Engineering Cross-Linkable Plasmonic Vesicles for Synergistic

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Engineering Cross-Linkable Plasmonic Vesicles for Synergistic Chemo-Photothermal Therapy Using Orthogonal Light Irradiation Kangning Zhu, Guhuan Liu, Guoying Zhang, 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

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ABSTRACT: Plasmonic vesicles combine the advantages of polymersomes and physiochemical properties of inorganic nanoparticles, exhibiting promising applications in theranostic nanovectors. However, although many of these plasmonic vesicles have been engineered as photothermal therapy (PTT) agents and drug carriers, they undergo structural destruction due to localized surface plasmon resonance (LSPR) effect. Notably, the disassembly of plasmonic vesicles leads to the burst release of encapsulated payloads and drastically decrease therapeutic efficacy. Herein, we report the fabrication of plasmonic vesicles that can be tracelessly cross-linked under 410 nm blue light irradiation, which can effectively avoid being disassembled when localized heat generation under light irradiation (e.g., 560 nm). Amphiphilic hybrid gold nanoparticles (AuNPs) functionalized with hydrophilic poly(ethylene oxide) (PEO) and hydrophobic poly(2-((((2nitrobenzyl)oxy)carbonyl)amino)ethyl methacrylate) (PNBOC) blocks were synthesized via ligand exchange process. The resulting amphiphilic AuNPs self-assemble into plasmonic vesicles capable of simultaneously carrying hydrophobic paclitaxel (PTX) and hydrophilic doxorubicin hydrochloride (DOX) drugs. Upon 410 nm blue light irradiation, primary amine moieties are generated due to the deprotection of NBOC moieties that subsequently undergo inter/intrachain amidation reactions, concurrently cross-linking and permeabilizing the vesicles. As such, the release of encapsulated PTX and DOX drugs could be actuated. Interestingly, the irradiated vesicles with highly densely packed AuNPs within the cross-linked bilayers generate localized high heat and can thus be used for PTT agents due to the LSPR effect of AuNPs under photoirradiation (e.g., 560 nm). Moreover, the hybrid vesicles present good X-ray attenuation capability and can be potentially used for computed tomography (CT) contrast agents. The fabrication of cross-linked plasmonic vesicle-based synergistic chemo-photothermal therapy agents using orthogonal light irradiation opens an avenue to explore new theranostic nanovehicles.

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INTRODUCTION The combination of several therapeutic modalities into one platform has emerged as a powerful tool in the treatment of cancers.1,2 For example, the integration of chemotherapy and photothermal therapy (PTT) can not only partially overcome the drawbacks of chemotherapy including nonspecific delivery and insufficient dosage in tumor tissues but also augment the cytotoxicity of chemotherapeutic drugs assisted by the hyperthermia effect.3−8 A feasible solution to impart chemotherapeutic platform with additional PTT performance is to incorporate chromophores with strong absorbance in the nearinfrared (NIR) window, exhibiting superior tissue penetration.9−13 However, the integrated NIR chromophores within drug carriers may suffer from photobleaching or heat-induced decomposition under NIR light irradiation, declining the PTT efficacy. To resolve this issue, inorganic materials (e.g., metal nanoparticles and graphene) with excellent antiphotobleaching and high absorbance extinction coefficient have been employed to replace organic chromophores to achieve a superior photothermal effect.14−16 Nevertheless, individually dispersed metal nanoparticles (e.g., AuNPs) commonly lack sufficient © XXXX American Chemical Society

absorbance intensities where light exhibits high tissue penetration (e.g., near-infrared region). Therefore, it is highly desirable to bathochomically shift the absorbance peaks of metal nanoparticles to longer wavelengths within the drug containers to achieve synergistic chemotherapy and PTT.17 To develop metal nanoparticle-based chemotherapy and PTT systems, metal nanocrystals have been modified with amphiphilic polymers, generating so-called amphiphilic nanoparticles, which allows for directing the self-assembly of the resulting amphiphilic nanoparticles by taking advantage of the grafted amphiphilic polymers.18−27 Because amphiphilic block copolymer can self-assemble into diverse nanostructures in a selective solvent,28 different hybrid nanostructures doped with metal nanoparticles could also be achieved. Indeed, hybrid micelles, nanotubes, and vesicles have been constructed by delicately adjusting the chain lengths, compositions, and densities of grafted polymers.29,30 To date, both “grafting Received: August 2, 2018 Revised: September 29, 2018

