Commentary

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Precision medicine based on nanoparticles:

the paradigm between targeting and
colloidal stability
Iris Renata Sousa Ribeiro1,2 , Flávia Elisa Galdino1,2 , Camila Pedroso Silveira1
& Mateus Borba Cardoso*,1,2
1
Brazilian Synchrotron Light Laboratory (LNLS), Brazilian Center for Research in Energy & Materials (CNPEM), Campinas, 13083
970, Brazil
2
Institute of Chemistry (IQ), University of Campinas (UNICAMP), Post Office Box 6154, Campinas, SP, 13083 970, Brazil
*Author for correspondence: cardosomb@lnls.br

“In our perspective, notwithstanding the synthesis improvement strategies are fundamental for
the production of NPs with the desired functionality, the evaluation of colloidal stability and
targeting in models that accurately simulate the human organism is also essential so that NPs can
get closer to medical translation.”

Keywords: biomolecular corona • colloidal stability • complex biological medium • multiple functionalization
• nanomedicine • nanoparticles • targeting

Conventional medicines present numerous drawbacks due to their ineffectiveness and imprecision. A recent report
has pointed out that the ten top-selling medications in the USA present efficiency treatment rates ranging from 2
to 25% [1]. For example, only 1 out of 20 individuals benefits from the most widely-used asthma treatment drug,
Advair Diskus R
[1]. These alarming low rates can be mainly attributed to the general lack of specificity inherent
to traditional medicines, which are not designed to discern between healthy and diseased cells. Particularly, they
have very limited potential to accumulate at specific disease sites, and therefore, much higher doses are needed to
ensure the drug will overcome biological barriers and end up at the target site at therapeutic levels [1,2]. The lack of
specificity becomes even more evident for chemotherapeutics due to their high toxicity and ability to induce drug
resistance [3]. Therefore, the development of new strategies that address the limitations of conventional therapies
remains an important and long-standing challenge as continuous efforts are still needed to generate more efficient
platforms.
Nanoparticle-based formulations have emerged as a great promise to overcome the aforesaid obstacles [2,3]. One
of the central ideas behind nanomedicine is that nanomaterials have comparable size with cellular components
and engage in biological interactions in a distinctive way compared with molecules or bulk materials. Moreover,
they present tunable physico-chemical properties that can be specifically adjusted for the desired application. For
instance, by tuning the material size, shape and surface coating, it is possible to modulate its cellular uptake [3–
5], toxicity [3–5], colloidal stability [3,4,6] and targeting ability [3,4]. The control of these parameters enables the
development of more efficient drug delivery systems, which in turn pave the way for the application of lower
medicine doses while reducing drug-associated toxicity [3].
Nanomedicine has witnessed compelling progress since the first approved nanoparticle-based system (commer-
cialized under the names of Doxil R
and Caelyx R
) [7–9]. Decrease in toxicity due to passive targeting and increase
in blood circulation time are benefits that currently-commercialized nanomaterials offer [10,11]. Nevertheless, these
systems lack active targeting, in other words, they are still unable to selectively accumulate at target sites [10,11].
In this sense, despite the numerous benefits of applying nanoparticles (NPs) in medicine, some challenges still
need to be overcome, particularly in relation to the maintenance of the NP physico-chemical integrity when ex-
posed to physiological environments. Once in contact with complex fluids, NPs interact with many physiological
components (e.g., amino acids, salts and proteins) that may induce colloidal destabilization or changes in their
original identity. Consequently, proteins and other biomolecules can adsorb on the NP surface and give them a

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Commentary Ribeiro, Galdino, Silveira & Cardoso

new biological identity (biomolecular corona formation) that masks their original functionality and hampers their
ability to reach the target site. This phenomenon might also decrease NP colloidal stability or lead to the activation
of unplanned biological pathways (e.g., the complement system), which contributes to low treatment efficiency
rates as well as the appearance of secondary undesirable effects [11–14].
A recent literature review has pointed out that NPs targeting efficiency has been kept at around 0.7% over
the last 10 years [15], which has severely delayed NP translation. It is essential to invest in NP systems that
combine both targeting and colloidal stability to fully benefit from NPs unique properties and potential to address
conventional medicine obstacles. Here, we shed light on the main approaches developed to obtain colloidally stable
and targeted NPs and discuss the strategies to enable nanomedicines that efficiently encompass the coexistence of
both characteristics.

