Photo- and Reduction-Responsive Polymersomes for Programmed

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Photo- and Reduction-Responsive Polymersomes for Programmed Release of Small and Macromolecular Payloads Ziqiang Sun, Guhuan Liu, Jinming Hu, and Shiyong Liu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00253 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

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Biomacromolecules

Photo- and Reduction-Responsive Polymersomes for Programmed Release of Small and Macromolecular Payloads Ziqiang Sun, Guhuan Liu, Jinming Hu,* and Shiyong Liu*

CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, iChEM (Collaborative Innovation Center of Chemistry for Energy Materials), Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China

ABSTRACT. We report the preparation of photo- and reduction-responsive diblock copolymers through reversible addition fragmentation chain transfer (RAFT) polymerization of a coumarin-based disulfide-containing monomer (i.e., CSSMA) using a poly(ethylene oxide) (PEO)-based macroRAFT agent. The resulting amphiphilic PEO-b-PCSSMA copolymers self-assembled into polymersomes with hydrophilic PEO shielding coronas and hydrophobic bilayer membranes. Upon irradiating the polymersomes with visible light (e.g., 430 nm), the coumarin moieties within the bilayer membranes were cleaved with the generation of primary amine groups, which spontaneously underwent inter/intrachain amidation reactions with the ester moieties, thereby tracelessly cross-linking and permeating the bilayer membranes. Notably, this process only gave rise to the release of small molecule payloads (e.g., doxorubicin hydrochloride, DOX) while large molecule encapsulants (e.g., 1

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Texas red-labeled dextran, TR-dextran) were retained within the cross-linked polymersomes due to the preservation of the integrity of the vesicular nanostructures. However, the cross-linked polymersomes were further disintegrated upon incubation with glutathione (GSH) due to the scission of disulfide linkages, resulting in the release of large molecule payloads. The dual-stimuli responsive polymersomes with tracelessly cross-linkable characteristics enable sequential release of payloads with spatiotemporal precision, which could be of promising applications in synergistic loading and programmed release of therapeutics.

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INTRODUCTION Chemotherapy, in addition to surgery and radiation therapy, has emerged as an important tool to treat severe diseases (e.g., cancer). Delivering therapeutics by taking advantage

of

nanocarriers

can

efficiently

optimize

the

pharmacokinetic

and

pharmacodynamic behavior of conventional small molecule drugs, exhibiting improved therapeutic performance.1-2 In this regard, much effort has been devoted to exploiting new drug carriers to precisely deliver therapeutics to pathological tissues and alleviate the systematic toxicities.3-4 Of these, polymersomes composed of hydrophilic aqueous interiors and hydrophobic bilayer membranes stand out as a category of appealing nanocarriers.5-12 The coexistence of both hydrophilic and hydrophobic domains within polymersomes allows for synergistic loading of small and large molecular therapeutics with distinct physicochemical properties.13-16 However, albeit more stable than liposomes, the bilayer membranes of conventional polymersomes lacked sufficient permeability, which remarkably limited their applications as drug carriers.17-18 To resolve this issue, a pyramid of strategies have been adopted to enhance the permeability of polymersomes such as the installation of channel proteins19 and the introduction of stimuli-responsive moieties20-25 within the hydrophobic bilayer membranes. In this context, stimuli-responsive polymersomes capable of responding to temperature,26-27 pH,28-30 light irradiation,31-33 hypoxia,34-35 and redox milieu36-39 and so on40 have been fabricated and engineered as drug containers. Although controlled release of encapsulated payloads could be achieved under specific stimuli, these polymersome-based nanocarriers 3



typically underwent structural disruption with concomitant release of the payloads at the same time. Thus, it is rather challenging to accomplish the sequential release of payloads from conventional polymersomes. Note that sequential release is highly desirable in some circumstances to maximize the therapeutic benefits. For example, polymeric carriers have been used for sequential release of vascular endothelial growth factor and platelet-derived growth factors for more efficient vessel recovery.41 To achieve sequential release of payloads from polymersome carriers, much attention has been paid to construct multicompartmental polymersomes.42 The innate spatial segregation within multicompartmental systems enables the stepwise release of diverse payloads. For instances, Lecommandoux and coworkers43-45 fabricated a number of multicompartment systems such as liposomes in polymersomes and polymersomes in polymersomes. They ingeniously switched the permeability of the inner components by temperature variations or other stimuli to achieve sequential release. Weitz et al.46 fabricated multiple polymersomes using a microfluidic approach and sequential disassociation of these polymersomes could be implemented by incorporation of hydrophobic homopolymers. In addition, Tsukruk and coworkers47 fabricated multicompartmental microcapsules instead of polymersomes from pH- and temperature-responsive star copolymers through a layer-by-layer technique. Sequential release of dextran (macromolecule) and Nile red (small molecule) could be achieved by tuning the solution temperature and pH. However, the fabrication procedures towards multicompartmental platforms were relatively tedious.

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Biomacromolecules

Moreover, these multicompartmental systems typically had rather large sizes (> 1 µm), remarkably limiting their drug delivery applications. Recently, we found amphiphiles containing caged carbamate linkages could self-assemble into polymersomes.31, 37, 48-49 Under specific stimuli (e.g., photoirradiation and hydrogen peroxide) the masking groups were removed and the carbamate moieties were transformed into highly reactive primary amine groups with simultaneous release of carbon dioxide (CO2). The in situ generated primary amine groups then facilitated intra/interchain amidation reactions through aminolysis of the ester bonds in the backbones, cross-linking and permeating the initially hydrophobic bilayer membranes. Similar to that of the Staudinger ligation that enables the formation of native amide bonds without the incorporation of unnatural phosphine oxide precursors,50 the current cross-linking process with the formation of amide bonds was thus coined as “traceless cross-linking”. This traceless cross-linking strategy rendered these polymersomes promising in the applications of controlled drug delivery and nanoreactors. Notably, the formation of cross-linked polymersomes only led to the release of small molecule payloads but encapsulants with large molecular weights would be retained. We reasoned that if other stimuli-responsive labile bonds could be incorporated into the design of tracelessly cross-linked polymersomes, the cross-linked polymersomes could be further disintegrated and the retained large molecule payloads could then be released. Therefore, sequential released of distinct payloads could be achieved in a conventional polymersome system without recourse to multicompartmental polymersomes.