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Scheme 1. Schematic Illustration of the Construction of Hybrid Vesicles from PEO-b-PMALA and PNBOC-b-PMALA Cofunctionalized Amphiphilic AuNPsa

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The resulting hybrid vesicles undergo traceless cross-linking under light irradiation because of light irradiation-actuated cascade deprotection reactions of PNBOC side Linkages and the in situ generation of primary amine moieties. Extensive amidation reactions then occur, leading to crosslinking of the hybrid vesicles and a hydrophobic-to-hydrophilic transition within the vesicle bilayers, thereby triggering the release of encapsulated payloads. Moreover, the cross-linked hybrid vesicles could be further employed for PTT.

from” and “grafting to” approaches have been employed to prepare amphiphilic nanoparticles, enabling greatly tuning the surface grafting densities of polymer chains, which plays a crucial role in the final morphologies of the assembled nanostructures.31,32 Remarkably, these hybrid nanostructures could be engineered as nanovehicles, and controlled release of encapsulated payloads could be realized upon structural destruction of hybrid assemblies. On the other hand, the as-assembled hybrid nanostructures embedded with inorganic nanoparticles naturally inherit the unique optical and electronic properties of metal nanoparticles. Moreover, in comparison with individual nanoparticles, the closely packed nanoparticles within the hybrid assemblies were imparted with enhanced localized surface plasmon resonance (LSPR) characteristics, exhibiting increased photothermal conversion efficiency.9,33,34 Therefore, the hybrid assemblies of amphiphilic nanoparticles represent a promising platform for synergistic PTT and chemotherapy. In this regard, several research groups have made tremendous achievements in the past few years, and a number of elegant plasmonic vesiclebased therapeutic platforms have been developed.7,17,29,31,35−37 On the other hand, gold nanoparticles can effectively attenuate X-ray and have been used for computed tomography (CT) contrast agents with the extra advantages of long circulation time and high accumulation in diseased tissues.38−41 Therefore, these hybrid nanoassemblies could be potentially utilized as multifunctional theranostic nanovectors. However, albeit promising, these hybrid nanoassemlies were likely subjected to disassembly after intravenous administration due to the high shear stress in blood flow and high dilution in the blood pool. Additionally, these nanoassemblies were collapsed/disassembled under light irradiation as well due to localized heat generation originating from the strong plasmonic coupling of AuNPs. Notably, the structural disruption of the hybrid

assemblies led to not only the burst release of encapsulated payloads but also a remarkable change in absorbance intensities, which was unfavorable for sustained drug release and PTT. We surmised that the as-assembled hybrid nanoassemblies could be in situ cross-linked and the crosslinked nanostructures could possibly avoid being disintegrated under localized heat generation. Moreover, if the cross-linking process could concurrently provoke a microenvironmental change within the hybrid assemblies, triggering the release of payloads could be achieved with the likelihood to avoid burst release as a result of structural disruption of the hybrid assemblies. Recently, we found that the photoreactive o-nitrobenzyl ester-caged carbamate moieties could generate primary amine moieties under UV light irradiation, which has been previously used for the design of photoresponsive systems.42−52 The in situ generated primary amines spontaneously underwent inter/ intrachain amidation reactions with the formation of amide bonds within nanoassemblies. As such, the as-assembled nanoassemblies concurrently underwent traceless cross-linking and a hydrophobic-to-hydrophilic transition.53−56 Moreover, the traceless cross-linking process could be readily coupled with triggered drug release and enhanced magnetic resonance imaging relaxivities.57 We reasoned that if the traceless crosslinkable residues and hydrophilic moieties could be simultaneously grafted onto the surface of AuNPs, the amphiphilicitydriven self-assembly of AuNPs allowed for the formation of hybrid plasmonic vesicles, which could be synergistically loaded with hydrophilic drugs in the interior aqueous lumen and hydrophobic payloads in the vesicle membranes. Under irradiation with the exposure of reactive primary amine groups, the plasmonic vesicles were cross-linked and loaded drugs were released. Moreover, the formation of cross-linked plasmonic vesicles rendered it possible to further augment therapeutic B

