-
regulation of the folate receptor.62 This threshold depends on
the targeting ligand, the LBNC hydrodynamic diameter, and
the targeted receptor type and density.63
In consequence, the ideal DSPE−PEG−ligand compromise
must be defined for each LBNC to lead to efficient cell binding
and uptake.
3. ANALYTICAL METHODS TO PURIFY AND
CHARACTERIZE FUNCTIONALIZED LBNCs
It is important to separate the coupled ligand from the free
fraction before performing further characterization of the
functionalized LBNCs. Adequate purification and character-
ization will play a fundamental role in the reproducibility of
functionalized LBNC production with a high purity but also
for control of the LBNC surface biomodification with the idea
to develop a scaling-up process. Purification of functionalized
LBNCs is a key issue before performing characterization.
Classical purification techniques are usually capable of
removing unbound ligands with, in some cases, the ability to
distinguish different populations. Final characterization of the
purified functionalized LBNCs will ultimately require a
combination of techniques to investigate all physicochemical
characteristics in order to reflect the reality of the studied
targeted nanocarriers (Table 3).
3.1. Purification Techniques. A range of processes have
been used for purification of nanoparticles (Table 3). The
complexity of the purification step is related to the nanometric
size of studied nanocarriers, which are subjected to Brownian
motion. Specific methods with more drastic conditions have
been used to increase density and dimensional differences
between each population in order to separate functionalized
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LBNCs from both free ligands and nontargeted LBNCs.
However, most of the methods are not able to separate two
populations of LBNCs (functionalized LBNCs from non-
targeted ones) except in the case of specific ligand with a
relatively big size (size exclusion chromatography could be
applied with highly sensitive methods for example).64 In this
section, techniques that are used to separate functionalized
LBNCs from free ligands will be described.
3.1.1. Centrifugation, Ultracentrifugation, and DCD. The
commonly used centrifugation and ultracentrifugation meth-
ods consist of the application of a centrifugal force to separate
nanoparticles from undesired material (uncoupled ligands,
additives, or surfactants).65 Ultracentrifugation can isolate
much smaller objects thanks to the use of high centrifugal
force. In the same way, diafiltration is a combination between
the use of centrifugal forces and filters with a known molecular
weight cut off (MWCO). These filters are added to centrifuge
tubes and allow a simple and easy separation process.
However, interactions between nanocarriers, more specifically
ligands at the LBNC surface, and membrane filters could
occur. The centrifugation force can impact nanostructure
properties and cause caking and difficulties in redispersing
nanoparticles.66 An insufficient centrifugal force can lead to an
important loss of nanoparticles in the supernatant, resulting in
low yield of nanoparticles in the final suspension.
3.1.2. Dialysis. Dialysis, which has also been extensively
used, consists of using a semipermeable dialysis bag with a
given MWCO. This membrane is filled with the sample to be
purified and immersed in a large excess volume of dispersant.
The liquid diffuses through the membrane from high to low
concentration. However, if dialysis is not performed under
appropriate controlled conditions, a reorganization of LBNCs
and a release of the active molecule could occur before
performing further characterization of the functionalized
LBNCs.
3.1.3. Chromatographic Techniques. Chromatographic
techniques, such as size exclusion chromatography (SEC) or
gel filtration, are also used to separate different population of a
same sample function of their size.19,67 Gel filtration is widely
used when LBNCs are functionalized with proteins or peptides
prior to further characterization. Barrajón-Catalán et al. used
gel filtration to eliminate the unbounded antibodies
(trastuzumab) at the surface of liposomes.67 The purification
step was performed passing the mixture through a Sephadex G-
25 (Sigma-Aldrich) column using histidine buffer for the
elution. The amount of antibody present at the surface of
immunoliposome was quantified using sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) separation.
For the quantification, the intensity of HER2 bands deriving
from immunoliposome sample was compared with the
intensity of pure antibody by densitometric analysis. One
disadvantage of the use of chromatographic methods is the
potential interaction between nanoparticles and the stationary
phase of the column.68 An alternative approach is the use of
asymmetrical flow field-flow fractionation (AF4). AF4 is
composed of a semipermeable membrane instead of a
stationary phase. During fractionation, molecules are separated
based on their molecular size. To this end, a cross-flow is
applied through the membrane, and a concentration gradient is
created. However, adsorption to the membrane could also
occur.69 SEC, gel filtration, or AF4 is usually used to separate
complex mixtures with antibodies/proteins ligands and nano-
particles before further characterization. These separation
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Table 4. Common Methods Used to Characterize Functionalized Nanoparticlesa

characterization
method entity characterized advantages limitation ref
in silico prediction • size • screen of different ligands • processing and analysis of massive data sets with 74−78
complex calculations and computerized models
• surface composition (ligand binding, • study of ligand conformational changes at
composition, and density) the nanoparticle surface in biological
media
DLS • size (Rh) • fast and easy analysis • dust free and dilute sample 8, 9, 48,
79−83
• size distribution • strong contribution of large particles
• zeta potential • monodisperse population
MALS • size (Rh and Rg) • multiple angle analysis
• size distribution
