Light-responsive polymersomes with a charge-switch for targeted drug delivery

Article abstract of DOI:10.1039/c9tb02411e

Unlike the traditional block amphiphilic polymersomes, we herein report a lipid-like amphiphilic polymer that self-Assembles into photo-responsive polymersomes for drug delivery. The introduction of a quaternary ammonium moiety not only provides a hydrophilic segment of the polymersomes, but also enables electrostatic adsorption with folic acid, thus achieving the targeting of cancer cells with overexpression of folate receptor. Upon light irradiation, the photocleavage reaction of the o-nitrobenzyl moiety disintegrates polymersomes by changing the polymer structure from cationic amphiphilic state to zwitterionic hydrophilic state, thus realizing photo-Triggered drug release. The data showed that anticancer drugs (doxorubicin hydrochloride, DOX·HCl) can be loaded into the hydrophilic cavity of polymersomes and controllably released by photo-induced disintegration of polymersomes. Cellular assay showed that the active targeting of folic acid and photo-Triggered release endowed the DOX-loaded polymersomes with a higher cytotoxicity to HeLa cells. Such cationic polymersomes provide a novel strategy for designing effective and intelligent drug carriers, and have potential application as a novel integrated platform for targeted drug delivery.

Full text of DOI:10.1039/c9tb02411e

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Materials Chemistry B
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Light-responsive polymersomes with a
charge-switch for targeted drug delivery†
Cite this:DOI: 10.1039/c9tb02411e
Yaowu Zhou, Rongrong Chen, Huiting Yang, Chunyan Bao, * Jinyan Fan,
Chenxi Wang, Qiuning Lin and Linyong Zhu
*
Unlike the traditional block amphiphilic polymersomes, we herein report a lipid-like amphiphilic polymer
that self-assembles into photo-responsive polymersomes for drug delivery. The introduction of a
quaternary ammonium moiety not only provides a hydrophilic segment of the polymersomes, but also
enables electrostatic adsorption with folic acid, thus achieving the targeting of cancer cells with
overexpression of folate receptor. Upon light irradiation, the photocleavage reaction of the o-nitrobenzyl
moiety disintegrates polymersomes by changing the polymer structure from cationic amphiphilic state to
zwitterionic hydrophilic state, thus realizing photo-triggered drug release. The data showed that anticancer
drugs (doxorubicin hydrochloride, DOXÁHCl) can be loaded into the hydrophilic cavity of polymersomes and
controllably released by photo-induced disintegration of polymersomes. Cellular assay showed that the
active targeting of folic acid and photo-triggered release endowed the DOX-loaded polymersomes with a
higher cytotoxicity to HeLa cells. Such cationic polymersomes provide a novel strategy for designing
effective and intelligent drug carriers, and have potential application as a novel integrated platform for
targeted drug delivery.
Received 30th October 2019,
Accepted 13th December 2019
DOI: 10.1039/c9tb02411e
rsc.li/materials-b
Although light stimuli can potentially control drug release in
specific space, active targeting often plays a more important
Introduction
Polymersomes, which are also referred to as polymeric vesicles, role in improving drug therapeutic efficacy and reducing toxic
have attracted growing interest because of their potential and side effects on normal tissues.14–16 For example, polymer-
applications in a broad range of biomedical areas including as somes modified with targeting folic acid (FA) demonstrate an
microreactors,1 drug carriers,2,3 and mimics of protocells.4 increased cellular uptake to cancer cells that overexpressed
Compared with micelles, liposome-like polymersomes show folate receptors, thereby improving therapeutic efficacy.8,17,18
superiorities when serving as drug carriers, providing a hydro- Therefore, the integration of active targeting and photo-
philic cavity for the encapsulation of water-soluble molecules regulated drug release can promote the therapeutic efficacy of
and hydrophobic membranes for loading oil-soluble species. polymersomes by elevating cellular uptake and the subsequent
Although polymersomes are considered more stable than lipo- light release.
somes, the conventional polymersomes lack sufficient permeability
and block the effective release of drug.