Nanoparticles colloidal stability

A fundamental concept within nanomedicine is that each NP entity individually interacts with the biological
machinery. Therefore, the biological outcome is a combinatorial of all the interactions from individual NPs. Even
a slight degree of heterogeneity in the nano-formulation can lead to different biological effects. This is a key idea to
understand the importance of keeping NP colloidal stability from the injection point to the target site. However,
the maintenance of NPs colloidal stability is not trivial and can be affected by biological components to form
irreversible aggregates characterized by large clusters of different sizes and morphologies. Besides driving random
biological processes, these structures are more susceptible to sedimentation and can be more often blocked by
biological barriers, which evidently affects their biodistribution, drug delivery and targeting efficiency. Also, NP
toxicity is size-dependent, and may increase depending on the aggregate size.
NPs keep their stability in aqueous media mainly by repulsive, steric or hydration forces. Repulsive stabilization
takes place by the presence of ionized groups on the NPs surface that avoid their approximation, while the NPs
stabilized by steric or hydration forces avoid the aggregation by steric repulsion between polymeric chains or by the
unfavorable desolvation energy, respectively [16]. Although NP stability in water is relatively easy to achieve, it can be
deeply compromised in complex biological media due to the high ionic strength and the proteins presence. Systems
stabilized by repulsive forces are the most affected in this sense. NP surface charges in a high ionic strength medium
are neutralized by the free ions, decreasing the repulsion and favoring aggregation [6]. Particularly, ions that have
high valency or salting-out ability are responsible for more intensified aggregation, as predicted by Schulze–Hardy
rule and Hofmeister series [17]. Regarding the proteins, these biomolecules are attracted by the NPs surface charge
and spontaneously adsorb on it, forming the biomolecular corona. In some cases, the adsorbed proteins can induce
aggregation, and this phenomenon speeds up and strengthens if the proteins present a net opposite charge to the
NP [6].
NP stabilization by steric or hydration forces seems to be more effective than by repulsive forces when complex
biological media are taken into account. One of the reasons is that the NP’s functionalization with polymers
or highly hydrophilic compounds can circumvent protein adsorption [18]. Additionally, these functionalization
strategies generally shift the NP surface charge closer to zero, reducing the electrostatic attraction of opposite-
charge proteins and molecules to the NP surface [6] The effect of ionic strength on the colloidal stability drastically
decreases when the NP surface charge is close to zero and aggregation usually occurs only in the presence of ions
with high salting-out abilities [19].
Polyethylene glycol (PEG) has been one of the most used compounds to avoid NP aggregation. This polymer is
present, for instance, in Doxil and Caelyx [18]. In addition to the steric barrier, PEG is highly hydrophilic, which
leads to the formation of a water passivation layer increasing the resistance to protein adsorption due to energetic
and physical barriers. Studies have shown that grafting the NP surface with high densities of high molecular weight
(2–20 kDa) PEG significantly enhances NP colloidal stability while the coupling strategy directly affects the grafting
density [20]. Additionally, it has been observed that the stealth effect is improved when cross-linked PEG is used
instead of the linear one [21]. Despite the PEG advantages, the anti-PEG antibody response makes its applicability
questionable [18]. In this context, zwitterionic groups have emerged as an alternative strategy. Zwitterionic moieties
are composed of equal units of cationic and anionic groups that result in an overall net neutral charge and in
the formation of a highly hydrated layer around the NP surface. Zwitterionic-functionalized NPs can be obtained
either by modification with zwitterionic molecules (charged groups in the same monomer) or by simultaneous
modification with anionic and cationic molecules (charged groups in different monomers). However, this latter
strategy is still not widely explored [22]. Other polymers can also be used as stabilizers and many have shown similar

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 Paradigm between targeting & colloidal stability Commentary

properties to zwitterionic groups and PEG, although used in smaller extension. The main limitation of these
above-mentioned stealth strategies relies, in some cases, on passivating the NP surface and consequently affecting
the ultimate targeting process.