NTA • size (Rh) • single particle resolution • dilution required 8, 81−83
• nanoparticle concentration • possibility of analyzing polydisperse
sample
SAXS • size (Rg) • high sensitivity • restricted access to X-ray scattering 84, 85
• size distribution • costly and time-consuming analysis
• shape
electron • size • morphological study • possible alteration of nanoparticles during 21,78−83
microscopy sample preparation
• shape • artifacts
chromatography • surface composition (ligand • low sample volumes • detectable moiety or bound is required 92
techniques composition and density)
• accurate quantitative measurements • in most cases indirect quantification
mass spectrometry • surface composition • high sensitivity • restricted mass range 93, 94
• easily coupled with separation techniques • sensitive toward interfering molecules
to obtain real-time information
electrophoresis • surface composition • low sample volumes • comigration of macromolecules with similar size 34, 93, 94
CD • ligand conformational changes • no extensive sample preparation • interferences with unbound ligands 96−99
• ligand structural information
FTIR • evaluation of protein or peptide • fast and easy analysis • low sensitivity 99, 100
aggregation and conformational
changes
• information about chemical bonds • artifacts with aqueous solution
• difficulties to interpret spectra with complex
mixtures
SPR • protein association to and • real time assay • need to immobilize either nanoparticles or 101, 102
dissociation from nanoparticles proteins that could affect the binding constants
measured
• sensitive
• label-free
NMR • surface composition • high resolution • need for high concentration to have enough 8, 93,
spectroscopy signal 103−112
• size • heterogeneous systems • identification of a specific hydrogen nuclei
ITC • intermolecular interaction • easy to use • ligand−ligand interactions are not considered 93, 101,
characterization between ligands and 115−120
nanoparticles
• high sensitivity • complex thermogram interpretation if multiple
interactions
• interaction mechanisms between ligands
and nanoparticles
• thermodynamic profile determination
a
Abbreviations used: DLS, dynamic light scattering; MALS, multiangle light scattering; NTA, nanoparticle tracking analysis; SAXS, small-angle X-
ray scattering; CD, circular dichroism; FTIR, Fourier-transform infrared spectroscopy; SPR, surface plasmon resonance; NMR, nuclear magnetic
resonance; ITC, isothermal titration calorimetry; Rh, hydrodynamic radius; Rg, radius of gyration.

techniques are coupled with light scattering methods to
characterize suspensions of polydisperse nanoparticles espe-
cially for the protein corona investigation.
3.1.4. Tangential Flow Filtration (TFF). TFF or cross-flow
filtration is widely explored in several applications for
biomacromolecules purification in the food industry but also
in the pharmaceutical field in order to overcome some
drawbacks associated with conventional purification techni-
ques.70 During the process, the purification is performed in a
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dynamic manner: a fluid containing solutes flows tangentially
to the filter surface and not perpendicularly compared to
conventional filtration methods. Thus, TFF allows to purify
and concentrate the suspension. Moreover, thanks to the
applied pressure and device geometry, the dispersion flow is
turbulent near the membrane allowing to reduce cake
formation. This approach can be transposed easily on an
industrial scale and has attached major attention notably for
the purification of liposomes71 but also for other LBNCs.
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For example, He et al. used TFF to remove the organic
solvent and concentrate DNA-delivering LBNC systems.72 In
fact, TFF provides a pressure-driven one-way permeation that
eliminates ethanol in contrast to dialysis method where the
ethanol concentration is the same inside and outside the
membrane. Different nanocarriers were considered: DNA-
loaded liposomes, lipo-complexes, and lipo-polyplexes. These
nanoparticles were formulated using a continuous production
process by flash nanocomplexation. Then, LBNCs went
through a TFF process for purification and concentration
before further characterization. Authors showed that after the
purification step, size, zeta-potential, and morphology of
LBNCs were preserved. Thus, the stability of nanoparticles
was ensured during the TFF process.
Hirsjärvi et al. used TFF and dialysis with comparable
membrane material as tools to evaluate LNCs behavior and
deformability.73 The loss of LNCs during these two
purification processes was also investigated. Results showed
that 10% of LNCs can be lost during the dialysis and may be
due to the surface deformability of nanostructures, but without
any evidence of the membrane passing. With the TFF method,
LNCs have been found in the filtrate even if the pore size of
membrane used was smaller than the LNC diameter. This
study highlights the importance of the nanostructure’s
rheological properties to choose the appropriate method. In
fact, the elastic nature of nanoparticles could contribute to
their loss during purification processes due to membrane
passing by deformation. Adequate purification and character-
ization will play a fundamental role in the reproducibility of
functionalized LBNC production with a high purity but also
for control of the LBNC surface biomodification with the idea
to develop a scaling-up process. Purification of functionalized
LBNCs is a key issue before performing characterization.
Classical purification techniques are usually capable of
removing unbound ligands with, in some cases, the ability to
distinguish different populations. Final characterization of the
purified functionalized LBNCs will ultimately require a
combination of techniques to investigate all physicochemical
characteristics in order to reflect the reality of the studied
targeted nanocarriers.
3.2. Physicochemical Characterization. Characteriza-
tion methods mentioned in the following sections are
summarized in Table 4 with the entity characterized, the
advantages, and limitations of each technique.