So far, the combination of active targeting and photo-
triggered drug release has been successfully applied to silica
Stimuli-responsive polymersomes in response to external nanoparticles19 and micelles;20,21 however, the same methods
(light,5 temperature,6 and magnetic effect7) or internal stimuli have rarely been achieved for polymersomes due to their
(pH,8 redox potential,9 and lysosomal enzymes10) provide a way complex structural design. In this work, we envisaged that a
to deal with this issue effectively. Among them, light-induced positively charged and light-responsive polymersome can serve
release is a particularly attractive option with remarkable as an effective targeted delivery platform for cancer cells (Scheme 1).
spatial and temporal resolution. In terms of light response, Accordingly, we synthesized an amphiphilic monomer, C12NB, in
drug-loaded polymersomes typically undergo structure disruption, which a cationic ammonium salt provides the hydrophilic segment
thus releasing the encapsulated drug on demand.5,11–13 and a didodecyl substituted o-nitrobenzyl derivative provides
hydrophobic and light-responsive functions. Upon mild UV
Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key
exposure (365 nm), photocleavage of an o-nitrobenzyl ester
moiety into 3,4-dimethoxy-6-nitrosoacetophenone and acid-
Laboratory of Bioreactor Engineering, East China University of Science and
Technology, 130# Meilong Road, Shanghai 200237, China.
terminated by-products permits a cleavage between hydrophilic
E-mail: baochunyan@ecust.edu.cn, linyongzhu@ecust.edu.cn
and hydrophobic segments.22–28 After polymerization (PC12NB),
† Electronic supplementary information (ESI) available: Synthesis procedures
and characterization of compounds 1–4. See DOI: 10.1039/c9tb02411e
the obtained amphiphilic polymer self-assembled into
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58.7, 52.33, 39.99, 31.8, 29.29, 26.04, 22.57, 21.76, 18.28, 14.37.
+
HRMS (ESI): m/z: calcd for C42H73N2O8 [M]: 733.5361. Found:
733.5366.
Polymerization of C12NB (PC12NB). Typically, monomer
C
12NB (1.1 g, 1.5 mmol) and AIBN (4 mg, 0.024 mmol) were
charged into a glass ampoule containing 3 mL of DMF. The
ampoule was then degassed via three freeze–pump–thaw cycles
and sealed under argon gas. It was then immersed into an oil
bath and heated at 75 1C to start the polymerization. After 48 h,
the reaction was quenched by putting the ampoule into liquid
nitrogen to terminate the polymerization and the solvent was
removed under vacuum. The residue was suspended in 1 mL of
DCM and the mixture was precipitated into an excess of
acetonitrile, and the above dissolution–precipitation cycle was
repeated three times. The final product was dried in a vacuum
oven overnight at room temperature, yielding the target poly-
mer PC12NB as a pale yellow solid (0.45 g, yield: 40.9%).
Photolysis of photo-responsive monomer C12NB. Photolysis
analysis of C12NB was performed as follows: C12NB (2.6 Â
10À4 mol mLÀ1) was dissolved in methanol–water solution
(9/1, v/v) using a quartz cuvette. Then, the homogeneous
solution was irradiated with a 365 nm light-emitting diode
(LED) light source (10 mW cmÀ2), and at specific time intervals,
the solution was analyzed by UV absorbance and high-
performance liquid chromatography (HPLC) spectra. Further-
more, liquid chromatography-mass spectrometry (LC-MS) spectra
were used to assign the components. For HPLC, the column was
eluted with a mixture of 90% MeOH and 10% water at a flow rate
of 1 mL minÀ1, and the chromatogram was obtained using
absorbance analysis at a wavelength of 295 nm.
Scheme 1 Schematics for (a) the self-assembly of polymersomes from
the amphiphilic polymer PC12NB and (b) the targeted and light-responsive
drug delivery to cancer cells.
polymersomes in aqueous solution, which could simultaneously
load hydrophobic and hydrophilic molecules and release them
upon light irradiation. At the same time, the positively charged
surface was conducive to FA adsorption, enabling the polymer-
somes to target cancer cells with overexpressed FA receptor
protein on the membrane surface.
Preparation of self-assembled polymersomes. A PC12NB THF
solution (10 mg mLÀ1, 1 mL) was added dropwise to 10 mL
of deionized water under vigorous stirring, which was kept
stirring for another 3 h in the dark. Thereafter, THF was
removed under reduced pressure and polymersomes were
collected through three ultrafiltration/dispersion cycles (Millipore,
MWCO 100 kDa), then redispersed in water (1 mL) and stored in
the dark at 4 1C.
