Erythrocyte-Platelet Hybrid Membrane Coating for Enhanced
Nanoparticle Functionalization
Diana Dehaini, Xiaoli Wei, Ronnie H. Fang, Sarah Masson, Pavimol Angsantikul, Brian T.
Luk, Yue Zhang, Man Ying, Yao Jiang, Ashley V. Kroll, Weiwei Gao, and Liangfang Zhang
*
Department of NanoEngineering and Moores Cancer Center, University of California, San Diego,
La Jolla, CA 92093, U.S.A
Table of content entry
Biomimetic dual membrane-functionalized nanoparticles, incorporating the natural properties of
two different cell types, are fabricated by a facile process employing fused cell membranes. The
resulting hybrid cell membrane-coated nanoparticles retain protein markers from each source cell
and combine the unique functions of both. The reported approach opens the door for the
fabrication of biocompatible nanocarriers with increasingly complex functionality.
Keywords
nanomedicine; biomimetic nanoparticle; membrane fusion; long circulation; targeted delivery
Nanotechnology has increasingly been employed for the design of drug delivery and
imaging modalities in order to further improve efficacy in the clinic. There are many classes
of nanoscale delivery vehicles, including liposomes, polymeric nanoparticles, and inorganic
zhang@ucsd.edu, Tel: +1-858-246-0999.
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systems among many others.
[1–4]
For applications such as cancer treatment, the use of such
nanoparticle platforms has the potential to enable greatly enhanced potency while
minimizing the systemic toxicity associated with traditional therapeutics.
[5–9]
Recently, a
new class of drug nanocarrier has been reported that is fabricated by combining synthetic
nanoparticulate cores with a biologically derived membrane coating.
[10–13]
These
biomimetic, cell membrane-coated nanoparticles directly leverage the versatility and
complexity of cellular membrane, which has been crafted by nature through the process of
evolution to perform specific functions, especially with regards to biointerfacing.
[14–17]
By
translocating the entire membrane from a cell onto the surface of a nanoparticle, all
biologically relevant surface moieties are transferred, including those that can potentially be
used for immune evasion and targeting, two highly desirable properties. Additionally, it may
be possible to take advantage of cell-specific functionalities that are known to exist, but have
not been well characterized on a fundamental biological level.
[18–20]
The first platform of this type employed red blood cells (RBCs), also known as erythrocytes,
as the source material in order to achieve lengthened blood residency, and the resulting RBC
membrane-coated nanoparticles (RBCNPs) displayed significantly enhanced circulation
half-lives compared with a nanoparticle stabilized by a layer of polyethylene glycol
(PEG).
[21]
It was later demonstrated that these cell membrane-coated nanoparticles exhibited
right-side-out membrane orientation and included immunomodulatory markers such as
CD47 at the same density as the original RBCs.
[22]
RBCNPs have also been used for novel
detoxification applications, including for toxic nerve agents and bacterial toxins.
[23–26]
More
recently, many different cell types other than RBCs, including platelets, white blood cells,
cancer cells, stem cells, and even bacteria, have been used to source the membrane material,
each with its own set of unique characteristics.
[14,18,27–29]
Of note, expansion of the cell
membrane-coated platform to other cells has enabled the fabrication of naturally targeted
drug carriers.
[30]
This includes the use of cancer cells for homotypic delivery to the tumor of
origin, or platelets, which play a role in a wide array of different diseases that range from
atherosclerosis to bacterial infection.
While cell membrane coating is a powerful method of enhancing nanoparticle utility, it is
often desirable to introduce additional functionality depending on the specific application.
For example, while RBCNPs can circulate for extended periods of time, the addition of a
targeting ligand can help to improve localization to the desired target, such as a tumor.
[31]
Herein, we report on an approach for increasing the number of functions that can be
performed by a single membrane-coated nanocarrier
via
the simultaneous derivation of
functionality from multiple cell types. This novel approach, which involves the fusion of
natural cell membranes from different origins, represents a facile and effective means of
fashioning nanoparticles that can perform increasingly complex tasks within biologically
relevant contexts. Specifically, we aimed to combine the functionality of human-derived
RBCs and platelets (Figure 1A). The resulting RBC-platelet hybrid membrane-coated
nanoparticles ([RBC-P]NPs) were thoroughly characterized, and it was demonstrated that
they retained functionality that is characteristic of each individual cell type. Finally, we show
that these dual membrane-coated nanoparticles displayed
in vivo
properties consistent with
expectations, warranting further study.
