Reversible Shielding between Dual Ligands for Enhanced Tumor

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Reversible shielding between dual ligands for enhanced tumor accumulation of ZnPc-loaded micelles Jing Cao, Xuefeng Gao, Mingbo Cheng, Xiaoyan Niu, Xiaomin Li, Yapei Zhang, Yang Liu, Wei Wang, and Zhi Yuan Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b04645 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 25, 2019

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Reversible shielding between dual ligands for enhanced tumor accumulation of ZnPc-loaded micelles Jing Cao,† Xuefeng Gao,† Mingbo Cheng,† Xiaoyan Niu,† Xiaomin Li,† Yapei Zhang,† Yang Liu,† Wei Wang,† Zhi Yuan*,†,‡ †Key

Laboratory of Functional Polymer Materials of the Ministry of Education, Institute of

Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China ‡Collaborative

Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300071, China KEYWORDS: Tumor targeting, reversible ligand shielding, pH-responsive, phenylboronic acid, galactose, photodynamic therapy

ABSTRACT. Herein, we report a ligand reversible shielding strategy based on the mutual shielding of dual ligands tethered to the surface of nanoparticles. To exemplify this concept, phenylboronic acid-functionalized poly(ethylene glycol)-b-poly(ε-caprolactone) (PBA-PEGPCL) and galactose-functionalized diblock polymer (Gal-PEG-PCL) were mixed to form dualligand micelles (PBA/Gal). PBA and Gal residues could form a complex at pH 7.4 and mutually shield their targeting function. At pH 6.8, the binding affinity between PBA and Gal weakened,

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and PBA preferred to bind with the sialic acid residues on the tumor cell surface rather than to Gal on the micellar surface; furthermore, the unbound Gal recovered its targeting ability toward the asialoglycoprotein receptor. When the pH decreased from 7.4 to 6.8, enzyme-linked immunosorbent assays (ELISAs) exhibited that the percentage of exposed Gal on the micellar surface increased 1.9-fold, and flow cytometry showed that HepG2 cellular uptake increased 4.3fold. More importantly, this process was reversible, confirming the reversible shielding and deshielding of dual ligands. With the encapsulation of a photosensitizer, zinc phthalocyanine (ZnPc), the reversible-shielding micelles showed a 48% improvement in the half-life (t1/2) in blood circulation, a 54% decrease in liver capture, a 40% increase in tumor accumulation and a 10.3% improvement in the tumor inhibition rate compared to the Gal-coated irreversible micelles. This dual-ligand mutual shielding strategy provides a new perspective on reversible tumor targeting.

Targeted drug delivery systems based on ligand-modified nanoparticles have emerged as powerful solutions to cancer therapy for enhanced tumor accumulation and reduced side effects.1-5 However, targeting ligands may lead to unexpected immune recognition by the reticuloendothelial system (RES), which shortens the blood circulation time and impedes the further accumulation of nanoparticles at the tumor site.4,6–9 To solve this problem, a tumorresponsive shielding/deshielding strategy was developed.10,11 The targeting ligands have been commonly shielded by stealth coronas or caging groups in the bloodstream and deshielded at tumor sites via stimuli-responsive cleavable linkages.12-20 Although these ligand shielding strategies enhanced the stability of the nanoparticles in blood circulation and facilitated the cellular uptake of the nanoparticles into tumors, it was reported that only 0.7% of an injected dose was found to be delivered to a solid tumor,21 which is far below expectations.