Experimental section
Synthesis of compounds
Synthesis procedures and characterization of compounds 1–4
are detailed in the ESI.†
Determination of critical vesicle concentration (CVC). In
addition to adding 20 mL of Nile red stock solution (1 mM in
Compound C12NB. To a solution of compound 4 (3 g, THF), the polymersomes encapsulated with Nile red (NR) were
3.05 mmol) in acetone (40 mL), dimethylaminoethyl methacrylate prepared as described above. To calculate the CVC, the Nile
(0.58 g, 3.66 mmol) was added. The reaction mixture was stirred red encapsulated PC12NB suspension was diluted to a series
and refluxed for 12 h. Then, the mixture was cooled to room of specific concentrations. The corresponding fluorescence emission
temperature and the solvent was removed under vacuum. The spectra were recorded with an excitation wavelength of 540 nm.
residue was purified by recrystallization from DCM/Et2O. This Then, fluorescence intensity at 605 nm as a function of log 10 of
process was repeated three times and the product was obtained as polymer concentration was plotted and CVC was determined at the
1
a white solid (1.27 g, 56.6% yield). H NMR (400 MHz, DMSO) intersection point of the two linear fittings of the curve.
d 7.57 (s, 1H), 7.08 (s, 1H), 6.34 (q, J = 6.4 Hz, 1H), 6.02 (s, 1H),
Photolysis of self-assembled polymersomes. The colloidal
5.69 (s, 1H), 4.83 (d, J = 17.0 Hz, 1H), 4.70 (d, J = 16.9 Hz, 1H), 4.53 solution (PC12NB, 0.634 mg mLÀ1) was added to a quartz
(t, J = 4.4 Hz, 2H), 4.26–4.10 (m, 2H), 4.09–4.01 (m, 2H), 3.94 (t, J = cuvette and irradiated with a 365 nm LED (10 mW cmÀ2).
10.0 Hz, 2H), 3.30 (d, J = 6.0 Hz, 6H), 1.86 (s, 3H), 1.81–1.66 Between certain specific time intervals, the irradiated solution
(m, 4H), 1.63 (d, J = 6.4 Hz, 3H), 1.41 (dd, J = 22.4, 6.2 Hz, 4H), 1.27 was analysed by UV absorbance. Meanwhile, the particle
(d, J = 31.9 Hz, 32H), 0.84 (t, J = 6.6 Hz, 6H). 13C NMR (100 MHz, sizes and zeta potentials of polymersomes were measured by
CDCl3) d (ppm): 166.24, 164.58, 153.86, 148.10, 139.85, 135.77, dynamic light scattering (DLS). The morphology was observed
130.87, 126.87, 109.99, 109.14, 70.19, 69.50, 69.27, 63.06, 61.55, via TEM.
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NR loading and release. The NR-loaded polymersomes were the dark. After 30 min of vigorous stirring at room temperature,
prepared as described in the CVC analysis and the amount of the resulting particles (designated as PC12NB + FA and PC12NB +
NR added was 2 mg. To determine the drug loading content DOX + FA) were collected by three ultrafiltration/dispersion
(DLC), the NR-loaded PC12NB suspension was lyophilized and cycles (Millipore, MWCO 100 kDa), and then the polymersomes
then dispersed in DCM. DLC was determined by the UV were redispersed in deionized water (1 mL), and kept in the
absorbance of NR at 540 nm against the standard calibration dark at 4 1C.
curve, and calculated according to the following equations:
DLC% = [Wloaded NR]/[WNR-loaded polymersomes] Â 100%.
