Graphene Quantum Dots-Mediated Theranostic Penetrative Delivery

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Graphene Quantum Dots-Mediated Theranostic Penetrative Delivery of Drug and Photolytics in Deep Tumors by Targeted Biomimetic Nanosponges Shou-Yuan Sung, Yu-Lin Su, Wei Cheng, Pei-Feng Hu, Chi-Shiun Chiang, Wen-Ting Chen, and Shang- Hsiu Hu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03249 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Graphene Quantum Dots-Mediated Theranostic Penetrative Delivery of Drug and Photolytics in Deep Tumors by Targeted Biomimetic Nanosponges Shou-Yuan Sung†, Yu-Lin Su†, Wei Cheng†, Pei-Feng Hu†, Chi-Shiun Chiang†, Wen-Ting Chen‡, Shang-Hsiu Hu†,*

†Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan. ‡Institute

of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan.

KEYWORDS: drug delivery, mesoporous structure, red blood cell (RBC) membrane, photoresponsive, graphene quantum dots, tumor therapy

Corresponding Author *Address correspondence to [email protected]

ACS Paragon Plus Environment

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ABSTRACT Dual targeted delivery of drugs and energy by nanohybrids can potentially alleviate side effects and improve the unique features required for precision medicine. To realize this aim, however, the hybrids which are often rapidly removal from circulation and piled up tumors periphery near the blood vessels must address the difficulties in low blood half-lives and tumor penetration. In this study, a sponge-inspired carbon composites-supported red blood cell (RBC) membrane that doubles as a stealth agent and photolytic carrier that transports tumor-penetrative agents (graphene quantum dots and docetaxel (GQD-D)) and heat with irradiation was developed. The RBC-membrane enveloped nanosponge (RBC@NS) integrated to a targeted protein that accumulates in tumor spheroids via high lateral bilayer fluidity exhibits an 8-fold increase in accumulation compared to the NS. Penetrative delivery of GQDs to tumor sites is actuated by near-infrared irradiation through a one-atom-thick structure, facilitating penetration and drug delivery deep into the tumor tissue. The synergy of chemotherapy and photolytic effects was delivered by the theranostic GQDs deep into tumors, which effectively damaged and inhibited the tumor in 21 days when treated with a single irradiation. This targeted RBC@GQD-D/NS with the capabilities of enhanced tumor targeting, NIR-induced drug penetration into tumors, and thermal ablation for photolytic therapy promotes tumor suppression and exhibits potential for other biomedical applications.


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Theranostic nanohybrids with highly integrated functionalities are currently of significant interest for enhancing the therapeutic effects of existing agents and accumulation of therapeutics at targeted sites.1–5 Despite current recent advance in amplified therapeutic efficiency, the difficulties of effective targeting and penetrating in the tumor still stem from the innate immune system and tumor heterogeneity. First, once the nanoparticles enter the bloodstream, the innate immune system readily recognize them as intruders and clear the particles by the reticuloendothelial system /mononuclear phagocyte system, leading to low delivery efficacy.6–8 Second, the enhanced permeability and retention (EPR) effect improves the accumulation of particles, but low proportion of an administered dose is achieved to deep tumor via this mechanism due to the dense physiological barrier of the tumor.9,10 This obstacle is led by high interstitial fluid pressure (IFP) of the tumor and to cancer-associated fibroblasts in tumors, which lower the transport of foreign matter and lead to the release of therapeutic agents around the perivascular cells of tumors.11–14 Thus, it is necessary to develop stealth and penetrating drug delivery systems to deliver therapeutic cargos for enhanced tumor therapy. A simple solution to address these challenges caused by the innate immune system is to engineer nanoparticles with anti-fouling surfaces to avoid nonspecific serum-protein adsorption. In this regard, polyethylene glycol (PEG) offers unique features, such as reduction of nonspecific interactions and suppression of reticuloendothelial system (RES) uptake between particles and blood components to protect particles from blood clearance via stealth coating.15–18 However, repeated injection of PEGylated particles has been documented to lead the immune rejection via the production of anti-PEG