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In fact, not all of the nanoparticles deshielded at the tumor site could be effectively internalized by tumor cells,22,23 and those deshielded nanoparticles which were not taken up by tumor cells returned to the bloodstream. However, once the cleavable linkages were broken at the tumor site, the shielding coronas or caging groups were removed, and achieving reshielding of the ligands in the bloodstream was difficult. This irreversible shielding strategy makes the deshielded nanoparticles easily recognized and eliminated by the RES, resulting in a limited shielding effect. Therefore, reversible ligand shielding of nanoparticles is urgently required to further prolong their circulation and enhance their tumor accumulation. Our recent work has proven the practicality and superiority of the reversible shielding strategy for improved tumor accumulation, imaging and therapy in vivo.24 The stimuli-responsive collapse of protective thermosensitive polymers25-29 or the “popping up” of targeting ligands along with pH-sensitive polymers30-34 are approaches frequently used to achieve reversible shielding of ligands. Taking advantage of both pH- and thermo-sensitivities, a pH-dependent thermo-responsive polymer was developed with its thermo-sensitivity spontaneously tailored by pH, and ultrasensitive ligand shielding and deshielding between the normal physiological environment (pH 7.4, 37C) and the tumor microenvironment (pH 6.5, 40C) was achieved.29 The reversible assembly/disassembly of nanoparticles provides another class of method for reversible ligand shielding.24,35,36 For example, ligands were hidden in an assembly of gold nanoparticles at pH 7.4 and exposed at pH 6.8 by disruption of the assembly. More importantly, this process was reversible and endowed the system with in vivo applications.24,35,36 However, the shrinkage and extension of polymers requires a prolonged transition time,29 and the assembly of nanoparticles is, in theory, concentration-dependent, thus making it difficult to trigger the assembly after several rounds of blood circulation. Here, we


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propose a new reversible shielding strategy. Assuming that the targeting ligand is protected by another kind of targeting ligand via dynamic covalent bonds and that both ligands are tethered to the surface of the same nanoparticle, both ligands would shield each other during blood circulation and be deshielded by stimuli at the tumor site without leaving the nanoparticle. As a proof-of-concept example of the dual-ligand mutually shielding strategy, phenylboronic acid-functionalized

poly(ethylene

glycol)-b-poly(ε-caprolactone)

(PBA-PEG-PCL)

and

galactose-functionalized poly(ethylene glycol)-b-poly(ε-caprolactone) (Gal-PEG-PCL) were mixed to prepare dual-ligand micelles, PBA/Gal. PBA can be utilized as a targeting ligand to recognize biologically relevant sialic acid residues (e.g., 2-O-methyl-α-D-N-acetylneuraminic acid, Me-SA) on tumor cells,20,34,37–41 and Gal is a commonly used targeting ligand acting toward asialoglycoprotein receptor (ASGPR)-overexpressed tumor cells.42-45 At the physiological pH of 7.4, PBA groups can form borates with Gal groups at the micellar surface,37,46 leading to the mutual shielding of their targeting abilities. At the tumoral acidic pH of 6.8, the binding affinity between PBA and Gal was weakened, and the PBA groups preferred to bind with Me-SA on the tumor cell surface rather than to Gal groups on the micellar surface;34,37,46 furthermore, the unbound Gal regained its targeting ability toward ASGPR. This process was reversible and independent from the micellar concentration. Enzyme-linked immunosorbent assays (ELISAs) and cellular uptake were performed to evaluate the pH-responsive shielding/deshielding ability of the dual-ligand system. Then, the photosensitizer zinc phthalocyanine (ZnPc) was encapsulated into the micelles for photodynamic therapy (PDT). The blood clearance, recognition and elimination by RES, and tumor accumulation and inhibition in vivo were also examined to verify the superiority of this intelligent reversible shielding strategy.


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Scheme 1. Schematic illustration of the reversible tumor targeting system based on a pHresponsive dual-ligand mutual shielding strategy. The synthesis routes of the targeted diblock copolymer PBA-PEG-PCL, Gal-PEG-PCL and nontargeted diblock copolymer mPEG-PCL are shown in Scheme S1. By calculating the proton ratio of the aromatic group of PBApin or the acetyl groups of Galpac relative to the ethylene units in PEG in 1H NMR spectra, it was determined that one HO-PEG3.4k-OH chain was installed onto one PBApin residue or one Galpac residue on average (Figure S1B and S2B). The other hydroxyl end of PEG was utilized to initiate the ring-opening polymerization (ROP) of caprolactone, and the number-average molecular weight of PCL was calculated to be 12000 for


