PG in Cosmetic Science

M Sc Cosmetic Science

UNIT 5: Interaction of nanomaterials with biomolecules

Dr. Asha Srinivasan

 Rational Bio-Nanoscience

Dispersion and
Nanoparticle synthesis Protein Corona
characterisation

Intracellular
Disease / therapy
Visualization

Functional impacts / toxicity

In vivo effects
 Interaction of Nanoparticles with
Membranes

What is a biomolecule?
Nanoparticle-Biomolecule Interaction

The formation of a protein corona

occurs when a nanomaterial is soaked
into a physiological environment.

Biomolecules with high affinity (green)

and low affinity (red) form a thin layer
of molecules on the nanomaterial
surface, which can be tightly bound
(“hard” corona) and/or reversibly
adsorbed (“soft” corona), or both.

The formation of the protein corona is

one of the key factors managing the
cellular response in terms of uptake,
accumulation and elimination.
 1
Liposome

Plasma
Membrane

3
2
 Computer Simulations for Understanding Nanoparticle-
biomolecule Interaction

Fusion of a liposome-coated nanoparticle with a lipid bilayer membrane

 Endocytosis
A nanoparticle placed in the external milieu of a cell can interact
with the exterior of the plasma membrane, which can lead to this
nanoparticle entry inside the cell through a process termed
“endocytosis”.

Endocytosis has an important role in diagnostics, allowing for the

selective uptake and labeling of cells by various medical imaging
agents (e.g., as contrast agents for identifying cancerous lesions
by MRI).
Classification of endocytosis based on endocytosis proteins that are
involved in the initial entry of particles and solutes.
Mechanisms of endocytosis and their relationship with size of either
endogenous or exogenous cargo.
 Endocytic Mechanisms

Cellular internalization models:

A) Phagocytosis;
B) Macropinocytosis;
C) Clathrin-mediated endocytosis;
D) Caveolae-mediated endocytosis;
E) Clathrin-independent and caveolin-independent endocytosis
 Endocytosis
Endocytosis is the major route for nanomedicines to transport across
the membrane. It is generally classified into phagocytosis and
pinocytosis. Phagocytosis was originally discovered in macrophages.
Pinocytosis is present in all types of cells in four forms, such as
clathrin-dependent endocytosis, caveolae-dependent endocytosis,
macropinocytosis, and clathrin- and caveolae-independent
endocytosis.
 Phagocytosis
Phagocytosis is a special endocytic pathway predominantly occurred in
phagocytes, such as macrophages, neutrophils and monocytes.
Relatively, large particles are more likely to take this way.

Nanoparticles which adopt this way of entry into cells need to be

recognized by the opsonin firstly, such as immunoglobulin (IgG and IgM),
complement component (C3, C4, and C5) and blood serum proteins.
Thereafter, the opsonized nanoparticles bind to the cell surface and
interact with the receptor, inducing the cup-shaped membrane
extension formation.
The membrane extensions enclose the nanoparticles and then
internalize them, forming the phagosomes which have a diameter of
0.5–10 μm. Finally, the phagosomes move to fuse with lysosomes.

But the cargo contained in the phagosomes will be destroyed by

acidification and enzymolysis in the lysosomes. Therefore, to produce
desired effects, nanomedicines must bypass this route to avoid
degradation
Phagocytosis
Phagocytic Pathway
Phagocytic pathway of cellular entry consists of three distinct
steps
1) recognition of the particles by opsonization in the bloodstream
2) adhesion of the opsonized particles onto the cell membrane
3) ingestion of the particle by the cells
 Phagocytic Pathway
Opsonization of nanoparticles occurs through adsorption of
proteins, such as immunoglubulins (Ig) G (and M), complement
components (C3, C4, C5), blood serum proteins (including laminin,
fibronectin, etc.) and others. The opsonized particle then
attaches to the macrophage surface through specific receptors.

The receptor-ligand interaction leads to signal cascades, which

result in actin rearrangement and formation of a phagosome. The
phagosome may have different sizes depending on the size of the
particles, which can range from as little as few hundred nanometers
to dozens of microns.
 Pinocytosis
Pinocytosis is a major route for the cells to drink fluid, solutes
and suspensions containing small particles.

It is classified to;
• Clathrin-dependent endocytosis
• Caveolae-dependent endocytosis
• Macropinocytosis
• Clathrin- and caveolae-independent endocytosis

based on the proteins involved in the pathways.