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immunoglobulin M (IgM) antibodies.19 Recently, the biomimetic strategy has been developed to prolong the blood circulating lifetime and reduce nonspecific macrophage uptake of nanoparticles by coating with membranes of red blood cells(RBCs) or macrophages.20–22 The RBCm-NPs exhibited a considerably longer circulation time than PEGylated NPs in a mouse model,23,24 which is mainly attributed to the transmembrane protein CD47, a “self-marker” on cell membranes that signals the phagocyte receptor CD172a and inhibits the immune response.25 Inspired by this concept, NPs surfaces integrally coated with natural RBCms exhibit a distinct top-down design, reproducing the surface antigenic characteristics of RBCs.26–28 Through RBCm coating, the effective shielding AuNP surfaces from thiolated probes have been demonstrated to cause a fourfold reduction in NP uptake by macrophages in vitro, and exhibited excellent in vivo blood circulation lifetime and significantly enhance tumor uptake.29 Furthermore, the biomimetic RBC-camouflaged iron oxide particles relied on CD47 is able to escape immune recognition via interacting with the SIRP-α receptor.30 However, most studies revealed the prolonged the circulation time of particles after RBCm coating yet focused on improve the enhanced tumor accumulation. Therefore, integrating RBCm to targeted delivery systems with multi-functional characteristics are received great interest for cancer therapy due to the improved targeting and therapeutic efficiency of these systems. In addition to boosting tumor accumulation, another key issue in tumor therapy is the penetrated ability of particles in deep tumor. Low penetration efficiency of particles suffers from the rapid removal of particles at the tumor site once the accumulation of particles only occurs via the EPR effect.


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There is increasing evidence indicating that particles with hundreds of nanometers in size are too large and impractical for deep penetration into tumors, and smaller-sized particles have shown superior tumor penetration capabilities.31,32 However, small particles may have shorter blood half-lives and may be distributed to normal tissues, resulting in adverse effects.33 Graphene oxide (GO) nanosheets with flexibility, large amounts of functional groups, and high electronic conductivity have been applied in delivery systems.34–36 The therapeutic cargos are typically tethered on GO for effective drug delivery. Upon NIR irradiation, the energy induced by photothermal conversion can not only release therapeutic cargos but also rupture the vesicle to suppress the tumor.37,38 Another nanosheets, graphene quantum dots (GQDs) possessing ultra-small size, versatile photoluminescence and mechanical characteristics, have also been noticed in physics and chemistry science. Furthermore, the unique corners or asperities of nanosheets exhibiting rich irregular edges facilitate the cell membrane penetration of GO.39,40 However, most current research has mainly issued by their preparations and physical properties.41 Few works have applied in biomedical application and were principally focused on cell interactions.42 With exceptional optical characteristics and transmembrane ability, the ultra-small GQDs taking advantage of imaging agents and labeling cell membrane are a potential agent for drug transportation for cancer therapy.43 Therefore, the integration of compact vesicles and theranostics GQDs to transport large payloads of drugs and overcome the obstacles of tumor physiology is able to probably increase the accumulation and permeability of drug in targeted tissue. In this study, a targeted RBC-membrane-enveloped nanosponge (RBC@NS) that combines the


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features of stealth and large payloads of functional agents to overcome the low EPR effect and heterogeneity of tumors was developed. Being biocompatible and light-responsive, this carbon-based porous particle functionally behaves as an RBC-camouflaged particle and exhibits superior tumor targeting and penetrating capabilities. The targeted nano-sponge, composed of protein/RBC membranes (targeting and stealth properties), porous carbon/silica (hydrophobic, capable of therapeutic agent transport) and graphene quantum dots (GQDs)/drug (photoresponsive, tumorpenetrating), was injected into a mouse model to deliver the anti-cancer drug docetaxel (DTX) and GQDs for penetrated delivery of drug to tumor (Figure 1). Cetuximab (Ct), which exhibits targeting properties, is anchored to the RBC layer to enhance particle accumulation at tumors. The nano-sponge loads and transports large payloads of GQD/DTX to the tumor and serves as a photopenetrative and photolytic agent for tumor penetration. While applying NIR, the release of GQD/DTX is triggered by the localized heat of NS, and thus, the tumor is damaged and the penetration efficiency of the therapeutic agents is improved. Furthermore, the synergistic effect of thermotherapy and GQD/drugpenetrated chemotherapy-suppressed tumors growth in NIR-irradiated as well as NIR-omitted areas of tumor cells prevent tumor recurrence.