3.2.1. Nanoinformatics/in Silico Prediction. The develop-
ment of in silico methods has gained in importance in recent
years since nanoinformatics can predict some interactions
between nanocarriers and biomolecules. It is possible to model
systems in their native environment, in contrast with several
characterization methods that require solvent removal or
deposition on a substrate, that can alter the nanostructure and
morphology. Nanoinformatics can investigate several parame-
ters of functionalized nanoparticles such as the coating ligand’s
length, grafting density and distribution, and rigidity but also
their affinity to receptors.74 Molecular dynamics (MD) have
been extensively used to describe protein and peptide
adsorption at a wide variety of solid/liquid interfaces. The
interactions between proteins and poly(ethylene glycol)- or
poly(phosphoester)-coated nanoparticles were also investi-
gated using MD simulations. The developed model, based on
the solvent-accessible surface area exposed by each amino acid
on the protein surface, allowed researchers to evaluate the
consequences of the coating by determining which binding
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epitopes will remain accessible for receptor binding.75
Carrasco-Sanchez et al. investigated the ligand conformation
at the surface of dendrimers using MD simulations.76 Authors
compared unfunctionalized poly(amidoamine) (PAMAM)
dendrimers with FA-functionalized dendrimers. Simulation
techniques showed similar sizes between unmodified and FA-
functionalized dendrimers because the folate groups were
oriented inward with a tendency to penetrate into the cavities
of the dendrimer. In the same manner, MD simulations were
used to study the influence of the ligand location on release of
guest molecules from the dendron-based supramolecular
assembly. Simulation showed a better availability for protein
binding when the ligand is linked at the periphery rather than
at the focal point or at the intermediate layer.77
The computational approach would allow estimation of
several parameters of functionalized LBNCs with a better
understanding of the ligand conformational changes at the
nanocarrier surface in biological media and in contact with
cellular membranes. Moreover, this technique allows screening
of functional ligands for rational design of surfaces with
resistance to nonspecific protein adsorption in order to obtain
the optimal response in vivo. However, nanoinformatics
required the processing and analysis of massive data sets,
with complex calculations and computerized models at the
atomic, molecular, and nano levels.78
3.2.2. Classical Characterization Methods. 3.2.2.1. Scatter-
ing Methods. One of the most significant factors of the
formulated LBNCs is size distribution (mean size and
polydispersity (PdI)). The predominant method is the light
scattering and more particularly dynamic light scattering
(DLS) or multiangle light scattering (MALS). DLS is a
classical and daily used technique to determine the hydro-
dynamic diameter of nanoparticles in suspension at a defined
scattering angle (usually 173°).9 In the same way, MALS is
based on the measurement of the scattering intensity as a
function of the scattering angles. Thanks to the Brownian
motion of nanoparticles in solution, this method relates the
diffusion coefficient to the hydrodynamic radius of a sphere
through the Stokes−Einstein equation. The hydrodynamic
diameter reveals not only the dimension of the nanoparticle
but also the corona layer linked to the nanoparticle surface.
The polydispersity index (PdI) could also be acquired by
cumulant analysis of the measured autocorrelation function.
This index reflects the heterogeneity of a sample based on size.
Moreover, DLS systems are able to measure zeta potential
which is determine from electrophoretic light scattering, also
known as laser Doppler electrophoresis. This parameter, which
is related to the electrostatic charge on nanoparticle surface,
could also be followed to control the surface functionalization.
Perrier et al. used DLS to follow the postinsertion of DSPE-
PEG-OCH3 into lipid nanocapsules (LNCs): the evolution of
hydrodynamic diameter and zeta potential was analyzed with
the amount of DSPE-PEG-OCH3. An increase of the size was
observed from 55 to 70 nm (27% increase) with an
incorporation of 5% of DSPE-PEG-OCH3. Then, increase of
size reached a plateau and 70 nm appears to be the limit. In
fact, when the amount is higher than 5%, the diameter was not
increased. In the other side, the zeta potential decreased with a
linear relationship with the amount of DSPE-PEG-OCH3. To
prove the DSPE-PEG-OCH3 shell formation, control experi-
ments were also performed with LNCs under the same
conditions but without DSPE-PEG-OCH3.48 In another study,
DLS was used to characterize curcumin-loaded nanoemulsions,
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Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
that were functionalized with nona-arginine (R9). A size
increase of about 20 nm was observed after the coupling of R9
to the nanoemulsion’s surface. The stability of functionalized
NE in PBS containing 50% serum was also investigated by
DLS, and results suggested that R9 was able to stabilize NE by
limiting interactions with serum components (no increase in
size and PdI).79 DLS is a very fast and inexpensive method that
needs a dust free and dilute sample with a monodisperse
population. However, this method, considered as a low-
resolution technique, cannot be used individually and must be
performed only for an initial analysis.80
Nanoparticle tracking analysis (NTA) could also be used to
determine not only the number size distribution but also the
nanoparticle concentration. By contrast with DLS, NTA does
not overestimate larger nanoparticles, allowing measurement of
particle size distribution of complex samples. The instrument is
able to capture the Brownian motion of each individual
nanoparticle and then to measure a single particle diffusion
coefficient.
However, for all light scattering methods, the results
obtained can be affected if nanoparticles are not scattering
by the laser beam due to their small size or because their
refractive index is very close to water. Methods usually require
sample dilution prior to the analysis that could affect the
nanoparticle structure. For this reason, it is important to
analyze the suspension stability after sample dilution. In order
to have reliable results in biological media, the use of light
scattering technique such as DLS as the only method was not
advised and a combination of high-resolution techniques (such
as AF4-DLS-MALS or tunable resistive pulse sensing (TRPS))
is required.81−83 The study of Guyon et al. highlighted the
essential and complementary contribution of each character-
ization methods to reflect the reality of the studied
nanostructure.8 In fact, DLS and transmission electron
microscopy (TEM) analysis revealed a 100 nm size population
with a narrow size distribution. However, high resolution
method (NTA, small-angle X-ray scattering and NMR
diffusometry) showed the presence of another population of
small objects (<2 nm) which represents more than 99% of the
matter.
The wavelength of visible light is in the 400−700 nm range
compared to X-rays wavelength (0.1−0.2 nm). From that, the
magnitude of the scattering wave vector q (= (4π/λ) sin(θ/2)
with θ being the scattering angle) is small for light scattering,
whereas small-angle X-ray scattering (SAXS) deals with larger
q-ranges. Moreover, this last method can also give data at the
light-scattering q-range by going to very small scattering angles.
