Nanocolloidosomes with Selective Drug Release for Active Tumor

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Nanocolloidosomes with Selective Drug Release for Active TumorTargeted Imaging-Guided Photothermal/Chemo Combination Therapy Hang Hu, Chen Xiao, Honglian Wu, Yihui Li, Qing Zhou, Yuxiang Tang, Chan Yu, Xiangliang Yang, and Zifu Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14796 • Publication Date (Web): 10 Nov 2017 Downloaded from http://pubs.acs.org on November 11, 2017

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ACS Applied Materials & Interfaces

Nanocolloidosomes with Selective Drug Release for Active Tumor-Targeted Imaging-Guided Photothermal/Chemo Combination Therapy

Hang Hu†,§, Chen Xiao†,§, Honglian Wu†, Yihui Li†, Qing Zhou†, Yuxiang Tang†, Chan Yu†, Xiangliang Yang*,†, and Zifu Li*,†,∥

†

National Engineering Research Center for Nanomedicine, College of Life

Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P. R. China ∥Wuhan

Institute of Biotechnology, High Tech Road 666, East Lake High Tech

Zone, Wuhan, 430040, P. R. China

*Corresponding authors: Professor Zifu Li Tel.: 86 27 87792234, Fax: 86 27 87792234, E-mail: [email protected] Professor Xiangliang Yang Tel.: 86 27 87792147, Fax: 86 27 87792234, E-mail: [email protected]

Author Contributions: §

Hang Hu and Chen Xiao contributed equally to this work.

ACS Paragon Plus Environment


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Abstract Selective drug release is highly desirable for photothermal/chemo combination therapy when two or even more theranostic agents are encapsulated together within the same nanocarrier. Conventional nanocarrier can hardly achieve this goal. Herein, doxorubicin and indocyanine green (DOX/ICG) loaded nanocolloidosomes (NCs), with selective drug release, were fabricated as a novel multifunctional theranostic nanoplatform for photothermal/chemo combination therapy. Templating from galactose functionalized hydroxyethyl starch-polycaprolactone (Gal-HES-PCL) nanoparticles stabilized Pickering emulsions, the resultant DOX/ICG@Gal-HES-PCL NCs had a diameter around 140 nm and showed outstanding tumor targeting ability, preferable tumor penetration capability, and promoted photothermal effect. Moreover, these NCs can be used for NIR fluorescence imaging and thus render real-time imaging of solid tumors with high contrast. Collectively, such NCs achieved the best in vivo anti-tumor efficacy combined with laser irradiation compared with DOX/ICG@HES-PCL NCs and DOX/ICG mixture. These NCs are valuable for active tumor-targeted imaging-guided combination therapy against liver cancer and potentially other diseases.

Keywords: Nanocolloidosomes, selective drug release, hydroxyethyl starch, active targeting, photothermal/chemo combination therapy


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1. Introduction Chemotherapy, although widely adopted in clinical settings, suffers from numerous drawbacks such as severe adverse reactions,1 low efficacies, and the possibility to induce cancer stem cell,2 multidrug resistance,3 and tumor metastasis.4 Therefore, great efforts have been devoted to the study of more effective

cancer

therapies,

including

photothermal

therapy

(PTT),5

photodynamic therapy (PDT),6-7 and immunotherapy.8 Due to the synergistic effect,

combinational

therapies

based

chemo/PTT,9-11

on

chemo/immunotherapy,12 PDT/PTT13-14 can further enhance the treatment efficacy and reduce side effects. Among these combinatory therapies, chemo/photothermal combination therapy received tremendous attention recently not only in fundamental research but also in the clinics.15-16 Accordingly, various nanocarriers have been developed to ensure that both therapeutic

drugs

and

photothermal

agents

can

be

co-delivered

simultaneously to the malignant cells.17-19 But spatial co-delivery only is far from enough. Different components, for example the Food and Drug Administration (FDA) approved theranostic agents, DOX and ICG, execute therapeutic effect via distinctive mechanisms. Once uptaken by cancer cells, DOX must be released free from the nanocarrier, whereas ICG are required to be encapsulated within the nanocarrier to achieve high stability and potent photothermal effect. This is because DOX has to enter the nucleus to interact with nuclear acids and exert its therapeutic effect,20 whereas free ICG is



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unstable and degrades quickly.21-22 It is, therefore, highly desirable to develop nanocarrier with selective drug release for efficient chemo/photothermal combination therapies. Colloidosomes, composed of a shell of densely packed colloidal particles, can allow for selective release via the interstitials formed by neighboring colloidal particles,23-25 and appear to be ideal carrier for chemo/photothermal combination therapies. The selective release can be easily tuned by modulating the size of colloidal particles on the shell, the colloidal particle packing density, and additional polymers.26-27 The selectivity can be further manipulated by constructing colloidosomes with stimuli responsive colloidal particles such as thermal responsive microgels28-29 and pH responsive nanoparticles.30 These colloidosomes are usually templating from Pickering emulsion droplets, where nanoparticles of a minimum size (˃10 nm) is required for favorable interfacial energy gain, and thereby making resultant colloidosomes typically larger than 500 nm.31-33 Nonetheless, for prolonged blood circulation as well as efficient tumor targeting, accumulation, and penetration, the ideal nanocarrier should have a diameter smaller than 200 nm.34-35 Up to now, despite recent progress in the exploration of colloidosomes for selective drug release,36-38 there is no colloidosomes achieving in vivo chemo/photothermal combination therapies against tumor. Here, we present, for the first time, the preparation of DOX/ICG laden nanocolloidosomes (NCs), with a diameter around 140 nm, for in vivo


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chemo/photothermal

combination

therapies

in

a

subcutaneous

hepatocarcinoma model. By templating from galactose functionalized hydroxyethyl starch-grafted-poly-caprolactone (Gal-HES-PCL) nanoparticles stabilized Pickering emulsions, the resultant NCs is composed of a PCL entangled hydrophobic core, with physically entrapped DOX and ICG, and a densely packed hydrophilic HES nanoparticles shell, Scheme 1. Owing to the distinctive structure, DOX/ICG@Gal-HES-PCL NCs possessed selective drug release behaviors, and also showed outstanding tumor targeting ability, preferable tumor deep penetration capability, and promoted photothermal effect. Moreover, these NCs can also be used for NIR fluorescence imaging and thus render real-time imaging of solid tumors with high contrast. Collectively, DOX/ICG@Gal-HES-PCL NCs achieved the best in vivo anti-tumor

efficacy

combined

with

laser

irradiation

compared

with

DOX/ICG@HES-PCL NCs and DOX/ICG mixture. To the best of our knowledge,

controlling

theranostic

agents’

selective

release

through

nanocarrier design for enhanced therapeutic effect has not been reported.