Results and discussion
Synthesis and photo-responsive characterization of C12NB
For photoirradiation mediated NR release, 3 mL of aqueous
NR-loaded polymersome dispersion (containing PC12NB 1 mg mLÀ1
)
In order to form a vesicular self-assembly, the design of C12NB
was inspired by the structures of amphiphilic lipids, in which
two hydrophobic alkyl chains and a hydrophilic ammonium
group were connected by a photolabile o-nitrobenzyl moiety
(as illustrated in Scheme 1). In addition, a methacrylate group
was introduced for further polymerization. After polymerization,
the amphiphilic and lipid-like structure of C12NB would facilitate
the formation of polymersomes for the polymer PC12NB. Upon
irradiation, the photocleavage reaction of the o-nitrobenzyl moiety
would cleave the hydrophobic tails and transfer the cationic
structures into zwitterionic structures, thus dissociating the polymer-
somes. The predicted structure and light-response would endow the
polymersomes with superiority as excellent carriers for drug delivery
systems. As shown in Fig. 1a, C12NB was synthesized from
3,4-dihydroxyacetophenone in five steps by (1) the dialkylation
of phenolic hydroxyl groups;29 (2) the nitration by conc. HNO3
under the catalysis of acetic anhydride;30 (3) the ketone reduc-
tion by NaBH4; (4) the esterification reaction; and (5) the
quaternarization reaction of the methacrylate functional group.
All compounds were confirmed by both NMR and mass spectra
(Fig. S1–S5, ESI†). The photolysis process of C12NB (Fig. 1b)
was detected by the evolution of UV-vis absorption spectra.
As shown in Fig. 1c, dramatic changes appeared for the
absorption of C12NB upon light irradiation, in which the
absorbance at 265 nm gradually increased, the absorbance at
348 nm increased and redshifted to 380 nm, and three isobestic
points generated at 320 nm, 290 nm, and 250 nm, respectively.
HPLC and LC-mass spectra were further used to identify the
photolysis products. As illustrated in Fig. 1d, light irradiation
resulted in the consumption of C12NB as well as the generation
of the ketone derivative 5 (Fig. S6, ESI†). All these results
indicated an efficient and relatively clean photolysis for
was irradiated with a 365 nm LED (10 mW cmÀ2) for varying
durations. The fluorescence intensities at 605 nm (lex = 540 nm)
were monitored. Moreover, the polymersomes without irradiation
were also analysed for comparison.
Loading and release of calcein and DOXÁHCl. Calcein (5 mg)
or DOXÁHCl (50 mg) was dissolved in deionized water (10 mL)
under magnetic stirring. Subsequently, 10 mg of PC12NB in
1 mL of THF was slowly added to the above aqueous dispersion
at a rate of 1 mL hÀ1 by a syringe pump. The mixed solution
was allowed to equilibrate in the dark for 3 h. Then, non-
encapsulated molecules were removed by three ultrafiltration/
dispersion cycles (Millipore, MWCO 100 kDa) and the polymer-
somes were redispersed in deionized water, and kept in the
dark at 4 1C. DLC was determined by the same method used for
the NR-loaded polymersomes. For photoirradiation-mediated
release, 5 mL of calcein or DOXÁHCl polymersome dispersions
(containing PC12NB 1 mg mLÀ1) was irradiated. Between certain
time intervals, aliquots of dispersions (0.5 mL) were taken out for
analysis. After the removal of the polymersomes by centrifugation,
the released amount of drugs was quantified by measuring the
fluorescence intensities at 510 nm (lex = 450 nm) for calcein or
595 nm (lex = 480 nm) for DOXÁHCl in the filtrates against a
standard calibration curve.
In vitro cytotoxicity assay. CCK-8 assay was used to evaluate
in vitro cytotoxicity of HeLa cells. HeLa cells were first cultured
in Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), penicillin (100 units mLÀ1),
and streptomycin (100 mg mLÀ1) at 37 1C in a 5% CO2 incubator
for 3 days. For cytotoxicity assay, HeLa cells were seeded in a
96-well plate at an initial density of 5000 cells per well in 200 mL
of complete DMEM medium. After incubating for 24 h, DMEM
was replaced with fresh medium, and the cells were treated
with samples at varying concentrations. For irradiation operation,
C
12NB upon light irradiation, and provided the premise for
the photo-triggered disassembly of polymersomes.
