Three-Component Sequential Reactions for Polymeric Nanoparticles with Tailorable Core and Surface Functionalities

Full text of DOI:10.1016/j.chempr.2019.09.001

Article
Three-Component Sequential Reactions for
Polymeric Nanoparticles with Tailorable Core
and Surface Functionalities
Bin Liu, S. Thayumanavan
thai@chem.umass.edu
HIGHLIGHTS
A simple one-pot multi-
component reaction was
developed for nanoparticle
synthesis
Methodology offers concurrent
surface and core functionalization
capabilities
Nanoparticles exhibit tunability in
size and encapsulation stability
Encapsulation is specifically
compromised due to the
triggerable release features
Functional organic nanoparticles were prepared through a one-pot, three-
component sequential reaction between small molecules and polymers with
concurrent functionalization and crosslinking. This simple, scalable, and green
process affords nanoparticles with size control, tunable surface and core
functionalities, and high encapsulation stability with triggerable molecular release
features.
Liu & Thayumanavan, Chem 5, 1–18
December 12, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.chempr.2019.09.001

Please cite this article in press as: Liu and Thayumanavan, Three-Component Sequential Reactions for Polymeric Nanoparticles with Tailorable
Core and Surface Functionalities, Chem (2019), https://doi.org/10.1016/j.chempr.2019.09.001
Article
Three-Component Sequential Reactions for
Polymeric Nanoparticles with Tailorable
Core and Surface Functionalities
Bin Liu1 and S. Thayumanavan1,2,3,4,
*
SUMMARY
The Bigger Picture
Nanomedicine offers a promising
approach for targeted
Efficient strategies for the preparation of nanostructures with tailorable func-
tionalities have implications in enhancing the repertoire of nanomaterials in
many applications. Multi-component reactions (MCRs) are very attractive
because they are synthetically simple while providing unique access to incorpo-
ration of functional groups onto a system. This highly efficient process has not
been brought to bear in the preparation of functional polymeric nanostructures.
In this paper, we report a three-component sequential reaction that is capable
of concurrently functionalizing the core and the surface of the nanoparticles
and crosslinking the polymeric assemblies, along with excellent control over
size (ꢀ10 nm to ꢀ1 mm). Variations in core offer the opportunity to optimize
the host-guest properties for non-covalent drug encapsulation, while the sur-
face features provide the ability to tune interfacial interactions and achieve
organelle targeting in cells. Encapsulation of drug molecules and their triggered
release features have been utilized for intracellular drug delivery.
therapeutics, such as in cancer.
The complexity of biological
barriers, however, presents a
significant challenge in designing
efficacious drug carriers. The key
criteria that define highly efficient
nanoparticles include (1) simple
preparative chemistry that
ultimately offers scalability, size
control, and surface
functionalization to optimally
interface with biological systems
to achieve the desired cellular
targeting and (2) host-guest
characteristics with good
INTRODUCTION
Nanoscale assemblies with sophisticated properties have attracted tremendous in-
terest because of the potential applications in different fields such as drug delivery,
biomedical diagnostics, and theranostics.1–5 Scaffolds such as inorganic nanopar-
ticles (NPs),6,7 liposomes,8 dendrimers,9 polymeric assemblies,10–15 and polymer-
based NPs10–16 have been explored for this purpose. Among these, amphiphilic
block copolymer-based assemblies have been extensively studied because of their
water solubility and non-covalent encapsulation capabilities of hydrophobic guest
molecules in aqueous phase.10–16 These features are particularly important for ther-
apeutic applications, where the ability to load hydrophobic drug molecules into a
water-dispersible nanoassembly is critical for overcoming some of the pharmacoki-
netic limitations of conventional drug formulations.17–20 Block polymeric micelles
contain well-defined core-shell structure with hydrophobic polymer cores and wa-
ter-soluble polymer shells. In order to achieve efficient drug encapsulation inside
the core of the block polymer micelles, it requires the guest molecules to overcome
the barrier formed by the shell layer and the soft-glassy core polymer block. There
are usually two methods for drug encapsulation, which is based on the dissolution
of the drug molecules and the block copolymers in a water-miscible organic solvent
together with subsequent addition of water and further removal of the organic sol-
vent by evaporation or dialysis to efficiently decrease the energy barrier for the drug
molecules to penetrate from the micelle shell to the glassy hydrophobic core.21–24
However, these methods are not suitable for scalable production as physical factors
(e.g., diffusion and solvent exchange rate) are influenced heavily by the scales during
encapsulation stability that
obviates premature drug release.
In this work, we present a simple
method for preparing functional
nanoparticles through a one-pot,
sequential multi-component
reaction that exhibits convenient
tunability in size, surface
functionalities, and high
encapsulation stability with
triggerable release
characteristics. The combination
of these features highlights the
potential of these nanoparticles in
biomedicine.
Chem 5, 1–18, December 12, 2019 ª 2019 Elsevier Inc.
