3. Parameters determining the NP-CM
interaction
3.1. Nanoparticle
Size
It is intuitive to believe that the difficulty for nanoparticles
entering the cell membrane will raise with the increasing particle size.
Essentially, many researchers have also found that some nanomaterials
with greater size will cause more critical changes in the morphology and
surface composition of cell membranes. Jirasak43 et
al. have found that small-sized nanoparticles similar to fullerenes can
quickly enter the hydrophobic region of the lipid-bilayer without
causing significant cell membrane deformation. Similarly, Rai
Beena44 et al. have used a rigid nanoparticle model to
measure the impact of the size effect of nanoparticles on cell membranes
by analyzing some structure factor of membrane plane projection area,
area compressibility, and order parameters. It has been found that
larger-sized nanoparticle will obviously cause changes in the morphology
and physical properties of the cell membrane (Figure. 4A). In addition,
it was also found that small graphene sheets can quickly enter the
inside of the bilayer, while large graphene sheets can only be partially
embedded in the cell membrane and lead to more complex cell membrane
deformations45. For graphene sheets adsorbed on the
cell membrane surface, the orientation of lipid molecules in the area
covered by the graphene sheets alters significantly driven by the
hydrophobic interaction, and there is the phenomenon of more obvious the
horizontal orientation of the lipid hydrophobic chains to be discovered
with enlarging the size of graphene sheet model46.
However, recent studies have shown that the size effect and the
difficulty of entering the cell membrane are not a simple linear
relationship, which indicates that there is theoretically a minimum and
an optimal radius for entering the cell membrane47,48.
As the radius is lower than the minimum radius, the cell membrane cannot
be endocytosed, entering the cell only through direct
penetration49. Meanwhile, for nanoparticles smaller
than the optimal radius, large-sized particles can be more easily
wrapped by cell membranes than small-sized particles (Figure.
4B)50.
The existence of the minimum radius lies in the competition of the
adsorption energy and the cell membrane bending
energy47. For larger nanoparticles, the number of
receptors on the surface will be relatively higher, resulting in higher
adsorption energy. Simultaneously, to wrap large-size nanoparticles, the
curvature of the cell membrane’s bending deformation should be larger,
the membrane bending energy barrier, which is negatively related to the
bending curvature51, would also be lower. On the
contrary, nanoparticles of too small size cannot provide sufficient
binding sites and small curvature, resulting in lower adsorption energy
and greater membrane bending energy, rendering it more difficult to be
wrapped by cell membranes. Therefore, only nanoparticles larger than the
critical size can be endocytosed by the cell membrane.
Gao47,50 et al. have also proposed another pair of
competing interactions, the diffusion kinetics of nanoparticles near the
cell membrane and the nanoparticle-cell membrane’s thermodynamic
attraction. Larger size nanoparticles may carry a larger number of
ligands, and their diffusion performance is weaker due to their size
effect, so they are more likely to be wrapped by cell membranes. In
comparison, the higher diffusion coefficient of small-sized particles in
the solution may give rise to an adverse effect on entering the cell
membrane. However, for nanoparticles with an excessively large size, the
wrapping time will increase significantly52, and the
cytotoxicity caused by larger deformations will also affect the
nanoparticles’ therapeutic efficiency, so there is supposed to exist an
optimal wrapping radius for the nanoparticles. To determine the optimal
radius, some researchers have already begun to put forward insights on
this issue from the perspective of the diffusion dynamics of
nanoparticles. Yan53 et al. have studied the diffusion
kinetics of graphene in cell membranes. They have pointed out that the
diffusion kinetics of graphene sheets in cell membranes are affected by
graphite interaction with hydrophobic phospholipid tails. Several
typical graphene diffusion dynamics models have also been summarized:
Brown dynamics-Levy dynamics-directional dynamics. Many studies have
established a variety of mathematical models for the diffusion of
nanoparticles, and their influencing factors are also be analyzing,
which would assist people to find the optimal nanoparticle
radius54.