Thus, small-angle X-ray scattering (SAXS) is a high sensitive
method that is able to provide not only the nanoparticle size
and size distribution but also the nanoparticle morphology
including the inner structure.84 The determination of the inner
structure is easier for inorganic nanoparticles. In fact, samples
that are composed of atoms with low scattering density (e.g.,
C, N, O, H, and P) lead to overall low scattering efficiency, and
a synchrotron radiation source is needed to provide orders of
magnitude higher than X-ray flux. With this technique,
Nascimento et al. demonstrated that an increased amount of
hyaluronic acid in the liposome formulation process led to
increasingly denser multilamellar structures. Moreover, the
SAXS patterns showed the reorganization of unilamellar
liposomes into oligolamellar vesicles when siRNA is added.85
3.2.2.2. Microscopy-Based Techniques. Electron micro-
scopic procedures (EM), and more specifically transmission
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electron microscopy (TEM) are usually employed to analyze
the morphology, size and stability of LBPNs.25 The size
increment after surface functionalization observed by DLS was
usually confirmed by TEM. In fact, an increase in nanoparticle
size observed by DLS was also verified by negative staining
TEM analysis after PEGylation of inorganic nanoparticles.86
Pelaz et al. studied the PEG shell at the surface of poly(maleic
anhydride-alt-dodecene) coated inorganic nanoparticles.88
TEM experiments revealed that the hydrodynamic radius
increase upon PEGylation was only between 1 and 6 nm with
three different length of PEG chains and not between 6 and 80
nm as expected.87 These results gave an indication about PEG
conformation: chains were not stretched but coil, fold, or twist
on the nanoparticle surface. EM allows visualization of
nanoparticles in 2D, but three-dimensional reconstitution
and 3D images could also be obtained.89 Visualization of the
nanoparticle shape is a crucial step before using other analytical
methods such as DLS, which assume a spherical morphology
to calculate the size distribution. Another example is the use of
cryogenic transmission electron microscopy (cryo-TEM) that
allows to observe the objects in their native form. Thanks to
this technique, the nanoparticle morphology but also the
siRNA localization into liposomes was studied.85 In fact, the
appearance of several small bubbles when the particles were
submitted to the electron beam for image acquisition revealed
the degradation of water molecules associated with the
siRNAs. Despite the fact that TEM is widely used to image
several nanoparticles such as inorganic nanoparticles but also
LBNCs, it is important to choose the appropriate sample
preparation method.90 In fact, depending on the sample
preparation (drying, negative staining, freeze-fracture, and
cryo-TEM), various artifacts such as structure deformation,
fractures, or particles aggregation should be considered during
the interpretation of the TEM images.91
3.2.2.3. Chromatographic Methods. Chromatographic
techniques, in particular, high-performance liquid chromatog-
raphy (HPLC), were also used to confirm the conjugation of
the peptide ligand to the nanoparticle surface. For instance,
Palekar et al. used this method to control the surface
modification of thrombin-targeted liposomes.92 Conjugation
of PPACK (a short chain peptide that inhibits thrombin) to
liposomes was investigated through HPLC quantification of
uncoupled peptide recovered from the supernatant after
centrifugation of predialysis PPACK-liposomes mixed with
cleanascite lipid adsorption reagent. This indirect quantifica-
tion was performed at a wavelength of 215 nm (detection of
amide bond). Because a lot of chemical functions can be
detected at this wavelength, it is important to check that there
is no interference with other compounds or LBNCs. The
disadvantage of this method is the indirect quantification of
bound peptide via detection of unbound ligand: if the
purification method is not robust, the amount of ligand at
the nanoparticle surface may be over- or underestimated.
3.2.3. Other Complementary Methods. 3.2.3.1. Mass
Spectrometry. Mass spectrometry is a high sensitive method
classically used to study interactions between larger proteins
(∼100 kDa) and nanoparticles.93 This technique can give
qualitative and quantitative data and can be used with
chromatographic methods to identify the composition of the
protein corona.94
3.2.3.2. SDS-PAGE. SDS-PAGE is a widely used technique
to separate nanoparticles−protein complexes according to the
electrophoretic mobility of each macromolecule. Menina et al.
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used this method for the quantification of Eap (bacteria-
derived invasion protein) associated with liposomal surfaces.34
SDS-PAGE gels showed a distinct Eap band at 55 kDa in both
Eap-functionalized liposomes via surface adsorption and via
covalent coupling. Gel analysis of Eap standard at different
concentrations allowed researchers to obtain the functionaliza-
tion efficiency (FE). This useful method was used to identify
the composition of the protein corona.94 However, in the case
of a complex mixture, comigration of several macromolecules
with a similar size could occur.93
3.2.3.3. Circular Dichroism (CD). CD permits investigation
of secondary or tertiary structure changes when proteins or
peptides are in contact with a nanoparticle surface.9596 Deng et
al. used this method to estimate the fibrinogen structure
evolution at the surface of poly(acrylic acid)-coated gold
nanoparticles (PAA-AuNP).97 Authors showed that nano-
particle−protein interactions change the fibrinogen conforma-
tion that becomes unfolded in contact with the nanoparticle
surface allowing it to enhance interaction with the integrin
receptor. In the same manner, several peptides that have
random coil conformation in aqueous media were shown to
adopt an α-helical secondary structure when associated with
DSPE-PEG2000 micelles.98 However, the risk of this technique
is to consider unbound protein that is frequently present in the
sample with nanoparticle and bound protein. To avoid
interferences with native protein, the purification step is
crucial before using CD. Moreover, it is important to notice
that the signal emitted by nanoparticles-bound protein could
be very weak.99
3.2.3.4. Fourier-Transform Infrared Spectroscopy (FTIR).
The FTIR technique is also used, in combination with CD, to
study protein tertiary structure change when it is adsorbed
onto a nanoparticle surface. Jiang et al. showed that the
addition of 16 nm gold nanoparticles to bovine heart
cytochrome c (cyt c) in 0.1 M PBS led to a decrease of β-
turn and β-sheet structures.99 On the contrary, the addition of
2−4 nm gold nanoparticles induced an increase of β-turn and
β-sheet structures. Thus, the opposite effect of different sizes of
gold nanoparticles was observed on the conformational
changes of the adsorbed cyt c. FTIR was also used to
determine the coating efficiency of liposomes with pectin and
whey proteins (WPI). Characteristic bands of liposomes
(symmetric and asymmetric stretching vibrations of CH2)
give conformational information on the lipid acyl chains.