A

OH

SO3

O

OH HO

H

OH OH

n

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O

O

O

OH

OH HN

OR

N

O

O

OH

OH

N

O

O

O O O OR

O

H 2N

O

OH O

HO

OR

OH O

O O OR

OH O

O OR

R = H, CH2CH2OH

SO3 Na

DOX

O O

O

ICG

Dandelion-like Gal-HES-PCL nanoparticle Galactose moiety

Pickering emulsion/evaporation

DOX

HES with diameter of 7.6 nm

PCL

ICG

DOX/ICG@Gal-HES-PCL NCs with diameter of ～140 nm

B DOX/ICG@Gal-HES-PCL NCs

In Vivo Imaging System

NIR laser

i.v. injection

In vivo NIR fluorescence imaging

C

ICG retained in NC

Imaging guided photothermal/ chemo combination therapy

DOX released from NC

Receptor-mediated endocytosis

NIR laser

Photothermal therapy

Chemotherapy

ASGPR

Photothermal/chemo combination therapy

Scheme 1. Schematic illustration of the structure of DOX/ICG loaded Gal-HES-PCL NCs (A) and their application for active tumor-targeted imaging-guided photothermal/chemo combination therapy (B, C).


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2. Results and discussion 2.1 Preparation and characterization of HES-PCL and Gal-HES-PCL nanoparticles Hydroxyethyl starch (HES) has been widely applied as a colloidal plasma volume expander in the clinics for numerous years.39-40 Due to their outstanding biocompatibility, high hydrophilicity, low immunogenicity, and in vivo well-tuned pharmacokinetics, HES has been applied as a drug carrier to deliver various therapeutic agents, including low aqueous solubility drugs and nuclear acids.41-43 By grafting HES with hydrophobic polymer PCL, we can obtain HES-PCL. Galactose functionalized HES-PCL (Gal-HES-PCL) can be easily synthesized with simple post modification. Galactose can bind specifically with asialoglycoprotein receptors (ASGPR), thus Gal-HES-PCL can target to HepG-2 or H22 cells with highly expressed ASGPR both in vitro and in vivo.44-45 HES-PCL was synthesized by grafting carboxy-terminated PCL onto HES through ester bond, as shown in Scheme S1. To afford Gal-HES-PCL, HES-PCL was first succinated and then conjugated with galactosamine through amide bond. The successful synthesis of HES-PCL and Gal-HES-PCL was validated by 1H-NMR and FT-IR spectra. The characteristic resonance signals of PCL at 3.99 (d), 2.28 (a), 1.55 (b), and 1.30 ppm (c) appear in 1

H-NMR spectra of HES-PCL, Figure 1 A, indicating the successful grafting of

PCL onto HES.46 The number of PCL chain per HES of the synthesized



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HES-PCL, as determined by 1H-NMR spectra, is 1.0. The 1H-NMR spectra of succinyl HES-PCL in DMSO-d6 and D2O show the characteristic resonance signals of succinyl at 2.45 and 2.64 ppm respectively, Figure 1 B. The molar substitution (MS) of succinyl of the synthesized succinyl HES-PCL, as determined by 1H-NMR spectra in D2O, is 9.4 %. The characteristic resonance signals of galactosamine at 5.15 ppm (f) appear in 1H-NMR spectra of Gal-HES-PCL in D2O, Figure 1 C, suggesting the successful coupling of succinyl

HES-PCL

and

galactosamine.

The

molar

substitution

galactosamine of the synthesized Gal-HES-PCL determined by

1

of

H-NMR

spectra in D2O is 9.6 %, which agrees well with the results determined by elemental analysis, 9.0 %, Table S1. The FT-IR spectra of HES-PCL show the characteristic band of C=O stretching vibration of ester bond of PCL at 1731 cm-1, Figure 1 D. This band is enhanced as for succinyl HES-PCL, which is ascribed to the newly formed ester bond between HES-PCL and succinyl anhydride. The characteristic band of N-H bending vibration of amide bond at 1565 cm-1 appears in the FI-IR spectra of Gal-HES-PCL, indicating the successful conjugating galactosamine onto succinyl HES-PCL by amide bond.47 In Gal-HES-PCL, the band at 1644 cm-1 is enhanced as compared to HES-PCL and succinyl HES-PCL, which is assigned to C=O stretching vibration of the newly formed amide bond. The 1H-NMR and FT-IR spectra consistently reveal that HES-PCL and Gal-HES-PCL are successfully synthesized with well-defined chemical structure. Due to the high molecular


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weight and the hyper-branched structure of amylopectin,48 single HES 130/0.4 molecule adopts nanoparticle structure in aqueous solution. The hydrodynamic diameter of HES 130/0.4 was measured by DLS and decreases with increasing concentration, Figure 1 E and F. This is because higher concentration leads to higher osmotic pressure, and thus smaller sizes of HES, Figure S2. Although HES has clinically used as colloidal plasma volume expander over 50 years, the colloid nature is not well characterized yet.39-40 We, for the first time, obtain the AFM images of HES single-molecule nanoparticles, Figure 1 G. It can be seen that HES single-molecule nanoparticles

are

near

spherical.