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the drug incorporation processes. Furthermore, there is also an environmental cost
associated with using organic co-solvents in a process that is primarily driven by the
propensity of the host assembly to self-assemble in water.21–24
Also, most disease models have disparate requirements in features such as size and
surface functionalities to optimize their circulation time as well as tissue, cellular, and
sub-cellular targeting.25–27 Note, however, that it is difficult to tune the size of mi-
celles from one polymeric precursor in the case of block copolymers, as the assembly
size here is controlled by the relative block composition and the block length under
equilibrium conditions23,24,28,29; i.e., a polymer with the specific structure usually af-
fords a micelle with a well-defined size.23,24,28,29 Therefore, when block copolymer
micellar assemblies with different sizes are desired, a series of different polymers
need to be designed and synthesized, which is time consuming and laborious.30
In fact, it is difficult to prepare block-copolymer-based micelles with small
(d < 20 nm) and big (d > 100 nm) sizes.28–32 Moreover, the surface functional groups,
which are often used for targeting purposes, must be specifically installed at the end
of the hydrophilic terminus.31,32 By the extension of this feature, for installing few
different surface functionalities, polymers with these different terminal functional
groups must be independently synthesized and co-assembled.31,32
In addition to size and surface functionalization features, these supramolecular as-
semblies also must exhibit excellent host-guest characteristics. For example, most
cancer chemotherapeutic molecules are highly toxic. Therefore, it is essential that
drug molecules are stably encapsulated within the interiors of these vehicles to pre-
vent premature release of drug molecules, when these systems undergo large dilu-
tion during biodistribution and when interacting with biological components such as
cellular membranes and serum proteins that can competitively sequester these mol-
ecules to cause off-target release.33–35 A consequence of stable encapsulation is
that these nanoassemblies must be programmed to release their hydrophobic guest
molecules in the presence of a bio-relevant trigger, such as a biomarker for a disease
so that the drug molecules are released at the location of a specific lesion.12,36 The
intrinsic instability of the micelles under diluted conditions will cause the disas-
sembly and premature release of the drug molecules especially under complex
serum condition. Therefore, it is difficult to achieve high encapsulation stability for
the block copolymer micelle.34,35,37–39 For instance, the widely used PEG-PDLLA
block polymer micelle has been found to rapidly release the encapsulated hydro-
phobic agents during circulation under in vivo conditions.40 Even the polyethylene
glycol-phospholipid micelles with enhanced stability were proposed, however, the
NPs can only be stable for 1 to 2 h under serum conditions.41 Even though the
increased polymeric hydrophobicity of the micelle may enhance the encapsulation,
the encapsulated drug would lack efficient release mechanism in these cases.34,36
Recent molecular design strategies can overcome one or more of these barriers to
increase the chemotherapy efficiency.42–45 For instance, layer-by-layer approaches
1Department of Chemistry, University of
have been used to prepare assemblies with tailorable crosslinking-based stabiliza-
tion and surface functionalization features.46 Similarly, shell-crosslinked micelles
have been developed for enhancement of the carrier stability and facile surface en-
gineering.47 Nonetheless, fulfilling all these structural and functional requirements,
mentioned above, in a single system continues to be a challenge.
Massachusetts, Amherst, MA 01003, USA
2Center for Bioactive Delivery, Institute for
Applied Life Sciences, University of
Massachusetts, Amherst, MA 01003, USA
3Molecular and Cellular Biology Program,
University of Massachusetts, Amherst, MA 01003,
USA
4Lead Contact
Moreover, introducing functional groups in NPs often involve multiple and often
tedious steps. Multi-component reactions (MCRs), involving three or more starting
materials performed in one pot for the preparation of a single final product, have
*Correspondence: thai@chem.umass.edu
https://doi.org/10.1016/j.chempr.2019.09.001
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been of interest for their atom economy and efficiency.48–53 MCRs were initially
developed for the synthesis of small molecules and have been launched for the prep-
aration of large libraries of molecules with the diversity and complexity that are crit-
ical for drug development and screening.51–53 More recently, these strategies have
been brought to bear for the synthesis of polymers with different topologies, struc-
tures, architectures, and properties.54–60 However, to our knowledge, MCRs have
not been exploited for the preparation of advanced nanostructures such as hierar-
chical polymeric assemblies with tailorable properties. In this paper, we use an effi-
cient one-pot multi-component sequential reaction for the preparation of the com-
plex supramolecular nanoassemblies with tunable features desired in a therapeutic
delivery vehicle, where the surface functionalization and crosslinking-based stabili-
zation of nanocarrier are achieved simultaneously in one pot with water as the
solvent.