5



Herein,

we

fabricated

dual-stimuli

responsive

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amphiphiles

comprising

reduction-responsive disulfide linkages36, 51 and photo-sensitive coumarin moieties52-53 in the hydrophobic blocks. The amphiphiles could self-assemble into vesicles capable of synergistically loading both small and large molecule payloads in the aqueous lumens. Note that photo stimulus could be operated in a non-invasive and spatiotemporally controlled manner and the irradiation wavelengths could be readily tuned while GSH is abundant within cellular cytosol, rendering it possible to regulate the responsive properties of as-assembled polymersomes within living cells. Upon photoirradiation, the hydrophobic coumarin moieties were deprotected with the generation of primary amines groups and the initially hydrophobic ester bonds in the backbones were subjected to aminolysis reactions with the formation of relatively hydrophilic amide bonds. These synergistic effects resulted in enhanced permeability of the bilayer membranes and the release of encapsulated doxorubicin hydrochloride

(DOX).

However,

the

macromolecular

drug

model

(i.e.,

Texas

red-labeled-dextran, TR-dextran) was retained within the cross-linked polymersomes. Upon further incubating the pre-irradiated polymersomes with glutathione (GSH), an abundant reducing agent within the cytosols of cancer cells, the cross-linked polymersomes were further disrupted due to the scission of disulfide linkages with the release of TR-dextran (Scheme 1). The tracelessly cross-linkable polymersomes may be of potential use in the treatment of diseases where the synergistic delivery and sequential dosage of distinct therapeutics is needed.

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Biomacromolecules

Scheme 1. Schematic illustration for the fabrication of photo- and reduction-responsive polymersomes for sequential release of small and large molecule payloads. Upon irradiation with visible light (e.g., 430 nm), the photo-sensitive coumarin moieties are cleaved with the generation of highly reactive primary amine groups, which primarily undergo (1) protonation, (2) intramolecular acyl migration, and (3-4) intra/interchain amidation reactions and the interchain amidation reactions thus crosslink the bilayer membranes (I). The traceless cross-linking process with a drastically enhanced permeability within the bilayer membranes leads to the selective release of small molecule payloads. A further addition of glutathione (GSH) results in the disruption of the polymersomes due to the scission of reduction-responsive disulfide linkages (II) and subsequent release of large molecule encapsulants.

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EXPERIMENTAL SECTION Materials. 2-Mercaptoethylammonium chloride, 2,2ʹ-dithiodiethanol, and trifluoroacetic acid

(TFA)

were

purchased

from

Energy

Chemical

Co.,

Ltd.

4-Methyl-7-diethylaminocoumarin was purchased from TCI (Shanghai) Development Co., Ltd. Methacryloyl chloride was vacuum distilled prior to use. Triethylamine (TEA) was dried over CaH2 and distilled before use. All other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. and used as received unless otherwise stated. Organic solvents (e.g., tetrahydrofuran and dichloromethane) were purified through a Pure-SolvTM 400 Solvent Purification

System.

BOC-NH2-SS-OH,54

coumarin-imidazole,55

and

PEO-based

macroRAFT agent56 were synthesized according to literature procedures.

BOC-NH2-SS-MA

BOC-NH2-SS-OH

Coumarin-imidazole

CSSMA

PEO45-b-PCSSMAn

n = 15, BP1 n = 22, BP2 n = 30, BP3

8


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Biomacromolecules

Scheme 2. Synthetic routes employed for the preparation of photo- and reduction-responsive CSSMA monomer and PEO-b-PCSSMA diblock copolymers.

Sample

Synthesis.

The

synthetic

routes

employed

for

the

preparation

of

BOC-NH2-SS-MA precursor, coumarin-based disulfide-containing CSSMA monomer, and PEO45-b-PCSSMAn amphiphilic diblock copolymers are shown in Scheme 2.

Synthesis of BOC-NH2-SS-MA Precursor (Scheme 2). Briefly, TEA (0.29 g, 2.84 mmol, 1.5 equiv.) and BOC-NH2-SS-OH (0.48 g, 1.89 mmol, 1 equiv.) were dissolved in dry THF (35 mL), cooled to 0 oC in an ice water bath, and then methacryloyl chloride (0.22 g, 2.08 mmol, 1.1 equiv.) in 10 mL of dry THF was added dropwise over a period of 0.5 h under vigorously stirring. After the addition was completed, the reaction mixture was warmed up to room temperature and stirred overnight. The insoluble salt was filtered off and the organic solvent was removed through vacuum distillation. The residues were diluted with ethyl acetate and washed twice with deionized water and saturated brine, respectively. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated and was further purified by gel column chromatography using ethyl acetate/petroleum ether (1/10, v/v) as the eluent, affording a colorless oil (0.50 g, yield: 82.4%). 1H NMR (CDCl3, δ, ppm, TMS; Figure S1a): 6.14 (s, 1H), 5.60 (s, 1H), 4.95 (s, 1H), 4.41(t, J =6.6 Hz, 2H), 3.47 (q, J =6.3 Hz, 2H), 2.96 (t, J =6.6 Hz, 2H), 2.81 (t, J =6.3 Hz, 2H), 1.95 (s, 3H), 1.45 (s, 9H).