This

can

be

attributed

to

their

hyper-branched structure. The diameter of the outlined HES single-molecule nanoparticle in Figure 1 G is 8.0 nm, Figure 1 H, which is very close to the hydrodynamic diameter measured at 100 mg/mL by DLS, 7.6 nm. The height of the outlined HES single-molecule nanoparticle in Figure 1 G is only 463 pm, Figure 1 H. This can be attributed to the structure collapse during the drying process. The DLS and AFM results corroborate that HES form single-molecule nanoparticles with near spherical morphology. Due to the single linear structure of PCL, it can be speculated that the synthesized HES-PCL and Gal-HES-PCL adopt dandelion-like nanoparticle structure, as illustrated in Scheme 1. Nonetheless, the height of linear PCL chain is below the detection limit of AFM, the AFM images of HES-PCL only show the spherical morphology of HES, Figure 1 I. Taken together, HES-PCL and Gal-HES-PCL nanoparticles



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have been successfully prepared and characterized.

Figure 1. Characterization of HES-PCL and Gal-HES-PCL. (A)

1

H-NMR

spectra of HES-PCL in DMSO-D6. (B) 1H-NMR spectra of succinyl HES-PCL in DMSO-D6 and D2O. (C) 1H-NMR spectra of Gal-HES-PCL in DMSO-D6 and D2O. (D) FT-IR spectra of HES-PCL, succinyl HES-PCL, and Gal-HES-PCL. (E) Size distribution of HES 130/0.4 with different concentration in deionized water. (F) Hydrodynamic diameters of HES 130/0.4 as a function of concentration. (G) AFM images of HES nanoparticles. (H) Height profile of single HES nanoparticle (Figure 1 G). (I) AFM images of HES-PCL nanoparticles. (J)


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Height profile of single HES-PCL nanoparticle (Figure 1 I).

2.2 Preparation and characterization of DOX/ICG loaded NCs



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Figure 2. Characterization of the Pickering emulsion, DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL NCs. (A) Confocal images of the Pickering


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emulsion droplets prepared with Cy5-HES-PCL (Red). (B) CLSM images of the formed Pickering emulsion droplets during the preparation of HES-PCL, Gal-HES-PCL, DOX/ICG@HES-PCL, and DOX/ICG@Gal-HES-PCL NCs (DOX: green; ICG: red). (C) Size distribution of DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL NCs. (D) TEM image of DOX/ICG@HES-PCL NC. (E) TEM image of DOX/ICG@Gal-HES-PCL NC.

Figure 1 reveals that both HES-PCL and Gal-HES-PCL are nanoparticles, however, they are distinctive from conventional rigid nanoparticles, they are structurally more akin to soft polymeric nanogels.49 Although conventional Pickering stabilizers, like silica or latex nanoparticles, have negligible interfacial activity,50-53 soft nanoparticles, for example microgels and bio-nanoparticles, based Pickering stabilizers can significantly decrease oil-water or air-water interfacial tensions.54-56 HES is intrinsically hydrophilic, in grafting HES with PCL, the resultant HES-PCL and Gal-HES-PCL are amphiphilic and interfacial active, and therefore can stabilize Pickering emulsions. To prepare HES-PCL or Gal-HES-PCL stabilized Pickering emulsions, the nanoparticles were first dispersed in deionized water, followed by adding chloroform and sonicating the mixed solution for 5 min. Confocal laser scanning microscope (CLSM) was used to characterize the prepared Pickering emulsion where HES-PCL nanoparticles were labeled by Cy5 to indicate their



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location.41 It can be seen that HES-PCL nanoparticles are absorbed at the oil-water interfaces, Figure 2 A, suggesting the successful preparation of HES-PCL stabilized Pickering emulsion. To prepare DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL Pickering emulsion, HES-PCL or Gal-HES-PCL nanoparticles were dispersed in deionized water followed by adding DOX and ICG aqueous solution and chloroform, and sonicating the above mixture for 5 min, Scheme S2. It can be seen that DOX and ICG fluorescence signals are co-localized within the Pickering emulsion droplets, Figure 2 B, revealing that DOX and ICG transfer from aqueous phase to oil phase during the sonication emulsification process. It is highly possible that DOX and ICG form molecular pair via electrostatic interactions, thus become hydrophobic, and transfer altogether to the oil phase.57 The sizes of as prepared Pickering emulsion are smaller (5 µm) than that prepared with HES-PCL or Gal-HES-PCL alone (9 µm), Figure S3. Notably, the sizes of DOX@HES-PCL Pickering emulsion (5 µm) are also smaller than HES-PCL Pickering emulsion, Figure S4, implying that

the

decrease

of

the

sizes

of

DOX/ICG@HES-PCL

and

DOX/ICG@Gal-HES-PCL Pickering emulsion is caused by DOX. It is highly possible that some DOX molecules are also absorbed at the oil-water interfaces and serve as co-surfactants, which help decrease the oil-water interfacial tension and the sizes of the formed Pickering emulsion.58-59 HES-PCL,

Gal-HES-PCL,

DOX/ICG@HES-PCL,

and

DOX/ICG@Gal-HES-PCL NCs were obtained by evaporation of the


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corresponding Pickering emulsions under vacuum. According to our rough estimation, the amount of HES-PCL nanoparticles on each single Pickering emulsion droplet is much higher (104 in magnitude) than that of single NCs, Figure S5-6. Arguably, it is highly possible that single Pickering emulsion divided into several smaller droplets and formed the final NCs during the vacuum evaporation process. The small sizes (around 10 nm) of HES-PCL and Gal-HES-PCL nanoparticles render their quick reorganization and therefore stabilization of smaller droplets. For this reason, the final obtained NCs can be as small as 140 nm. During the vacuum evaporation process, the hydrophobic PCL polymers entangle together and compose the hydrophobic core while HES densely pack at the periphery and form the shell, with theranostic agents DOX and ICG physically trapped within the hydrophobic core. The DLS and drug loading characters of the prepared NCs are summarized in Table S2. The DOX and ICG loading content of DOX/ICG@HES-PCL NCs (DOX: 5.4%, ICG: 5.0 %) are very close to those of DOX/ICG@Gal-HES-PCL NCs (DOX: 5.1%, ICG: 4.6 %) and the weight ratio of DOX to ICG are close to 1.1 for both NCs. The hydrodynamic diameters of DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL NCs are 147.1 ± 2.7 and 134.6 ± 1.9 nm respectively, which are slightly larger than empty HES-PCL (109.2 ± 5.7 nm) and Gal-HES-PCL NCs (100.2 ± 5.8 nm). This is because DOX and ICG inside the NCs occupy considerable volume. The PDI of DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL NCs are 0.10 ± 0.01 and