Clathrin-dependent endocytosis
(Classical degradable pathway)

• Clathrin-dependent endocytosis is present in all mammalian cells,

occupying an important part in cellar entry. After nanomaterials
interact with receptors on the cytomembrane, a kind of cytosolic
protein named clathrin-1 polymerizes on the cytosolic side of the
plasma where the cargo is internalized.
• After wrapping the nanoparticles inside, the vesicle is pinched off
through the GTPase activity of dynamin, forming a clathrin coated
vesicles (CCV).
• With energy supplied by actin, CCVs move towards inside the cells,
and the route is regulated by the cytoskeleton. The clathrin coat is
shed off in the cytosol.
Clathrin-dependent endocytosis
Clathrin mediated endocytosis (CME) is the “classical route” of
cellular entry, which is present and inherently active in all
mammalian cells.
CME appears to be defined as the most prominent mechanism for the
cellular entry.

Essential nutrients such as

- cholesterol = LDL receptors
- Iron = transferrin receptors

CME = engulfment of receptors associated with their ligands to a

coated pit. The coated pit forms due to polymerization of a cytosolic
protein called clathrin-1 (AP2 receptors).

The assembled vesicle (120 nm) is pinched off from the plasma
membrane by a small GTPase called dynamin.
Formation of Coated Pit

3
 Formation of Coated Pit
1) The assembly proteins, AP-2 and AP180 are targeted to the
plasma membrane where they mediate clathrin assembly. Clathrin
polymerizes into which helps to deform the plasma membrane into
a coated pit.

2) Dynamin, a multidomain GTPase, is recruited to the necks of

coated pits, where it assembles into a spiral collar. Upon hydrolysis
of GTP dynamic collar promotes scission of the membrane and
release of the vesicle known as CCV.

3) The next step involves uncoating of CCV and formation of an

early endosomes, which are then routed towards the lysosomes .
The coat constituents are recycled for reuse.
Receptor-Mediated Endocytosis and Exocytosis

1 Ligand binds to membrane receptor. Extracellular fluid

9 Exocytosis
2 Receptor-ligand migrates to
8 Transport vesicle clathrin-coated pit.
and cell membrane
fuse (membrane Clathrin-
recycling). coated pit 3 Endocytosis

Receptor

Clathrin

7 Transport vesicle
with receptors moves
to the cell membrane.

44 Vesicle loses
5 Receptors clathrin coat.
To lysosome or and ligands
Golgi complex separate.

6 Ligands go to lysosomes Endosome Intracellular fluid

or Golgi for processing.
Receptor-Mediated Endocytosis
and Exocytosis
1 Ligand binds to membrane receptor. Extracellular fluid

Receptor

Intracellular fluid

Step 1
Receptor-Mediated Endocytosis
and Exocytosis
1 Ligand binds to membrane receptor. Extracellular fluid

2 Receptor-ligand migrates to
clathrin-coated pit.
Clathrin-
coated pit

Receptor

Clathrin

Intracellular fluid

Steps 1–2
Receptor-Mediated Endocytosis
and Exocytosis
1 Ligand binds to membrane receptor. Extracellular fluid

2 Receptor-ligand migrates to
clathrin-coated pit.
Clathrin-
coated pit 3 Endocytosis

Receptor

Clathrin

Intracellular fluid

Steps 1–3
Receptor-Mediated Endocytosis
and Exocytosis
1 Ligand binds to membrane receptor. Extracellular fluid

2 Receptor-ligand migrates to
clathrin-coated pit.
Clathrin-
coated pit 3 Endocytosis

Receptor

Clathrin

44 Vesicle loses
clathrin coat.

Intracellular fluid

Steps 1–4
Receptor-Mediated Endocytosis
and Exocytosis
1 Ligand binds to membrane receptor. Extracellular fluid

2 Receptor-ligand migrates to
clathrin-coated pit.
Clathrin-
coated pit 3 Endocytosis

Receptor

Clathrin

44 Vesicle loses
5 Receptors clathrin coat.
and ligands
separate.

Endosome Intracellular fluid

Steps 1–5
Receptor-Mediated Endocytosis
and Exocytosis
1 Ligand binds to membrane receptor. Extracellular fluid

2 Receptor-ligand migrates to
clathrin-coated pit.
Clathrin-
coated pit 3 Endocytosis

Receptor

Clathrin

44 Vesicle loses
5 Receptors clathrin coat.
To lysosome or and ligands
Golgi complex separate.

6 Ligands go to lysosomes Endosome Intracellular fluid

or Golgi for processing.