Moreover, with this technique authors demonstrated that WPI
had preferential interactions with pectin compared to lip-
osomes. Thus, WPI was the second coating of the lip-
osomes.100
3.2.3.5. Surface Plasmon Resonance (SPR). SPR allows
quantitative analyses of the interaction between functionalized
nanoparticles and their biological targets. This technique can
provide details on the formation of the protein corona.
Typically, either nanoparticles or proteins are immobilized on
a sensor chip surface, and the other partner is flowed through a
microfluidic system in contact with the coated surface. Binding
is revealed in real time as a change of mass at the surface, and
the interaction can be characterized in terms of kinetics and
affinity. Using this technique and copolymer-based nano-
particles immobilized on gold surface, Cedervall et al. showed
that albumin and fibrinogen exhibit higher association and
dissociation rates than apolipoprotein A-I and many other
plasma proteins.101 Moreover, results revealed that the
exchange rates and the degree of surface coverage were
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affected by the nanoparticle hydrophobicity. These results
were confirmed using other complementary methods (ITC in
the next section). Selected proteins (immunoglobulin G and
bovine serum albumin, both involved in the formation of the
corona) were also immobilized in the sensor surface that was
subjected to the flow of SLN solutions. In this case, SPR was
successfully used to monitor protein−nanoparticle interactions
involved in the formation of the corona and to mimic different
circulations times in the bloodstream.102
In summary, different analytical tools have been developed
to characterize the surface decoration of nanoparticles,
specifically for the study of biocorona formation on inorganic
nanoparticles. Each of the methods mentioned above has their
own advantages and drawbacks. The choice of the analytical
technique depends not only on the nanoparticle type and
ligand used but also on the physical properties being
monitored. In the next section, two emerging analytical
techniques will be described: NMR spectroscopy and ITC.
3.2.4. Promising Characterization Techniques. In this
section, described techniques are mainly developed for
inorganic and polymeric nanoparticles. To date, they were
not used for functionalized LBNCs, but they could be of
interest for the characterization of surface functionalization.
3.2.4.1. Nuclear Magnetic Resonance (NMR) Spectrosco-
py. The NMR technique appears as a powerful and challenging
technique to separate and identify sample components. Several
parameters, depending on the dynamics and conformation of
the free and bound ligand, are exploited: the chemical shifts,
relative intensities, and line widths for NMR-visible spin
systems, which are related to the speed of nuclei relaxation. If
ligands are bound to nanoparticles, chemical exchanges create
a modulation in a nuclei’s chemical environment, associated
with a chemical shift and a relaxation change. In 1H NMR, the
signal can be used to quantify the binding capacity of
nanoparticles. This experiment is usually performed to study
the spontaneous protein corona formation at the nanoparticle
surface such as the GB3 protein adsorption at the surface of
gold nanoparticles. To determine the bound protein
concentration and hence, the binding capacity of nanocarriers,
protein is titrated with increasing concentrations of nano-
particles associated with a 1H signal intensity decrease.103
Additionally, a 1D half-filter experiment can be used to
evaluate the competitive binding of multiple isotopically
enriched 13C or 15N-proteins to the nanoparticle surface. For
instance, Wang et al. used this approach to evaluate the
apparent binding capacities of the AuNPs for two proteins
(wild-type GB3 and ubiquitin) using an NMR measure-
ment.104 This study revealed that several parameters were
involved at different steps of protein adsorption: the net
charge, binding association constant but also size of protein
have to be considered. Thanks to 1D half-filter assays, and if
separate isotopic labeling with a well distinct peak dispersion
can be used, it is also possible to detect two different types of
ligand functionalized nanoparticles in the same suspension.93
Two-dimensional NMR techniques, such as heteronuclear
single-quantum coherence spectrum (HSQC)105 or transverse
relaxation optimized spectroscopy (TROSY),106 can also be
used to identify the orientation and conformational changes of
proteins upon binding to nanoparticles but also measure the
binding capacity of nanoparticles.
Because of the structural similarity of liposomes with the
plasma membrane, protein interactions with LBNCs have been
studied using NMR spectroscopy. Relaxation based methods
Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Figure 4. Scheme of the ITC instrument with cells and injection syringe. Right side: representative raw ITC data from titration experiment.
can be applied with such systems thanks to the fast exchange
and dynamic between protein and liposomes that generate
measurable relaxation rates.107 Ceccon et al. developed several
NMR experiments to understand the interactions between
ubiquitin, used as a protein model, and negatively charged
liposomes.108 From the different contributions and relaxation
fitting, it appeared that the protein rotates on the low
microsecond time scale around an internal axis to the
nanoparticle surface. Moreover, from paramagnetic relaxation
enhancement (PRE) used with Gd3+-paramagnetically tagged
liposomes, it was demonstrated that the central hydrophobic
region principally interacts with the negatively charged surface
of liposomes. This study proved that an NMR approach allows
researchers to characterize the dynamics behavior and
exchange kinetics of an interaction between liposome and an
NMR visible compound.