Our approach to prepare NPs and its features that highlight the key advantages in
drug delivery applications include the fact that (1) the synthetic procedure is simple
to perform multistep reaction for NPs in one pot, which is beneficial for scalability; (2)
the reaction is performed with high atomic efficiency in a green solvent, water; (3) the
NPs exhibit high encapsulation stability because of their crosslinked nature; (4) the
NPs are triggerable for molecular release, as the crosslinks are based on disulfide
bonds that are susceptible to the reducing intracellular conditions;61 and (5) the
nature of the self-assembly process and the multi-component feature allows for an
unprecedented and convenient tunability in size, hydrophobicity of the interior,
and surface functionalities. We elaborate the details of our preliminary findings
and details in this paper.
RESULTS AND DISCUSSION
Three-Component Sequential Reaction for NPs with Controllable Sizes
Simple synthetic procedure to prepare the NPs, while offering the flexibility in en-
dowing them with functional characteristics, is the key for a scalable impact. Cascade
chemical reactions in a single pot offer that unique opportunity. The three-compo-
nent sequential reaction that is used in the formation of the nanoassembly involves
two reactions, where the reactive functionality for the second step is generated in the
first step and the functional groups involved in the first and the second step are
mutually compatible. The molecular design that offers this possibility is shown as
P1 in Figure 1. We note here that the synthesis of the random copolymer itself
requires much lower levels of synthetic efforts, compared to the block copolymer,
which is good for scale up.22–24 This random copolymer contains a hydrophilic poly-
ethyleneglycol (PEG) methacrylate and two different hydrophobic methacrylate
monomers containing an amine-reactive g-thiolactone unit and a thiol-reactive pyr-
idyldisulfide unit. In this case, an externally added amine (R-NH2) would specifically
react with the g-thiolactone unit to cause a ring opening of the latter to reveal a thiol
moiety. This thiol moiety would then react with the pyridyldisulfide moiety to
generate disulfide bonds. We hypothesized that in a polar environment, this random
copolymer would self-assemble into a nanoscale structure, which can be captured
using the three-component reaction induced crosslinking process. The initial amide
bond formation using the substituted primary amine allows for incorporation of var-
iations in the functional groups present within the interior of the polymer assembly,
as well as on the surface of the NPs (Figure 1). The ring opening and the disulfide
bond formation reactions provide the crosslinking required for the formation of
the NP. Moreover, if the crosslinking process occurs exclusively within the initially
formed aggregate, i.e., without any crosstalk among the nanoassemblies, then the
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Figure 1. Three-Component Sequential Reaction for the Preparation of the NPs
(A) Schematic illustration of preparation of the NPs and their responsive release process.
(B) The chemical transformation of three-component sequential reaction for NPs.
(C) NMR spectra for monitoring the three-component sequential reaction process in D2O. The stars
represent the byproduct (pyridinethiol) from the reaction as the readout.
ability to achieve variations in non-covalent supramolecular assemblies would
directly offer access to various crosslinked NPs.
The thiolactone-containing monomer synthesis started with the reaction of DL-ho-
mocysteine thiolactone hydrochloride with succinic anhydride in the presence of
sodium bicarbonate. The resultant carboxylic acid moiety was coupled to hydroxye-
thylmethacrylate (HEMA) monomer under 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide (EDC)-coupling conditions to obtain the targeted monomer (see Supple-
mental Information for details). Note that the amine moiety of the starting
material could indeed be directly coupled with methacrylic acid to obtain a mono-
mer with the thiolactone on the side chain. However, the solubility of this monomer
was not compatible for copolymerization with other monomers in this reaction. Poly-
mer P1 was obtained through copolymerization of the thiolactone monomer above
with PEG-methacrylate and pyridyldisulfidyl-ethyl-methacrylate monomers under
RAFT polymerization conditions. To label the polymer P2, a P1 analog with a fluoro-
phore label, these three monomers were copolymerized with a small percentage of
an additional monomer containing rhodamine B. Synthetic and characterization de-
tails for the monomers and the polymers are described in Schemes S1–S4 and Fig-
ures S1–S11. Molecular weight (Mn) of P1 is ꢀ14 k with a dispersity of 1.1. The ratio
of the chemical compositions (PEG: thiolactone [TLt]: pyridyldisulfide [PDS]) was
found to be about 30: 35: 35 based on the NMR integration of their characteristic
peaks respectively at 3.37, 4.61, and 8.46 ppm (Figure S7). The dye labeled polymer
(P2) has almost the same composition with ꢀ1% of rhodamine B label.
The next step was to use this polymer precursor to test the idea of using the three-
componentsequential reaction to prepare crosslinked polymeric NPs. To qualita-
tively assess whether the amphiphilic random copolymer forms assemblies in
aqueous phase, 1H NMR of P1 was assessed in D2O. Indeed, the aromatic peaks
that correspond to the PDS units in the polymer were found to be very broad (Fig-
ure 1C) in relation to the same polymer in an organic solvent (see Supplemental
4
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Information). This observation was taken to indicate the formation of a nanoscale
aggregate in aqueous phase, where the PDS units are mostly buried within the inte-
rior of the assembly with limited segmental mobility. Upon the addition of 1 equiv of
octylamine, there was a clear emergence of sharp aromatic NMR peaks that are
attributed to the formation of the pyridothione byproduct of the anticipated
three-component reaction.