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both of PBApin-PEG-PCL and Galpac-PEG-PCL by 1H NMR (Figure S1C and S2C) based on the proton ratios of the ethylene units in PEG and the ethylene units in PCL. Finally, the deprotection of PBApin was achieved by acidic hydrolysis and confirmed by the 1H NMR spectrum of PBA-PEG-PCL (Figure 1A), which shows a decrease in the proton integral of the four methyl groups (1.38 ppm) compared with that of PBApin-PEG-PCL (Figure S1C). The deprotection of Galpac was achieved by hydrazine hydrolysis and confirmed by the disappearance of the characteristic peaks of acetyl groups (2.14, 2.05–2.03, and 1.98 ppm) in the 1H

NMR spectrum (Figure 1B). The nontargeted mPEG-PCL was synthesized by the ROP of

commercial mPEG2k-OH, and its structure was confirmed by 1H NMR (Figure 1C). The polydispersity index of PBA-PEG-PCL, Gal-PEG-PCL, and mPEG-PCL measured by gel permeation chromatography (Figure S3) were 1.23, 1.26, and 1.18, respectively.

Figure 1. 1H NMR spectra of PBA-PEG-PCL (A), Gal-PEG-PCL (B) and mPEG-PCL (C) in CDCl3 (400 MHz). (D) CMC of PBA/Gal micelles. (E) Hydrodynamic diameter distribution of PBA/Gal micelles at pH 7.4 and 6.8. (F) TEM image of PBA/Gal micelles.


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The self-assembly of amphiphilic diblock copolymers is an approach commonly used to prepare complex micelles with functionality. Dual-ligand co-modified reversible micelles, PBA/Gal, were self-assembled by the mixing of targeted PBA-PEG-PCL and Gal-PEG-PCL with nontargeted mPEG-PCL. Dynamic light scattering (DLS) measurements showed that the diameters of PBA/Gal at pH 7.4 and 6.8 were 124.2  2.2 nm with a PdI of 0.13 and 120.2  1.8 nm with a PdI of 0.14, respectively, both of which are appropriate for enhanced permeability and retention (EPR) effects in vivo. The zeta potential of PBA/Gal was -13.5  2.2 mV at pH 7.4 and slightly increased to -11.2  1.5 mV at pH 6.8, implying changes of PBA and Gal binding at different pH values. The TEM image of PBA/Gal micelles exhibited a spherical morphology and a smaller average diameter compared with those from DLS measurements, probably due to the loss of the hydrated layer during TEM measurements. The critical micelle concentration (CMC) of the PBA/Gal micelles was 3.13 mg/L (Figure 1D). This low value of the CMC ensures the stability of the micelles after dilution in vivo. Zinc phthalocyanine (ZnPc) is hydrophobic photosensitizer with near-infrared absorption and a high singlet oxygen quantum yield. Therefore, ZnPc was used as a model drug to study the reversible targeted drug delivery system. ZnPc was encapsulated into the hydrophobic core of micelles, and the drug loading content (DLC) of ZnPc was 7.9% (Table S1) according to fluorescence measurements. As a control, irreversible micelles, GalcoPBA, were prepared by adding Gal into PBA micelles at pH 7.4 via the formation of borate between PBA and Gal. The diameter and zeta potential of GalcoPBA were 121.7  3.4 nm with a PdI of 0.12 and -14.3  1.8 mV, respectively, which were comparable with those of the PBA/Gal micelles. The DLC of ZnPc in the GalcoPBA micelles was 7.8% (Table S1), showing no significant difference compared with that of PBA/Gal micelles.