Two-dimensional NMR pulsed field gradient (PFG)
acquisition was also used to evaluate the diffusion coefficients
of several components present in a sample. These coefficients
are related to a chemical shift by the study of the exponential
decay of the NMR signal to the self-diffusion behavior
occurring between the two magnetic field gradients.109 Smaller
components, such as ligands, should diffuse faster compared to
larger nanoparticles and consequently should provide the
major contribution in terms of protons. Thus, free ligands and
ligand-functionalized nanoparticles can be separated based on
its diffusion coefficient. This approach requires not only
precise data acquisition but also an appropriate processing
method.110 In fact, the precision of the measure depends on
the acquisition parameters and the signal decay sampling.111
Inverse Laplace transform (ILT) on diffusion ordered
spectroscopy (DOSY) signals were used in combination with
a constrained regularization calculation algorithm called
REPES112 to characterize cell-penetrating peptides-ferrocifen
nanoparticles. In this study, NMR diffusometry allowed
researchers to differentiate nanoparticles from soluble ligands
thanks to the diffusion weighted factor. Then, hydrodynamic
diameters were calculated from the estimated diffusion
coefficient according to the Stokes−Einstein equation.
Interestingly, the results revealed the presence of small
nanoparticles that were not visible by classical methods
pubs.acs.org/molecularpharmaceutics Review
showing the potential of the NMR approach to obtain reliable
characterization.8
The NMR approach is a promising tool for complex mixture
analysis. However, the capacity to resolve the different
contributions is challenging, and the acquisition parameters
have to be adjusted as a function of the mixture composition.
3.2.4.2. Thermal Analysis - Isothermal Titration Calorim-
etry (ITC). Thermal analysis (TA) is linked to a change in a
sample property which is related to an imposed temperature
alteration. For example, calorimetry means the measurement of
the heat flux as a function of time or temperature. Differential
scanning calorimetry (DSC) and isothermal titration calorim-
etry (ITC) are the only methods for the direct determination
of the enthalpy (ΔH). During a DSC assay, the difference in
the amount of heat required to increase the temperature of a
sample and a reference is measured. This method is commonly
used for studying LBNC molecular organization and protein
stability. ITC can be used to directly measure the variation of
enthalpy when ligands interact with nanoparticles. This
promising method has been applied to characterize inter-
molecular interactions with high sensitivity allowing the
determination of several parameters: the association constant
(K), the stoichiometry, the enthalpy (ΔH), and the entropy
(ΔS) from which the Gibbs free energy (ΔG) can be
calculated.113,114
During an experiment, the ligand is titrated in the
nanoparticle solution in an isothermal measurement cell. The
heat difference, that needs to be added to the sample and
reference cells to keep both cells at the same temperature, is
then monitored. ITC instrument is described in Figure 4. If the
reaction is exothermic, the power applied to the control heater
decreases in order to keep the sample and reference at a
constant temperature, and a negative signal is obtained. A
classical power compensation ITC, which represents the power
in μcal/s as a function of time, is shown in Figure 4. If the
concentrations of both the nanoparticles and injected ligands
are known, data obtained after an experiment can provide
information on the number of ligands bound per nanoparticle,
the apparent affinity, and the enthalpy change.114
ITC has been more and more used to investigate the
mechanism behind protein corona formation.115,116 In fact, this
method allows researchers to evaluate the protein corona in
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Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
situ, without the need to separate protein-bound nanoparticles
from the media before further characterization. Moreover,
another important advantage of ITC is that experiments do not
require labeling ligands or nanoparticles, allowing character-
ization without any modification of the compounds or the
interactions. Finally, this promising technique gives informa-
tion not solely on the binding affinity but also on the
thermodynamic parameters of the interaction.
By ITC, Cedervall et al. analyzed kinetic and equilibrium
parameters, i.e., enthalpy, stoichiometry, affinity, implied in the
interactions between human serum albumin (HSA) and
various N-isopropylacrylamide (NIPAM): N-tert-butylacryla-
mide (BAM) copolymer based-nanoparticles.101 From experi-
ments, the equilibrium association constant between HAS and
the nanoparticle surface was evaluated to 2 × 106 M−1. Thanks
to gel filtration assays, a comparison of association and
dissociation constants between several plasma proteins
revealed that albumin and fibrinogen exhibit higher exchange
rates than apolipoprotein A-I. By comparing ITC results,
authors demonstrated the thermodynamic nature of the
nanoparticle−protein interactions and the evolution of protein
conformation upon binding. This study showed the strong
influence of protein identity and nanoparticle characteristics
(size and more or less hydrophobic surface) on the protein
corona formation and morphology. Indeed, it was suggested a
higher degree of surface coverage at equilibrium and the
presence of a single layer of albumin adsorbed at the surface of
the bigger and most hydrophobic nanoparticles.
ITC was also used with 4-(N)-trisnorsqualenoylgemcitabine
(SQdFdC) nanoparticles.117 These nanocarriers were func-
tionalized with CKAAKN peptide by the thiol-maleimide
Michael addition coupling strategy. The conjugation was
achieved either with preformed nanoparticles or by coupling
the peptide with a squalenoyl derivative prior to nanoparticle
formulation. Then, the resulting CKAAKN-SQdFdC nano-
particles obtained by these two different approaches were
characterized using several analytical tools. For functionalized
nanoparticles obtained by coupling CKAAKN to preformed
nanoparticles, ITC thermograms revealed that the CKAAKN/
maleimide molar ratio (1:0.25) led to the coexistence of both
noncovalent interactions and covalent conjugation of the
CKAAKN onto the nanoparticle surface. Higher CKAAKN/
maleimide molar ratios have been shown to promote the
Michael addition reaction, but these higher ratios led to
nanoparticle aggregation probably due to peptide instability at
the surface. Valetti et al. demonstrated the importance of
performing in-depth analysis of targeted nanoparticles in order
to understand how different compounds collaborate and
interact together to reach the higher cell targeting capacity.