Results and discussion. The versatile particles are prepared by applying scalable approaches introducing GQDs as tumor-penetrative drug delivery platforms (Figure 2a). GQDs were prepared from artificial graphite (AG) via a modified process of bottom-up fabricated AG approach.44 First, the hexaphenylbenzene (HPB) containing six benzyl groups was bonded via oxidation to form


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hexabenzocoronene (HBC), and then, HBCs led to the formation of artificial graphite (AG). Then, the AG was oxidized and exfoliated by Hummers’ method to obtain artificial graphene oxide (AGO). Raman spectroscopy was used to evaluate the structure of AGO (Figure S1a, Supporting Information). Both in-phase sp2 bond vibration (G band) at 1580 cm−1 and the disorder band of the graphene edge functional group (D band) at 1350 cm−1 were detected for graphite and were characteristic of the presence of GO. The ratio of the D-band and G-band intensities (ID/IG) of AGO determined by Raman spectrum was approximately 1.1. Furthermore, the ID/IG were also indicative of the fragile structure of ultra-small GQDs through the degree of disorder in GO. Subsequently, AGO was reduced by hydrazine to obtain the resulting GQDs. The size of resulting GQDs ranged from 2 to 6 nm, as evaluated by TEM in Figure 2b. In Figure 2c, the photoluminescence of the GQDs showed an emission wavelength of approximately 450 nm, indicating the strong blue photoluminescent emission of GQDs.44 With the variation of excitation from 319 to 382 nm, the emission wavelength of the tunable photoluminescence correspondingly shifted from 381 to 461 nm (Figure 2c). The atomic force microscopic (Figure S1b, Supporting Information) images displayed that the thickness of GQD is about 1 nm. According to literature, single-layered graphene quantum dots or graphene oxide nanosheets possess the thickness of 0.7 nm by AFM measurements. The thickness of GQD may be attributed to the low content of defects or hydroxyl group on its surface. On the other hand, GQDs served as potent drug-absorbents, incorporating the hydrophobic drug docetaxel (DTX) via π-π interactions. To obtain the silica/carbon vesicle, mesoporous silica with large pores was synthesized in advance.


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The silica spheres were prepared by tetraethylorthosilicate (TEOS) hydrolysis via a template-based mechanism by using cetyltrimethyl ammonium bromide (CTAB) as a surfactant.45 Meanwhile, styrene monomers were polymerized with the CTAB template during silica formation to obtain a porous composite structure. Then, the polystyrene was pyrolyzed at 800 °C in an inert gas atmosphere (N2) to form an ultra-thin carbon layer on the surfaces of the pores. The particle was termed as NS. The carbon surface of NS exhibited hydrophobicity, which facilitated the encapsulation of GQD/DTX in the NS. Then, the particles were capped with RBCm and Ct via fusion.46 The application of RBCms and targeting agents to the shell of the porous particles was considered by several reasons. First, the stability of the lipids and proteins of RBCms on porous particles is better than that of lipids on liposomal particles due to high adhesion energy. Second, compared to RBCms on non-porous templates, the porous surfaces of the particle exhibit excellent lateral bilayer fluidity, potentially improving targeting efficacy. Third, particle recognized by immune system and undesired serum/protein adsorption in vivo is able to be decreased through RBCms coating. The development of this Ct-RBC@NS is an overture at applying this strategy to fabricate targeted RBCm-based nanosponges with integrated functions. Based on scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the carbon/silica spheres with an average diameter of 120 nm exhibits porous structures with high densities, forming a carbon composite that was named nanosponge (NS) (Figure 2d and 2e). To investigate the porous structures, the particles were measured by Brunauer-Emmett-Teller (BET) analysis via nitrogen


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absorption/desorption isotherms, and the results revealed that the NS had a surface area of ~130 m2g1

and a pore size of ~12 nm (Figure S2a and S2b, Supporting Information). Upon the removal of

polystyrene from the preparation, the mean size of pore reduced to 1.7 nm, implying that the large pores of the NS were induced by polystyrene incorporation (Figure S2c, Supporting Information). Similar effects of polymers on the formation of pores in silica has also been demonstrated by Nandiyanto et al., who showed that styrene concentrations affect pores sizes.47 After GQD/DTX loading, the GQD/DTX could be observed in the pores of the NS by TEM (Figure 2f and 2g), and the pore volume decreased by approximately 50 %, as evaluated by the N2 adsorption isotherm, indicating that the pores had been partially filled (Figure S2a, Supporting Information). Figure 2h and 2i show TEM images of the NS capped by RBCm and Ct. Due to the uniform capping of the RBCm and the proteins, the porous NS adopted a core-shell structure, and the thickness of shell was approximately 7 nm. The porous structure of silica becoming invisible after RBC membrane coating was induced by the large amounts of protein and lipid covering which affected the electrons passing and reduced the resolution. Figure 2j displayed the size distribution of Ct-RBCm@NS (260±120 nm) after the NS (170±80 nm) was coated with RBCm/Ct, measured by dynamic light scattering. Furthermore, the surface zeta potential of NS changed from −16.1 mV to −10.2 mV after RBCm/Ct fusion (Figure 2k). The shift in surface charge was caused by the RBC membranes, the surfaces of which possessed a weak negative charge. To evaluate the RBCms on the NS, the RBCms removed from the NS were analyzed by SDS-PAGE. The results showed the presence of spectrin cytoskeletal proteins (peripheral