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0.07 ± 0.04 respectively, showing that they are highly monodispersed. The zeta potential of DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL NCs are -2.23 ± 0.22 and -2.48 ± 0.50 mV respectively. Due to the hydrophilic and slightly

negatively

charged

surfaces,

DOX/ICG@HES-PCL

and

DOX/ICG@Gal-HES-PCL NCs show excellent colloidal stability in PBS and PBS with 15 mg/mL BSA, Figure S7. The TEM images show that DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL NCs are spherical and the diameter matches well with the DLS data, Figure 2 D and E. Notably, it can be seen that the surfaces of the NCs are densely packed with small nanoparticles. These small particles are HES-PCL and Gal-HES-PCL nanoparticles in which PCL chains are entangled in the inner cavity of the NCs. The entangled PCL chains in the core physically trap the loaded theranostic agents DOX and ICG and therefore govern not only their release but also their theranostic performance. After being encapsulated into NCs, the characteristic absorption peak of ICG shifts from 780 to 800 nm, the peak shape of ICG is also changed significantly, Figure 3 A. Similar results have also been observed by Chiu et al.60 In addition, the XRD patterns show that the crystallization of DOX and ICG was strongly restricted in the NCs, Figure S8. These results suggest that there are multiple interactions between DOX, ICG, and the hydrophobic PCL chains inside the NCs, including hydrophobic and π-π stacking interactions. The fluorescence of DOX and ICG is significantly quenched in the NCs, Figure S9, which is due to the homo Förster resonance


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energy transfer (homo-FRET) effect considering that they are aggregated in the inner cavity of the NCs. Our results are in the same line with Chen et al.61 The UV/Vis spectra, fluorescence spectra, and XRD patterns corroborate that DOX and ICG are successfully loaded into NCs. It is known that free ICG shows poor stability in aqueous solution due to aggregation and degradation.62 The maximum absorbance of free ICG (at 780 nm) in deionized water and PBS decreases 77.5 % and 73.1 % respectively after 5 days’ incubation, Figure 3 B and C. In contrast, the absorbance of ICG (at 780 nm) in DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL NCs only decreases slightly (less than 20 %). The enhanced stability of ICG in NCs can be attributed to the protective effect of hydrophobic PCL matrices, which prevent ICG from degradation.63 The photothermal response of free ICG and DOX/ICG loaded NCs was evaluated under 808 nm laser (2 W/cm2), Figure 3 D and E. The photothermal response of DOX/ICG@HES-PCL (∆T = 15.0 ℃) and DOX/ICG@Gal-HES-PCL NCs (∆T = 15.4 ℃) is significantly enhanced as compared to free ICG (∆T = 10.6 ℃). The enhanced photothermal efficiency of the NCs can be ascribed to two reasons. First, the characteristic absorption peak of ICG shifts from 780 nm to 800 nm which matches better with 808 nm laser.60 Second, the NCs have enhanced nonradiative transition of ICG due to the self-quenching of ICG fluorescence.61 The photothermal response of free ICG decreases significantly after 24 h incubation. In contrast, the photothermal response of the NCs does not change. This can be attributed to the enhanced stability of ICG in NCs,



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Figure 3 C. The NCs exhibit both concentration-dependent and laser power-dependent photothermal response, Figure 3 F and G. The temperature decline 200 s (1.5 W/cm2) and 140 s (2 W/cm2) after irradiation is caused by the high power laser induced photo-bleaching of ICG.60 The DOX and ICG release

profiles

show

that

DOX/ICG@HES-PCL

and

DOX/ICG@Gal-HES-PCL NCs have similar drug release behaviors, with DOX released selectively, Figure 3 H and I. For example, there is more than 60 % of DOX released from the NCs within 24 h, whereas the release of ICG from the NCs is less than 30 %. This unique release behavior is correlated with the structure of the NCs. As illustrated in Scheme 1 and Figure 1, the NCs templating from Pickering emulsion have a hydrophobic core composed of PCL and hydrophilic shell of HES nanoparticles. The release behavior is controlled by the interstitials formed by neighboring HES nanoparticles at the shell and the size of the diffusants. DOX is smaller than ICG and therefore easier to diffuse from the PCL core and the interstitials of HES nanoparticles.23-25 Such selective release behavior has not been reported previously but is highly desirable for photothermal/chemo synergistic effect. Because the relatively fast release of DOX is beneficial for the cytotoxicity of the NCs against tumor cells, while the slow release of ICG from the NCs is beneficial for the stability of ICG, which results in enhanced photothermal response. Otherwise, released ICG will be unstable whereas encapsulated DOX cannot execute immediate therapeutic effect against cancerous cells.


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Figure 3. UV/Vis spectra, stability, photothermal response, and drug release profiles

of

NCs.

(A)

UV-Vis

spectra

of

free

ICG,

DOX/ICG,

DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs (5.6 µg/mL of DOX, 5.0 µg/mL of ICG) in PBS buffer (6.7 mmol/L, pH 7.4). (B) UV-Vis stability of free ICG, DOX/ICG@HES-PCL, and DOX/ICG@Gal-HES-PCL NCs in deionized water. (C) UV-Vis stability of ICG, DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs in PBS buffer. (D) Photothermal response of ICG, DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs under NIR laser (808 nm, 2.0 W/cm2) after incubation for 0 and 24 h in PBS buffer. (E) Thermal images after irradiated with NIR laser for 5min. (F) Photothermal



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response of DOX/ICG@Gal-HES-PCL NCs with different ICG concentration in PBS

buffer

under

NIR

laser.