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In order to fabricate the [RBC-P]NPs, the first step was to verify that it was possible to fuse
RBC membrane and platelet membrane together, the product of which would then be used as
the base material for downstream nanoparticle preparation. To test for fusion, platelet
membrane was doped with two different dyes that constituted a Förster resonance energy
transfer (FRET) pair and added to increasing amounts of RBC membrane under elevated
temperature and stirring (Figure 1B). It was observed that, as the amount of RBC material
increased, there was a recovery of fluorescence at the lower emission wavelength around
534 nm, indicating the interspersing of the two membrane materials weakening the FRET
interactions in the original platelet membrane. Using a 1:1 protein weight ratio of RBC
membrane to platelet membrane for further study, we fabricated a batch of the hybrid [RBC-
P]NPs. RBC membrane labeled with a red fluorescent dye was fused with platelet
membrane that was labeled with a green fluorescent dye, and the resultant material was
coated onto preformed poly(lactic-
co
-glycolic acid) (PLGA) cores. When a dilute solution
of the [RBC-P]NPs was immobilized in glycerol and viewed under confocal microscopy,
significant colocalization of fluorescent signals was observed (Figure 1C). In stark contrast,
a mixture of RBCNPs and platelet membrane-coated nanoparticles (PNPs) fabricated with
the individual fluorescently labeled membranes exhibited distinct red and green punctates. It
was further demonstrated that the RBC and platelet membranes were retained on the [RBC-
P]NPs at a ratio nearly identical to the 1:1 input (Figure 1D). The results indicate that it was
indeed possible to fuse the two types of natural cell membrane and incorporate the material
of both onto the same nanoparticle.
Dynamic light scattering (DLS) was used to compare the [RBC-P]NPs with each single
membrane formulation of RBCNPs or PNPs (Figure 2A,B). It was observed that bare PLGA
cores, which were originally 80 nm in size, exhibited a very uniform increase in size of
approximately 20 nm after coating with each membrane. Additionally, the surface zeta
potential increased by over 10 mV for each of the three nanoparticle types compared with
PLGA cores, approaching the value of approximately −25 mV observed for each of the
different membrane vesicles. This phenomenon is commonly seen after membrane coating
and indicates shielding of the highly negative cores with the less negative outer membrane
surface.
[14,21]
When visualized under transmission electron microscopy (TEM), the
RBCNPs, PNPs, and hybrid [RBC-P]NPs all displayed a characteristic core-shell structure
that was consistent with the sizing results (Figure 2C). A set of assays were then carried out
to assess nanoparticle stability in various media. To determine stability of the hybrid
formulation in solution over time, samples were stored in either water or 1× phosphate
buffered saline (PBS, pH 7.4) and the size was measured over time (Figure 3A,B). The
[RBC-P]NPs, in addition to RBCNPs and PNPs, exhibited stable size over the 3 week
duration of the study in both solutions. In contrast, bare PLGA cores, which are only
stabilized by charge repulsion, immediately aggregated in PBS to the micron range.
Additionally, [RBC-P]NPs displayed little change in absorbance before and after incubation
with 100% serum, while bare PLGA cores again exhibited a large increase (Figure 3C).
Regarding long-term storage, the [RBC-P]NP formulation exhibited near identical size both
before freeze-drying and after resuspension (Figure 3D). Taken together, the characterization
data give strong physical evidence for successful coating by the fused membrane material,
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with results in line with what would be expected given the properties of the single
membrane-coated RBCNPs and PNPs.
To analyze the overall protein content of the [RBC-P]NPs, SDS-PAGE was used to run RBC
membrane, platelet membrane, hybrid membrane, and all corresponding nanoparticle
formulations followed by Coomassie staining for visualization (Figure 4A). Compared with
RBCNPs and PNPs, the [RBC-P]NPs had a profile that represented the union of the two
single membrane formulations. As expected, all nanoparticles had protein profiles that
largely mirrored that of the corresponding membrane. To analyze specific protein markers,
western blotting analysis was carried out (Figure 4B). CD235a, a major RBC
sialoglycoprotein also known as glycophorin A,
[32]
as well as the blood group A antigen,
were present on RBCNPs and also to a lesser degree on [RBC-P]NPs. CD41 and CD61,
which collectively form integrin αIIbβ3,
[33]
are important for platelet adhesion as well as
activation, and both were present on PNPs and also to a lesser degree on [RBC-P]NPs.
Meanwhile, CD47,
[34,35]
an immunomodulatory protein responsible for inhibiting
macrophage uptake that is expressed by both cell types, was found at a near equivalent
degree on RBCNPs, PNPs, and [RBC-P]NPs. Additionally, immunogold staining followed
by TEM imaging provided visual evidence that a single [RBC-P]NP could simultaneously
present both RBC-specific and platelet-specific markers (Figure 4C). Ultimately, the protein
analysis carried out here indicates that functionalization with the fusion membrane can
bestow both RBC and platelet surface proteins onto the hybrid [RBC-P]NPs, although the
exact nature of the intraparticle distribution of individual protein markers at the molecular
level remains unknown.
Upon confirming successful transference of markers unique to both source cells, we next
sought to characterize the hybrid membrane coating on a functional level. Both RBCNPs
and PNPs, given the presence of immunomodulatory markers on their surface, have been
shown to be adept at minimizing macrophage uptake.