13

C NMR (CDCl3, δ,

ppm, TMS; Figure S1b): 167.16, 155.73, 136.01, 126.03, 79.57, 62.59, 39.17, 38.68, 36.98, 28.38, 18.28. HPLC analysis (Figure S1c): 4.9 min (MeOH/H2O = 85/15, v/v). ESI-MS (m/z): calcd for [M+H]+, 322.107; found, 322.114 (Figure S1d). 9



Synthesis of CSSMA monomer (Scheme 2). BOC-NH2-SS-MA (1.02 g, 3.2 mmol, 0.9 equiv.) and TFA (5 mL) were dissolved in 5 mL of DCM. The mixture was stirred at room temperature and continuously monitored by TLC until the reaction was completed (~1 h). Subsequently, the mixture was concentrated and dried under vacuum for ~3 h to remove any volatile impurities. Afterwards, coumarin-imidazole (1.21 g, 3.55 mmol, 1.0 equiv.) and TEA (0.72 g, 7.1 mmol, 2.0 equiv.) were dissolved in DCM (40 mL), and then the solution of deprotected BOC-NH2-SS-MA in DCM (10 mL) was added dropwise. After stirring at room temperature for 12 h, the reaction mixture was washed with saturated brine and dried over anhydrous Na2SO4. The organic solvent was removed under reduced pressure and the crude product was further purified by flash chromatography (hexane/ethyl Acetate=1/1, v/v), affording a brown solid (1.01 g, yield: 63.8%). 1H NMR (CDCl3, δ, ppm, TMS; Figure 1a): 7.31 (s, 1H), 7.28 (s, 1H), 6.60 (d, J = 2.5 Hz, 1H), 6.58 (d, J = 2.5 Hz, 1H), 6.52 (d, J = 2.5 Hz, 1H), 6.13 (d, J = 4.3 Hz, 2H), 5.65 – 5.58 (m, 1H), 5.40 (t, J = 5.9 Hz, 1H), 5.23 (s, 2H), 4.43 (t, J = 6.7 Hz, 2H), 3.59 (q, J = 6.2 Hz, 2H), 3.40 (q, J = 7.1 Hz, 4H), 2.97 (t, J = 6.7 Hz, 2H), 2.86 (t, J = 6.1 Hz, 2H), 1.95 (s, 3H), 1.21 (t, J = 7.1 Hz, 6H). 13C NMR (CDCl3, δ, ppm, TMS; Figure 1b): 167.21, 161.97, 156.20, 155.49, 150.60, 150.21, 135.96, 126.15, 124.38, 108.63, 106.09, 105.99, 97.76, 62.59, 61.88, 44.74, 39.81, 38.30, 36.86, 18.29, 12.43. HPLC analysis (Figure 1c): 20.0 min (MeOH/H2O = 70/30, v/v). ESI-MS (m/z): calcd for [M+H]+, 495.155; found, 495.161 (Figure 1d).

Synthesis of Dual-Stimuli Responsive PEO45-b-PCSSMAn Amphiphilic Diblock Copolymers via RAFT Polymerization. Using the preparation of PEO45-b-PCSSMA15 (BP1) 10


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Biomacromolecules

as an example, briefly, PEO macroRAFT agent (23 mg, 0.01 mmol, 1 equiv.), CSSMA (100 mg, 0.2 mmol, 20 equiv.) were dissolved in 1,4-dioxane (0.15 mL) containing AIBN (0.33 mg, 0.002 mmol, 0.2 equiv.) and were charged into a reaction tube equipped with a magnetic stirring bar. The tube was carefully degassed by three freeze-pump-thaw cycles, sealed under vacuum and stirred in an oil bath at 70 oC. After 8 h, the reaction was quenched by immersing the reaction tube into liquid nitrogen. The reaction mixture was precipitated into an excess of ether and the precipitate was collected and dissolved in THF again. The above dissolution-precipitation cycle was repeated three times. The final product was dried in a vacuum oven overnight at room temperature, yielding a yellowish solid (60 mg, yield: 49%). The molecular weight and molecular weight distribution of the as-synthesized diblock copolymers were determined by GPC using DMF as the eluent, revealing a number average molecular weight (Mn) of 10.9 kDa and a polydispersity index (Mw/Mn) of 1.22 (Figure 2a). The degree of polymerization (DP) of the PCSSMA block was determined to be 15 according to 1H NMR analysis (Figure 2b). The obtained diblock copolymer was thus denoted as PEO45-b-PCSSMA15 (BP1,

Table

1).

Similarly,

PEO45-b-PCSSMA22

(BP2)

and

PEO45-b-PCSSMA30 (BP3) were also synthesized by elevating the CSSMA monomer to PEO-based macroRAFT agent ratios. The structural parameters of BP1, BP2, and BP3 copolymers are summarized in Table 1.