Steps 1–6
Receptor-Mediated Endocytosis
and Exocytosis
1 Ligand binds to membrane receptor. Extracellular fluid

2 Receptor-ligand migrates to
clathrin-coated pit.
Clathrin-
coated pit 3 Endocytosis

Receptor

Clathrin

7 Transport vesicle
with receptors moves
to the cell membrane.
44 Vesicle loses
5 Receptors clathrin coat.
To lysosome or and ligands
Golgi complex separate.

6 Ligands go to lysosomes Endosome Intracellular fluid

or Golgi for processing.

Steps 1–7
Receptor-Mediated Endocytosis
and Exocytosis
1 Ligand binds to membrane receptor. Extracellular fluid

2 Receptor-ligand migrates to
8 Transport vesicle clathrin-coated pit.
and cell membrane
Clathrin-
fuse (membrane 3 Endocytosis
recycling). coated pit

Receptor

Clathrin

7 Transport vesicle
with receptors moves
to the cell membrane.
44 Vesicle loses
5 Receptors clathrin coat.
To lysosome or and ligands
Golgi complex separate.

6 Ligands go to lysosomes Endosome Intracellular fluid

or Golgi for processing.

Steps 1–8
Receptor-Mediated Endocytosis
and Exocytosis
1 Ligand binds to membrane receptor. Extracellular fluid
9 Exocytosis
2 Receptor-ligand migrates to
8 Transport vesicle clathrin-coated pit.
and cell membrane
Clathrin-
fuse (membrane 3 Endocytosis
recycling). coated pit

Receptor

Clathrin

7 Transport vesicle
with receptors moves
to the cell membrane.
44 Vesicle loses
5 Receptors clathrin coat.
To lysosome or and ligands
Golgi complex separate.

6 Ligands go to lysosomes Endosome Intracellular fluid

or Golgi for processing.

Exocytosis is the opposite of endocytosis Steps 1–9

Where is the destination of the vesicles?
It may be associated with the receptor that nanoparticles' ligands
attach to.

For example, low-density lipoprotein particles are internalized

through LDL receptor and transferred to lysosomes for degradation;
while, iron-loaded transferrin is engulfed via transferrin receptor and
recycled to the cell surface. This route can be blocked by its inhibitors
or some other factors, such as chlorpromazine, a hypertonic medium
or potassium depletion
Caveolae-dependent endocytosis
• Caveolae-dependent endocytosis is also a common cellular entry
pathway. It could bypass lysosomes, thus many pathogens
including viruses and bacteria select this way to avoid lysosomal
degradation.
• This route is believed to be beneficial for enhancement of
concentration of targeting position and improvement of
therapeutic effect.
• In this pathway, caveolin, a protein exist in most cells, plays a
dominate role. There are three isoforms of caveolin in mammalian
cells. Caveolin-3 is muscle specific, while caveolin-1 and -2 are
abundant in most nonmuscle cells (such as endothelial cells,
fibroblasts and adipocytes) and absent in neurons and leukocytes.
• By binding to the receptors on the plasma membrane,
nanoparticles or pathogens, like Simian virus 40 and cholera toxin
can interact with the receptors to induce the formation of the
flask-shaped vesicles, which are cut off from the membrane by
dynamin.
Caveolae-mediated endocytosis (Avoid degradable
pathway)
They are a subset of lipid rafts, the
cholesterol-rich plasma membrane
regions that cluster endocytosis and
signal transduction functionalities. The
definitive characteristic of caveolae is
the presence of the hairpin-like
membrane protein, caveolin-1, which is
necessary for biogenesis of caveolae.

Caveolae are abundant in muscle,

endothelial cells, fibroblasts and
adipocytes and absent in neurons and
leukocytes.
 Calveolae
Due to this protein, caveolae assume their hallmark flask-shaped
structure (60–80 nm) and can engulf cargo molecules, which bind to
caveolae surface.

• Calveolin
– high-affinity cholesterol binding protein

Other proteins
Cavin – indues membrane curvature
Dynamin - enables vesicle scission
VAMP-2 – mediate vesicle fusion
SNAP – mediate vesicle fusion
 Calveolae
After budding of the plasma membrane the caveolae
vesicles transport and fuse with caveosomes that have neutral
pH.