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AuNPs (∼3 nm) and sodium citrate-coated AuNPs (∼13 nm) were synthesized and were characterized by UV−vis spectroscopy (Figures S5a and S6a) and TEM (Figures S5c and S6c). AuNPs with small diameters had much weaker SPR absorbance, in accordance with previous reports.59 Although OAm-stabilized AuNPs could directly exchange the surface ligands in organic solvent (e.g., chloroform) with lipoic acidcontaining polymers (BP1 and BP5), the citrate-coated AuNPs were not readily dispersible in organic solvent, and the ligand exchange process with BP2 and BP4 copolymers was conducted in a mixture solvent of water/DMF/THF. The free polymers were removed by repeated centrifugation, and the resultant amphiphilic AuNPs (denoted as AuNPx@PEOm/ PNBOCn, where x is the average diameter of AuNPs and m and n are the DPs of the PEO and PNBOC blocks, respectively) were achieved. AuNP3@PEO113/PNBOC27 (PV-1) and AuNP13@PEO45/ PNBOC50 (PV-2) could be readily redispersed in THF, suggesting a successful surface modification of AuNPs. Moreover, a typical FT-IR spectrum of the dried PV-1 sample clearly indicated the coexistence of both BP1 and BP5 chains after ligand exchange process (Figure S4d), although UV−vis absorbance (Figures S5b and S6b) and TEM observations (Figures S5d and S6d) revealed negligible changes as compared to that of the AuNPs prior to ligand exchanges. To further confirm the successful surface modification of AuNPs, we conducted the NMR spectra of PV-1 and PV-2 in CDCl3 (Figure S7a,c), in which the amphiphilic nanoparticles could be facilely dispersed and the surface polymer grafts should be discernible. Notably, both the characteristic peaks of PEO (∼3.6 ppm) and phenyl rings (7−8 ppm) of NBOC blocks were observed, suggesting that both the hydrophobic polymers (BP1 and BP2) and hydrophilic polymers (BP4 and BP5) were unambiguously attached to the surfaces of AuNPs, considering that the free polymer chains have been removed by exhaustive centrifugation process. Moreover, in combination with the TGA results, the grafting numbers of BP1 and BP5 on PV-1 were calculated to be 18 and 7 and BP2 and BP4 on PV2 were determined to be 148 and 100, respectively (Figure S7). As such, the grafting densities of PV-1 and PV-2 were estimated to be 0.884 and 0.467 chain/nm2. With the amphiphilic AuNPs in hand, next, we investigated the self-assembly behavior of PV-1 and PV-2. Overall, two methods were applied to self-assemble the amphiphilic AuNPs, namely, slow water addition and flash nanoprecipitation. Using PV-1 as an example, the former approach involved the slow addition (6 mL/h) of water (a selective solvent of PEO blocks) into the THF (a cosolvent of PEO and PNBOC blocks) that led to the formation of hybrid vesicles with an average diameter ∼160 nm (Figure 1a), while the latter gave rise to the formation of hybrid vesicles with a much larger size (∼320 nm) through quickly injecting the THF dispersion of PV-1 into aqueous solution in one shot (Figure S8a,c). This discrepancy suggested that the self-assembling approaches played a crucial role in the fabrication of nanostructures. Notably, these hybrid vesicles composed of hydrophilic PEO corona and hybrid PNBOC/AuNPs monolayer membranes could be potentially cross-linked by taking advantage of the in situ generated primary amine moieties of NBOC moieties via inter/intrachain amidation reactions.53,57 Next, we investigated the photoresponsive behavior of the vesicular nanoparticles of the resulting hybrid vesicles under UV irradiation. TEM observation revealed that the vesicular