(G)

Photothermal

response

of

DOX/ICG@Gal-HES-PCL NCs in PBS buffer under different laser power (at 808 nm). (H) DOX release profiles of NCs in pH 7.4 and pH 5.0 PBS buffer (10.0 mmol/L). (I) ICG release profiles of NCs in pH 7.4 and pH 5.0 PBS buffer.

Taken together, DOX/ICG@Gal-HES-PCL and DOX/ICG@HES-PCL NCs are successfully prepared with similar size, zeta potential, morphology, and drug loading content. Moreover, these two NCs also have very close stability, photothermal response, and selective drug release behaviors. Next, the active tumor-targeting ability of DOX/ICG@Gal-HES-PCL NCs can be studied both in vitro and in vivo by using DOX/ICG@HES-PCL NCs as a control.

2.3 In vitro antitumor assay of DOX/ICG loaded NCs The cellular uptake of DOX/ICG loaded NCs was studied by CLSM. The intracellular DOX and ICG content was also determined. HepG-2 cells with highly expressed ASGPR were selected, and MCF-7 cells with no expression of ASGPR were used as a negative control.44,

64

In comparison with

DOX/ICG@HES-PCL NCs, DOX/ICG@Gal-HES-PCL NCs show enhanced cellular uptake for HepG-2 cells, Figure 4 A, B, and C, but very similar cellular uptake for MCF-7 cells, Figure 4 D, E, and F. In addition, pretreatment of HepG-2 cells with galactosamine significantly inhibit the cellular uptake of


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DOX/ICG@Gal-HES-PCL NCs, Figure 4 A, B, and C. These results corroborate

the

ASGPR

receptor-mediated

endocytosis

of

DOX/ICG@Gal-HES-PCL NCs by HepG-2 cells. Similar results were also reported by Zhang et al.64 Before conducting the in vitro antitumor activity study, we had assessed the biocompatibility of empty HES-PCL and Gal-HES-PCL NCs in HepG-2 and MCF-7 cells, Figure 4 G and H. The cell viability is over 90 % for both NCs even at concentration up to 2 mg/mL after 24 h incubation, indicating the good safety of these two NCs. To investigate the in vitro antitumor activity of DOX/ICG loaded NCs, HepG-2 and MCF-7 Cells were incubated with different formulations for 6 h, followed by replacing the drug-containing medium with fresh drug-free medium and irradiated with 808 nm laser (2 W/cm2) for 3 min (or no irradiation). Then, the cells were incubated for another 18 h before adding MTT dyes. The results show that DOX/ICG@Gal-HES-PCL NCs exhibit enhanced cytotoxicity against HepG-2 cells, Figure 4 I and K, but similar cytotoxicity against MCF-7 cells, Figure 4 J and L, as compared to DOX/ICG@HES-PCL NCs, DOX/ICG, and free DOX. The enhanced cytotoxicity of DOX/ICG@Gal-HES-PCL NCs against HepG-2 cells can be the result of the enhanced cellular uptake by ASGPR receptor-mediated endocytosis (Figure 4 A and B). Due to limited cellular uptake and poor stability, free ICG + irradiation exhibits no antitumor effect, and DOX/ICG mixture + irradiation exhibits almost the same antitumor effect as compared to single chemotherapy with DOX/ICG mixture (Figure 4 I and J).



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Notably,

DOX/ICG@HES-PCL

and

Page 22 of 49

DOX/ICG@Gal-HES-PCL

NCs

+

irradiation significantly enhance the antitumor effect as compared to single chemotherapy with corresponding NCs (Figure 4 I and J). The enhanced photothermal cytotoxicity of the NCs can be the combination effect of enhanced cellular uptake (Figure 4 C and F), enhanced stability (Figure 3 C), and promoted photothermal efficiency (Figure 3 D), as compared to free ICG. As a result, DOX/ICG@Gal-HES-PCL NCs + irradiation achieves the highest antitumor activity against HepG-2 cells, with IC50 values (of DOX) 2.0 and 1.8 times

lower

than

those

of

DOX/ICG

mixture

+

irradiation

and

DOX/ICG@HES-PCL NCs + irradiation (Figure 4 K and L). Together, Figure 4 reveals the active targeting ability of DOX/ICG@Gal-HES-PCL NCs to ASGPR high expression tumor cells

and their superior

photothermal/chemo

combination therapeutic effect in vitro, suggesting these NCs can be translated for in vivo investigations.


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Figure 4. In vitro cell viability assays and cellular uptake of NCs. (A) CLSM images of HepG-2 cells incubated with free DOX, DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs (5.0 µg/mL of DOX) for 6 h and HepG-2 cells pre-incubated with 250 µmol/L galactosamine for 1 h followed by incubation with DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL NCs for 6 h. (B) Cellular uptake of DOX by HepG-2 cells after incubation with each group (5.0 µg/mL of DOX) for 6 h. (C) Cellular uptake of ICG by HepG-2 cells after incubation with each group (4.5 µg/mL of ICG) for 6 h. (D) CLSM images of MCF-7 cells incubated with free DOX, DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs for 6 h. (E) Cellular uptake of DOX by MCF-7 cells after incubation with each group for 6 h. (F) Cellular uptake of ICG by MCF-7 cells after incubation with each group for 6 h. The nucleus was stained



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Page 24 of 49

with DAPI (blue). DOX fluorescence color is red. (G) In vitro cytotoxicity of empty HES-PCL and Gal-HES-PCL NCs against HepG-2 cells. (H) In vitro cytotoxicity of empty NCs against MCF-7 cells. (I) In vitro cytotoxicity of free DOX,

free

ICG,

DOX/ICG,

DOX/ICG@HES-PCL

NCs,

and

DOX/ICG@Gal-HES-PCL NCs against HepG-2 cells without and with laser irradiation for 3 min (808 nm, 2.0 W/cm2). (J) In vitro cytotoxicity of free DOX, free ICG, DOX/ICG, DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs against MCF-7 cells without and with laser irradiation for 3 min. (K) IC50 values (DOX) of each group against HepG-2 cells. (L) IC50 values (of DOX) of each group against MCF-7 cells. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. as not significant.