3.2. Nanoparticle
Shape
Shape is also a significant factor in determining how nanoparticles
enter cells. For nanoparticles with good isotropy, such as spherical and
cubic nanoparticles, the way they enter the cell is determined by their
size: small-sized nanoparticles will directly penetrate through the cell
membrane31,55. In contrast, larger nanoparticles need
to be wrapped by the cell membrane to accomplish cell
internalization56. For non-isotropic materials, the
researchers have found that, under a strong driving force (concentration
gradient, ligand-receptor interaction, etc.), the sharp end of materials
can also penetrate the cell membrane57. By adjusting
the surface chemistry of such nanoparticles, the kinetic process of
nanoparticles piercing the cell membrane can be optimized (detailed
discussion will be carried out in the next section). For the process of
endocytosis, anisotropic materials possess a particular dynamic process:
orientation-wrapping-reorientation58. Their initial
conformation plays an extremely important role for anisotropic
materials, such as rod-shaped, disc-shaped, and elliptical
nanoparticles. Taking rod-shaped particles as an example, if the
particles’ initial conformation is perpendicular to the cell membrane,
direct wrapping needs to overcome a large amount of membrane bending
energy. Therefore, the nanoparticle will preferentially adjust its
original orientation so that its morphological long axis could be
parallel to the cell membrane, resulting in the lower bending energy
(Figure. 5A). After being wrapped by the cell membrane, some
nanoparticles may continue to rotate due to the residual rotational
momentum (Figure. 5B)19. As reported in the previous
study, this special internalization kinetics will reduce the efficiency
of nanoparticles entering cells. Li59 et al. have used
DPD simulations to verify that spherical and cubic nanoparticles with
better isotropy can be wrapped by cell membranes within simulation time
of 7 - 9 ns, while rod-like and disc-like particles with poor isotropy
would not finished the internalization within 13.5 ns (Figure. 5C).
However, this preferentially oriented dynamic behavior can help cell
membranes abate the membrane bending energy needed to overcome in the
process of the endocytosis59. Chen et al. have also
used Langevin’s molecular dynamics simulation to demonstrate that some
deformable spherical nanoparticles will also deform into an asymmetric
structure and then be wrapped by cell membranes (Figure.
5D)26.
From the above discussion, it can be seen that the study of the rotation
dynamics of nanoparticles is very indispensable for understanding
nanoparticle internalization. Chen60 and his
colleagues have studied the diffusion mechanism of nanoparticles by
analyzing the mean square displacement. It has been found that charged
particles possess better diffusion ability than neutral particles.
Simultaneously, the nanoparticles with randomly distributed electric
charges conform to the law of super diffusion (the nonlinear power-law
relationship between the mean square displacement and the simulation
time61,62), while the diffusion of uniformly
distributed nanoparticles is more in line with the law of linear
diffusion. By analyzing the typical trajectory and turning angle
distribution of nanoparticles, it has been found that nanoparticles with
randomly distributed charges would be inclined to diffuse in a specific
direction due to more complex asymmetric forces and torques to be given
to nanoparticles with random charge distribution, while nanoparticles
with uniform charge distribution have almost the same probability of
diffusing in all directions. Ji63 et al. have also
found that nanoparticles modified with more or shorter ligands are more
likely to interact with cell membranes, since longer modifiers may
destroy the anisotropy of the nanoparticles, leading to the inhibition
of the nanoparticle rotation59. However, the rotation
of nanoparticles can also bring disadvantages. Both experiments and DPD
simulations have indicated that the rotation of nanoparticles will
damage the surface structure of the cell membrane64.
Balancing the relationship between cytotoxicity caused by the rotation
of nanoparticles and optimizing the energy barrier of cell
internalization is the next essential research topic in this field.
3.3. Chemical property of nanoparticle
surface
The surface chemistry of nanoparticles is supposed to be the most
critical factor affecting the interaction with cell membranes. In this
review, more emphasis would be laid on three aspects: hydrophilic /
hydrophobic properties, charge properties, and ligand-receptor
interaction. In every aspect, the density surface pattern and the
covalent bonding of the modification would have an important influence.
The hydrophilic surface modification of nanoparticles is the basis for
successful drug delivery. For one thing, hydrophobic nanoparticles would
reduce the biocompatibility of the drug delivery
system65,66. For another, under the protection of the
hydrophilic modifier, the bioavailability of nanoparticles will be
guaranteed by avoiding being adsorb to plasma
proteins30. However, it can be seen from Figure. 6A
that the hydrophilic surface also affects the interaction between
nanoparticles and cell membranes. Driven by the hydrophobic attraction,
the nanoparticles can be trapped into the cell membrane faster, while
nanoparticles with hydrophilic surfaces can only adsorb on the
phospholipid head layer of the cell membrane, requiring to overcome a
higher free energy barrier due to the incompatibility between the
nanoparticle and the phospholipid tail67. Similarly,
in the study of graphene and cell membranes, researchers have also found
that graphene can form a sandwich shape with cell membranes, while
graphene oxide can be adsorbed on the cell membrane surface or
vertically pass through the cell membrane
conformation46,68. Therefore, researchers should
appropriately adjust the hydrophilic / hydrophobic ratio of the
nanoparticle surface. Furthermore, many experimental and theoretical
studies have indicated that the hydrophilic / hydrophobic surface
pattern can also have bearing on the cell
internalization69. Compared with Janus particles, the
nanoparticles in which two kinds of surface patterns alternately
distributed will increase the probability of entering the cell. The
study of Wang and his colleagues25 has pointed out
that the more alternate surface pattern stratification, the higher the
efficiency of cell internalization. Zhang67 et al.