outcome by taking advantage of the photothermal therapy of the embedded AuNPs. To verify our hypothesis, diblock copolymers of poly(ethylene oxide)-b-poly(2-(methacryloyloxy)ethyl 5-(1,2-dithiolan-3-yl)pentanoate) (PEO-b-PMALA) and poly(2-((((2nitrobenzyl)oxy)carbonyl)amino)ethyl methacrylate)-b-poly(2-(methacryloyloxy)ethyl 5-(1,2-dithiolan-3-yl)pentanoate)) (PNBOC-b-PMALA) were synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerizations. The as-synthesized block copolymers comprising PMALA blocks firmly attached to the surface of AuNPs due to the presence of lipoid acid residues that can form multivalent Au− S dative bonds with AuNPs, while hydrophilic PEO blocks and hydrophobic photoreactive PNBOC blocks rendered the functionalized AuNPs amphiphilic. The resulting amphiphilic AuNPs self-assembled into hybrid vesicles with hydrophilic PEO coronas and closely packed AuNPs and photoreactive PNBOC moieties within the vesicle membranes. Chemotherapeutic drugs, hydrophilic doxorubicin hydrochloride (DOX) and hydrophobic paclitaxel (PTX), were embedded within the aqueous lumen and hydrophobic membrane, respectively. Under visible light irradiation (e.g., 410 nm), the originally hydrophobic PNBOC moieties turned to be hydrophilic as a result of in situ generation of reactive primary amine groups, which spontaneously implemented intra/ interchain amidation reactions and thus cross-linking the plasmonic vesicles. The formation of cross-linked hybrid vesicles could be used for computed tomography (CT) contrast agents as well. Moreover, the cross-linking process dramatically permeabilized the vesicle membranes and thus triggered the release of encapsulated CPT and DOX drugs. In addition to chemotherapy, the therapeutic performance of the hybrid vesicles could be further elevated by taking advantage of the photothermal effect of embedded Au NPs (Scheme 1).

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RESULTS AND DISCUSSION Amphiphilic AuNPs can self-assemble into diverse nanostructures by taking advantage of the amphiphilicity of surface grafted polymer chains, bathochromically shifting the SPR peaks as compared with individual nanoparticles due to the enhanced LSPR effect. The resulting hybrid nanoassemblies can not only be employed for loading chemotherapeutic drugs but also exert a photothermal effect, serving as a promising platform for synergistic chemotherapy and photothermal therapy. The polymer chains could be tethered onto the surface of AuNPs via the formation of dative Au−S bonds through either “grafting from” or “grafting to” approach.17 Notably, the compositions of grated polymers and the grafting densities could be rationally designed and delicately tuned, which can effectively alter the packing of nanoparticles within the assemblies and further optimize the photothermal conversion efficiency, thereby elevating the therapeutic outcomes.58 First, hydrophobic photoresponsive diblock copolymers with or without NR-labeling (PNBOC-b-PMALA; BP1−BP3, Table S1) bearing lipoic acid residues were synthesized via consecutive RAFT polymerizations. The structural parameters of the homopolymer precursors and the target block copolymers are summarized in Table S1. Meanwhile, PEO-bPMALA diblock copolymers (BP4, BP5) were prepared via RAFT polymerization using PEO-based macroRAFT agents with varying PEO chain lengths (Schemes S1 and S2, Figures S1−S4, Table S1). In addition, oleylamine (OAm)-stabilized C

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Figure 1. (a, b) TEM images recorded for hybrid PV-1 vesicles (a) before and (b) after UV 365 nm irradiation (∼1.0 mW/cm2). The insets show enlarged AuNP hybrid vesicles. (c, d) TEM images of (c) untreated and (d) UV-irradiated hybrid PV-1 vesicles after dilution with THF (THF/water, v/v, 9/1).

Figure 2. (a) Evolution of UV/vis absorbance spectra recorded for the aqueous dispersion of hybrid PV-1 vesicles under UV 365 nm irradiation (∼1.0 mW/cm2). The inset in (a) shows absorbance changes at 260 nm upon UV 365 nm (∼1.0 mW/cm2) or 410 nm blue light irradiation (∼1.0 W/cm2). (b) Irradiation durationdependent evolution of intensity-average hydrodynamic diameter, ⟨Dh⟩, and scattered light intensities of hybrid PV-1 vesicles. (c) FT-IR spectra recorded for hybrid PV-1 vesicles before and after UV light irradiation; the samples were obtained via lyophilization of vesicle dispersions. (d) Irradiation duration-dependent changes of fluorescence emission spectra (λex = 520 nm) of Nile red-loaded AuNP hybrid vesicles upon UV irradiation. The inset shows emission intensity changes (λex = 520 nm, λem = 630 nm) under 365 nm (∼1.0 mW/cm2) and 410 nm (∼1.0 W/cm2) irradiation.