Self-Assembly of Amphiphilic Diblock Copolymer PEO45-b-PCSSMAn and Drug Loading. Supramolecular assemblies of PEO45-b-PCSSMAn were fabricated via a cosolvent self-assembly approach. Typically, 1 mg of PEO45-b-PCSSMA22 (BP2) was dissolved in 1 11



mL of 1,4-dioxane and 9 mL of deionized water was then slowly injected at a rate of 9 mL/h by a syringe pump under stirring (600 rpm). After the water addition was completed, the colloidal solution was stirred for another 30 min prior to dialysis against deionized water using a cellulose membrane with a molecular weight cutoff (MWCO) of 3.5 kDa. For the encapsulation of hydrophobic Nile red probe within the hydrophobic bilayer membranes, 1 mg of BP2 amphiphilic diblock copolymer was dissolved in the Nile red (0.01 g/L) solution in 1,4-dioxane and 9 mL deionized water was added dropwise at 9 mL/h by a syringe pump. Unloaded Nile red was removed by exhaustive dialysis against deionized water. For the encapsulation of doxorubicin hydrochloride (DOX), 1 mg of BP2 diblock copolymer was dissolved in 1 mL of 1,4-dioxane. Then, DOX solution (10 mg/mL, 1 mL) was slowly added to the polymer solution at a rate of 9 mL/h through a syringe pump, followed by the addition of 8 mL of deionized water at the same speed. After water addition, the colloidal solution was stirred for another 30 min and was subjected to dialysis against deionized water for 24 h to remove residual 1,4-dioxane and unloaded DOX using a cellulose membrane (MWCO: 3.5 kDa). Using a similar procedure, hydrophilic Texas red-labeled dextran (TR-dextran, MW ≈ 10 kDa) was also encapsulated. Free TR-dextran was removed by ultrafiltration (Millipore, MWCO: 100 kDa) instead of dialysis. The loading contents of DOX and TR-dextran were calculated to be 15 wt% and 5.8 wt%, which were quantified using standard calibration curves by measuring the fluorescence intensities of DOX and TR fluorophores at 595 nm and 622 nm, respectively.

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Biomacromolecules

In Vitro Release Profiles of DOX and TR-Dextran. For photoirradiation-mediated DOX release, 0.36 mL of DOX-loaded polymersome dispersions (0.1 g/L; PBS buffer, pH 7.4, 10 mM) was first irradiated for 0 min, 10 min, 20 min, and 30 min, respectively, which were then charged into dialysis tubes (cellulose membrane; MWCO: 3.5 kDa) and were immersed into 3.6 mL PBS medium under gently shaking at 37 oC. Periodically, the external buffer solutions were taken and replaced with fresh medium. The DOX concentrations in the dialysates were measured by fluorescence spectrophotometer and the release amounts were determined using a standard curve. For GSH-triggered release of TR-dextran, 1 mL of TR-loaded polymersome dispersions were first irradiated for 0 min or 30 min, which were subjected to incubation with 10 mM GSH. After removal of the polymersomes by ultrafiltration (Millipore, MWCO: 100 kDa), the release amounts of TR-dextran, at pre-determined time intervals, was quantified by measuring the fluorescence intensities in the filtrates against a standard calibration curve. Characterization. All nuclear magnetic resonance (NMR) spectra were recorded on a 300 MHz Bruker instrument and deuterated chloroform (CDCl3) was used as the solvent. Electrospray ionization mass spectrometry (ESI-MS) experiments were conducted on Thermo Scientiﬁc LTQ Orbitrap Mass Spectrometer equipped with an electrospray interface. Molecular weights and molecular weight distributions were determined by gel permeation chromatography (GPC) equipped with Waters 1515 pump. The eluent was DMF at a flow rate of 1.0 mL/min. A series of low polydispersity polystyrene standards were employed for calibration. Fluorescence spectra were recorded on an F-4600 (Hitachi) spectrofluorometer. 13



Page 14 of 45

All UV/vis spectra were acquired on a TU-1910 double-beam UV/vis spectrophotometer (Puxi General Instrumental Company, China). Dynamic light scattering (DLS) measurements were collected on a commercial spectrometer Zetasizer Nano ZS90 (Malvern). Scattered light was collected at a fixed angle of 173o for a duration of ~3 min. All data were averaged over three consecutive measurements. Static light scattering (SLS) was conducted on a commercial spectrometer (ALV/DLS/SLS-5022F) equipped with a multi-tau digital time correlator (ALV5000) and a cylindrical 22 mW UNIPHASE He-Ne laser (λ0 = 632 nm) as the light source. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis was performed on a Shimadzu HPLC system, equipped with an LC-20AP binary pump, a Symmetry C18 column, and an SPD-20A UV-vis detector. Transmission electron microscopy (TEM) were conducted on a JEOL 2010 electron microscope at an acceleration voltage of 200 kV. The samples for TEM observations were prepared by dipping 10 µL of colloidal dispersions on copper grids successively coated with thin films of Formvar and carbon. No staining procedures were applied.

Table 1. Structural Parameters of Photo- and Reduction-Responsive Amphiphilic Diblock Copolymers and Corresponding Self-Assembled nanostructures in Aqueous Media.

Mn

Mn

Entry Samples

a

b

(kDa)

(kDa)

Mw/Mnb (nm) c µ2/Γ2 c Morphology d

BP1

PEO45-b-PCSSMA15

9.8

10.9

1.22

220

0.15

LCM

BP2

PEO45-b-PCSSMA22

13.2

13.9

1.18

400

0.17

LCV

14


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Biomacromolecules

BP3

PEO45-b-PCSSMA30

17.2

17.7

1.13

440

0.05

LCV

a

Determined by 1H NMR analysis; b Obtained from GPC analysis using DMF as the eluent; c Determined by dynamic light scattering analysis and the parameter, µ2/Γ2, represents the polydispersity index of the as-assembled nanostructures; d Determined by TEM observations (LCM = large compound micelles; LCV = large compound vesicles). RESULTS AND DISCUSSION