The reaction was deemed efficient as all the PDS groups in the polymer aggregate
seem to have been transformed to the pyridothione within 12 h even at low reactant
concentrations. For comparison, when the same three-component reaction was also
performed in an organic solvent, where no self-assembly is anticipated, the reaction
was found to be much slower (Figure S18). This is understood as the hydrophobicity-
driven nanoaggregate formation in water would significantly increase the local con-
centration of the reactively complementary moieties.
Next, we were interested in controlling the properties of the NPs, specifically their size.
The ability to vary size of the NPs was motivated by the fact that the impact of nanoas-
semblies in many biological applications, such as drug delivery, is dictated by size.25
These precise size requirements do also vary with different disease models.62 Therefore,
an ideal nanoscale platform would allow control over size of the NPs. To this end, we at-
tempted to vary the self-assembly conditions that would offer variations in the size of the
nanoassemblies. When P1 was assembled in water at 10 mg/mL concentration, the
resultant aggregate was found to be ꢀ15 nm at room temperature (Figure S19).
Whenthisaggregatewascrosslinkedatambienttemperaturewithhexylamine, the poly-
mer NP was found to be ꢀ10 nm, suggesting that the intermolecular crosslinking pro-
cess could cause collapse of the aggregate (Figure 2A). We also used different amines
to trigger the three-component sequential reaction for NPs under different solvents at
ambient temperature. All the NPs revealed similar sizes in the range of 10–20 nm (Figure
S20). We further explored the possibility of utilizing the propensity of PEG-based poly-
mers to exhibit variations in aggregate sizes, because of their temperature-dependent
dehydration possibilities.63,64 Indeed, as we increased the temperature to 70ꢁC, the size
of the aggregates increased, which was also found to exhibit a systematic dependence
on the concentration of the polymer. With this observation, we were able to tunethe size
of the NPs over a broad range from 30 nm to 40, 60, 170, or even ꢀ1,000 nm by using
polymer concentrations of 2 to 5, 10, 20, and 30 mg/mL (Figures 2 and S21), when the
crosslinking reaction was carried out at 70ꢁC.
Size of these NPs were mainly determined by dynamic light scattering (DLS), where all
assemblies exhibited excellent correlation function. Although these particles were
formed at 70ꢁC, DLS measurements were performed at ꢀ25ꢁC, where the initial assem-
blies were found to be substantially smaller. The fact that the sizes of the crosslinked
assemblies did not change at 70ꢁC (Figure S21C) suggests that the crosslinking process
can efficiently lock the size of the NPs. The NPs were also characterized using transmis-
sion electron microscopy (TEM), as shown in Figure 2. The NPs were spherical and ex-
hibited a slight decrease in size, relative to that from DLS measurements, suggesting
that these soft NPs collapse in dry state. Finally, it is noteworthy that all these NPs
were found to be stable in water for long periods of over a month (Figure S22).
Functionalization of the NPs
Functionalization of the NPs affords us the opportunity to tune the properties for a
wide range of applications. A key feature of the three-component sequential reac-
tion here is that the primary amine, used as the initiating reactant of the reaction
cascade, offers the opportunity to incorporate diverse functionalities on the NP
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Figure 2. Size Control of the NPs
(A) Size of the NPs by DLS from different preparation methods (measurements were performed at
25ꢁC). The size of the NPs was varied from 10 nm to 30, 40, 60, and 170 nm.
(B) Correlation function of the NPs from DLS. All the NPs exhibited good correlation functions.
(C) Unstained TEM images of corresponding NPs with different sizes from different preparation
methods. The names represent the preparation conditions for different NPs. For example,
10mg@70C means the NPs were prepared with 10 mg polymer in 1 mL water at 70ꢁC.
surface. To this end, we systematically varied the alkyl group in the alkylamine,
R-NH2. Accordingly, we initiated the crosslinking reaction with alkyl moieties
containing a negatively charged carboxylic acid moiety or a positively charged qua-
ternary ammonium group, along with a control that does not bear any charge (hexyl-
amine). The charged nature of the first two functionalities should dictate the surface
charge of the NP, which was assessed with zeta potential measurements (Figure 3).
The surface charge of the functionalized particles was indeed found to be negative
(À42.8 eV) for the carboxylic-acid-functionalized NP, while the ammonium-function-
alized NP exhibited a positively charged surface (+11.4 eV). Interestingly, the hexyl-
amine-functionalized control NPs were found to be negatively charged, albeit with a
lower magnitude. This observation is, however, consistent with other PEG-function-
alized NPs reported in the literature.65
A reasonable expectation, among these structural variations, is that the surface
exposure of the modifying functionalities would be dictated by their solvophobicity.