morphology was retained after irradiation with UV light (Figure 1b). Although the nonirradiated hybrid vesicles could be disassembled after dilution with 9-fold THF as evidenced by a drastic decrease in hydrodynamic diameters, ⟨Dh⟩, from 164 to 7 nm (Figure S9), the irradiated vesicles can be no longer disintegrated into dispersed nanoparticles, and the vesicular morphology remained unchanged under otherwise identical conditions (Figure 1c,d). This discrepancy clearly suggested the formation of cross-linked vesicles after UV light irradiation. Moreover, our previous studies indicated that the presence of iron oxide nanoparticles or upconversion nanoparticles within PNBOC-cored micelles did not adversely affect the crosslinking process either.57,60 Under UV light irradiation, the NBOC moieties underwent a tandem decaging reaction with the generation of primary amine groups, which then in situ implemented inter/intrachain amidation reactions, thereby cross-linking the hybrid vesicles and permeating the bilayer membranes (Scheme 1). Interestingly, although slow water addition and flash nanoprecipitation methods resulted in different ⟨Dh⟩ of the PV-1 assemblies, both of them underwent photomediated cross-linking processes (Figure S8). To further follow the photomediated cross-linking process, UV−vis absorbance spectra were recorded. Under both 365 and 410 nm light irradiation, the absorbance intensities of NBOC moieties on PV-1 vesicles underwent gradual decreases within 25 min, indicating the consumption of o-nitrobenzyl ester moieties (Figure 2a and Figure S10). Although there were no significant changes in the sizes of irradiated PV-1 vesicles, the corresponding scattered intensities experienced a monotonous drop subjected to the light irradiation within 25 min (Figure 2b). This result was in good agreement with the loss of hydrophobic nitrobenzene moieties under light irradiation. Moreover, FT-IR spectra revealed that the simultaneous decrease of ester moieties (1726 cm−1) and the formation of amide residues (1629 cm−1; Figure 2c). The formation of amide bonds was further corroborated by XPS analysis (Figure S11). Notably, there was a remarkable transition in the polarity of bilayer membranes under light irradiation as probed by Nile red (NR) fluorogen (Figure 2d), whereas the fluorescence of the nonirradiated vesicles remained unchanged without light irradiation (Figure S12). These results concurred quite well with the previous reports

and unequivocally supported the formation of cross-linked hybrid vesicles under irradiation. However, we found that there were no significant differences between the SPR peaks of PV-1 vesicles before and after light irradiation (Figure S13). As mentioned above, hybrid PV-1 vesicles with larger sizes could be fabricated through a flash nanoprecipitation procedure. However, the photoresponsive behavior and light irradiation-induced cross-linking of the bilayer membranes were not affected by the self-assembly procedures. For the hybrid vesicles obtained by the flash nanoprecipitation technique, upon 365 nm UV light irradiation for 30 min, a steady decrease in the absorbance spectra centered at ∼260 nm was observed, while the ⟨Dh⟩ kept unchanged and the relative scattered intensities gradually decreased (Figures S14). This result was quite similar to that of hybrid vesicles fabricated through the cosolvent self-assembling approach (Figure 2). Taken together, amphiphilic PV-1 could self-assemble into hybrid vesicles with diverse diameters via either a cosolvent or flash nanoprecipitation procedure. The resulting hybrid vesicles can be further tracelessly cross-linked under both UV light (e,g., 365 nm) and visible light (e.g., 410 nm) irradiation. Giving that AuNPs could be potentially used for CT contrast agents and AuNPs with small sizes were more beneficial for CT contrast agent,61 we further estimated the potency of PV-1 vesicles as CT contrast agents. Notably, iodinated small molecules have been widely used in clinical practice for CT scanning. These small molecule-based contrast agents were subjected to quick renal clearance after administration, exhibiting a very narrow imaging window.62 We envisioned that the cross-linkable hybrid vesicles could overcome this drawback by taking advantage of the elongated D