Synthesis of Dual-Stimuli Responsive Coumarin-based CSSMA Monomer and Amphiphilic PEO-b-PCSSMA Diblock Copolymers. Recently, we found that o-nitrobenzyl moiety-caged carbamate linkages could be unmasked under UV 365 nm light irradiation with the generation of primary amine residues, which could subsequently crosslink and permeate the bilayer membranes of as-assembled vesicles via amidation reactions.31-32, 48 Inspired by this chemistry, we envisioned that 4-hydroxymethyl coumarin moieties could probably be employed as an alternative photo-responsive caging moiety of carbamate linkages as well. Notably, coumarin chromophores exhibited longer absorbance wavelengths than that of

o-nitrobenzyl derivatives, rendering them advantageous in biomedical applications with decreased irradiation damages and enhanced tissue penetration.52 Indeed, 4-hydroxymethyl coumarin moieties have been confirmed to be responsive to both UV and two-photon irradiation and have been engineered as drug containers.57 On the other hand, we hypothesized that if reduction-responsive disulfide linkages could simultaneously be incorporated, the cross-linked polymersomes could be further degraded. To this end, coumarin-based disulfide-containing CSSMA monomer was designed and synthesized (Scheme 2). Specifically, disulfide linkage-containing precursor, BOC-NH2-SS-MA, was synthesized via an esterification reaction of methacryloyl chloride and BOC-NH2-SS-OH. 15


Biomacromolecules

The chemical structure of the BOC-NH2-SS-MA precursor was confirmed by a combination of NMR, HPLC, and ESI-MS analysis (Figure S1). Subsequently, the as-synthesized BOC-NH2-SS-MA

precursor

was

further

treated

with

TFA

to

remove

the

tert-butyloxycarbonyl (BOC) protecting group; the released primary amine moieties were then reacted with coumarin-imidazole derivatives to afford the target CSSMA monomer (Scheme 2). Similarly, the chemical structure of CSSMA monomer was unequivocally confirmed by NMR, HPLC, and ESI-MS analysis (Figure 1).

a)

b) a CDCl3

m

o

g

i

l j

d

a

b

a

e

k

h

n

g

c+e f+n

o

b j

u

i

l k

r

c)

d)

MeOH/H2O(v/v,70/30) 1 mL/min 365 nm

5

10

15

n m

o

q

20

25

l ie g j k

b

c f h a b

H2O

h

0

p

r

t

f m

d

d

s

c

30

m+h k c+i

Relative abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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t u

q

g+d

e j

f

s

op l

n

495.161

100

Calcd. for [M+H]+, 495.155 found, 495.161 Calcd. for [M+Na]+, 517.143 found, 517.143

80 60

517.143

40 20 0

420

Elution Time / min

450

480

510

540

570

600

m/z

Figure 1. (a) 1H and (b) 13C NMR spectra, (c) HPLC elution profile (detection wavelength: 365 nm; eluent: MeOH/H2O = 70/30; flow rate = 1 mL/min) , and (d) ESI-MS spectrum recorded for CSSMA monomer.

16


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Biomacromolecules

With CSSMA monomer in hand, RAFT polymerization of CSSMA monomer was then conducted using a PEO-based macroRAFT agent, affording amphiphilic PEO-b-PCSSMA diblock copolymers. GPC analysis revealed that the obtained diblock copolymers exhibited single unimodal peaks and relatively narrow polydispersities (Figure 2a; Table 1). The DP of the PCSSMA block was calculated from the NMR result by comparing the integral areas of the PEO units (peak a) and the methylene groups (peak c) in the CSSMA monomers (Figure 2b). Moreover, the repeating units of the PCSSMA blocks could be readily tuned by adjusting the feeding ratios of CSSMA monomer to the macroRAFT agent and three PEO-b-PCSSMA diblock copolymers (BP1-BP3) were prepared and their structural parameters are summarized in Table 1.

17



a)

Page 18 of 45

PEO45-b-PCSSMA15 (BP1) PEO45-b-PCSSMA22 (BP2) PEO45-b-PCSSMA30 (BP3)

13

14

15

16

17

18

Elution Time / min i

a

b)

c a f h

l

e g

b j

k

d

h

CDCl3 f k+l

j

b

g c

d+e

i

Figure 2. (a) GPC traces recorded for BP1, BP2, and BP3 diblock copolymers, respectively. (b) Typical 1H NMR spectrum recorded in CDCl3 for BP1 copolymer.

Supramolecular Self-Assembly and Stimuli-Responsive Behavior of PEO-b-PCSSMA Assemblies. Subsequently, we investigated the self-assembly behavior of the as-synthesized diblock copolymers. The amphiphilic diblock copolymers were first dissolved in a good solvent (e.g., 1,4-dioxane), deionized water was then slowly injected to trigger the formation of colloidal assemblies. After dialysis against deionized water to remove any residual organic solvent, the as-assembled nanoparticles were first analyzed by DLS, revealing the intensity 18


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average hydrodynamic diameters, , of the BP1, BP2, and BP3 copolymers being 220 nm, 400 nm, and 440 nm, respectively (Figure 3a). TEM observations demonstrated the formation of micellar nanoparticles for BP1 copolymer and large compound vesicles (LCVs) for BP2 and BP3 copolymers (Figure 3b-f). Moreover, static light scattering measurement revealed that the ratio (/) of mean square radius of gyration, , and hydrodynamic radius, , was close to 1.0, further confirming the formation of hollow structures. The formation of LCVs should be attributable to the increased hydrophobic blocks of BP2 and BP3 copolymers that led to increased packing parameters as compared to that of

BP1.58 Moreover, a similar self-assembled morphology was observed in previous reports.59-61

19



Figure 3. (a) Intensity-average hydrodynamic diameter distributions, f(Dh), recorded for the aqueous dispersions of BP1, BP2, and BP3 assemblies. (b-d) Typical TEM images obtained for (b) BP1 LCMs, (c) BP2 LCVs, and (d) BP3 LCVs (scale bars: 1 µm).