An interesting study used ITC to evaluate the thermodynamics
of the interaction between bovine serum albumin (BSA) and
squalene-adenosine (SQAd) nanoparticles.118 The stoichiom-
etry of the complex and the thermodynamic parameters were
calculated using a one-binding site fitting model. Results
demonstrated, in addition to other analytical methods (small-
angle neutron scattering, cryo-TEM analysis, circular dichro-
ism, steady-state fluorescence spectroscopy, and molecular
dynamic simulations) that BSA partially disassembles SQAd
nanoparticles by extracting individual SQAd monomers from
them. The removal of SQAd from the nanoparticle and
subsequent binding to BSA were enthalpy driven. Moreover,
the SQAd binding to BSA compensated for the entropy loss
arising from the nanoparticle disassembly. Consequently, the
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SQAd nanoparticles could act as a circulating reservoir in the
bloodstream. Huang et al. analyzed the drug−lipid interactions
of weak-hydrophobic drug-loaded solid lipid nanoparticles by
ITC.119 In this study, benzoic acid (BEA) and salicylic acid
(SAA) were chosen as the model weak-hydrophobic drugs, and
stearic acid (SA) was selected as the lipid material. Results
revealed that BEA−SA interaction exhibited negative ΔH and
negative ΔS indicating hydrogen bonds. SAA−SA interactions
possessed slightly negative ΔH and positive ΔS, which
suggested hydrophobic forces. Interestingly, in this study
SLN was not studied directly with ITC in nanoparticle form. In
fact, just a small part of this nanoparticle was used to
investigate the interactions between drugs and lipid.
Several challenges exist when using ITC to study
interactions between ligands and LBNCs. Indeed, the classical
ITC method requires molar sample concentrations and
relatively high volumes (∼1 mL). Therefore, a method
known as micro-isothermal titration calorimetry analysis (μ-
ITC) uses the same principle as ITC, but the mass of the
sample investigated is on the order of 10 μg, with the ability to
measure millimolar to nanomolar affinities. However, the study
of a complex protein system and the determination of the
molar concentration of nanoparticles, which is often based on
the assumption of a monodisperse population with perfectly
spherical objects, can be challenging. In most cases, fits of the
adsorption isotherms are based on the independent binding
model, assuming that interacting or bound ligands do not
influence the binding of other ligands such as proteins.120 This
mathematical model does not consider potential cooperative
effects and ligand−ligand interactions. Moreover, if no
measurable heat accompanies adsorption even at high
nanoparticle concentrations, it becomes impossible to analyze
the data. Finally, if interactions imply multiple steps, binding
and then anchoring, the interpretation of ITC thermograms
may become challenging.93
To conclude, ITC has historically been used to study
biomacromolecular interactions in order to quantify the
binding of ligands to receptor molecules or to the lipid matrix
of biomembranes. In most cases, nanoparticles which were
studied in their full form using ITC or NMR are inorganic
nanoparticles. Limited ITC applications for the study of the
interaction mechanisms between ligand and LBNCs could be
due to several parameters.121 If nanoparticles are produced by
using organic solvents within the formulation process, the
resulting traces may hamper the signal, which renders the
signal treatment difficult. ITC would be well adapted for
characterization of some LBNCs such as lipid nanocapsules or
also as lipid nanoemulsions, that are generated from
spontaneous emulsification without use of any organic
solvent.55,122 Moreover, ITC can require solution volumes
and concentrations that are difficult to obtain in some cases
due to either the high cost or limited quantities of the
compounds. To address this issue, some ITC instruments (μ-
ITC) offer lower volume calorimeters. However, it is important
to note that smaller volumes also result in a smaller heat signal,
which can affect the signal-to-noise ratio. Moreover, every
chemical process that occurs in the sample cell will contribute
to heat. In this case, it could become challenging to
deconvolute the effects of multiple contributors to the overall
heat, particularly in the case of LBNC with multicomponent
chemical system. In this case, it can be tricky to optimize the
parameters to obtain reliable results and to find the appropriate
mathematical model. Despite these considerations, ITC
Mol. Pharmaceutics XXXX, XXX, XXX−XXX
 Table 5. Decorated LBNCs: Considerations and Development of Methods Used for the Functionalization, Purification, and Characterization Steps
key considerations
Functionalization • Require adjustment function of the LBNC and ligand type
• Different functionalization strategies can lead to different
biological results.
• Ideal ligand density must be defined for each LBNC to lead
to efficient cell binding and uptake.
Purification • Separation of functionalized LBNCs from free ligands
• Importance of the nanostructure’s rheological properties to
choose the appropriate method
Characterization • The choice of the characterization method depends not only
on the nanoparticle type and ligand used but also on the
physical properties being monitored.
• Require a combination of techniques to investigate all
physicochemical properties
P
Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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advances
• Four strategies can be used:
- Direct ligand conjugation with preformed LBNC
- Postinsertion of lipid−PEG−ligand micelles
- Incorporation of lipid−PEG−ligand within the LBNC formulation process.
- Noncovalent adsorption of the ligand.
• Development of methods with high resolution and reproducibility: size exclusion
chromatography (SEC) and tangential flow filtration (TFF)
• The computational approach would allow estimation of several parameters of
functionalized LBNCs.