2.4 NIRF imaging and biodistribution of NCs ICG is approved by FDA not only as a photothermal agent but more importantly as an optically diagnostic tool for in vivo NIR fluorescence imaging.65-66 The in vivo NIR fluorescence imaging and biodistribution of DOX/ICG loaded NCs was studied on subcutaneous H22-tumor models, since ASGPR is also highly expressed on the surfaces of H22 hepatoma cells.67 The fluorescence signals of free ICG at tumor regions stay at low level during 96 h, Figure 5 A and B. Free ICG is reported to be quickly eliminated from the blood after intravenous injection, accounting for its limited tumor accumulation.21-22 The fluorescence signals of DOX/ICG@HES-PCL NCs at tumor regions are


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significantly higher than those of free ICG, and DOX/ICG@Gal-HES-PCL NCs exhibit the highest fluorescence signals at tumor regions, Figure 5 A and B. For example, the fluorescence signals of DOX/ICG@Gal-HES-PCL NCs at tumor regions 24 h post injection are 1.6 and 3.3 times higher than those of DOX/ICG@HES-PCL

NCs

and

free

ICG.

Even

after

96

h,

DOX/ICG@Gal-HES-PCL NCs still get 1.4 and 2.2 times higher fluorescence signals at tumor regions than DOX/ICG@HES-PCL NCs and free ICG. Obviously, DOX/ICG@Gal-HES-PCL NCs possess the capability of imaging H22 tumors with high contrast in long term. The enhanced tumor accumulation of DOX/ICG@HES-PCL NCs as compared to free ICG may result from the EPR

effect

of

the

NCs,

and

the

best

tumor

accumulation

of

DOX/ICG@Gal-HES-PCL NCs can be the combination result of EPR effect and

specific

binding

between

the

galactose

moieties

on

DOX/ICG@Gal-HES-PCL NCs and ASGPR highly expressed on H22 cells. The ex vivo DOX/ICG double channel imaging was also used to characterize the biodistribution of DOX and ICG in organs and tumors, as shown in Figure 5 C, D, and E. At 12 h post injection, the tumor accumulation of DOX from DOX/ICG@Gal-HES-PCL NCs is 1.5 and 1.2 times higher than those from free DOX and DOX/ICG@HES-PCL NCs, while the tumor accumulation of ICG from DOX/ICG@Gal-HES-PCL NCs is 2.8 and 1.9 times higher than those from free ICG and DOX/ICG@HES-PCL NCs. The ratio in terms of ICG is higher than that in terms of DOX. This can be ascribed to the different release



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behaviors between DOX and ICG, Figure 3 H and I. DOX is released relatively fast from the NCs. So it can be rapidly eliminated from the blood, resulting in minor enhanced tumor accumulation. In contrast, the slow release of ICG from the NCs is beneficial for the stability and circulation of ICG in the blood, which leads to significantly enhanced tumor accumulation by EPR effect and active targeting. The accumulation of DOX/ICG@Gal-HES-PCL NCs in tumors is also higher than those in major organs including heart, liver, spleen, lung, and kidney, indicating DOX/ICG@Gal-HES-PCL NCs exhibit outstanding tumor targeting ability in vivo. The immunohistochemical staining of tumor sections was used to characterize the intratumoral distribution of DOX 48 h post injection. The nucleus was stained with DAPI (blue), and the blood vessels were stained by CD31 antibody (green). The fluorescence signals of DOX (red) are mainly co-localized with the tumor blood vessels for free DOX and DOX/ICG@HES-PCL NCs, Figure 5 F and G. In contrast, the fluorescence signals of DOX from DOX/ICG@Gal-HES-PCL NCs spread all over the entire tumor tissues, suggesting DOX/ICG@Gal-HES-PCL NCs have better tumor penetration capacity than free DOX and DOX/ICG@HES-PCL NCs. Figure 5 demonstrates that DOX/ICG@Gal-HES-PCL NCs exhibit enhanced tumor retention and penetration capability as compared to free DOX and DOX/ICG@HES-PCL NCs.


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Figure 5. In vivo NIR fluorescence imaging and biodistribution of NCs. (A) In vivo NIR fluorescence images of mice intravenously injected with free ICG, DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs (equivalent to 3.6 mg ICG /kg bodyweight). (B) Quantification of ICG fluorescence signals of tumor regions. (C) Ex vivo DOX/ICG double channel fluorescence images of heart, liver, spleen, lung, kidney, and tumor 12 h post injection (equivalent to 4.0 mg DOX and 3.6 mg ICG /kg bodyweight). (D) Quantification of DOX fluorescence signals of each organ and tumor 12 h post injection. (E) Quantification of ICG fluorescence signals of each organ and tumor 12 h post injection. (F) Fluorescence images of tumor sections 48 h post injection (equivalent to 4.0 mg DOX /kg bodyweight). Cell nucleus was stained with



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Page 28 of 49

DAPI (blue). Tumor blood vessels were stained with CD31 marker (green). DOX fluorescence color is red. (G) Quantification of DOX and CD31 antibody fluorescence signals on the white line. * p < 0.05, ** p < 0.01, *** p < 0.001.

2.5 Imaging guided photothermal/chemo combination therapy of DOX/ICG loaded NCs Next, the in vivo photothermal/chemo response of DOX/ICG loaded NCs was investigated. The in vivo biodistribution has shown that the tumor accumulation of the NCs reached a peak value at 24 h, Figure 5 A and B. The in vitro drug release also demonstrates that over 60% of DOX has been released from the NCs while over 70% of ICG is retained within NCs at 24 h (Figure 3 H and I). Therefore, laser irradiation was conducted 24 h post injection to achieve the optimum in vivo photothermal/chemo effect. The tumor temperature treated with free ICG + irradiation and DOX/ICG mixture + irradiation increase to 39.5 and 39.7 ℃ respectively, which are not high enough to cause irreversible thermal damage (43 ℃),68 Figure 6 A and B. Notably,