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To evaluate whether the dual ligands of PBA and Gal could be mutually shielded or deshielded at different pH values, the percentage of exposed Gal, defined as the ratio of exposed Gal to the total amount of Gal on the micelle surface, was calculated, reflecting the holistic variation of exposed ligands on the micellar surface. The percentage of exposed Gal on the PBA/Gal micelle surface increased as the pH decreased (Figure S5). When the pH decreased from 7.4 to 6.8, the percentage of exposed Gal on the micellar surface increased 1.9-fold (Figure 2A and S5), indicating that the PBA and Gal ligands were shielded at pH 7.4 and deshielded at pH 6.8, confirming the pH-responsive mutual shielding of the dual ligands. With the addition of different concentrations of Me-SA, the percentage of exposed Gal on the PBA/Gal micelle surface increased rapidly at pH 6.8 and slowly at pH 7.4 (Figure 2B). This was because PBA preferred to bind with Me-SA compared with Gal at pH 6.8, leading to the exposure of Gal, whereas PBA preferentially bound with Gal rather than with Me-SA at pH 7.4, and at this pH, binding between PBA and Gal still dominated. The result proved the pH-responsive binding of PBA and Gal in the presence of Me-SA. More importantly, after switching the pH values between 7.4 and 6.8 for five cycles, the percentage of exposed Gal on the PBA/Gal micelle surface at pH 7.4 was still lower than that at pH 6.8 during the cycles, and a reversible transition behavior responding to pH changes between the normal physiological environment and the acidic tumor microenvironment was demonstrated (Figure 2A). These results confirmed the reversible shielding nature of the dual-ligand mutual shielding system.


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Figure 2. Percentage of exposed Gal on the PBA/Gal micelles surface at pH 7.4 and 6.8 (A) in five cycles of pH adjustment and (B) after the addition of different concentrations of Me-SA. Flow cytometric histogram (C) and CLSM images (D) of HepG2 cells incubated with ZnPcloaded PBA/Gal micelles under different conditions: (a) at pH 6.8, (b) at pH 6.8 in the presence of free PBA, (c) at pH 6.8 in the presence of free Gal, (d) at pH 7.4, and (e) at pH 6.8 in the presence of both free PBA and Gal. Scale bar, 100 m. (E) HepG2 cellular uptake of PBA/Gal micelles after different cycles of pH adjustment. (F) RAW 264.7 cellular uptake of PBA/Gal and GalcoPBA micelles at pH 7.4 after different cycles of pH adjustment.


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The pH-responsive shielding and deshielding capacity of the PBA/Gal micelles was examined by the cellular uptake of HepG2, a human hepatocellular carcinoma (HCC) cell line with overexpressed biological sialic acid derivatives (e.g., Me-SA)20,34,40 and asialoglycoprotein receptors (ASGPR) on the surface42-44. The flow cytometer analysis (Figure 2C) shows that the cellular uptake of the PBA/Gal micelles at pH 6.8 was 4.3 times higher than that at pH 7.4. However, the un-shielded PBA micelles and Gal micelles showed no significant difference in HepG2 cellular uptake between pH 7.4 and 6.8 (Figure S6), indicating that the cell targeting was facilitated by the deshielding of the ligands rather than by the pH variation. Furthermore, the addition of free PBA or free Gal to the cell culture media reduced the cellular uptake of the PBA/Gal micelles at pH 6.8, demonstrating that the cellular uptake of PBA/Gal micelles is due to the interaction of PBA moieties and Gal moieties with the cells. The addition of both free PBA and free Gal further reduced the cellular uptake of PBA/Gal micelles compared with the separate addition of free PBA or free Gal, indicating the synergistic effect of dual-targeting. Images from confocal laser scanning microscopy (CLSM) exhibit the fluorescence signal variation of the PBA/Gal micelles under different conditions (Figure 2D), which were consistent with the above flow cytometry results and further proved the pH-responsive shielding and deshielding of the PBA/Gal micelles. For comparison of the reversible ligand shielding capacity, the irreversible ligand shielding micelles GalcoPBA were used as the control. As shown in Figure 2E, after five cycles of pH adjustment, the cellular uptake of the PBA/Gal micelles was still effectively inhibited at pH 7.4, and their mean fluorescence intensity (MFI) significantly increased once the pH value was changed to 6.8, exhibiting the reversible ligand shielding and deshielding behavior. In contrast, although the amount of internalized GalcoPBA at pH 7.4 was lower than that at pH 6.8 in the