the cells were irradiated for 1 min (365 nm LED light, 5 mW cmÀ2
the safe dose without cytotoxicity) after the coculture for 2 h. After
,
Preparation and characterization of polymersomes
the coculture for 12 h and 48 h, the medium in each well was By free radical polymerization, PC12NB was prepared by using
removed and replaced by 100 mL of fresh DMEM. Then, CCK-8 AIBN as the photoinitiator in DMF. GPC analysis revealed a Mn
reagent (10 mL) was added to each well and the cells were further of 3294 and a Mw/Mn of 1.22, relative to linear polystyrene
incubated for 3 h at 37 1C. Finally, the absorbance at 450 nm was standards. Polymersomes were prepared by dispersing the THF
recorded by a microplate reader, and the data were averaged from solution of PC12NB into water (Fig. 2a) and the vesicular
at least quadruple trials.
structure was verified by TEM images with diameters ranging
Electrostatic adsorption of folic acid (FA) on polymersomes. from 80 to 150 nm, which was in good agreement with the DLS
In brief, 4 mL of aqueous FA (0.735 mg mLÀ1) was dropwise data (inset in Fig. 2b). The CVC was estimated using hydro-
added into the PC12NB colloidal solution (8 mL, 1 mg mLÀ1) in phobic NR as
a
fluorescent probe,31 in which the NR
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Fig. 1 Synthesis and characterization of the photo-responsive monomer C12NB. Schematic of the (a) synthesis procedure and (b) the photolysis reaction
of C12NB. The evolution of (c) UV-vis and (d) HPLC spectra of C12NB upon 365 nm LED light irradiation (10 mW cmÀ2) for varying durations; the sample
concentration was 2.6 Â 10À4 mol LÀ1 in methanol.
encapsulated polymersomes were diluted and the corres- as expected. TEM investigation (inset in Fig. 2e) further confirmed
ponding fluorescence emission spectra were recorded. Fig. 2c that the irradiation dissociated the polymersomes into an
shows the NR fluorescence intensity at the emission maximum amorphous morphology.
(lmax = 605 nm) versus the polymer concentrations. The CVC
of PC12NB was calculated as 50 mg mLÀ1 by using the antilog
Drug loading and photo-triggered release
of the intersection point value of the baseline and the steep To explore the potential application as a photo-responsive drug
curve. The relatively low CVC value indicated the stability of the delivery vehicle, both hydrophobic (NR) and hydrophilic
polymersomes.
(calcein) model drugs were selected to evaluate the drug payloads
Subsequently, a series of methods was employed to examine and the photo-triggered release of polymersomes (Fig. 3a). Based
the photo-triggered performance of the polymersomes. Unlike on the fluorescence spectra, the drug loading content was calcu-
the photolysis of C12NB monomer, the irradiation of polymer- lated as 0.23% for hydrophobic NR and 1.65% for hydrophilic
somes exhibited a slight decrease and redshift at 355 nm calcein. Fig. 3b shows the NR release profile under varying photo-
(Fig. 2d), which should be attributed to the self-assembled irradiation durations; the longer the irradiation time, the greater
structure of the polymersomes. The variation of the absorption the cumulative release, whereas only negligible release was
showed the maximum after 240 s of irradiation, suggesting that exhibited from the polymersomes kept in the dark. The color
the photolysis of the o-nitrobenzyl group in the polymersomes change from pink to yellow further confirmed the efficient
should be completed efficiently. In order to confirm whether release of NR from the polymersomes (inset in Fig. 3b).
photolysis induces the surface charge of polymersomes to Fig. 3c exhibits the precise control of NR release under alter-
change from cationic to zwitterionic state, the change in nating light and dark conditions; the stepped profile revealed
z-potential values of polymersomes as a function of the irradiation that NR release only proceeded under light irradiation. Fig. 3d
time was recorded. As illustrated in Fig. 2e, light irradiation and e exhibit the cumulative calcein release over irradiation
reduced the z-potential of polymersomes from +52 mV to +6 mV time, and almost 75% calcein was released after 240 s of
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Fig. 2 Synthesis, self-assembly and characterization of the photo-responsive poymersome PC12NB. (a) Schematic of self-assembly and photo-induced
disassembly. (b) Typical TEM image of polymersomes, and the inset represents the hydrodynamic radius distribution of polymersomes determined by DLS
assay. (c) CVC test of PC12NB: the relative fluorescence intensity at 605 nm of NR as a function against concentration. The evolution of (d) UV-vis spectra
and (e) zeta potential of PC12NB polymersomes in water upon 365 nm LED light irradiation (10 mW cmÀ2). The inset in (e) represents the TEM image of
disintegrated polymersomes upon 3 min light irradiation.