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Macromolecules circulation time. More importantly, after cross-linking, the AuNPs were held together by covalent bonds within the crosslinked vesicles that should avoid being disassembled into small nanoparticles upon high shear stress and high dilution after administration. X-ray phantom images revealed that the signals became brighter upon increasing the concentration of crosslinked PV-1 vesicles. A similar phenomenon was observed for the commercially available Xenetix 300, but a much higher concentration was needed to achieve the same Hounsfield unit (HU) values. Quantitative analysis revealed that the slopes of cross-linked PV-1 vesicles and Xenetix 300 were 90.5 and 28.2, respectively. Therefore, the cross-linked PV-1 vesicles were more efficient in attenuating X-ray and could be potentially used for CT contrast agents. Moreover, the slope of crosslinked PV-1 vesicles was also higher than that of CTABstabilized Au NPs (81.3) and untreated PV-1 vesicles (86.4), further confirming that the cross-linkable of PV-1 vesicles were more advantageous for in vivo CT applications (Figure S15). Besides high X-ray attenuation, Au NPs could be used for photothermal agents as well by taking advantage of the high absorption coefficient.17 However, the SPR peak of PV-1 vesicles labeled with small AuNPs (e.g., 3 nm) was rather weak, impeding their photothermal therapy application. To explore the potential application of the cross-linkable hybrid vesicles in synergistic PTT and chemotherapy, AuNPs with an average diameter of ∼13 nm were used, and hybrid PV-2 amphiphiles were synthesized. Interestingly, TEM and SEM observations revealed that PV-2 amphiphiles could self-assemble into hybrid vesicles as well using a cosolvent self-assembly procedure (Figure 3a,b). Moreover, the vesicular nanostructure was also retained after light irradiation (Figure 3c,d), akin to that of PV1 vesicles, although the degradation of NBOC moieties was not readily discerned by UV−vis spectroscopy since the formation of o-nitrosobenzaldehyde was masked by the intense absorbance of AuNPs (Figure S16). However, the light irradiation process (e.g., 365 nm for 30 min) led to a slight increase in the ⟨Dh⟩ from 194 to 212 nm (Figure 3e and Figure S17a). Meanwhile, the zeta potential of PV-2 vesicles increased from −16.8 to −10.4 mV under 30 min irradiation (Figure S17b). This phenomenon could be interpreted by the generation of positively charged primary amines moieties under UV irradiation, which partially compensated negatively charged potential of the PV-2 vesicles.53 The irradiated vesicles cannot be disassociated into unimers either and the size of the irradiated PV-2 vesicles swelled to 308 nm after dilution with 9-fold THF (Figure 3e). Notably, the formation of cross-linked PV-2 vesicles could also be validated by the SPR changes. Specifically, the SPR peak of monodispersed PV-2 amphiphiles in THF was centered at 527 nm, which was bathochromically shifted to 560 nm after the formation of hybrid PV-2 vesicles due to the LSPR effect of AuNPs within the hybrid membranes.33,34,36,63 However, the maximum SPR peak of nonirradiated PV-2 vesicles shifted to the original position after dilution with THF (Figure 3f). By sharp contrast, once irradiated with 365 nm light for 20 min, the SPR peaks shifted to 545 nm even after dilution with THF, indicating the formation of cross-linked vesicles that prevented the AuNPs from being disintegrated into dispersed nanoparticles. Nevertheless, there were no significant shifts in the SPR peaks before and after light irradiation in aqueous solutions, rendering the hybrid vesicles suitable for a stable plasmonic photothermal agent (Figure 3f).

Figure 3. (a, c) TEM and (b, d) SEM images recorded for PV-2 vesicles (a, b) before and (c, d) after UV 365 nm irradiation (∼1.0 mW/cm2). (e) Hydrodynamic distributions of (i) sodium citratestabilized AuNPs in water, (ii) PV-2 in THF, and (iii) untreated PV-2 vesicles and UV-irradiated PV-2 vesicles (iv) before and (v) after dilution with THF (THF/water, v/v, 9/1). (f) UV/vis absorbance spectra recorded for PV-2 in THF and untreated and UV-irradiated PV-2 vesicles before and after dilution with THF (THF/water, v/v, 9/1).