• Nuclear magnetic resonance spectroscopy (NMR): challenging technique to
separate and identify sample components.
• Isothermal titration calorimetry (ITC): a powerful tool to optimize the LBNC
surface functionalization and to enhance the understanding of the interaction
mechanisms between targeting ligands and LBNCs
current limitations
• The ligand conformation and availability could change the
function of the functionalization strategy, resulting in a
modification of the targeting efficiency.
• Conditions used could induce detrimental destabilization of the
nanocarriers, denaturation of the targeting ligands, and/or
degradation of the encapsulate drugs.
Molecular Pharmaceutics
• Difficulties to separate two LBNC populations (functionalized
LBNCs from nontargeted ones)
• Reorganization of LBNCs and a release of the active molecule
could occur.
• Caking and difficulties in redispersing LBNCs function of the
method used.
• Several methods cannot be used individually and must be
performed only for an initial analysis (such as scattering
methods).
• NMR and ITC are mainly developed for inorganic and polymeric
nanoparticles and have to be adapted to LBNCs for the
characterization of surface functionalization.
pubs.acs.org/molecularpharmaceutics
Review
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
appears as a promising powerful tool to optimize the LBNC
surface functionalization and to enhance the understanding of
the interaction mechanisms between targeting ligands and
LBNCs. An attractive area is the integration of ITC data with
information from complementary experimental techniques
such as DLS and/or NMR.
The use of NMR could also appear as a challenging
technique due to the complexity of the system. First of all, the
formulation process or the suspension of the colloidal
formulation needs to be performed with deuterated solvents.
Assuming that each component diffuses differently from the
faster small species to the slower large compounds, each
species can be separated based on its diffusion coefficient.
Additionally, when a molecule is involved in several molecular
associations, signal overlapping appears, and this phenomenon
can complicate the interpretation of results. The accuracy of
the results will strongly depend on both the quality of the
acquired data set and the choice of the processing method.
According to the NMR method (1D, NOESY), or the NMR
equipment (liquid or solid sensor), it is possible to obtain
more or less precise, fast and decisive information about the
structural organization of the LBNC or the peptide, and about
their interactions. All types of NMR equipment are not always
available in the same research unit, or in an easy manner
through collaborative works. Besides, some contradictory
studies report a potential effect of the spectrometer magnetic
field on the shape of liposomes.123 To our knowledge, there is
no final view on this matter yet. However, we have to keep in
mind that this powerful method allows researchers to analyze
small populations that are not visible using classical techniques.
The dynamics behavior and exchange kinetics of the
interaction could also be investigated.
Both ITC and NMR experiments, still underutilized so far,
would complement other analytical methods and permit more
accurate overall characterization of targeting ligand−LBNC
interactions.
4. CONCLUSION
Surface functionalization of LBNCs with targeting ligands have
received much attention in the field of nanomedicines for their
ability to overcome the current limitations such as biological
barriers and physiological factors. Special attention should be
given to the different steps of the LBNC decoration: the
functionalization process and the purification stage in order to
have a reliable characterization of the ligand corona. All the key
considerations, advances, and current limitations covered in
this review are summarized in Table 5.
It is important to select a suitable surface functionalization
approach to avoid the inactivation of the targeting ligands.
Moreover, several parameters have to be adjusted all along the
formulation and functionalization processes to obtain the
optimal response. The choice of ligand type and excipients but
also ligand’s density has demonstrated to impact the
functionalization efficiency. It is not possible to identify the
best coating strategy that allows the higher selectivity for all
LBNCs. Optimization of the functionalization approach is
strictly case-dependent and has to be investigated for each new
ligand-LBNC development.4,5
Prior to the characterization step, purification of nano-
particles after surface functionalization is a significant challenge
to obtain a reliable characterization of functionalized LBNCs.
Incomplete characterization impacts several aspects of nano-
particles development, including the reproducibility of results
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but also difficulties to correlate their physicochemical proper-
ties with their biological effects in order to predict their
efficacy.81 In parallel, different analytical techniques need to be
used to prove the benefit of the surface functionalization and
demonstrate the efficacy. The optimal ligand/LBNCs ratio
could be determined thanks to other methods such as receptor
binding affinity assessment through flow cytometry or
fluorescence spectroscopy. The analytical methods have to
be selected based on the ligand type, LBNC, and the biological
target.
The choice of the features to follow and the appropriate
analytical methods could be tricky. The use of numerous
techniques is often required for evaluating well and completely
even a single feature such as the nanoparticle size.124 In
addition to the low-resolution technique, the use of the
complementary method has been developed in the nano-
medicine field in order to obtain reliable characteriza-
tion.8,80,82,83 In recent years, promising methods, such as
solution NMR spectroscopy and ITC, have been used as
powerful tools for characterizing the protein corona formation,
allowing researchers to monitor the structure, thermodynam-
ics, and dynamics.93,113 These techniques, mainly developed
for inorganic and polymeric nanoparticles, can be adapted to
LBNCs for the characterization of surface functionalization.
■ AUTHOR INFORMATION
Corresponding Author
Aurélie Malzert-Fréon − CERMN, UNICAEN Université de
Caen Normandie, F-14000 Caen, France; orcid.org/
0000-0001-6023-1861; Email: aurelie.malzert-freon@
unicaen.fr
Authors
Léna Guyon − CERMN, UNICAEN Université de Caen
Normandie, F-14000 Caen, France; orcid.org/0000-
0002-1752-5255
Anne-Claire Groo − CERMN, UNICAEN Université de Caen
Normandie, F-14000 Caen, France
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.molpharmaceut.0c00857
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
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U https://dx.doi.org/10.1021/acs.molpharmaceut.0c00857
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