irradiation. These exciting phenomena result due to the stored under dark conditions, whereas light irradiation resulted
disintegration of polymersomes by the photocleavage of hydro- in steady rises of the fluorescence in dialysate as a function of
phobic o-nitrobenzyl groups in the membrane and the subse- irradiation time, indicating the light-triggered release of DOXÁHCl
quent formation of zwitterionic and hydrophilic polymers.
via the disintegration of the polymersomes.
Cationic nanoparticles may induce toxic effects to cells by
disrupting the plasma membrane.34 Hence, the cytotoxicity of
DOXÁHCl loading, release and cytotoxicity studies
As is well-known, doxorubicin hydrochloride (DOXÁHCl) is a the polymersomes was investigated using CCK-8 assay. The
widely used chemotherapeutic drug for the treatment of several cytotoxicity assay revealed that both the polymersomes (PC12NB) and
types of cancers; however, the clinic applications were limited irradiated polymersomes (PC12NB + hn, post-irradiation for 1 min)
because of its severe side effects. To reduce the fast clearance have no significant cytotoxic effect on HeLa cells after 12 h and 48 h
from the blood system and the severe systemic toxicity, smart of incubation up to a concentration of 333 mg mLÀ1, and the cell
drug delivery systems are always required for DOXÁHCl release.32,33 viability was higher than 90% (Fig. 5a). This indicated that the
Herein, the encapsulation of DOXÁHCl in polymersomes was the polymersomes from PC12NB can be used as an excellent carrier for
same as that of calcein, and the drug loading content was drug delivery. In addition, the cytotoxicity of the DOXÁHCl encap-
calculated as 1.5%. As illustrated in Fig. 4a and b, less than 15% sulated polymersomes on HeLa cells after 12 h and 48 h of
DOX was released spontaneously when the polymersomes were incubation was investigated and compared with the cytotoxicity
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Fig. 3 Loading and photo-triggered release of model molecules from polymersomes. (a) Schematic representation of photo-triggered release of
hydrophobic NR and hydrophilic calcein from polymersomes. (b) Release profile and (c) partial progress of NR from hydrophobic bilayers upon light
irradiation and in the dark control. The times mean the cumulative irradiation time. Evolution of (d) fluorescent spectra and (e) the corresponding release
profile of calcein (determined by the relative fluorescence intensity at 510 nm) from the aqueous interiors of polymersomes upon light irradiation.
A 365 nm LED light (10 mW cmÀ2) was used for irradiation.
of the free DOXÁHCl. After 12 h of incubation (Fig. 5a), PC12NB + loaded with PC12NB + DOX polymersomes showed clearly visible
DOX decreased the cell viability gradually with increasing concen- red fluorescence in the cells; however, the cells loaded with free
tration of encapsulated DOXÁHCl, and exhibited lower cell viability DOXÁHCl showed much lower fluorescence, indicating the
(IC50 E 1.85 mg mLÀ1, the content of DOX) than the free DOXÁHCl efficient cellular uptake of PC12NB + DOX polymersomes. Notice-
(IC50 E 2.5 mg mLÀ1). This should be due to the passive targeting of ably, upon cellular uptake and UV irradiation for 1 min followed by
the cationic polymersomes, which helps the drug-loaded poly- 10 h of incubation (total 12 h incubation), it was found that the
mersomes to be enriched in the cells. This enrichment can be polymersomes (PC12NB + DOX + hn) exhibited a dose-response with
verified by the cellular uptake assay since the red fluorescence of further improved cellular cytotoxicity (IC50 E 1.25 mg mLÀ1, 2-fold
the DOXÁHCl enabled the visualization of the cells. Fig. 5b shows enhanced cytotoxicity to free DOXÁHCl). Here, the irradiation
the representative confocal images of HeLa cells treated with condition (5 mW cmÀ2 for 1 min) has been shown to be safe for
PC12NB + DOX and DOXÁHCl. After 2 h of incubation, the cells the cells. This confirmed that the phototriggered drug release
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Fig. 4 Release assay of DOXÁHCl from irradiated polymersomes.