This pathway appears to be slower compared to the CME in

vitro but, importantly, in some cases it can bypass lysosomes.

Hence, several pathogens including viruses and

bacteria exploit this pathway to prevent lysosomal degradation.

For the same reason this pathway is believed to be beneficial

for cellular delivery of proteins and DNA.
 Calveolae pathway for Nanoparticle
- Polymeric micelles with cross-linked anionic core
- DOXIL®
- Polysiloxane nanoparticle
- QDs
- Abraxane®
- Surface-modified nanoparticles that target caveolae

Abraxane® nanoparticles take advantage of caveolae-mediated

transcytosis for efficient delivery of the drug to the tumor sites.
They bind to GP60, the albumin receptor present in caveolae of
endothelial cells, and transport across the vascular walls to the
tumor interstitial spaces. After entering the interstitial spaces the
Abraxane® nanoparticles are captured by SPARC (secreted protein,
acidic and rich in cysteine) that is selectively secreted by the
tumors. The SPARC-nanoparticles complexes are taken up in tumor
cells resulting in selective tumor cytotoxicity.
Abraxane – mechanism of uptake
Abraxane – mechanism of action
Clathrin- and Caveolae-independent endocytosis
This is a distinct pathway, which relies on cholesterol and requires
specific lipid compositions
Cellular entry occurs in cells devoid of both CME and caveolin-1.
Multiple effectors molecules such as Arf6-dependent, flotillin-
dependent, Cdc42-dependent and RhoA-dependent.

Arf6
Flotillin require specific lipid compositions
Cdc42 and are dependent on cholesterol
RhoA
There are not many nanomaterials documented to utilize different
subtypes of the clathrin and caveolae-independent endocytosis.
However, polymeric nanoparticles modified with folate binds to GPI-
anchored folate receptor (FRα) which is overexpressed in tumor
cells.
Clathrin- and Caveolae-independent
endocytosis for nanoparticles
Many nanomaterials and polymers are conjugated with folate
including liposomes, protein toxins, biodegradable nanoparticles,
and water soluble N-(2-hydroxypropyl) methacrylamide (HPMA).

These materials were used targeted intracellular delivery of

cytotoxic drugs, imaging agents and also for boosting immune
response.
 Macropinocytosis
Macropinocytosis is commonly defined as a transient, clathrin- and
caveolin-independent, growth factor-induced, actin-driven
endocytosis that internalizes the surrounding fluid into large
vacuoles.

Macropinocytosis is a special case of clathrin-, caveolae- and

dynamin-independent endocytosis, which is initiated by transient
activation of receptor tyrosine kinases by growth factors.

The receptor activation mediates a signaling cascade that leads to

changes in the actin cytoskeleton and triggers formation of
membrane ruffles. These membrane ruffles protrude to engulf the
surrounding fluid and nutrients in the extracellular milieu.
 Macropinocytosis

TEM images showing macropinocytosis of protein nanoparticle in A549 cells

upon incubation with nanoparticles for 60 min
 Fig. 1 Uptake and transcytosis of 50 nm SiO2
nanoparticles in hCMEC/D3 cell monolayers. Cells
were exposed to 100 µg/ml nanoparticles in assay
medium containing 2 % FBS for 4 hours. Arrows
indicate nanoparticles.

(a) A single SiO2 nanoparticle entering a cell

(possibly in a clathrin coated pit).
(b) A cluster of SiO2 nanoparticles entering a cell.
(c) A SiO2 nanoparticle in an endosomal vesicle and
clusters of nanoparticles outside the cells.
(d) SiO2 nanoparticles in an endosome (E);
(e) SiO2 nanoparticles in a multivesicular body
(MVB);
(f) SiO2 nanoparticles in a lysosome (L).
(g) SiO2 nanoparticles were found close to
membrane invaginations between two cells,
suggesting possible transcytosis or export.
(h) A few SiO2 nanoparticles were found beneath
the basolateral cell membrane close to the filter
membrane, also suggesting transcytosis. In the
same image we also
observe large numbers of SiO2 nanoparticles
accumulated into a multivesicular body (MVB) and
single nanoparticles in other intracellular
structures.
(i) A large cluster of SiO2 nanoparticles was
observed between the BBB layer and the filter, with
the cell basal membrane engulfed around it

Dawson and team, 2013

 Methods to study endocytic pathways
How to study these processes and identify certain pathway
that nanomedicines employ?