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first cycle of pH adjustment, cellular uptake significantly increased once the pH value returned to 7.4 in the second cycle and was even comparable to cellular uptake at pH 6.8 (Figure S7A), indicating that the targeting ligands were no longer shielded at pH 7.4. The same situation was also observed for irreversible PBAcoGal micelles (Figure S7B). However, for the PBA/Gal dualligand micelles, the PBA and Gal groups were tethered to the micellar surface, ensuring that the dual ligands would not depart from the micelles, even when deshielded at the acidic tumoral pH, and retained the reshielding ability upon being returned to the normal physiological environment. The ELISA test and HepG2 cellular uptake confirmed the successful construction of the intelligent dual-ligand reversible shielding system. Due to the hydrophobicity, immunogenicity and exogenous origin of the ligands used to modify the surface of the nanoparticles, effective targeting and drug delivery to tumor cells would inevitably be hampered by recognition and endocytosis by macrophages. Therefore, the internalization of the reversible PBA/Gal micelles and the irreversible GalcoPBA micelles into macrophages was studied using RAW 264.7 cells at pH 7.4 by flow cytometry. As shown in Figure 2F, during cycle 1 of pH 7.4, the targeting ligands on the surface of the PBA/Gal and GalcoPBA micelles were in the shielded state, leading to weak recognition and internalization by RAW 264.7 cells. However, after one cycle of pH adjustment (switching the pH from the original 7.4 to 6.8 and then back to 7.4), the MFI of the irreversible GalcoPBA micelles was significantly increased during cycle 2 of pH 7.4, whereas the reversible PBA/Gal micelles retained the low capture rate by RAW 264.7 cells. The irreversible PBAcoGal micelles exhibited similar RAW 264.7 uptake behavior to that of the irreversible GalcoPBA micelles (Figure S8). The protective molecules of the irreversible micelles were removed once the pH was changed to 6.8, and the targeting ligands remained exposed on the nanoparticle surface, no matter whether at


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pH 6.8 or 7.4, facilitating capture by macrophages, which would impede blood circulation and tumor accumulation. In contrast, the dual targeting ligands of the PBA/Gal micelles remained in vicinity on the nanoparticle surface and were mutually shielded by each other, retaining the reversible shielding capacity to suppress recognition and capture by macrophages in the normal physiological environment. The

1O

2

generation of the ZnPc-loaded PBA/Gal micelles was confirmed by a 1,3-

diphenylisobenzofuran (DPBF) assay based on the oxidation of DPBF, as indicated by the decrease in the characteristic absorption at 410 nm.47 Figure S9A shows the typical absorption spectra of DPBF treated with ZnPc-loaded PBA/Gal micelles for different irradiation times, and the absorption decrease at 410 nm (ln(A0/At)) as a function of the irradiation time was finely fitted with the first-order plot. Next, the capacity of the ZnPc-loaded PBA/Gal micelles to produce effective 1O2 in cancer cells was determined by using 2,7-Dichlorofluorescein diacetate (DCFH-DA) as the intracellular 1O2 indicator, as previously described.48 DCFH-DA, a cellpermeable non-fluorescent probe, could be de-esterified intracellularly and then oxidized to green-emissive dichlorofluorescein (DCF) in the presence of intracellular 1O2. Intense green fluorescence was clearly observed for ZnPc-loaded PBA/Gal micelles at pH 6.8 (Figure 3A). Extremely low green fluorescence was detected in the micelles-treated HepG2 cells without 660 nm laser irradiation and in untreated cancer cells in the presence and absence of laser irradiation (Figure 3A). These results confirmed effective 1O2 generation by ZnPc-loaded PBA/Gal micelles in cancer cells. However, when HepG2 cells were incubated with ZnPc-loaded PBA/Gal micelles at pH 7.4, weaker green fluorescence was observed (Figure 3A). The difference in cellular uptake between pH 6.8 and 7.4 caused this difference in fluorescence, confirming the pHresponsive shielding and deshielding effect of PBA/Gal micelles.