Thus, when the modification is based on a hydrophilic functionality, these function-
alities would be mostly presented on the exterior of the NP. Conversely, the
6
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Figure 3. Functionalization of the NPs
(A and B) Functionalization of the NPs with different charged species on the surface: (A) chemical illustration of the functionalization and (B) zeta
potential of the NPs.
(C and D) Interior functionalization: (C) chemical illustration and (D) emission spectra of the NPs with different concentrations (unit: mg/mL).
hydrophobic moieties would mainly become part of the core of the NP. While the
zeta potential measurements support this hypothesis for the positively and nega-
tively charged NPs, we further explored the hydrophobic modifications with pyrene-
methylamine, a spectroscopic probe. The absorption and emission spectra of this
modification are shown in Figures S23 and 3D, respectively. The characteristic ab-
sorption peak for pyrene showed the functionalization of the NP with the pyrene
functionality. On the other hand, the presence of the excimer emission peak along
with the monomer emission peak in the fluorescence spectrum suggests that these
functionalities are most likely present within the interior of the NP. To further explore
whether these excimer peaks are due to an aggregation of surface exposed pyrene
moieties, the NP solution was diluted by about 20 times and the excimer emission
peak could still be seen. These data further support the idea that the hydrophobic
modifications mostly affect the interior of the NP. This feature has implications in
the host-guest properties of these NPs, which we explored further (vide infra).
In order to demonstrate the utility of the facile surface functionalization capabilities,
we targeted the decoration of the NPs with functionalities that can specifically target
a sub-cellular compartment in a cell. Prior to carrying out any cellular experiments, it
is imperative that we test whether these NPs are cytotoxic, as this might preclude any
meaningful sub-cellular targeting. Accordingly, toxicity of the NPs was assessed us-
ing an MTT assay. The NPs did not exhibit any discernible cytotoxicity, even at a con-
centration of 0.2 mg/mL (Figure 4A). The data shown in Figure 4A are based on
60 nm NPs. To assess whether size variations have any effect on the cytotoxicity,
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Figure 4. Cytotoxicity and Cell Uptake of the NPs
Cytotoxicity (A–C) and cell uptake (D–F) of the NPs (size: 60 nm) in (A and D) HeLa cells, (B and E) MDA-MB-231 cells, and (C and F) MCF-7 cells. The
cytotoxicity was performed by MTT assay. The confocal images were recorded after 4 h incubation with the concentration of 30 mg/mL of NPs. The red
color represents the rhodamine-B-dye-labelled NGs. The blue color represents the nucleus after hoechst 33342 staining.
NPs of different sizes were assessed and were also found to be non-toxic (Fig-
ure S25). To even further test these NPs, the cytotoxicity of the 60 nm NPs was
also studied in two more cell lines, MDA-MB-231 and MCF-7 (Figures 4B and 4C).
Here too, the NPs were found to be quite cyto-compatible. A potential issue with
a typical cytotoxicity analysis is that a NP might be deemed non-toxic, because it
never was taken up by the cell and thus does not induce any toxicity. To test this pos-
sibility, we tested whether the NPs are taken up by the cells using rhodamine-B-
labeled NPs (Figures 4D, 4F, and S26–S28). The presence of the bright red color,
from the covalently attached rhodamine B, throughout the cells indicates that these
NPs are taken up by the cells after a short incubation time of 4 h. This feature too was
found to be true for MDA-MB231 and MCF-7 cell lines (Figures 4E and 4F) and for
NPs of different sizes (Figures S27 and S28).
To investigate the possibility of sub-cellular targeting using functionalities presented
on the surface of the polymer NPs, triphenylalkylphosphonium moieties were incorpo-
rated on the surface of the NPs, as these functionalities are known to target mitochon-
dria. Structure of the polymeric NP is shown in Figure 5. Synthetic and characterization
details of the ligand molecules and their incorporation on the surface of the NPs are
detailed in Scheme S5 and Figures S12–S17. The intracellular distribution of the NPs
and the potential mitochondrial targeting was studied using a high-resolution confocal
microscope. In this experiment, the mitochondria were stained with a green mito-
tracker dye. NP localization in the mitochondria was then assessed by evaluating the
colocalization of the green color from this dye with the red color of the rhodamine-
labeled NP. As shown in Figure 5, the triphenylalkylphosphonium-decorated NPs ex-
hibited excellent mitochondrial localization, while the unfunctionalized NP exhibited
8
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Figure 5. Mitochondria Targeting Experiment
(A) Schematic illustration of the preparation of ligand-functionalized NPs.