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membrane proteins, CD47), observed as a band at approximately 51 kDa (Figure S3a, Supporting Information). To investigate specific markers, western blotting analysis was also carried out for characteristic RBC markers CD47 (Figure S3b, Supporting Information). Other proteins on RBC membrane including Band 3 and glycophorin were also labelled on RBC@NS. These proteins have been documented to improve the biocompatibility of RBC-derived drug delivery systems.48,49 When a dilute solution of RBC@NS was detected by confocal microscope, the clear colocalization of fluorescent dots was monitored, suggesting the coating of RBC membrane on NS (Figure S4a to S4e, Supporting Information). Furthermore, CD47, Band 3 and glycophorin were labelled on RBC@NS and evaluated by fluorescence changes. The results potentially indicated that the proteins of RBC membrane on NS still maintained their orientation (Figure S4g, Supporting Information). Next, fluorescence recovery after photobleaching (FRAP) evaluated the fluidity of RBCms and Ct on the porous carbon surface through the different degrees of fluidity (Figure S2l). The Ct@NS sample was prepared by Ct conjugation, and RBCm@NS was fabricated by fusion. The RBCms on the NS displayed much faster fluorescence recovery than Ct or the fluorescent dye (FITC) on the NS at 37 °C, suggesting the excellent long-range motion of lipid bilayers of RBCms. Ct-RBC on NS displayed slower fluorescence recovery than RBC on NS, suggesting that the Ct delayed the motion of RBCms on the identical particle. Such in-phase fluidity of bi-layered lipids on porous silica has also been documented in literature.50 To verify that it was possible to fuse Ct and RBC membrance, fluorescence recovery after photobleaching (FRAP) was also applied to evaluate the fluidity of Ct-RBC on the


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porous carbon surface. As shown in Figure S2l, the Ct-RBC on NS displayed slower fluorescence recovery than RBC on NS, suggesting that the Ct delayed the fluidity of the lipid bilayers of RBCms on the identical particle since the protein possessed large molecular size and low fluidity on particle when compared to lipid bilayer.50 Furthermore, Ct-RBC@NS was prepared to examine the fusion. RBCm stained by red fluorescence was fused by Ct which is appeared in green fluorescence. Observed via a confocal microscopy, the colocalization of two signals was detected (Figure S5a). However, a mixture of Ct@NS and RBC@NS represented distinct locations. The result suggested that both Ct and RBCm resided on NS and indicated the fusing Ct to RBC on one NS. The inorganic/organic ratio and UV-vis absorption spectrum of the NS and Ct-RBC@NS are also provided (Figure S5b and S5c, Supporting Information). Furthermore, both NS and Ct-RBC@GQD-D/NS displayed slight size changes within 2 days in D.I. water (Figure S6a, Supporting Information). Some aggregation of NS was observed after 3 days, but Ct-RBC@GQD-D/NS still exhibited good colloidal stability in water over 7 days. In cell culture medium (DMEM supplemented with 10% FBS serum), the aggregation of NS was diminished, and the Ct-RBC@GQD-D/NS exhibited excellent stability after 7 days (Figure S6b, Supporting Information). The colloidal stability of NS and Ct-RBC@GQD-D/NS were evaluated in fresh homologous serum over 7 days. The aggregation of NS was detected after 3 days, and CtRBC@GQD-D/NS maintain the superior stability in in fresh homologous serum over 7 days (Figure S6c, Supporting Information). The DLS measurement suggested that RBC membrane prevented the aggregation of NS.