1. Molecular probes or Markers:

There is a method that uses proper molecular probers or markers
to study the intracellular fate of nanomedicines. Mark the specific
probers or markers which can show specific fluorescence or color
on the nanomedicines, the marked nanomedicines in the
intracellular compartments or organelles can be viewed intuitively
with the help of the confocal imaging technology. It is important to
confirm the destination of the cargo and the pathway employed by
nanomedicines.

Combined with a three dimensional confocal technology, we can

get more intact information of the whole cell layer by layer.
Confocal Microscopy

Confocal imaging of nanoparticle uptake by GI-1 cells

 Cellular uptake and
intracellular distribution of
the nanoparticles studied in
bEND-3 cells.

Drug loaded chitosan nanoparticles surface modified with Herceptin

https://www.youtube.com/watch
?v=Va1jaBGwoT8
 Markers or Probes
Some classical probers or makers are known to be internalized
through specific endocytic pathway.

• Low density lipoprotein (LDL) and transferrin (Tf) enter cells

through clathrin dependent endocytosis (CME), so they are
commonly used as markers of CME.

• Cholera toxin (CTBs), Shiga Toxin and even caveolin-1 are

usually used as markers of caveolae dependent endocytosis,
and dextran is the marker of macropinocytosis.

• Proteins contained in specific endocytic vesicles or

intracellular organelles, can be used with fluorescent proteins,
and the formed fusion proteins can show the exact position of
the nanomedicines in the cells, like Rab5 in the early
endosome, Rab7 in the late endosome.
 Inhibitors
Inhibitors of endocytosis can be used to block the specific endocytic
pathway to confirm whether it is employed by the nanomedicines to
enter cells.

As almost all endocytic pathways are energy dependent

processes, they can be inhibited by low temperature and an
ATPase inhibitor (like sodium azide) at the same time.

Specific endocytosis mechanisms,

• Hypertonic sucrose (0.4- 0.5 M), chlorpromazine (50-100 mM)
and potassium depletion can be used to inhibit the clathrin
dependent endocytosis;
• Methyl-beta-cyclodextrin , filipin, nystatin and cholesterol oxidase
can be used as the inhibitors for caveolae dependent endocytosis,
amiloride, cytochalasin D and rottlerin can block
macropinocytosis.
 Cellular Trafficking using chemical inhibitors
OR Effect of transport inhibitors on cellular uptake
Genistein - an inhibitor of tyrosine kinases involved in caveolae-mediated
endocytosis.

Chlorpromazine - inhibits clathrin disassembly and receptor recycling to the

plasma membrane during clathrin-mediated endocytosis.

Dynosore – GTPase inhibitor and block dynamin dependent endocytosis.

Nacodazol – A reversible anti-neoplastic agent which exerts its effect by interfering

with the polymerization of microtubules.

Cytochalasin A - an actin-disrupting agent that is used widely to study the role of

actin filaments in different biological systems.

Filipin – is a polyene macrolide antibiotic and antifungal. The antifungal

mechanism of action may be due to altering membrane permeability and
associated functions by binding to membrane sterols. Filipin inhibits prion protein
(PrP) endocytosis and causes the release of PrP from the plasma membrane.

Sodium Azide (NaN3) /2deoxyglucose – Energy dependent pathway.

Confocal images of A549 cells showing the F-actin morphology in normal control cells and, following
incubation with each of the different inhibitors at 37°C or 4°C for 2 h30 min.

Effects of Transport Inhibitors on the Cellular Uptake of Carboxylated Polystyrene Nanoparticles in Different Cell Lines (2011).
PLoS ONE 6(9): e24438. doi:10.1371/journal.pone.0024438
 Factors affecting endocytic pathways
After engulfed, the intracellular fate of the nanoparticles is
dependent upon the selected endocytic pathway.

Research show that selection of nanomaterials & transport pathway

is affected by the physicochemical characteristics of nanoparticles
(size, charge, shape, etc.) and the different endocytic machinery in
various cell type.

1. Size - the particles should be small enough to enter the vesicles,

and the size range from 10 nm to 500 nm and limited up to 5
mm. The large particles are most likely to be engulfed via
macropinocytosis. The size of vesicle involved in clathrin
mediated endocytosis is about 100 nm, while the size involved
in caveolae mediated endocytosis is about 60-80 nm.
2. Surface Charge
Cytomembrane possess negative charge. Therefore, the cationic
nanoparticles may show a strong electrostatic interaction with the
cells, which result in a rapid entry.