[14,22,36]
An uptake study using human
THP-1 monocytes differentiated into macrophage-like cells was thus carried out to evaluate
this property in [RBC-P]NPs (Figure 5A). Using flow cytometric analysis after incubation
with the macrophage-like cells, [RBC-P]NPs had low uptake consistent with both RBCNPs
and PNPs, whereas PLGA cores, without immunomodulatory membrane coatings, exhibited
a high degree of uptake. As an RBC-specific functional marker, we next assessed the level of
acetylcholinesterase activity (Figure 5B). The enzyme is important for regulating
neurotransmitter concentrations, and RBCNPs have been shown to be potent decoys capable
of neutralizing lethal doses of poisonous compounds that can deactivate the protein.
[25]
It
was confirmed that [RBC-P]NPs had an intermediate level of activity compared with
RBCNPs and PNPs, the latter of which had a negligible amount of activity. This was further
reflected in detoxification capacity, as the amount of dichlorvos, a model organophosphate,
left in solution after incubation with each nanoformulation was inversely related to
acetylcholinesterase activity (Figure 5C). Regarding platelet-specific functionality, the
cancer targeting properties of the hybrid [RBC-P]NPs was first evaluated. PNPs, given the
large number of disease-relevant binding markers present on their surface, have been shown
to be effective at targeting certain metastatic cancers.
[37]
Using both flow cytometry as well
as fluorescent imaging, it was demonstrated that [RBC-P]NPs bound to highly metastatic
MDA-MB-231 human breast cancer cells within the spectrum between RBCNPs and PNPs,
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which exhibited low and high amounts of binding, respectively (Figure 5D,E). On the other
hand, normal HFF-1 human foreskin fibroblasts showed significantly lowered binding by all
nanoparticle types (Figure 5D), and this was further confirmed by a co-culture imaging
study using [RBC-P]NPs (Figure 5F). After binding to cancer cells, it is expected that the
hybrid nanoparticles would be endocytosed via the clathrin-dependent pathway.
[37]
To assess
in vivo
targeting ability, a mouse model of atherosclerosis, which platelets are known to
interact with, was employed. Fluorescence imaging of aortas collected after intravenous
nanoparticle administration revealed a trend consistent to what was observed for the
in vitro
studies (Figure 5G). Overall, the ability of [RBC-P]NPs to incorporate the functional
properties of both RBCs and platelets is encouraging and provides a strong indication that
other cell-specific functions should likewise be present on the dual membrane-coated
nanoparticles.
Finally, to evaluate the [RBC-P]NP formulation for potential
in vivo
application, the
circulation and biodistribution of the nanoparticles were evaluated using a mouse model.
Murine-derived RBCs and platelets, along with fluorescently labeled PLGA cores, were
used as the starting materials to fabricate RBCNPs, PNPs, and [RBC-P]NPs. To test the
circulation half-life, nanoparticles were administered intravenously via the tail vein, and
blood was sampled at increasing timepoints to evaluate remaining nanoparticle
concentration (Figure 6A). After fitting to a two-phase decay model, it was apparent that all
three formulations exhibited very similar circulation profiles. Indeed, both RBCNPs and
PNPs have previously been reported to circulate for extended periods of time upon
administration.
[21,37]
Numerical analysis indicated that RBCNPs, PNPs, and [RBC-P]NPs
had one-phase half-lives of 5.7, 5.7, and 6.4 hours and two-phase elimination half-lives of
42.4, 38.3, and 51.8 hours, respectively. All values were within error of each other (see
Experimental Section). To analyze biodistribution, nanoparticles were again administered
intravenously, and the mice were euthanized 24 hours afterwards in order to collect the
heart, lungs, kidneys, liver, and spleen for fluorescence analysis (Figure 6B). All three
nanoformulations, including the [RBC-P]NPs, displayed similar organ-level localization,
with the majority of the nanoparticles found in the liver and spleen as a result of
reticuloendothelial uptake. This pattern is consistent with what has been previously reported
for similar nanoformulations.
[14,21]
The uniformity in circulation and distribution profiles
between all experimental groups can likely be explained by the fact that, under normal
conditions, both RBCs and platelets are inherently designed to persist in the bloodstream for
extended periods of time; in healthy individuals, there is an absence of reactive substrates
and molecules that can contribute to accelerated clearance of either cell type.
In conclusion, we have successfully fabricated a new class of cell membrane-coated
nanocarrier that combines the function of two different cell types. It was confirmed that it is
possible to fuse RBC membrane and platelet membrane together, using it as a coating
material to fabricate hybrid [RBC-P]NPs. Physically, the resultant particles were similar to
pure, single membrane formulations of either RBCNPs or PNPs. On a protein and functional
level, the [RBC-P]NPs represented a cross between the two parent nanoparticle types,
retaining properties once only exclusive to either. The reported method of bestowing
nanoparticles with enhanced functionality provides a facile and natural alternative to
synthetic post-functionalization strategies. Importantly, the dual membrane-coated
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