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When the particles were subjected to NIR irradiation (1.5 W cm-2) for 10 min, the temperature was increased by photothermal conversion in GQDs (0.5 mg/mL), GQD/NS (3.8 mg/mL), RBCm@GQD/NS (4.6 mg/mL), and Ct-RBC@GQD-D/NS (4.8 mg/mL) in aqueous solution; the particle solutions had similar concentrations to the NS (Figure 3a). Since water (ddH2O) kept the temperature after irradiation, the heat generation was caused from material absorption. Similar to reduced graphene oxide, the GQDs were seen to be superb photothermal agents, exhibiting strong photothermal conversion and heating the solution to approximately 70 °C within 5 min. When compared to GQDs, GQD/NS, RBC@GQD/NS and Ct-RBC@GQD-D/NS had a slower heating rates. The difference in photothermal efficiency is caused by irradiation absorption, which was weaker for RBC@GQD/NS owing to deflection by the organic shell. Porous silica particles (PSPs), prepared by pyrolyzing the polystyrene/silica spheres at 800 °C, exhibit no heating ability under NIR irradiation owing to the carbon-free structure of these particles. The docetaxel (DTX, a hydrophobic drug)-loading capacity of the GQDs@NS, NS and PSP was much higher than that of the GQDs and PS@NS (Figure 3b). The excellent loading capacity potentially reflects the affinity between DTX and the large hydrophobic surface of the NS. Carbon-based porous materials have been used as potent moleculeabsorbent vehicles. The release patterns of the PS@NS, PSP, GQD/NS and RBC@GQD/NS in DMSO also supported this observation (Figure 3c). While the non-porous PS@NS and porous PSP released over 30 % drug within 42 h, the GQD/NS and RBC@GQD/NS released less than 9 % of the corresponding DTX loads. While subjecting the GQD/NS and RBC@GQD/NS to NIR irradiation at


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1.5 W cm-2 for 1 min, distinct enhancement of drug release by photon treatment was exhibited in Figure 3d. With a 1 min exposure to NIR radiation, both GQD/NS and RBC@GQD/NS demonstrated bursts of drug release, and then, after the removal of radiation, these particles showed much slower release rates. The triggered release may be contributed by loosening of the hydrophobic-hydrophbic interactions between drug the materials by thermal heating. Furthermore, no changes in DTX structure were seen after the DTX was released from the NS, as estimated by HPLC (Figure S7, Supporting Information). To verify whether the GQDs will be released from the NS, the GQDs@NS was subjected to NIR irradiation at 1.5 W cm-2 for 3 min. High-resolution TEM (Figure S8a, Supporting Information) showed that the NS were surrounded by the small particles, indicating that thermal heating can cause GQD release from the NS. Figure S8b in Supporting Information shows the size distribution of the released GQDs after NIR irradiation at 1.5 W cm-2 for 3 min. To evaluate the release of GQD from CtRBC@GQD-D/NS, the Ct-RBC@GQD-D/NS solution was irradiated by NIR irradiation at 1.5 W cm2 for 3 min, and then, the particles were separated by centrifugation at 4,000 rpm for 5 min. The TEM image of suspension displayed amounts of GQDs surrounded by Ct and RBCm, indicating the effective release of GQDs from Ct-RBC@GQD-D/NS (Figure S8c, Supporting Information). Furthermore, the strong fluorescence signal of rhodamin-labeled GQDs can be detected in the suspension after NIR irradiation (Figure S8d, Supporting Information). Once the organic components of collected suspension were removed by calcination at 800 oC in an inert gas atmosphere (N2) and re-dissolved into de-ionic water, the size distribution of particles exhibited the similar profile as GQDs, suggesting


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the GQD releasing (Figure S8e, Supporting Information). To quantify the release of free DTX and DTX attached on GQD, free DTX and DTX attached on GQD were separated by the centrifugation at 20,000 rpm for 20 min after NIR irradiation treating RBC@D-GQD/NS. Subsequently, free DTX and DTX attached on GQD was extracted by chloroform and the concentration of DTX was determined by HPLC. As shown in Figure S9a, when RBC@DGQD/NS was subjected to NIR irradiation for 1 min, the release amounts of free DTX and DTX attached on GQD reached more than 30 %. Increasing the irradiation time to 10 min, the 53% of free DTX release was observed and the amount of DTX attached on GQD was decreased to 21%. The mild release of free DTX under 10 min of NIR irradiation was probably caused by the weak affinity of DTX and PBS solution. In this condition, more than 20 % of DTX can still attach on the GQD after 10 min of NIT irradiation. Based on this result, part of DTX was still adsorbed on GQD. Furthermore, the enhanced DTX penetration effect mediated by GQD were also evaluated by a tissue-like prosthesis (10 wt% collagen) when DTX released from RBC@D-GQD/NS (Figure S9b, Supporting Information). Briefly, RBC@D-NS and RBC@GQD-D/NS were placed on the tissue-like prosthesis and irradiated by NIR irradiation for various time. After the irradiation treatment, the particle and free drug were removed by washing the gel surface. Then, the DTX was extracted by chloroform and the concentration of DTX was determined by HPLC. The results exhibited that the increase of DTX amount in a tissue-like prosthesis was observed while increasing the irradiation time. However, the concentration of DTX in the gel released from RBC@D-NS was lower than that from RBC@D-