Nanoparticles which possess different charge or no internalization into cells.

a) Cationic particles strongly interact with the membrane and enter cells rapidly.
b) Anionic particles bind the positive site at the membrane and enter cells.
c) Neutral nanoparticles also can get into cells.
1. Positively charged nanoparticles can escape from endosomes after
internalization and exhibit perinuclear localization because of the
‘proton-sponge’ effect.

2. The nanoparticles without any charge at physiological pH may

interact with the cells with the aid of hydrophobic and hydrogen bond
interactions.

3. Neutral particles coated with hydrophilic polymers can prevent

interaction with the cytomembrane leading to less absorption.

4. The anionic nanoparticles may be endocytosed through the

interaction with the positive site of the proteins in membrane, and
they can be highly captured by cells because of their repulsive
interactions with the negatively charged cell surface.

5. For cationic nanoparticles, the majority of the reports indicated

they mainly enter cells through CME.
What is proton sponge effect?
The “proton-sponge effect”. A) Cationic particles bind with high
affinity to lipid groups on the plasma membrane and are
endocytosed. Once these nanoparticles enter into a lysosomal
compartment, the unsaturated amino groups are capable of
sequestering protons that are supplied by the v-ATPase (proton
pump). This process keeps the pump functioning and leads to the
retention of one Cl− ion and one water molecule per proton.
Subsequent lysosomal swelling and rupture leads to nanoparticle
release in the cytoplasm.
3. Surface hydrophobicity
Hydrophobic nanoparticles have higher affinity for the cell
membrane than hydrophilic ones, leading to an improvement
of cell uptake in the kinetics and the amount.
Hydrophilic polymers used to modified nanoparticles,
such as polyethylene glycol (PEG), poly (N-vinyl-2-
pyrrolidone) (PVP), poly(amino acids), and dextran, form
a ‘cloud’ to suppress the interaction between the
nanoparticles and lipid bilayer of cells.

On the other hand, it can prolong nanoparticles life in

blood to reach specific site.

The chemical composition at the surface nanoparticles

determines the surface hydrophobicity which can
promote or suppress the interaction with cells, thereby
influencing the route of cell uptake.
Nanoparticles with
different hydrophobicity
present different affinity
with the cell membrane .
4. Shape

The effect of particle shape on phagocytosis.

Ω is defined as the angle between the membrane normal at the
point of attachment and the line defining the particle curvature at
this point. Particles are internalized successfully at Ω ≤ 45° ; the
internalization of particles can be inhibited at Ω > 45 °.

Review paper: The endocytosis and intracellular fate of

nanomedicines: Implication for rational design (2013).
Shape matters when engineering mesoporous silica-based
nanomedicines

This article is part of the themed collection: Biomaterials Science review articles 2016
Note: Organelle target will be a hotspot

Drugs can be delivered to lesion site in the specific organelles and

unwanted side effects will be minimized.
As for the design of nanomedicines, it is critical to understand their
uptake pathways because many therapeutic sites are located in the
cells.

Endocytic pathways and the tools to dissect the specific mechanism

and the factors affecting the selection of pathway are employed by
nanomedicines.

Except for caveolae-mediated endocytosis, the other pathways all

have a relationship with lysosomes, and it is a great idea for drugs to
target lysosomes.
Effect of Nanoparticle size and shape on its
interaction and cellular uptake
Nanoparticle Shape and Size
Unlike spherical nanoparticles;

(a) non-spherical particles, such

as those possessing discoidal
geometries, are more prone to
tumbling and oscillatory effects
in vasculature, increasing greatly
the propensity of nanoparticle–
cell wall contact and potential
extravasation through
fenestrations in vasculature.
Nanoparticle Shape and Size
Once in contact with endothelial cells,
the small size and surface area of
conventional spherical nanoparticles
reduce the number of binding and
contact points compared with larger
discoidal nanoparticles as well as
other non-spherical geometries),
which can affect tumor accumulation
and active targeting strategies.
Intravenous Administration

Upon intravenous administration, drug-containing nanoparticles

encounter a number of sequential obstacles hindering efficacious,
site-specific delivery to tumors. Nanoparticles undergo opsonization
and subsequent uptake by resident macrophages.
Opsonization – a process of identifying the invading particle by a phagocyte. Without the opsonization
process the “recognition” and destruction of invading agents such as bacteria would be inefficient.
Nanoparticle Uptake
NPs may enter the human body via inhalation, ingestion or through the
skin. In the extracellular fluid, NPs are coated by proteins and other
biomolecules.
The protein corona
determines how the
NP interacts with a
cell.