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Figure 3. (A) Intracellular 1O2 detection by an inverted fluorescence microscope using DCFHDA as indicator after HepG2 cells were treated with PBA/Gal micelles at pH 7.4 with 660 nm laser irradiation (a), at pH 6.8 with 660 nm irradiation (b), at pH 7.4 without irradiation (c), and at pH 6.8 without irradiation (d). Cells treated with DCFH-DA under irradiation (e) or dark (f) as a control. Scale bar, 200 m. Cytotoxicity of ZnPc-loaded PBA/Gal micelles against HepG2 cells in the absence (B) and presence (C) of 660 nm laser irradiation at pH 7.4 (gray) and 6.8 (red). Cells without any treatment were used as the control.


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After the 1O2 generation ability of the ZnPc-loaded PBA/Gal micelles was validated, the PDT efficacy against HepG2 cancer cells was studied using an MTT assay. Blank PBA/Gal micelles without ZnPc loading showed no toxicity toward HepG2 cells after laser irradiation (Figure S10), and ZnPc-loaded PBA/Gal micelles were not toxic to HepG2 cells without irradiation (Figure 3B), suggesting the biocompatibility of the micelles and the safety of laser irradiation. In sharp comparison, when exposed to a 660-nm laser for 3 min, HepG2 cells treated with ZnPc-loaded PBA/Gal micelles exhibited dose-dependent cytotoxicity due to the PDT (Figure 3C). Moreover, the IC50 at pH 6.8 was 5.7  0.3 M, which was much lower than that at pH 7.4. The ligand mutual shielding of the PBA/Gal micelles at pH 7.4 led to the decrease in HepG2 cellular uptake, resulting in reduced photodynamic toxicity at pH 7.4. However, for PBA/Gal micelles at pH 6.8, the ligands tended to be exposed and facilitate cellular uptake, exhibiting more efficient photodynamic inhibition of cell growth. It is hypothesized that the dual-ligand re-shieldable targeting micelles maintained the ligand shielding capacity, even if they were not taken up by tumor cells and returned to the blood circulation, which is beneficial for evading the RES and extending the circulation time. However, the targeting ligands of irreversible micelles could not be shielded after the protective coating was removed, thus facilitating capture by the RES and shortening the circulation time. To test this theory, Cy5.5-labeled micelles were prepared by the introduction of a certain amount of Cy5.5-PEG-PCL (Scheme S1D and Figure S11) to the polymer solution, and their blood clearance were evaluated following intravenous injection to SD rats. As expected, the residual amount of Cy5.5-labeled PBA/Gal micelles in the blood (expressed as the percentage of the injected dose, %ID) decayed much slower than that of Cy5.5-labeled GalcoPBA micelles (Figure 4A) and PBAcoGal micelles (Figure S12), which verified the superior blood circulation of