(B and C) The resonant scanning confocal with structured illumination super-resolution (SIMe)
images of NP uptake by HeLa cell after 4 h incubation for (B) ligand-functionalized NPs and (C)
control NPs without ligand.
no localization in the mitochondria (Figures 5B and 5C). The ability to deliver NPs to
specific sub-cellular organelle offers many opportunities for improving therapeutic
approaches through targeting.66
Non-covalent Encapsulation and Triggered Release of Guest Molecules
Stable encapsulation of the guest molecules with a triggerable release modality is the
key feature to ultimately implement these NPs in practical applications. In addition to
the surface functionalization, we also surmised that when modified with hydrophobic
amines, the interior of these NPs would be affected. To test this idea further, we tested
the non-covalent encapsulation capability of the NPs. Accordingly, we systematically
varied the hydrophobicity of the modifying functionality in the NP using hexylamine,
octylamine, decylamine, and benzylamine. The ability of these NPs to non-covalently
bind to guest molecules was assessed using Nile red as the probe. All NPs exhibited
the ability to non-covalently bind the spectroscopic probe, where the extent of
guest loading inside the NPs seemed to depend on the functional group. Interestingly,
octylamine-modified NPs exhibit an optimal ability to bind Nile red, compared to both
hexylamine and decylamine (Figure S24). These results could be rationalized by the pos-
sibility that there is a balance between the hydrophobicity of the modifying functionality
and its sterics-induced volume exclusion.
An equally important criterion for a good host, especially in the context of a drug
carrier, involves the encapsulation stability of the guest molecule in the NP. The
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Figure 6. FRET Experiment of NPs from Different Amines
(A) No amine.
(B) PEG8-amine.
(C and D) Hexylamine.
(E and F) Octylamine.
(G and H) Decylamine.
(A–C, E, and G) In the absence of DTT.
(D, F, and H) In the presence of 10 mM DTT.
The fluorescent spectra were measured by excitation wavelength at 450 nm.
encapsulation stability of the NP for hydrophobic guest molecules should directly
correlate with the hydrophobicity of the modifying amine functionality. We used
the previously developed fluorescence resonance energy transfer (FRET)-based
method67 to evaluate the encapsulation stability of the NPs. In brief, a solution con-
taining a FRET donor molecule as the guest in the NP host is mixed with a similar so-
lution with the corresponding FRET acceptor molecule as the guest. The kinetics of
FRET evolution provides direct insights into the encapsulation stability; faster FRET
evolution indicates poor encapsulation stability.
Hydrophobic cyanine dyes, DiO and DiI, were used as the FRET donor and acceptor
molecules, respectively, for this study. First, the encapsulation stability of the amphi-
philic polymer assembly, before crosslinking, was assessed as the control. The non-
crosslinked polymeric nanoaggregate showed very fast FRET increase rate with a
rapid increase of the acceptor emission at 550 nm and decrease of the donor
emission at 510 nm (Figure 6A), with a leakage coefficient of >18/h, suggesting a
leaky nanoaggregate. Next, the crosslinked NPs based on the reaction with hexyl-
amine was evaluated. Here too, there was a temporal evolution of the FRET ratio;
however, the evolution was found to be much slower with a leakage coefficient of
4.57 3 10À3/h (Figures 6C and S29). As the hydrophobicity of the amine increases
from the hexylamine to octylamine and decylamine-based NPs, the encapsulation
stability substantially increased with no discernible temporal evolution of FRET for
the latter two NPs (Figures 6E and 6G). The alkylamine-induced crosslinking reaction
can provide enhanced encapsulation stability due to two different reasons—
enhanced hydrophobicity of the interior endowed by the incorporation of the alkyl
moiety and the crosslinking of the interior that offers increased steric barrier for
molecular diffusion out of the NPs. The increased encapsulation stability with
increasing hydrophobicity of the alkyl chain suggests that the former is certainly a
factor. To test whether there is contribution from the latter, the NP crosslinking
10
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Figure 7. Serum Stability Test toward NPs Prepared from Different Amines
All the FRET experiments were performed in serum (90%).
(A and B) No amine.
(C and D) Hexylamine.
(E and F) Octylamine.
(G and H) Decylamine.
(A, C, E, and G) In the absence of DTT.
(B, D, F, and H) In the presence of 10 mM DTT.
The fluorescent spectra were measured by excitation wavelength at 450 nm.
was initiated using a hydrophilic polyethyleneglycol-amine (PEG8-amine). The resul-
tant NP exhibited better encapsulation stability, compared to the uncrosslinked con-
trol nanoaggregate, with a leakage coefficient of ꢀ4.2/h. However, this encapsula-
tion is much poorer compared to that from the hexylamine. These results clearly
show that both crosslinking-based steric barrier and hydrophobicity-based encapsu-
lation play critical roles in the encapsulation stability of these NPs.
If these NPs were to be useful, it is also essential that this stable encapsulation is
compromised in the presence of a specific biologically relevant trigger. Because
of the presence of the disulfide bonds, these are likely to be susceptible to the pres-
ence of a reducing agent such as dithiothreitol (DTT). Indeed, the FRET evolution
was found to be significantly faster in the presence of mM concentration of DTT (Fig-
ures 6D and S30). Interestingly, however, no change in FRET evolution was observed
for octylamine- and decylamine-based NPs even in the presence of DTT (Figures 6F
and 6H). The main reason is the increase in the hydrophobicity to stabilize the NPs
under pure water solution. When we use serum as the medium (vide infra), the dye
can still be released out from the NPs in the presence of DTT. Based on the results,
we conclude that crosslinking can efficiently enhance the stability of the NPs.