Nanoparticle targeting
The use of targeted NP formulations has been considered as a promising strategy for theranostics by acting in
particular sites in the human body [23]. The rationale behind this strategy is that surface receptors present in
cells [13,24], bacteria [12] and viruses [25] can be used as targets for nanoparticle-based systems. Tumor cells for
example, overexpress specific receptors (e.g., transferrin receptor) that can be explored when designing a NP system
(e.g., transferrin-functionalized NPs) [24]. Thus, NP surface modification with biologically active groups, such as
antibodies and other biomolecules, is a potential approach to enable NP binding to particular receptors on the
target cells [23,24,26]. This specific interaction decreases the NP off-target tissues concentration, which allows lower
doses administration as well as reduced side effects compared with conventional treatments [3].
Research has proven considerable targeting improvement in in vitro conditions (e.g., serum-free). However,
the complexity of real biological environments and the number of biological barriers (from gastro-intestinal and
encephalic barriers to cellular membranes) and defense mechanisms impair the ability of nano-formulations to
reach the target site and deliver the payload [23,24,26]. Moreover, biotransformations such as biomolecular corona
formation or sample aggregation confer a new biological identity to the NP, which becomes the interface between
the bare particle surface and the biological apparatus [12,23,26,27]. Thus, peptide-containing sequences engage
with cellular receptors in a key-lock mechanism to activate or deactivate cellular pathways. Consequently, each
protein in the biomolecular corona can potentially engage in endogenous processes if they meet their respective
receptors and generate random signaling processes while preventing the original targeting moiety to be available
for the interaction [25]. Salvati and colleagues [24] showed that transferrin-functionalized silica nanoparticles lost
their tumor-targeting ability in complex biological media. According to the authors, this phenomenon occurs due
to the adsorption of proteins on the NPs surface that hides the targeting molecules. Another important aspect
concerns NPs aggregation, in which not only the targeting is impaired, but also the NP uptake process [5]. Albanese
and collaborators [5] demonstrated that gold nanoparticle’s exposure to biological media led to their aggregation,
generating structures with different sizes and shapes. Such aggregates are asymmetrical in relation to the targeting
moieties surface distribution and density when compared with individualized NPs. This heterogeneity creates
multiple cellular responses that affect the nano-formulation efficiency and makes impossible to correlate the cell
response with the material properties [5].
The development of new strategies for the synthesis of colloidal nano-entities capable of maintaining stability
and targeting ability in complex media is essential to promote advances in diagnoses and clinical treatments.
Currently, the literature discusses two main strategies to achieve these goals. The first is based on the use of
a single functionalizing group that guarantees colloidal stability and specific targeting [12]. The second one, in
turn, is based on a multiple functionalization approach, with at least one group responsible for the NPs colloidal
stability and another group for targeting [13,28]. The single functionalization strategy has been scarcely explored by
the scientific community due to the challenge in finding a molecule capable of simultaneously performing both
functions. In addition, this approach is not versatile and, consequently, its ultimate nanomedicine application is
very questionable. Herein, we focus our attention on NP multiple functionalization that, despite the inherent
obstacles to their synthesis and characterization process, represents a more flexible platform that can be tuned to
distinct medical-related applications.

Colloidal stability versus targeting

Strategies for NP multiple functionalization that are able to produce colloidally stable and targeted dispersions
have been widely investigated over the recent years [12,13,28,29]. As previously mentioned, several molecules are able
to improve NP stability in complex biological media by preventing protein adsorption [6]. However, achieving
functionalization strategies that preserve both targeting and colloidal stability in complex media has proved to be a
major challenge [13,28,29].
Recently, our group produced silica nanoparticles functionalized with a zwitterionic molecule and biologically
active groups (amino, mercapto or carboxylic functionalities) to verify their interaction with cells, viruses and
bacteria [28]. According to our results, kinetic stability was obtained since the zwitterionic molecule acted as a
shield, preventing nonspecific protein adhesion. However, this shielding effect was also responsible for hiding the

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Commentary Ribeiro, Galdino, Silveira & Cardoso