DOX/ICG@HES-PCL

NCs

+

irradiation

and

DOX/ICG@Gal-HES-PCL NCs + irradiation exhibit a temperature rise to 45.4 and 52.6 ℃

respectively. The enhanced photothermal response of

DOX/ICG@HES-PCL NCs as compared to free ICG and DOX/ICG mixture is owing to their enhanced tumor accumulation by EPR effect (Figure 5 B) and encapsulation within NCs (Figure 3 I and D). The best photothermal response


Page 29 of 49

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of DOX/ICG@Gal-HES-PCL NCs is attributed to their best tumor accumulation by a combination of EPR effect and active-tumor targeting, Figure 5 B, as well as encapsulation within NCs. To investigate the photothermal damage of each group, the tumors were harvested 24 h post irradiation and analyzed by H & E staining. The tumors treated with each formulation without laser irradiation were used as control. PBS +irradiation, free DOX + irradiation, free ICG + irradiation, and DOX/ICG mixture + irradiation all exhibit almost the same histologic features as compared to corresponding formulation without laser irradiation, Figure 6 C, indicating that these formulations with laser irradiation cause no thermal damage. Imposingly, DOX/ICG@HES-PCL NCs + irradiation exhibits significant thermal damage, while DOX/ICG@Gal-HES-PCL NCs + irradiation causes the most significant thermal damage, Figure 6 C. These results are consistent with the in vivo photothermal response of each group, Figure 6 B. Due to the chemotherapeutic effect of DOX, free DOX, DOX/ICG mixture, DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs also exhibit significant tumor tissue damage as compared to PBS and free ICG, and DOX/ICG@Gal-HES-PCL NCs exhibit the most significant tumor tissue damage, Figure 6 C. The enhanced photothermal and chemo effect of DOX/ICG@Gal-HES-PCL NCs are benefited from their enhanced tumor accumulation, retention, and penetration, Figure 5 and selective release of DOX, Figure 3 H.



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Figure 6. In vivo photothermal response under laser irradiation (808 nm, 2.0 W/cm2 for 5 min). (A) Tumor temperature profiles of mice receiving different formulations and exposed to NIR laser 24 h post intravenous injection (equivalent to 4.0 mg DOX and 3.6 mg ICG /kg bodyweight). (B) Thermal images of mice receiving different formulations and exposed to NIR laser 24 h post intravenous injection. (C) Tumor sections (stained by H&E) of mice 48 h post intravenous injection without and with laser irradiation at 24 h for 5 min.

2.6 In vivo antitumor efficacy of DOX/ICG loaded NCs Finally, the in vivo antitumor activity of DOX/ICG loaded NCs was evaluated on subcutaneous transplanted H22-tumor models. Mice of each group were given a total of two doses, and the tumor volume changes of each


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group

were

monitored

up

to

16

days

post

administration.

DOX/ICG@Gal-HES-PCL NCs exhibit enhanced tumor inhibition rate (63.1 %) as compared to DOX/ICG@HES-PCL NCs (47.2 %), DOX/ICG mixture (48.5 %), and free DOX (44.2 %), Figure 7 A. The enhanced chemotherapeutic effect of DOX/ICG@Gal-HES-PCL NCs can be ascribed to their enhanced tumor accumulation, retention, and penetration, Figure 5, and selective release of DOX, Figure 3 H. Due to the limited in vivo photothermal response and poor stability, free ICG + irradiation shows no antitumor effect, and DOX/ICG mixture + irradiation exhibits almost the same antitumor effect as compared to DOX/ICG mixture without laser irradiation, Figure 7 A. Importantly, DOX/ICG@HES-PCL NCs + irradiation and DOX/ICG@Gal-HES-PCL NCs + irradiation exhibit significantly enhanced antitumor effect as compared to corresponding NCs without laser irradiation, Figure 7 A. This can be attributed to their enhanced in vivo photothermal response, which causes significant thermal damage, Figure 6. Combining the superior chemotherapeutic effect and photothermal damage, DOX/ICG@Gal-HES-PCL NCs + irradiation achieves the best in vivo tumor inhibition rate (100 %) as compared to DOX/ICG mixture + irradiation (46.7 %) and DOX/ICG@HES-PCL NCs + irradiation (89.9 %), Figure 7 A. The tumor weight and tumor images of each group are summarized in Figure 7 B and C, which are in good agreement with the tumor growth inhibition depicted in Figure 7 A. The H & E staining of tumor sections was also used to evaluate the therapeutic effect of each group, as



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Page 32 of 49

shown in Figure 7 D. The cell density of the tumors treated with free DOX, DOX/ICG mixture, DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs is significantly lower than those treated with PBS and free ICG, and DOX/ICG@Gal-HES-PCL NCs exhibit the lowest cell density among these formulations,

validating

the

enhanced

chemotherapeutic

effect

of

DOX/ICG@Gal-HES-PCL NCs, Figure 6 C. The cell density of the tumors treated with PBS + irradiation, free DOX + irradiation, free ICG + irradiation, and DOX/ICG mixture + irradiation is very close to that treated with corresponding

formulation

without

laser

irradiation.

In

contrast,

DOX/ICG@HES-PCL NCs + irradiation exhibits significantly lower cell density as compared to DOX/ICG@HES-PCL NCs without laser irradiation, confirming the enhanced photothermal damage of the NCs, Figure 6 C. The tumor slices were also stained by Ki67 and DAPI to evaluate the tumor proliferation of each group, Figure S12 A and B, and the results agree well with the tumor growth inhibition depicted in Figure 7 A.


Page 33 of 49

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Figure 7. In vivo antitumor effect. (A) Relative tumor volume changes of mice intravenously injected with different formulations (equivalent to 4.0 mg DOX and 3.6 mg ICG /kg bodyweight). Blue arrows represent the day on which intravenous injection was conducted. Red arrows represent the day on which laser irradiation was performed (808 nm, 2.0 W/cm2 for 5 min). (B) Tumor weight of mice from each group at the end of the test. (C) Images of excised tumors of each group at the end of the test. (D) Tumor sections (stained with H&E) of mice from each group at the end of the test. * p < 0.05, ** p < 0.01, *** p < 0.001. n.s. as not significant.