The studies above show that the encapsulation stability in these NPs is dependent
on the hydrophobicity of the modifying functionalities and the crosslinks in the NP
in aqueous solution. An even more rigorous study of the encapsulation stability in
the context of biological applications would involve evaluating it under serum con-
ditions. We adapted a previously reported method68 for this purpose, where the
FRET pairs are co-encapsulated in the NP. In this case, the solution already exhibits
excellent FRET (Figure S31). If the encapsulation stability is poor, the hydrophobic
guests would leave the interior of the NP and accumulate in the reservoir offered
by the serum proteins that are capable of binding to hydrophobic molecules,
Chem 5, 1–18, December 12, 2019
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Core and Surface Functionalities, Chem (2019), https://doi.org/10.1016/j.chempr.2019.09.001
Figure 8. In-Cell FRET Experiment toward Different NPs from Different Amines
(A) No amine.
(B) Hexylamine.
(C) Decylamine inside cells (HeLa cell line) after 24 h incubation.
The confocal images were recorded by excitation wavelength of 488 nm, and the images were
obtained with two different filters in the ranges of 505–525 and 585–605 nm.
such as serum albumin. Interestingly, the leakage coefficient of the uncrosslinked
nanoaggregates and the hexylamine-based NPs was found to be higher in serum
(Figure 7). Here too, the octyl- and the decyl-NPs were still found to exhibit stable
encapsulation. However, when the experiments were performed in the presence of
10 mM DTT, even the octylamine- and decylamine-based NPs exhibited a lower
FRET ratio over time, as discerned by the increase of the donor peak and decrease
of the acceptor emission peak (Figures 7F and 7H). These results also show that our
strategy can efficiently endow the particles with high encapsulation stability without
any leakage of the guest molecules under serum conditions. On the other hand,
there have been recent reports that show that micelles based on many block copol-
ymers can stably encapsulate hydrophobic molecules in water but exhibit fast
leakage under serum conditions at the time scales used in the present work.38–41
These results also highlight that encapsulation stability in water is not a sufficient
measure for evaluating NPs for biological applications such as targeted drug
delivery.
Next, we evaluated whether the observed trend in encapsulation stability trans-
lated to molecular release kinetics variations inside cells, as the cytosolic environ-
ments of most cells are highly reducing. If we were to use the encapsulated
FRET pairs, we might not be able to discern the difference between cellular uptake
12
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Core and Surface Functionalities, Chem (2019), https://doi.org/10.1016/j.chempr.2019.09.001
Figure 9. In-Cell FRET Study of Different NPs from Different Amines (No Amine, Hexylamine, and Decylamine)
The images and spectra were obtained from the spectral confocal microscope with excitation wavelength at 488 nm after 24 h incubation.
followed by molecular release and leakage of molecules outside the cells where
the hydrophobic molecules can then passively diffuse inside the cells. To circum-
vent this, the NPs were covalently labeled with rhodamine B, which can act as
the FRET acceptor for the donor dye DiO. The latter was non-covalently encapsu-
lated as the hydrophobic guest molecules within the NPs (Figure S32). The princi-
ple of this method is similar to the experiments in the serum, where there would be
a high FRET because of the concurrent presence of both the donor and the
acceptor in the NP. However, if the non-covalently encapsulated molecules were
to be released, then the decreased proximity between these molecules will sub-
stantially impact the FRET. We used the spin disk fluorescent microscope and
A1R spectral microscope to study the encapsulation stability in HeLa cells after 4
and 24 h incubation times. As the donor DiO is a green dye, we used the
488 nm laser for excitation. Two different filters were monitored for emission: the
505–525 nm channel present the DiO emission and the 585–605 nm channel pre-
sent the rhodamine B acceptor emission (Figures S33 and 8). The relative intensity
of the green channel and the red channel, which also dictates the color in the
merged channel, provides insights into the relative kinetics of molecular release
from the NPs. For the non-crosslinked nanoaggregate, there is a significant emis-
sion from the green channel and the merged image is quite green, suggesting
that the DiO guest molecule has been released from the NP. The hexylamine-
based NP shows a mixture of the green and red fluorescence in the merged image
indicating slow release of the guest molecule, while the domination of the red fluo-
rescence in the decylamine NP suggests high encapsulation stability with very little
to no molecular release in 24 h.
Chem 5, 1–18, December 12, 2019
13

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Core and Surface Functionalities, Chem (2019), https://doi.org/10.1016/j.chempr.2019.09.001
Figure 10. Time-Dependent In-Cell FRET Study of NPs (from Hexylamine)
The images and spectra were obtained from the spectral confocal microscope with excitation wavelength at 488 nm after different time incubation
(4, 27, and 51 h).