(a) Evolution of fluorescent spectra and (b) the release profile upon light
irradiation and in the dark control. The data in (b) were determined by the
relative fluorescence intensity at 590 nm of DOX. A 365 nm LED light
(10 mW cmÀ2) was used for irradiation.
further enhanced cytotoxicity of the drug-loaded polymersomes.
After 48 h of culture (Fig. 5a), no significant difference in the
viability of HeLa cells can be observed when cultured with PC12NB
+ DOX polymersomes, PC12NB + DOX + hn polymersomes and
DOXÁHCl. This was reasonable since all encapsulated and free
drugs were retained in the culture system, and the drug concen-
tration would reach the same level after a long period of culture. In
view of the fact that drugs are easily metabolized and cleared
in vivo, it is of great significance to enrich and enhance toxicity of
drugs in the early stage as our polymersomes do.
Fig. 5 Cytotoxicity assay of HeLa cells. (a) Cell viability determined by
CCK-8 assay at 12 h and 48 h. (b) The confocal images of HeLa cells
after incubation with free DOX and PC12NB + DOX nanoparticles for 2 h.
The scale bars are 50 mm.
loading exhibited excellent cytocompatibility with cell viability
higher than 90% after 12 h of incubation. And cell viability
decreased to 37% for PC12NB + DOX and 27% for PC12NB +
DOX + hn polymersomes, respectively. Both of them showed
FA-induced active targeting to HeLa cells
Although the nanosized and positively charged polymersomes
exhibited passive targeting to cells, ligand-induced active targeting
is believed to play a greater role in targeting cancer therapy.35,36
Thanks to the positive charge character, PC12NB polymersomes
are expected to adsorb FA due to the electrostatic effect. DLS data
(not shown) exhibited that the particle size changed little with the
addition of FA to the polymersomes, while the z-potential value
changed from +52 mV to +4.5 mV, verifying the FA adsorption to
the polymersomes. To evaluate the active targeting effect of FA,
cell viability of HeLa cells was evaluated and compared under
comparable concentrations (2.5 mg mLÀ1, the content of DOX). As
illustrated in Fig. 6, all the polymersomes (PC12NB, PC12NB + hn,
and FA adsorbed polymersomes PC12NB + FA) without drug
Fig. 6 Viability of HeLa cells after 12 h of incubation with samples at compar-
able concentrations (polymersomes: 167.0 mg mLÀ1, DOX: 2.5 mg mLÀ1).
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cytotoxicity by promoting cellular uptake via a receptor-mediated
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Conclusions
In summary, we fabricated an amphiphilic cationic polymer,
which can self-assemble into polymersomes to encapsulate
drugs, regulate drug release upon photo-irradiation and adsorb
targeted ligands to enhance cytotoxicity. The polymersomes
were verified to encapsulate a hydrophobic model drug (NR)
into the membrane, and a hydrophilic model drug (calcein) and
an anticancer drug (DOXÁHCl) in the aqueous cavities. And
the involved photocleavage reaction of o-nitrobenzyl groups
switched the polymersomes from amphiphilic cationic state
to hydrophilic zwitterionic state, thus dissociating the polymer-
somes and releasing the encapsulated drugs. Cytotoxicity
studies revealed that DOXÁHCl encapsulated polymersomes
subjected to light irradiation exhibited higher cytotoxicity than
the free DOXÁHCl due to the efficient cell uptake and photo-
triggered drug release, while the blank polymersomes showed
no cytotoxicity effect even at 333 mg mLÀ1. In addition, the FA
adsorption further improved cytotoxicity by the active targeting
action. Overall, the reported photo-responsive and cancer cell-
targeting polymersomes performed well showing their promising
application in a drug delivery system, which represents a potent
strategy for the efficient treatment of cancers.
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
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This work is supported by the NSFC (Grant No. 21472044,
21425311), the National Key Research and Development Project
(2019YFA0110500) and the Open Project of State Key Laboratory
for Supramolecular Structure and Materials (SKLSSM 201901).
The authors thank the Research Centre of Analysis and Test of
East China University of Science and Technology for its help
with the characterization.
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