Cellular internalization
may involve active
(receptor-mediated)
or passive transport
across the cell
membrane.
The nanoparticle journey in the body: from synthetic identity to
physiological response through biological identity.
 (A) The bare NP (synthetic identity) that has a specific shape, size
and charge is injected into the body.
(B) Once exposed to biological fluids, synthetic NPs come into
contact with active biomolecules that surround them thus giving
rise to the NP–PC complex (biological identity). These NP-
bound blood proteins can influence the immune system by
mediating subsequent immune cell responses
C) These NP–PC complexes are responsible for the interaction with
biological barriers and cells.
The NP can reach the target site using a specific receptor endowed
upon them by the PC as part of their new biological identity
(physiological response).
The experimental steps associated to the study of bare NPs, NP–PC
complexes and their physiological behaviors are also reported.

NP: Nanoparticle; PC: Protein corona

 Biomolecule-Nanoparticle Interaction

• Blood plasma contains several roughly 4,000 different proteins, their

abundance in the plasma does not correspond to their abundance in
the protein corona (PC).
• The affinity based competition between proteins for adsorption on
the NP surface is responsible for the changes in the PC composition
over time.
• Abundant proteins are attached first followed by higher affinity
proteins – Vroman’s Effect.
• Based on the exchange time of its composition, the PC is classified
into hard and soft.
• The hard corona is considered the first tightly bound layer of
proteins that has a long exchange time (many hours), while the
soft corona is represented by the second layer of proteins (not
directly bound to the NP) that undergoes fast exchanges over time
(seconds or minutes) .
 The “Vroman effect”

The proteins adsorbed on NPs are considered to be in a continuous

flux of desorption/adsorption mainly controlled by the so-called
‘Vroman effect.

According to this, ‘effect’, an initially attached protein can, at any

time, desorb from a NP and be replaced by a different one with
major affinity.

OR

The competitive displacement of earlier adsorbed proteins by

other proteins with stronger binding affinities results in
undesired layer instabilities that are difficult to control.
 Nanoparticle–Corona complex
Proteins that adsorb with high affinity form what is known as the
“hard” corona, consisting of tightly bound proteins that do not readily
desorb, and proteins that adsorb with low affinity form the “soft”
corona, consisting of loosely bound proteins

Hard corona proteins interact directly with the nanomaterial surface.

The soft corona proteins interact with the hard corona via weak
protein–protein interactions.
Nanoparticle - Protein Corona
What does the cell see?
Immediately on contact with
biological fluid, nanoparticles
take on a corona of proteins
(red–yellow-helices and red–
blue-sheets) that exchange
with their surroundings.

Proteins that reside on the

nanoparticle surface for much
longer, can be identified by
cells.
 Nanoparticle –
protein corona
complexes:
characteristics of
hard and soft
coronas.
The protein corona
represents the
biological entity of
a nanoparticle

Hard corona proteins are directly adsorbed on the nanoparticle surface

due to their strong binding affinity. These proteins also have a slow
exchange time.
Soft corona proteins associate with the hard corona via weak protein–
protein interactions, thus showing a short residence time around the
nanoparticle and a fast exchange time.
The majority of adsorbed biomolecules on the surface of
nanoparticles in blood plasma are proteins, but minor traces of lipids
have also been reported. The adsorption of proteins on the surface
of nanoparticle is governed by protein–nanoparticle binding
affinities as well as protein–protein interactions.
 Protein Corona
In the physiological environment, NPs selectively bind proteins to
form a ‘protein corona’. This is always a first step when NPs enter
a biological fluid and this corona likely determines the fate of the
NPs in vivo.
Structure and composition of the protein corona depends on;

1. Physicochemical properties of the nanomaterial (size, shape,

composition, surface functional groups, and surface charges)
2. Nature of the physiological environment (blood, interstitial
fluid, cell cytoplasm, etc.)
3. Duration of exposure
 Protein Corona
The protein corona alters the size and interfacial composition of a
nanomaterial, giving it a new biological identity which is what is seen
by cells.