Next, given the presence of photo-sensitive coumarin moieties, we examined the photo-responsive performance of the assemblies using BP2 vesicles as an example. To unravel the underlying molecular mechanism of the photo-responsive characteristics, we first monitored the photo-degradation behavior of CSSMA monomer in methanol under blue light (e.g., 430 nm) irradiation. Note that the o-nitrobenzyl moieties only exhibited a rather weak absorbance at this wavelength.62 Upon irradiation, the coumarin moieties were cleaved with the formation of coumarin cations,63 which could be hydrolyzed by water or react with the solvent (e.g., methanol) in the presence or absence of oxygen with the formation of a number of degraded products (1-3, Figure S2a). The proposed degradation products were supported by the ESI-MS analysis (Figure S2a), in accordance with previous studies regarding the degradation process of coumarin derivatives.64 Moreover, HPLC analysis revealed that the CSSMA signal was gradually attenuated while the signals of degraded products (1-3) were steadily intensified. Moreover, CSSMA monomer was invisible after 30 min irradiation, suggesting that photoirradiation-mediated degradation process was accomplished within 30 min. Moreover, we found the photo-responsive performance of CSSMA moieties were highly dependent on the irradiation wavelengths as well. For example, the half-lives of CSSMA extended from ~12 min under 430 nm irradiation to ~35 min under 440 nm irradiation, which was further elongated to over 120 min under 460 nm irradiation. By contrast, there were no 20


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significant changes in the half-lives of CSSMA monomer if 410 nm irradiation was applied (Figure S2c). Therefore, to avoid long-time exposure to photoirradiation and the potential risk of short wavelength irradiation, the subsequent studies were conducted under 430 nm light irradiation (230 mW/cm2) to examine the photo-responsive behavior of the photo- and reduction-responsive assemblies.

Figure 4. (a) Time-dependent UV-vis absorbance spectra of BP2 vesicles (0.1 g/L, [CSSMA] = 0.18 mM) under 430 nm light irradiation. Inset: absorbance intensity changes at 398 nm against irradiation time. (b) Time-dependent evolution of scattering intensities and intensity average hydrodynamic diameters, , of the aqueous dispersion of BP2 vesicles under 430 nm light irradiation. TEM images of BP2 vesicles (c) before and (d) after irradiation with 430 nm light for 40 min (scale bars: 0.5 µm). 21



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The non-irradiated BP2 vesicles had an intensive absorption peak centered at ~390 nm, in line with the absorbance of 7-diethylamino-4-hydroxymethylcoumarin derivatives.57 Upon irradiation the vesicle dispersion with 430 nm light, the absorption peak gradually became weak and a hypochromic shift of the maximum absorption peak to ~370 nm was observed (Figure 4a). The absorbance intensities stabilized out after 40 min irradiation, suggesting the completion of the photolysis reaction of coumarin derivatives within the vesicle bilayer membranes. On the other hand, although there was only a slight decrease in the under irradiation, the scattering intensities underwent a constant drop likely due to the removal of hydrophobic coumarin moieties after light irradiation (Figure 4b). Interestingly, the vesicular nanostructures remained unchanged after irradiation for 40 min (Figure 4c,d); this result may suggest the formation of cross-linked vesicles after photoirradiation. To testify whether the BP2 vesicles was cross-linked or not under 430 nm irradiation, we diluted the irradiated vesicles with THF, a good solvent for BP2 copolymers. Theoretically, if the irradiated vesicles were not cross-linked, they should be disintegrated into unimers after dilution with a large amount of THF (e.g., THF/H2O = 9/1). As expected, the non-irradiated BP2 vesicles possessed a rather low scattering intensity

(Figure 5a). This

phenomenon clearly indicated that the formation of vesicles was dominated by noncovalent hydrophobic interactions, which could be disrupted by the addition of THF. However, once the irradiation process was initialized, the scattering intensities experienced a monotonous increase and reached a plateau after ~10 min irradiation (Figure 5a). TEM studies revealed the appearance of spherical nanoparticles after 3 min irradiation (Figure 5b), suggesting that 22


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partially cross-linked structures were already formed. The formation of cross-linked nanostructures was likely ascribed to the deprotection of coumarin moieties and the subsequent release of primary amine groups that further implemented inter/intrachain amidation reactions (Scheme 1), forming branched chains at the beginning and finally cross-linked the entire nanoassemblies.37 Although the vesicular morphology cannot be maintained after 3 min irradiation after dilution with THF, the initial vesicular nanostructures were kept after irradiation for 40 min, as evidenced by TEM observation (Figure 5c). This drastic discrepancy was ascribed to the increased cross-linked densities upon extending the irradiation time and a sufficient cross-linked density was necessary to maintain the vesicular nanostructures to resist the solvent-mediated disassembly process. Since the interchain aminolysis of ester moieties contributed to the cross-linking process, which was accompanied with the release of small molecule primary amine functionalities (Scheme 1), it was possible to roughly calculate the cross-linking degree by quantifying the concentration of released primary amine moieties. Using ninhydrin as a colorimetric probe,65 the concentration of released primary amine moieties in the dialysate of

BP2 vesicles (0.1 g/L) under 430 nm irradiation for 30 min was determined to be 35.7 µΜ (Figure S3). Note that the theoretical amount of primary amine moieties corresponding to the carbamate linkage concentration in BP2 vesicles (0.1 g/L) was calculated to be 168 µΜ, assuming that the intermediate products of photoreactions did not react with the amine groups. The maximum cross-linking degree was thus lower than 21.2%, considering that intrachain aminolysis reactions did not efficiently result in the cross-linking process but the 23



primary amine moieties generated from intrachain aminolysis reactions could not be excluded by the ninhydrin assay. Interestingly, there was a pronounced transition in the polarity of bilayer membranes of BP2 vesicles under 430 nm light irradiation as probed by Nile red fluorogen (Figure 6). During the course of photoirradiation, the hydrophobic bilayer membranes were transformed into hydrophilic mesh structures that only allowed for the passage of small molecule payloads. Therefore, the tracelessly cross-linkable vesicles could be employed to deliver small molecule therapeutics and the controlled release could be actuated by visible light irradiation.