have also proved that nanoparticles with randomly distributed
hydrophilic / hydrophobic surfaces can penetrate cell membranes, while
Janus particles can only be trapped in cell membranes. In addition to
affecting the efficiency of cell internalization, the surface properties
of nanoparticles will also change cell membranes’ physical and chemical
properties. On the one hand, passing through the cell membrane will
cause the cell membrane deformation. On the other hand,
Lin70 et al. have discovered that hydrophobic
nanoparticles will not change the cell membrane’s phase transition
temperature. As the degree of surface modification strengthens, the
phase transition temperature would first increase with the subsequent
decrease, leaving the final phase transition temperature higher than the
original value.
Compared with normal cells, the cell membrane of cancer cells contains a
higher density of negative charges71. Hence, the
charge modification of nanoparticles is also an effective alternative
means to enhance drug delivery efficiency. For example, researchers have
proved that nanoparticles modified with zwitterionic polymers are better
capable of consummating the cell internalization72. It
has been reported that different charge types determine distinct cell
membrane deformation mechanisms: neutral nanoparticles will not
significantly change the lateral curvature of the cell membrane.
Positively charged nanoparticles would cause the cell membrane to bulge
upwards, while negatively charged particles cause the cell membrane to
dent downwards (Figure. 6B)60. Moreover, for pH
amphoteric drug-loaded nanoparticles, the charge effect is particularly
significant. As the degree of ionization increases, the charge effect
can help improve the efficiency of nanoparticles penetrating through the
cell membrane. However, nanoparticles would disintegrate before being
internalized by the cell with too much mounting degrees of
ionization23. Therefore, these simulation results
suggest that people need to adjust the relationship between nanoparticle
cohesion and ionization. Another major factor, manifested from several
studies, lies in choosing the covalent bond and non-covalent bond
modification73. Rigid nanoparticles covalently linked
to the modified polymer containing ligands can be wrapped by the cell
membrane more productively due to the strengthening connection between
nanoparticles and modifiers. Adversely, non-covalently attached
nanoparticles cannot be wrapped by the cell membrane as the modification
polymer detaching from the nanoparticle. Under the pH trigger, the
modification polymer connected by non-covalent bonds has a significant
increase in hydrophilicity then falls apart from the surface of the
nanoparticle so that the core of the hydrophobic nanoparticle has the
opportunity to enter the middle layer of the cell membrane.
Another key chemical modification is the ligand-receptor distribution,
which assists nanoparticles specifically recognize targeted cells
without damaging normal cells. Only sufficient ligand density and
ligand-receptor interaction strength can induce cell membrane
deformation and encapsulate nanoparticles. Therefore, understanding the
relationship between the strength of the nanoparticle ligand-receptor
interaction and the membrane tension can provide more insights to
understand the mechanism of nanoparticle-cell interaction. As shown in
Figure. 6C, the stronger the ligand density (f ) on the surface of
the nanoparticle, the greater extent of the cell membrane deformation,
which results in more complete membrane wrapping. Nevertheless, the
mounting cell membrane tension (σ ) increase the difficulty of the
membrane deformation, the nanoparticles thus tend to penetrate the cell.
Therefore, by adjusting the relationship between f and σ ,
five nanoparticle-cell membrane interaction mechanisms can be observed:
(1) Direct penetration (lower f and higher σ ). (2)
Penetration or partial wrapping (lower f and moderate σ ).
(3) Full wrapping (higher f and lower σ ). (4) Wrapping
following penetration (higher f and appropriate σ ). (5)
Wrapping- penetration-rewrapping (lower f and lower σ ).
3.4. Nanoparticle
concentration
The concentration gradient is also one of the driving forces for the
interaction between nanoparticles and cell membranes. Therefore, both
experimental and theoretical studies have indicated that nanoparticle
concentration has a huge impact on internalization
efficiency43. Multiple nanoparticles will aggregate or
disperse on the cell membrane surface, which adjusts the nanoparticles’
size and shape to affect the nanoparticle-cell membrane interaction
mechanism.
For example, high-concentration nanoparticles lead to larger aggregates.
As described in section 3.1 , large size aggregates can
reduce the membrane bending energy, thereby promoting the cell membrane
to encapsulate the nanoparticles. Sometimes, the aggregates of
nanoparticles will rearrange and recombine into various shape to
increase the required curvature of the cell membrane, improving the
efficiency of nanoparticle cell internalization. Hu74et al. have explored the effect of peptide concentration alteration, and
it can be seen from the simulation results that peptides can be adsorbed
on the surface of the cell membrane and can open perforations in the
cell membrane to enter the cell as the concentration is high. Bundling
multiple small peptides into large individuals will also accelerate the
pore-forming effect, which is similar to the concentration effect.