Subsequently, we examined the potential application of the hybrid PV-2 vesicles as a drug container and plasmonic PTT agent. First, both hydrophobic PTX and hydrophilic DOX were synergistically encapsulated into PV-2 vesicles (Figure 4a). Considering that 365 nm UV light may be detrimental to living organisms, 410 nm blue light was applied throughout the cell studies. Upon irradiation of the hybrid vesicles with 410 nm light, sustained corelease of PTX and DOX could be achieved (Figure 4b,c). Moreover, the drug release contents were highly dependent on the irradiation time, and a longer irradiation time led to more drug release. Specifically, after irradiation of the PV-2 vesicles for 20 min, ∼83.7% DOX and ∼85.3% PTX were released within 16 h incubation period, whereas ∼22.4% DOX and ∼14.2% PTX were released without irradiation under otherwise identical conditions. CLSM studies revealed that the PV-2 vesicles could be efficiently taken up by HepG2 cells. The red fluorescence of loaded DOX could be discerned after 2 h incubation that was E

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Figure 4. (a) Schematics of phototriggered corelease of hydrophobic PTX and hydrophilic DOX from hybrid PV-2 vesicles. (b, c) In vitro release profiles of (b) DOX and (c) PTX from hybrid PV-2 vesicles without and with 410 nm light irradiation for varying times. (d) Representative CLSM images recorded for HepG2 cells after incubation with DOX and PTX coloaded hybrid PV-2 vesicles for 2 h, followed by rinsing with PBS buffer and 410 nm light irradiation for 10 min; the cells were further incubated for an additional 2, 6, and 10 h. Late endosomes/lysosomes and cell nuclei were stained with LysoTracker green (green channel) and DAPI (blue channel). (e) Changes of colocalization ratios between the red channel (DOX) and the green channel (LysoTracker green) and (f) normalized fluorescence intensities of DOX within cell nuclei and with and without 410 nm light irradiation for 10 min quantified from CLSM images.

5a). Interestingly, the photothermal effect of the PV-2 vesicles did not compromise after 410 nm light irradiation that resulted in the formation of cross-linked hybrid vesicles (Figure 5a). This was in line with the UV−vis spectroscopy, demonstrating that the photoirradiation process did not shift the SPR peaks (Figure 3f). Note that the photothermal effect of the PV-2 vesicles was better than that of dispersed AuNPs and PBS buffer control under the same condition, which was attributable to the LSPR of AuNPs within the PV-2 vesicles. Importantly, the AuNPs were confined within the cross-linked networks and exhibited a stable photothermal conversion capability, which was in sharp contrast to previously reported plasmonic vesicle-based photothermal agents that were disrupted or collapsed after light irradiation due to the local high heat.31,34 Cell viability tests revealed that neither 410 nor 560 nm light irradiation incurred significant cell death in the absence of PV2 vesicles (Figure 5b). Moreover, PV-2 vesicles with or without 410 nm irradiation were nontoxic to HepG2 cells, and over 90% cells survived at a concentration of 266 μg/mL (Figure S19). By contrast, ∼50% of cells survived after irradiation with 560 nm light for 30 min in the presence of preirradiated PV-2 vesicles (410 nm for 10 min) without DOX/PTX loading. Therefore, the cell death, in this case, was primarily ascribed to the photothermal effect of PV-2 vesicles under 560 nm irradiation. Moreover, in the presence of DOX/ PTX-loaded PV-2 vesicles, the cell viability further dropped to

gradually intensified along with increasing the incubation time (Figure 4d). However, the internalized PV-2 vesicles were primarily trapped within endolysosomes, and the loaded DOX drug cannot be efficiently released without light irradiation (Figure S18). The colocalization ratio between the red channel (DOX) and the green channel (Lysotracker green) was calculated to be ∼85% and remained almost a constant within 10 h (Figure 4e). However, once the PV-2 vesicles were irradiated with 410 nm light for 10 min, DOX could be released from the plasmonic vectors, and the colocalization ratio of the red/green channels dropped to 45% (Figure 4e) after the same incubation time (e.g., 10 h). Moreover, normalized fluorescence intensities within the nuclei suggested that the DOX fluorescence exhibited negligible changes without light irradiation, whereas a significant fluorescence increase was observed after light irradiation (Figure 4f). This result was in good agreement with the retention of DOX within endolysosomes without light irradiation and subsequent light-actuated DOX release. Therefore, spatiotemporal release of encapsulated drugs from the plasmonic vesicles can be manipulated by light irradiation. On the other hand, in addition to chemotherapy, the hybrid vesicles loading with AuNPs rendered it suitable for PTT as well by taking advantage of the LSPR effect. Next, we investigated the photothermal effect of the PV-2 vesicles. Upon irradiation of the PV-2 vesicles with a 560 nm lamp, there was a temperature rise of 9.5 °C within 30 min (Figure F