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Figure 5. (a) Time-dependent evolution of scattering intensities recorded for BP2 vesicles (0.1 g/L, [CSSMA] = 0.18 mM) upon irradiation for varying times and subsequently diluted by THF (THF/water = 9/1, v/v). (b,c) TEM images of BP2 vesicles (0.1 g/L) upon irradiation (λ = 430 nm) for (b) 3 min and (c) 40 min, followed by dilution with THF (THF/water = 9/1, v/v).

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FL Intensity

a)

3000

Increasing the irradiation time

2000

1000

0

b) FL Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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600

650

700

750

Wavelength / nm 3000

2000

1000

0

0

10

20

30

40

Irradiation time / min Figure 6. Irradiation time-dependent (a) fluorescence emission spectra (λex = 550 nm) and (b) relative fluorescence intensities at 638 nm of Nile red (NR)-loaded BP2 vesicles (0.1 g/L) under 430 nm irradiation.

After the photo-responsive behavior of the BP2 vesicles being confirmed, subsequently, we further testified the reduction-sensitive performance of the cross-linked BP2 vesicles by taking advantage of the disulfide linkages. Upon incubating the pre-irradiated cross-linked vesicles with 10 mM GSH, mimicking the intracellular GSH concentration within the cytosol, the scattering intensities of irradiated BP2 vesicles were gradually decreased upon extending 26


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the incubation time, indicating the disassociation of the cross-linked vesicles. The exhibited a slight increase within the first 12 h, followed by a monotonous drop within the subsequent 80 h (Figure 7). Time-dependent evolution of morphological transitions, monitored by TEM observations, revealed that the cross-linked BP2 vesicles were gradually degraded, with the formation of irregular aggregates, and finally a small population of micellar nanoparticles (Figure 7b-f). As such, we anticipated that the GSH-triggered disruption of the cross-linked polymersomes should enable the release of large molecule encapsulants from the cross-linked polymersomes if the payloads could be previously embedded.

27



Figure 7. (a) Time-dependent evolution of scattering intensities and intensity average hydrodynamic diameters, , of BP2 vesicles after irradiation with 430 nm light upon incubation with 10 mM GSH at 37 oC. (b-f) Typical TEM images of irradiated BP2 vesicles under 430 nm light for 30 min upon further incubation with 10 mM GSH for (b) 0 h, (c) 24 h, (d) 48 h, (e) 72 h, (f) 92 h, respectively (scale bars: 1 µm).

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Photoirradiation- and GSH-Triggered Sequential Release of Small and Large Molecule Payloads. In the previous section, we investigated the photo- and reduction-responsive characteristics of the dual-stimuli responsive vesicles. The dual-stimuli responsive polymersomes were first tracelessly cross-linked under photoirradiation and the cross-linked vesicles were subsequently disintegrated upon further incubation with GSH, which could be of potential use in releasing encapsulants with different sizes in a sequential manner. To testify whether the BP2 vesicles could be employed for sequential release of payloads or not, DOX was chosen as a model of small molecule therapeutics while TR-dextran (Mn = 10 kDa) was used as a model of large molecule therapeutics. First, we assessed the independent release behavior of DOX and TR-dextran from BP2 polymersomes upon photoirradiation or GSH treatment, respectively. Although it was quite advantageous to trigger DOX release using visible light, we found that DOX was drastically bleached under 430 nm irradiation (230 mW/cm2) within 30 min. Thus, a hand-held UV lamp (365 nm, 1 mW/cm2) was used to mediate the release of DOX. Notably, the DOX release profiles were highly dependent on the irradiation time and a longer irradiation time gave rise to an accelerated release (Figure 8a). Specifically, less than 20% DOX was spontaneously released within 48 h without photoirradiation, whereas the release amounts increased to 41%, 51%, and 92% after 10 min, 20 min, and 30 min irradiation, respectively. This result concurred quite well with the previous conclusions31, 37 that the traceless cross-linking process could not only reinforce the vesicular nanostructures but also enhanced the permeability of bilayer membranes (Figures 4 and 5). By sharp contrast, the formation of cross-linked vesicles did not evoke the release of 29



TR-dextran in the absence of GSH (Figure 8b). The TR-dextran release could only be achieved in the presence of GSH that cleaved the disulfide linkages and in turn disintegrate the BP2 vesicles (Figure 7), regardless of with or without pre-irradiation for 30 min. Notably, it was feasible to accomplish the release of small molecule therapeutics (e.g., DOX) while retaining the large ones (e.g., dextran) in our dual-stimuli responsive vesicles (Figure 8a,b), which, to our knowledge, has not been achieved in previously reported polymersome systems. This encouraging result spurred us to further explore the potential application of dual-stimuli responsive polymersomes in sequential release of payloads. Next, DOX and TR-dextran were synergistically encapsulated within the aqueous lumens of BP2 vesicles and the co-release profiles were evaluated. Upon irradiation the coloaded BP2 vesicles for varying times (0-30 min) in the absence of GSH, we can only observe increased DOX release while the release of TR-dextran was negligible (Figure 8c), in good agreement with the individual loading results (Figure 8a,b). Specifically, after irradiating the coloaded