The biological identity determines the physiological response

including agglomeration, cellular uptake, circulation lifetime,
signaling, kinetics, transport, accumulation, and toxicity.
 Structure and Composition of Corona
The size , shape and surface charge together with the surface
modification are key factors in determining the composition
of the PC.
The characteristics of the biological environment also play a
determinant role in the formation of a PC:

• the type of plasma (e.g., human or murine),

• Incubation
• Time and temperature,
• pH and
• the physiological state of the plasma (alterations due to
disease/medical conditions) may also affect the protein
adsorption on the NP surface.
 Composition of Protein Corona
• Composition of corona - The composition of the protein corona is
unique to each nanomaterial and depends on many parameters.
• Variations in the corona are affected by the physicochemical
properties of nanomaterials (e.g., size, shape, surface charge,
surface functional groups and hydrophilicity/hydrophobicity.
• Besides the nanomaterials effects, the role of environment
factors, such as protein source and slight temperature variations
also effect this interaction.
• The thickness of protein corona can be a factor of many
parameters such as protein concentration, particle size, and
surface properties of particle. Most plasma proteins present a
hydrodynamic diameter of about 3–15 nm; thus, the coronas on
these nanoparticles are too thick to be composed of only a single
layer of adsorbed protein and are composed of multiple layers
Table 1. Comprehensive overview of serum/plasma proteins adsorbed on the surface of
different types of nanomaterials with varied size and surface chemistries .
Protein adsorption, physical characteristics of the NPs and the
properties of interacting cells may influence NP uptake. Kinetics of
uptake of the same nanomaterial has been shown to differ with
different cell types. Adsorption of proteins on the NP surface can
take place almost instantly. Therefore, it can be assumed that
interaction of the NP with cellular structures is indirect and occurs
mostly via the NP-PC and not the bare NP surface .
Interaction of nanoparticles with the cellular interface. NPs interact with cells via the protein
corona. (A) Uptake of large sized NP-protein complexes, agglomerates of NP may be ingested by
specialized cells such as macrophages and neutrophils via phagocytosis. It involves folding of the
plasma membrane over the NP complex to form the phagosome. (B) Non-specific uptake of
extracellular fluid containing aggregates of NP may also be taken up by cells via macropinocytosis
which involves ruffling of the plasma membrane to form vesicles which ultimately fuse to form
lysosomes. Endocytosis of NP complexes may also be directed by specific receptors involving
formation of (C) caveolae that are plasma membrane indentations consisting of cholesterol binding
proteins called caveolins or (D) clathrin-coated vesicles. (E) Apart from these other endocytic
mechanisms, independent of clathrin or caveolae may also facilitate uptake of NP.
Mechanisms of adsorption of proteins on the
surface of nanoparticles

Entropy-driven binding - The mechanism of entropy-driven-

bonded proteins such as fibrinogen, lysozyme, ovalbumin, and
human carbonic anhydrase II is the release of bound water from
the surface of the nanoparticle.

The increase in entropy of released water molecules is larger than

the decrease in the entropy of adsorbed proteins. Entropy-driven
mechanism usually does not change the conformation of the
protein.
 Protein Conformation
Upon adsorption of the protein on the nanoparticles, proteins
may undergo structural rearrangements called “conformational
changes” and are typically irreversible after desorption.

Example : conformational changes in the iron-transport protein

transferrin are not recovered after desorption from iron oxide
nanoparticles.

 Conformation of adsorbed proteins is altered more in the

presence of charged
or hydrophobic nanomaterials
 These changes are thermodynamically favorable if they allow
a hydrophobic or charged sequence within a protein to
interact with a hydrophobic or charged nanomaterial surface,
respectively.
 Parameters Affecting Protein Corona
Protein Corona - Various parameters such as nanoparticle size,
shape, curvature, surface charge (zeta potential), solubility, surface
modification, and route of administration of nanoparticles to the
body affect the composition, thickness, and conformation of
protein corona.
Nanoparticle: - Among the nanoparticle (NP), parameters which
affect the protein corona, the surface properties such as
hydrophobicity and surface charge have more significant role than
other parameters

Advantage:
An understanding of role of each physicochemical parameter on
the protein corona is promising for design of targeting
nanomaterial, long-circulating drug carriers, or for decreasing the
toxicity.