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The potential side effects of cancer therapy is a big concern for clinical translations. As shown in Figure S13 A, DOX, DOX/ICG mixture, DOX/ICG@HES-PCL and DOX/ICG@Gal-HES-PCL NCs all exhibit significant bodyweight loss after administration. This can be caused by the acute toxicity of DOX.20 The bodyweight of these groups are gradually recovered afterwards, Figure S13 A. The blood biochemical test was used to evaluate the toxicity of each group 16 days post administration. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured to evaluate the functions of liver, blood urea nitrogen was measured to evaluate the functions of kidney, creatinine kinase (CK) was measured to evaluate the functions of heart. All treatment groups show no significant changes of ALT, AST, LDH, BUN, and CK levels as compared to PBS group, Figure S13 B, C, D, E, and F. In addition, H & E staining of organ slices was also used to evaluate the toxicity of each group at the end of the test. All treatment groups exhibit no obvious pathological changes on heart, liver, spleen, lung and kidney as compared to PBS group, Figure S14. Both blood biochemical test and H & E staining results corroborate that all treatment groups exhibit no obvious toxicity at the end of the test. These results also support that the DOX-induced acute toxicity here dose not lead to organic lesions.


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3. Conclusions In summary, we have successfully prepared DOX/ICG laden NCs, with diameters around 140 nm, and explored their applications for active tumor-targeted imaging-guided photothermal/chemo combination therapy against liver cancer for the first time. Owing to the distinctive structure, DOX/ICG@Gal-HES-PCL NCs possessed selective drug release behaviors, which are beneficial for potent chemotherapy and thermal therapy, and also showed outstanding tumor targeting ability, preferable tumor deep penetration capability, and promoted photothermal effect. Moreover, these NCs can also be used for NIR fluorescence imaging and thus render real-time imaging of solid tumors. Collectively, DOX/ICG@Gal-HES-PCL NCs achieved the best in vivo tumor inhibition rate (100 %) as compared to DOX/ICG mixture (46.7 %) and DOX/ICG@HES-PCL NCs (89.9 %), combined with laser irradiation. The reported results here are significant because, first we have demonstrated selective drug release is beneficial for photothermal/chemo combination therapy, for example, released DOX for chemotherapy and encapsulated ICG for enhanced stability and promoted thermal response of ICG; and second, DOX/ICG@Gal-HES-PCL

NCs

templating

from

dandelion-like

nanoparticles-stabilized Pickering emulsion droplets represent a simple and robust drug loading approach and can be used for co-loading of a variety of theranostic agents to fight against numerous cancers and potentially other diseases as well.



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Page 36 of 49

4. Experimental section The synthesis of Gal-HES-PCL and other experimental procedures are available in the supporting information.

Supporting Information Materials and methods; schematic illustration of the synthetic routes of HES-PCL and Gal-HES-PCL; schematic illustration of the preparation procedure of DOX/ICG loaded NCs; 1H-NMR spectra of HES-PCL in D2O; elemental analysis of HES, HES-PCL, succinyl HES-PCL, and Gal-HES-PCL; diameters of HES 130/0.4 as a function of osmotic pressure; size distribution of the formed Pickering emulsion droplets during the preparation of HES-PCL, Gal-HES-PCL, DOX/ICG@HES-PCL, and DOX/ICG@Gal-HES-PCL NCs; CLSM images and size distribution of the formed Pickering emulsion droplets during the preparation of DOX@HES-PCL NCs; DLS and drug loading characters of the prepared NCs; calculation of the number of HES-PCL or Gal-HES-PCL dandelion-like nanoparticles per Pickering emulsion droplet; calculation of the number of HES-PCL or Gal-HES-PCL dandelion-like nanoparticles

per

NC;

stability

of

DOX/ICG@HES-PCL

and

DOX/ICG@Gal-HES-PCL NCs in PBS buffer and BSA containing PBS puffer; XRD patterns of free DOX, free ICG, DOX/ICG, DOX/ICG@HES-PCL NCs, and DOX/ICG@Gal-HES-PCL NCs; fluorescence spectra of free DOX, free ICG, DOX/ICG, and DOX/ICG loaded NCs; representative UV/Vis spectra


Page 37 of 49

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changes

of

free

ICG,

DOX/ICG@HES-PCL

NCs,

and

DOX/ICG@Gal-HES-PCL NCs in deionized water and PBS buffer; CLSM images of HepG-2 cells incubated with different formulations for 0.5 h; tumor proliferation analysis; bodyweight changes and blood biochemical test; tissue sections (stained by H & E).

Acknowledgement This work was financially supported by grants from National Basic Research Program of China (2015CB931802), National Science Foundation of China (Grant No. 81627901, 81473171, 31700867), PCSIRT (Grant No. IRT13016), and Scientific Research Foundation of Huazhong University of Science and Technology (Grant No. 3004170130). We also thank the HUST Analytical and Testing Center for allowing us to use its facilities.

Conflict of Interest The authors declare no conflict of interest.



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L.; Ma, A.; Cui, H.; Ma, Y.; Cai, L., Cancer Cell Membrane–Biomimetic Nanoparticles for Homologous-Targeting Dual-Modal Imaging and Photothermal Therapy. Acs Nano 2016, 10 (11), 10049-10057.


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TOC OH

SO3 O

OH HO

H

OH OH

n

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


O

O

O

OH

OH HN

OR

O

N O

O

OH

OH

O

N

O

O O OR

O

H 2N

O

OH O

HO

OR

OH O

O O OR

OH O

O OR

R = H, CH 2CH2OH

SO3 Na

DOX

O O

O

ICG

Dandelion-like Gal-HES-PCL nanoparticle Galactose moiety

Pickering emulsion/evaporation

DOX

HES with diameter of 7.6 nm

PCL

ICG

DOX/ICG@Gal-HES-PCL NCs with diameter of ～140 nm

ICG retained in NC

DOX released from NC

Receptor-mediated endocytosis

NIR laser

Photothermal therapy

Chemotherapy

ASGPR

Photothermal/chemo combination therapy


Nanocolloidosomes with Selective Drug Release for Active Tumor

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