(A, E, F, C, G, and K) Spectral images.
(B, F, and J) Emission spectra from (A), (E), and (I), respectively.
(D, H, and J) Emission spectra from the selected areas in (C), (G), and (K), respectively.
To more quantitatively compare the encapsulation stability inside cells, we used
spectral confocal microscope to obtain the emission spectra inside the cells. Three
different nanocarriers (non-crosslinked nanoaggregate, hexylamine-functionalized
NPs, and decylamine functionalized NPs) were studied by cell uptake after 24 h
incubation (Figure 9). There is very little FRET in the cells (FRET ratio Ia/(Ia + Id) =
0.26) for non-crosslinked nanoaggregates, which is attributed to the possible
release of the guest molecules even prior to cellular uptake. After the hydrophobic
functionalization of the NPs with hexylamine to decylamine, the FRET ratios were
found to be much higher with values of 0.49 and 0.62.
To further assess whether there is a time-dependent intracellular release of the guest
molecules, the FRET ratio was monitored for the hexylamine-based NPs at 4, 27, and
51 h. As shown in Figure 10, the emission at ꢀ560 nm decreased, with a concurrent
increase in the emission at ꢀ510 nm. This change corresponded to the FRET ratios
0.68, 0.47, and 0.33 at 4, 27, and 51 h, respectively. Moreover, the site-selected
spectra obtained from inside the cells also showed similar results (Figures 10D,
10H, and 10L). These results clearly show that the intracellular reducing environment
of the cells, due to high glutathione concentration, can cause molecular release
14
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Core and Surface Functionalities, Chem (2019), https://doi.org/10.1016/j.chempr.2019.09.001
Figure 11. Cytotoxicity of the Drug-Loaded NPs
(A) Schematic illustration for the preparation of drug (CPT)-loaded NPs.
(B) DLS of CPT-loaded NPs.
(C) Concentration and time-dependent cytotoxicity of the drug-loaded NPs.
(D) Cytotoxicity of the free drug.
inside cells, the rate of which is correlated with the encapsulation stability obtained
in test tube experiments.
As the NPs exhibit many tunable features that are translatable inside cells, we carried
out preliminary experiments to assess the possibility of these NPs acting as a carrier
for chemotherapeutic drug molecules to be released under intracellular conditions.
We used camptothecin (CPT) because of its hydrophobicity and well-established phar-
macology. A ꢀ60 nm hexylamine-based NP was synthesized with 13% loading capacity
(1.3 mg drug/10 mg polymer) of the drug (Figures 11B and S34). Cell viabilities were
then evaluated at different concentrations of CPT-loaded NPs in three different cancer
cells (HeLa, MDA-MB-231, and MCF-7). The extent of cell death was investigated after
different times (24 h, 48 h, and 72 h). The cell death efficiency was comparable with free
drug in most cases for the NP and was even better in some cases suggesting that the NP
is capable of making the hydrophobic drug molecules more bioavailable.
Chem 5, 1–18, December 12, 2019
15

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Core and Surface Functionalities, Chem (2019), https://doi.org/10.1016/j.chempr.2019.09.001
Conclusions
A facile strategy for the preparation of NPs with tailorable properties through concur-
rent functionalization and stabilization has been demonstrated using a sequential
three-component reaction. By tuning the polymeric structure and self-assembly condi-
tions, we have shown that the non-covalent assembly and thus the resultant NP sizes
can be conveniently tuned by 2 orders of magnitude. Similarly, the functionalization
of the NPs can be used to decorate the outer shell of the NP to tune its interfacial inter-
action or to functionalize the interior to optimize its host-guest properties. The former
feature has been used to vary the surface charge of the NPs and to target specific or-
ganelles in cells. The latter option has been exploited to evaluate variations in the hy-
drophobicity that offer an optimal encapsulation stability, where the guest molecules
are robustly encapsulated in one set of conditions but are reliably released in a target
environment. Three different FRET-based methods, including an in-cell FRET experi-
ment, have been used to test the effect of core structure variations upon encapsulation
stability in aqueous solutions, in serum, and inside cells. The ability to tightly tune the
physiochemical properties of NPs has implications in a variety of applications, including
in drug delivery and diagnostics. As a preliminary demonstration, we have shown that
these NPs can encapsulate hydrophobic drug molecules and can be released in the
target intracellular environment with high fidelity.
EXPERIMENTAL PROCEDURES
Full experimental procedures are provided in the Supplemental Information.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.chempr.
2019.09.001.
ACKNOWLEDGMENTS
We thank the United States National Science Foundation (CHE-1307118) for
support.
AUTHOR CONTRIBUTIONS
B.L. and S.T. designed this project. B.L. performed the experiments. B.L. and S.T.
wrote the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: May 3, 2019
Revised: June 25, 2019
Accepted: September 10, 2019
Published: October 14, 2019
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