BP2 polymersomes for 30 min, the DOX and TR-dextran release amounts were determined to be ~ 36% and ~3%, respectively. This result potently demonstrated that the photoirradiation process that led to traceless cross-linking of the bilayer membranes could mediate selective release of DOX. Notably, the DOX release amounts in the coloaded vesicles were slightly lower than that of DOX-loaded BP2 vesicles without TR-dextran loading (Figure 8a), which was probably ascribed to the noncovalent interaction between DOX and TR-dextran. Although photoirradiation only enabled the release of DOX, the TR-dextran release could be actuated by incubating the coloaded polymersomes with GSH 30


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that could cleave the disulfide linkages. For example, the TR-dextran release amounts increased to approximately 20% without irradiation and 45% with irradiation for 30 min after treatment with 10 mM GSH for 12 h (Figure 8c). This discrepancy was likely due to the increased GSH accessibility by taking advantage of the formation of hydrophilic cross-linked networks after photoirradiation. Meanwhile, it should be mentioned that the DOX release amounts after treatment with GSH were higher than that of vesicles without GSH addition, regardless of with or without photoirradiation. We tentatively ascribed this phenomenon to the disintegration of entire vesicles upon GSH incubation; this is in striking contrast with the formation of cross-linked mesh nanostructures under light irradiation. Taken together, sequential release of small molecule (e.g., DOX) and large molecule (e.g., TR-dextran) encapsulants could be achieved in the current dual-stimuli responsive polymersomes by first irradiation the polymersomes, followed by treatment with GSH. Moreover, concomitant release of diverse payloads could be achieved as well by straightforwardly incubating the dual-stimuli responsive polymersomes with GSH.

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b)

80 60 40 20 0 0

12

24

36

48

80 60 40 20 0

Incubation Time / h c)

DOX Release / %

Irradiation 30 min + 10 mM GSH Irradiation 0 min + 10 mM GSH Irradiation 30 min Irradiation 20 min Irradiation 10 min Irradiation 0 min

100

Irradiation time 0 min 10 min 20 min 30 min

0

6

w/ GSH

w/o GSH

100

12

100

80

80

60

60 DOX TR-dextran

40

40

20 0

18

20 0

10

24

Incubation Time / h

20

30

0

30

0

TR-dextran Release / %

100

Cumulative Release / %

a)

Cumulative Release / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Irradiation Time / min Figure 8. (a) In vitro release profiles of DOX from DOX-loaded BP2 vesicles (0.1 g/L; PBS buffer, pH 7.4, 20 mM; 37 oC) under varying photoirradiation durations (365 nm, 1 mW/cm2). (b) In vitro release profiles of Texas Red-dextran (MW ≈ 10 kDa) from TR-dextran-loaded BP2 vesicles (0.1 g/L; PBS buffer, pH 7.4, 10 mM; 37 oC) under varying irradiation durations with or without GSH addition. (c) Release extents after 12 h incubation of DOX and TR-dextran from DOX- and TR-dextran-coloaded BP2 vesicles (0.1 g/L; PBS buffer, pH 7.4, 20 mM, 37 oC) under varying irradiation times with or without GSH (10 mM).

CONCLUSIONS

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In summary, a novel photo- and reduction-responsive monomer composed of coumarin moieties and disulfide linkage was designed and synthesized. The dual-stimuli responsive monomer could be polymerized to form amphiphilic diblock copolymers using a PEO-based macroRAFT agent through RAFT polymerization. The resulting amphiphiles could self-assemble into vesicular nanostructures that naturally inherited the photo- and reduction-responsive characteristics. Upon irradiation the vesicle dispersion with 430 nm light, the coumarin moieties were cleaved with the generation of reactive primary amines that further cross-linked the vesicles through extensive amidation reactions. This process gave rise to a remarkable polarity transition within the bilayer membranes and the release of encapsulated small molecule payloads while retaining the large ones. Moreover, the cross-linked vesicles could be further disassembled upon incubation with GSH, resulting in the release of large molecule encapsulants. The dual-stimuli responsive vesicles capable of being tracelessly cross-linked enable sequential release of synergistically loaded small and large molecule therapeutics. We expect these dual-stimuli responsive vesicles could be of important use in the treatment of diseases where distinct therapeutic agents should be concomitantly administrated and sequentially dosed.

ASSOCIATED CONTENT Supporting Information. Additional NMR result, ESI-MS, HPLC elution traces, and wavelength-dependent photolysis of CSSMA monomer. This material is available free of charge via the Internet at http://pubs.acs.org AUTHOR INFORMATION 33



Corresponding Author [email protected] (S.L.) [email protected] (J.H.) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

Financial support from the National Natural Science Foundation of China (NNSFC; projects 51690150, 51690154, 21674103, 51722307, and 51673179), the International S&T Cooperation Program of China (ISTCP) of MOST (2016YFE0129700), the Natural Science Foundation of Anhui Province (1708085QB34), and the Fundamental Research Funds for the Central Universities (WK3450000003 and WK2060200023) is gratefully acknowledged.

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Biomacromolecules

For Table of Contents Use Only

Photo- and Reduction-Responsive Polymersomes for Programmed Release of Small and Macromolecular Payloads

Ziqiang Sun, Guhuan Liu, Jinming Hu,* and Shiyong Liu*

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Photo- and Reduction-Responsive Polymersomes for Programmed

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