Inspired by the concentration effect, the researchers have proposed a
method of cooperative internalization of multiple
nanoparticles75. As mentioned in section
3.3 , hydrophilic nanoparticles can be adsorbed on the hydrophilic
surface of the cell membrane, but it is challenging to enter the
hydrophobic inner layer of the cell membrane. With the aid of a partner
nanoparticle, the hydrophilic nanoparticle can smoothly penetrate cell
membranes under their synergistic effect, and the difference in
nanoparticle surface properties can produce different degrees of
cooperative internalization (Figure. 7A). A single hydrophilic
nanoparticle is adsorbed on the cell membrane surface, while four
hydrophilic nanoparticles can penetrate the cell membrane together. A
single nanoparticle with a random hydrophilic / hydrophobic surface
distribution can penetrate the cell membrane but can only be embedded in
the cell membrane after increasing the concentration.
Zhang74 et al. have analyzed the force spectrum of
nanoparticles and the distance between nanoparticles. They have
concluded that nanoparticle synergistic effect is greatly relevant to
the dispersion behaviour of the nanoparticles. The aggregation of
nanoparticles is more conducive to penetration, which is related to the
difference in force status of the nanoparticles on the horizontal plane.
Although the influencing factors and mechanisms of synergistic
interaction are given, there are still counter-intuitive aspects. Cite
an instance, the diffusion performance of hydrophilic nanoparticles in
an aqueous solution should be better than that of hydrophobic
nanoparticles. However, according to their simulation results, the
distance between hydrophilic nanoparticles is smaller than that of
hydrophobic nanoparticles. Li76 et al. have also
studied the influence of nanoparticle charge on the synergistic
penetration effect. It has been found that two positively charged
nanoparticles can still enter the cell membrane since the interaction
between the nanoparticle and the cell membrane weakens the electrostatic
repulsion. In addition to the nanoparticle surface properties, the
difference in their shapes will also cause different synergistic
effects. For example, vertically-oriented asymmetric nanoparticles can
further promote cell membrane penetration28. In order
to promote the cooperative internalization between the nanoparticles,
many researchers would choose to use polymers to connect the two
nanoparticles. According to Figure. 7B, two nanoparticles connected by a
polymer can increase the degree of engulfment by the cell membrane under
the synergistic effect. The chemical properties, length, and molecular
rigidity of the polymer connecting the nanoparticles should be
systematically optimized when designing such a system. If too long and
rigid polymer molecular chain has been utilized, it will increase the
distance between the nanoparticles and reduce the synergistic effect of
the nanoparticles77.
3.5. Nanoparticle elastic
modulus
Although most researchers use rigid nanoparticle models to simulate the
cell internalization process, with the diversification of modelling
methods, more emphasis has been paid to nanoparticle mechanical
properties, especially the elastic modulus78. As
illustrated in Figure 8A, rigid hydrophilic nanoparticles have a higher
free energy barrier to penetrate the cell membrane due to the
incompatibility between the hydrophilic nanoparticle surface and the
interior of the lipid bimolecular membrane. After appropriately reducing
the nanoparticle elastic modulus, the nanoparticles would undergo a
certain degree of deformation, which reduces the contact area between
the nanoparticles and the lipid tail. Therefore, a lower energy barrier
is supposed to be overcome by the softer hydrophilic nanoparticles. On
the contrary, since the good attractive interaction with the middle part
of the cell membrane for rigid hydrophobic nanoparticles, the free
energy will decrease during the process of the nanoparticle penetrating
the cell, which reaches a minimum as the nanoparticle is trapped in the
middle of the cell membrane. The deformation of the softer nanoparticles
changes the contact state between the nanoparticles and the cell
membrane, enlarging the minimum value of free energy, making it more
arduous for the nanoparticles to penetrate the cell
membrane25. For the process of endocytosis, the effect
of elastic modulus is also twofold. For one thing,
Yi79 et al. have used the framework of Helfrich theory
to propose a theoretical model for the interaction between elastic
nanoparticles and cell membranes. From the numerical simulation results,
it can be found that soft nanoparticles are more challenging to be
wrapped by cell membranes than rigid nanoparticles because the
deformation of nanoparticles will bring more energy. For another, the
nanoparticle deformation will adjust the nanoparticle shape. If the
deformation leads to an appropriate shape, which needs enlarged cell
membrane curvature, thereby improving the nanoparticle internalization
efficiency (Figure. 8B)26. Excessively lower
nanoparticle elastic modulus will bring more negative effects, such as
low nanoparticle stability under the shear of fluid and even the
disintegration before being internalized by cells80.