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Figure 5. (a) Temperature variations of PBS buffer and aqueous dispersions of (0.8 mg/mL) sodium citrate-stabilized AuNPs and untreated and preirradiated (410 nm) PV-2 vesicles under 560 nm light irradiation. (b) Cell viability determined by MTT assay of HepG2 cells under different conditions: without PV-2 vesicles and light irradiation (black bar), without PV-2 vesicles with 410 nm (red bar) or 560 nm (green bar) light irradiation, with drug free PV-2 vesicles under 410 and 560 nm irradiation (blue bar) and DOX/PTX coloaded PV-2 vesicles under 410 and 560 nm irradiation (cyan bar). Error bars represent mean ± SD, n = 4. (c) Chemotherapy and PTT-induced death of HepG2 cells were evaluated by Live/Dead assay (carboxyfluorescein diacetate/propidium iodide). Representative CLSM images of HepG2 cells: (I) without PV-2 vesicles under 410 nm light irradiation for 10 min and 560 nm light irradiation for 30 min; (II) after incubation with drug free PV-2 vesicles for 2 h, followed by irradiation with 410 nm for 10 min and 560 nm for 30 min; (III) after incubation with DOX/PTX coloaded PV-2 vesicles for 2 h, followed by irradiation with 410 nm light for 10 min and 560 nm light for 30 min.

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∼5% after irradiation with 410 and 560 nm light, which could be facilely observed by CLSM images probed by a live/dead assay (Figure 5b,c) and the cellular morphology changes (Figure S20). The remarkable decrease in cell viability should be ascribed to the chemotherapy effect of the released DOX and PTX drugs under 410 nm light irradiation and PTT effect of PV-2 vesicles under 560 nm irradiation. Apparently, the combination of chemotherapy and plasmonic PTT gave rise to a synergistic therapeutic performance.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b01653.

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CONCLUSIONS

In conclusion, amphiphilic AuNPs with varying diameters modified with hydrophobic PNBOC chains and hydrophilic PEG chains have been prepared. The resultant hybrid amphiphiles self-assembled into plasmonic vesicles, which can simultaneously encapsulate both hydrophobic PTX and hydrophilic DOX drugs. Under 410 nm light irradiation, intra/ interchain amidation reactions occurred within the bilayer membranes, concurrently cross-linking and permeabilizing the plasmonic vesicles. As such, the synergistic release of encapsulated drugs can be realized under 410 nm light irradiation. In addition, the cross-linked hybrid vesicles loaded with highly densely packed AuNPs can generate localized high heat under 560 nm irradiation, exerting extra PTT function in addition to chemotherapy. Moreover, the cross-linkable plasmonic vesicles can be used for CT contrast agents as well. The cross-linkable hybrid vesicles could potentially be employed as a multifunctional theranostic nanovector, and the synergistic therapeutic performance of chemo-phototherapy could be in situ reported by CT imaging.

Additional schemes, 1H NMR spectra, 13C NMR spectra, FT-IR spectra, UV/vis absorbance, fluorescence spectra, TEM images, TGA measurements, DLS, XPS, drug release profiles, confocal images, and cytotoxicity assay (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.H.). *E-mail: [email protected] (S.L.). ORCID

Kangning Zhu: 0000-0002-2020-4495 Jinming Hu: 0000-0002-6969-1343 Shiyong Liu: 0000-0002-9789-6282 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support from Natural Science Foundation of China (NNSFC) project (51690150, 51690154, 21674103, 51722307, 51673179, and 51773190), Natural Science Foundation of Anhui Province (1708085QB34), the International S&T Cooperation Program of China (ISTCP) of the MOST (2016YFE0129700), and the Fundamental Research G

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DOI: 10.1021/acs.macromol.8b01653 Macromolecules XXXX, XXX, XXX−XXX