biologically active functionalization, reducing its interaction and activity in cells, bacteria and viruses. Probably, the
hydration layer formed by the zwitterionic group mitigated the diffusion charge effect, making interactions between
the surface groups and the target difficult. The use of targeting molecules long enough to pass the hydration layer
is a promising alternative to solve this problem [16,28,29]. Therefore, the coupling of long spacers, polymer chains
and proteins must be enough to surpass the hydration layer and make the interaction domain available for specific
interactions [16,28].
The opposite scenario was also observed by our group, in which targeted NPs were successfully produced,
but without full stability capacity [13]. Basically, we synthesized zwitterionic-functionalized silica nanoparticles
combined with Ki-1 surface grafting, an antibody capable of recognizing the overexpressed TNFRSF8 receptor on
lymphomas [13]. Although targeting for lymphomas was achieved, the NP colloidal stability was below expectations.
Even using the zwitterionic molecule as a colloidal stabilizer, the NPs showed some aggregation degree when exposed
to serum-supplemented culture medium. This phenomenon indicates a possible unbalance in the zwitterionic-
antibody ratio added. In the synthesis process, relatively high amounts of zwitterionic and Ki-1 were used, in
nonoptimized quantities. Such disbalance may have impaired the formation of an effective hydration layer by
the zwitterionic molecules, causing aggregation of the NPs in complex medium. On the contrary, the amount of
antibody added proved to be enough for targeting, even considering some NP aggregation.
According to the previous studies [13,28], we suggest that parameters such as the stabilizer and biologically active
groups lengths, along with their absolute concentrations and proportions, play a fundamental role to obtain NPs
with the desired functionality. Additionally, the orientation, flexibility and distribution of the active groups on the
surface are other factors that have been lately considered by the scientific community [30,31]. Although the role
of precise engineering on the interactions between receptor and active molecule is yet unclear, promising results
have been addressed in the literature. Recently, Thalhauser and colleagues [30] showed that functionalized NPs with
active groups in a multivalent, oriented, and dense manner are essential for the development of efficient targeting
systems. In addition, Shaw and collaborators [32] verified that the use of flexible functional groups (e.g., proteins)
might be necessary to facilitate interaction with the required target. Finally, double-function NP production is not
trivial and must be optimized and evaluated individually as a function of the modifying groups, whose selection
depends on the application goal.

Conclusion & future perspective

Herein, we addressed the main challenges for the development of efficient nanomedicine, namely the maintenance of
NPs colloidal stability when exposed to complex media and the ability to target the desired site. Although strategies
that achieve one goal or another have been successful, the synthesis of NPs that present both characteristics is still a
challenge. Multiple functionalization is a promising alternative; however, many obstacles need to be overcome. The
use of kinetic stabilizers can often mask biologically active groups, decreasing the targeting efficiency. In addition,
nonoptimized proportions between stabilizer-targeted groups may not be enough to prevent the biomolecular
corona formation and, consequently, NP aggregation. In our opinion, current synthesis strategies should consider
the careful tuning of the surface groups concentrations and proportions, as well as the use of targeting molecules
that are prominent in relation to the stabilizers. Moreover, precise NP surface engineering must also be taken into
account, with the control of the active molecule orientation, as well as its distribution over the surface. We believe
that all these factors contribute to the development of efficient platforms.
Furthermore, it is worth highlighting that most studies on NP stability and targeting are performed in vitro
or in animals. While these assays are important steps for the approval and commercialization of nanomedicines,
they do not reliably represent the environment found in the human body. Cell cultures and animal models
deliver different responses than cells in the human organism. For the first case, the discrepancy becomes more
evident for studies performed on 2D cell culture, for which one side of the cells adheres to a rigid plastic surface.
Additionally, in vitro experiments are carried out in serum or cell culture medium, characterized by the absence
of coagulation factors, which can establish a different composition of biomolecular corona and different extension
of NP aggregation [23]. Regarding the use of animals, although they are an essential stage before clinical studies,
differences in relation to human responses are often observed due to: the differences in blood composition, which
affects biomolecular corona and stability; the difficulties in developing an adequate animal disease model [33,34];
the expression of tumors with different complexity and heterogeneity from human tumors [34,35]; the expression of
different targeting biomolecules [34,35]; the use of immunosuppressed or immunodeficient animals [34,35] etc. More
recently, humanized experimental animals have been developed in order to obtain higher accuracy in mimicking

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 Paradigm between targeting & colloidal stability Commentary

the human system, for example, applying human tumor cells from the patient to mice and enabling the expression
of T- and B-cells [34,35]. In our perspective, notwithstanding the synthesis improvement strategies are fundamental
for the production of NPs with the desired functionality, the evaluation of colloidal stability and targeting in models
that accurately simulate the human organism is also essential so that NPs can get closer to medical translation.

Author contributions
IRS Ribeiro and FE Galdino contributed toward literature review and manuscript writing. CP Silveira and MB Cardoso contributed
toward the discussion and manuscript review. MB Cardoso was further responsible for the supervision of all the process.

Financial & competing interests disclosure

The authors gratefully acknowledge the financial support of the Fundação de Amparo à Pesquisa do Estado de São Paulo (pro-
cesses 2015/25406-5, 2017/21318-0, 2019/14007-3, and 2018/09555-9). MB Cardoso acknowledges CNPq for a productivity
fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding
agencies. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial
interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

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