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GQD/NS. After 10 min of irradiation, more than 30% of released DTX can be detected from RBC@DGQD/NS, but only 6 % of DTX was resided in the gel for RBC@D-NS, suggesting the enhanced penetration of DTX by GQD. Beside, three-dimensional confocal laser scanning microscope (CLSM) images of a tissue-like prosthesis revealed that the penetration depth of DTX/GQD can reach about 300 m after 10 min of NIR irradiation. To evaluate the effects of coating the surface of the NS on cell viability, the GQDs/NS, RBC@GQD/NS and Ct/RBC@GQD/NS were co-cultured with A549 cells (a lung cancer cell line) for distinct concentrations. When the concentration of the drug-free particle reached 20 g/mL, cell viability was approximately 92 % for the GQDs/NS and 90 % for the RBC@GQD/NS and Ct/RBC@GQD/NS, suggesting the low toxicity of these particles toward cells (see Figure S10 in Supporting Information). For effective drug delivery and photothermal therapy, vesicles that can transport cargo into cancer cells are desirable. Cetuximab (Ct), an antibody, targets epidermal growth factor receptor (EGFR)-overexpressing cancer cells and increases survival rates in clinics for EGFRpositive colorectal cancer, metastatic non-small cell lung cancer, and head/neck cancer.47 Once EGFRexpressing cancer cells bind to Ct, complement activation is initiated, which can directly lead to the deposition of complement components and to complement-mediated cell death. To demonstrate the internalization of RBC@NS, we first anchored Ct onto the surface of the NS via extrusion with RBC. Quantum dots were embedded in the hydrophobic pores of the RBC@NS and Ct/RBC@NS to evaluate effects on cell uptake (Figure 3e). Since A549 cells overexpress Ct receptors on the surface, incubation


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of these cells with the Ct/RBC@NS led to strong cell-uptake efficiency, as estimated by flow cytometry (Figure 3f). Compared to the Ct/RBC@NS, without the assistance of Ct targeting, the cellular uptake of the particles was weak. On the other hand, the flow cytometry was also used to estimate the quantification of RBC@NS with various numbers of Ct after 30 min of incubation to A549 cells (Figure S11). A significant increase of fluorescence signals of Ct-RBC@NS could be detected when increasing the amount of Ct on NS, suggesting that Ct maintain the strong affinity to A549 after inserting in RBC@NS. Figure 3g displays the cell-uptake results of another cell line, RAW 264.7 (a murine macrophage cell line), with low EGFR expression, and exhibits the weak cell-uptake efficiency for all groups. Fluorescence microscopy of A549 cells incubated for 30 min shows that most of the Ct/RBC@NS remained in the cytoplasm (Figure S12, Supporting Information), but only a few of RBC@NS were observed in the cell. No obvious cellular uptake of either the Ct/RBC@NS or RBC@NS was detected in RAW 264.7 cells after 30 min of incubation. By increasing the incubation time to 2 h, effective cellular uptake of Ct/RBC@NS was observed in the cells, suggesting potent internalization of these particles (Figure 3h and 3i). However, over time, no significant increase in cell uptake was observed in RAW 264.7 cells (Figure 3j and 3k). Therefore, the Ct/RBC surfaces on the NS can enhance EGFR-expressing cancer cells in vitro. A control experiment confirmed that the red fluorescence is from the QDs, since no signal was observed from A549 cells incubated with QD-free NS (Figure S13, Supporting Information). To investigate tumor penetration by particles and therapeutic agents, a tumor on a chip containing


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hundreds of uniform multicellular tumor spheroids was used. With heterogeneous cell morphologies similar to those of an in vivo tumor, and the compact cancer cells at the periphery of tumor spheroids latently decrease the penetration of most therapeutic agents into the inner regions of tumors. Uniform 3D tumor spheroids built on microfluidic chips display green fluorescence from the cell skeleton, approximately 200 m in diameter, and exhibit ultra-dense structures and clear boundaries under CLSM (Figure S14, Supporting Information). Without the assistance of Ct, most of the particles were dispersed from the tumor spheroids, and only few NS were absorbed on the spheroids (Figure 4a and 4b). In contrast, when the spheroid was incubated with the Ct/RBC@NS, the surface cells of the tumor spheroid exhibited a large amount of signal from the particles, indicating that most of the particles resided on the surface of the tumor (Figure 4c). To estimate the rate of particle accumulation, the fluorescence intensity was monitored over time. Although both the Ct-RBC@NS and Ct@NS exhibited a rapid increase in fluorescence signal at the beginning of the 2 h, the intensity exhibited by the Ct-RBC@NS was twice as strong as that exhibited by Ct@NS (Figure 4d); this result was caused by the fluidity of RBCs. When particles transporting GQD and DTX were in incubated with tumor spheroids, the fluorescence intensities of tumor were seen to be improved by approximately 5 %, indicating the absence of a significant effect on cargo loading (Figure 4e). Next, the tumor spheroids were subjected to NIR irradiation to evaluate the effect of NIR radiation on particle penetration. The tumor spheroids preserved the spheroidal morphology of the compact cells after 20 sec of NIR irradiation at 1.5 W cm-2, but the red fluorescent dots can be observed due to the release of QDs from


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the Ct-RBC@NS (Figure 4f). To track the GQD penetration in tumor spheroids, GQD labeled with Rhodamine is displayed in red. Upon high resolution of the CLSM image (Figure 4g), the bright red fluorescence was seen to be present in the inner regions of the tumor and resided in the dense cell skeletons. Even though the heat did not damage the boundary of the tumor spheroid, a high density of QDs can still be observed in the core of the tumor spheroids. This result suggested that the release of QDs, which was induced by NIR irradiation in a few seconds, substantially affected the intercellular structure and penetrated the periphery of the compact cells in the tumor spheroid. The TEM image of RBC@GQD/NS was also evaluated to clarify the desorption of membranes from NS after 1 min of NIR irradiation (1.5 W cm-2), possibly reducing the blocking effects of RBCm for GQD/drug penetration (Figure S15, Supporting Information). Cell membrane penetration of GO-based materials can be mediated by corners or asperities of these ultrathin 2D structures that have abundant irregular edges.26 The shape and ultra-small size of the GQDs improved their cellular uptake efficiency as well as improved their tumor penetration ability. Furthermore, RGD-based drug delivery system is known to bind to αvβ3/αvβ5 integrins overexpressed on the endothelial cells of tumor angiogenic vessels and cancer cells. Compared to Ct-RBC@GQD/NS, cyclic RGD (cRGD)-conjugated NS was also subjected to multicellular tumor spheroids (MTS) with an average diameter of 200 µm on a chip to investigate the tumor penetration effect (Figure S16a, Supporting Information). After 24 h of incubation, most cRGD-NS was surrounded on the surface of MTS. Furthermore, the MTS treated by cRGD-NS was subjected to 20 sec of NIR irradiation at 1.5 W cm -2; only few particles can be observed in the MTS,


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suggesting the low penetration of cRGD-NS (Figure S16b, Supporting Information). The weak penetration effect was probably caused by large size of NS. Compared to cRGD@NS, the GQDs was detected in core of MTS after 24 h of incubation (Figure S16c, Supporting Information) without any NIR irradiation. An in vitro study on photolytic therapy was conducted on tumor spheroids incubated with 10 μg/mL Ct-RBC@GQD-D/NS and subjected to NIR irradiation for various durations. After 2 h of incubation, cells were subjected to NIR irradiation (1.5 W cm– 2) for 0, 2, 4 and 10 min and then stained with Alexa Fluor 488 Phalloidin to investigate the cell morphology: F-actin appeared green, and nuclei appeared blue (see Figure S17a to S17d in Supporting Information). The morphology of the tumor spheroids was maintained before NIR irradiation (Figure S17a, Supporting Information). Upon NIR irradiation (1.5 W cm-2) of the Ct-RBC@GQD-D/NS for 2 min, the combination of photothermal GQD penetration and chemotherapy caused significant deformation of the tumor spheroids (Figure S17b, Supporting Information). With increased irradiation time, rupturing and cracking of the tumor spheroids was observed, and the cells were partially separated from the spheroids (Figure S17c, Supporting Information). Figure S17d in Supporting Information shows the strong photothermal and chemotherapeutic penetration effects on tumor spheroids with sufficient irradiation time, resulting in most of the cancer cells losing binding affinity for neighboring cells. Figure 4h and 4i demonstrate the quantification of different treatments in terms of percent cell viability. Compared to other groups, the RBC@GQD-D/NS exhibited lower toxicity to A549 cell line at a concentration of 20 g/mL.


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Meanwhile, the Ct@GQD-D/NS and Ct-RBC@GQD-D/NS exhibited increased cytotoxicity with increasing concentration. Consistent with the outcomes from the cellular internalization experiment, the particles that exhibited high cellular uptake were able to effectively transport DTX to cancer cells and induce chemotherapy (Figure 4h). Figure 4i reveals the results of cell viability upon subjecting various vesicles to 10 min of NIR irradiation (1.5 W cm-2). Few conclusions can be drawn from these outcomes. First, photothermal conversion enhanced the cell-killing effects when the GQD-D/NS and RBC@GQD-D/NS were subjected to cells via the combination of thermo- and chemotherapy. At identical concentrations, NIR irradiation for targeted and penetrated drug delivery by the CtRBC@GQD-D/NS was extremely lowered the cell survival rate to only 3 %. In particular, the CtRBC@GQD-D/NS can distinctly enhance cell viability while applying irradiation at low concentrations. These results revealed that the photothermal-conversion approach via the CtRBC@NS with the GQD and DTX improved for the suppression of tumor spheroids in vitro. To assess the in vivo targeting efficacy, 100 µL of Cy5.5-labelled RBC@NS and Ct-RBC@NS at 1.5 mg/mL was injected into nude mice bearing A549 tumor cells through the tail vein. Twenty-four hours post-injection, the biodistribution of the particles (Cy5.5, Ex-Max 640 nm/Em-Max 700 nm) was investigated by an in vivo imaging system (IVIS, Caliper Lifesciences, USA). The fluorescence intensity revealed that the accumulation of the Ct-RBC@NS was greater than that of the RBC@NS (Figure 5a), indicating accumulation enhancement by targeting. Next, photothermal conversion was carried out by subjecting particle-treated tumor-bearing mice to 1.5 W/ cm2 of NIR irradiation for 10


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min. Figure 5b shows that the tumor treated with the Ct-RBC@GQD/NS can be heated to 68 °C, which is accessible for thermal tumor ablation. Furthermore, the temperature in the tumor treated with the Ct-RBC@NS and Ct@NS increased to 62 °C and 53 °C, respectively. The saline-treated mice exhibited no apparent temperature increase after irradiation. The better photothermal conversion of the Ct-RBC@GQD/NS could be associated with the higher accumulation and to the photothermal combination effects of GQD and NS. Next, major organs and tumors were spectrally examined by the IVIS. Even though fluorescence in the liver and lungs could be detected for both the RBC@NS and Ct-RBC@NS (Figure 5c), the accumulated particles at the tumor exhibited strong fluorescence since the clearance organs possessed the ability of particle metabolism. The cumulative efficiency of the CtRBC@NS in the tumor quantified by the IVIS was 60 % higher than that of the RBC@NS. To investigate the passive targeting effect, NS, PEG@NS and RBC@NS were injected intravenously via the tail vein to the tumor-bearing mice with similar NS concentration, respectively. PEG coated-NS showed the greater passive targeting effect at tumor site than NS along (Figure S18, Supporting Information). Next, the biodistribution and tumor targeting efficiency of two types of particles were also investigated. 100 μL of solution containing with 0.5 wt% RBC@NS or Ct-RBC@NS labeled by CdSe quantum dots was injected into nude mice intravenously. Quantitative determination of Cd in clearance organs and tumors by ICP‐MS. As shown in Figure S19a in Supporting Information, CtRBC@NS exhibited greater accumulated in the tumor than RBC@NS. Even though the RBC@NS exhibit accumulation in the tumor in first day via EPR effects, the intensity decreased after 2 days,


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suggesting the weak bonding efficacy of non-targeted NS to cancer cell. In parallel, Ct-RBC@NS showed clear accumulation at targeted site after 24 h and 48 h post injection, exhibiting the increased tumor targeting of Ct-RBC@NS through the cell internalization. To quantify the amount of DTX in tissue and organ, the amount of DTX in each organ and tumor were also evaluated by HPLC. As shown in Figure S19b, both Ct-RBC@D/NS and Ct-RBC@GQD-D/NS could transport the DTX to targeted tumor site. When applying NIR irradiation to tumor at 30 min postinjection for 10 min, the increase of DTX amount at tumor was observed for two group. Remarkably, the concentration of DTX transported by Ct-RBC@GQD-D/NS+NIR exhibited about 8-fold greater than that particle without NIR treatment at tumor. It potentially indicated that the treatment enhanced the accumulation of drug at the tumor site. To evaluate the systemic circulation time of each type of particle, the CdSe quantum dots were loaded to all three types of particles through the strong hydrophobic interactions and displayed minimal release (

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