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reversible PBA/Gal micelles. Moreover, the pharmacokinetics of ZnPc in ZnPc-loaded reversible and irreversible micelles were also evaluated. Before this experiment, the stability, drug loading and leakage of the different types of micelles were tested, and the results (Figure S13 and Table S1) confirmed that there was no significant difference in the stability, drug loading and leakage between systems. Figure S14 shows the concentration of ZnPc in the plasma versus time. The ZnPc concentration of PBA/Gal micelles was higher than that of irreversible GalcoPBA micelles in the whole time region. For example, at 24 h, the ZnPc concentration of PBA/Gal micelles in the plasma was 0.80 g/mL and 2.8 times of that for GalcoPBA micelles (0.29 g/mL). Additionally, non-compartment model was used to calculate the pharmacokinetic parameters of ZnPc-loaded micelles. The calculated area under curve (AUC0-) (Figure S14B) and terminal elimination half-life (t1/2) (Figure S14C) of PBA/Gal micelles were 1.8- and 1.5-fold those of the GalcoPBA micelles, respectively. Furthermore, the blood clearance (Cl) of the PBA/Gal micelles (0.036 mL/h/g) was much slower than that of the GalcoPBA micelles (0.062 mL/h/g) (Figure S14D). Therefore, the reversible PBA/Gal micelles exhibited a prolonged circulation time compared with that of the irreversible GalcoPBA micelles. The pharmacokinetic parameters of ZnPc-loaded PBAcoGal micelles were similar to those of ZnPc-loaded GalcoPBA micelles (Figure S14). Based on these observations, we considered that the increased blood clearance exhibited by the GalcoPBA micelles or PBAcoGal micelles could be attributed to the irreversible deshielding and fast clearance of PBA or Gal ligands from the micelle surface during blood circulation, respectively. Furthermore, the enhanced circulation time of the PBA/Gal micelles was because of the high concentration of dual ligands on the PBA/Gal micelles that ensured a high mutual shielding effect, allowing them to efficiently elude capture by the RES and rendering them with extended bloodstream circulation.


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Figure 4. Blood clearance profiles (A) and accumulation in liver and tumor (B) of Cy5.5-labeled PBA/Gal micelles and GalcoPBA micelles. Data are presented as means with standard deviations (n = 3) (mean ± SD).  p < 0.05. The in vivo application of PBA/Gal micelles was studied using Kunming mice bearing a H22 heterotopic grafted tumor.40,49,50 The tumor accumulation of Cy5.5-labeled micelles after tailvein injection was monitored by fluorescence imaging using a Maestro system. Cy5.5-labeled PBA/Gal micelles reached peak fluorescence at 36 h post-injection, and their fluorescence was consistently higher than those of Cy5.5-labeled GalcoPBA and PBAcoGal micelles (Figure S15). The photosensitizer ZnPc has the dual functions of use for fluorescence imaging and photodynamic therapy (PDT).51 Therefore, the non-invasive living imaging technique was also employed to investigate the tumor accumulation of ZnPc-loaded PBA/Gal micelles. As shown in Figure 5A and 5B, within the first 24 h, the fluorescence signals in tumors corresponding to ZnPc gradually become stronger in both PBA/Gal and GalcoPBA groups. The group receiving irreversible GalcoPBA micelles reached peak fluorescence at 24 h post-injection, and then the fluorescence began to decrease. Another group receiving irreversible PBAcoGal micelles showed a similar trend of fluorescence variation versus time to that of the GalcoPBA micelles


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group (Figure S16). However, the corresponding fluorescence intensity of PBA/Gal micelles continued to increase until 36 h post-administration and was consistently higher than those of irreversible groups over the experiment period of 48 h. The stronger fluorescence of PBA/Gal micelles demonstrated the better accumulation and retention of PBA/Gal micelles in the tumors, owing to the re-shieldable targeting ability and long circulation of PBA/Gal micelles. As the tumor extracellular pH (pHe) can be decreased by 0.4 pH units after glucose infusion52,53, tumorbearing mice with a pHe perturbated by glucose infusion were utilized as controls for tumor accumulation evaluation. As shown in Figure 5A and 5B, the tumor-bearing mice with glucose infusion showed a stronger fluorescence intensity than those without glucose infusion, demonstrating key roles of tumor acidification and pHe-dependent cellular uptake in vivo. Due to the hydrophobic, immunogenic, and exogenous nature of targeting ligands, tumortargeting nanoparticles injected into blood are prone to be captured by the RES organs such as the liver. This undesired uptake by RES would be harmful to blood circulation and tumor accumulation. The livers and tumors were excised after sacrificing the mice at the moment of the peak fluorescence, and ex vivo imaging was examined (Figure 5C). The liver exhibited weak fluorescence, and the fluorescence of the ZnPc-loaded PBA/Gal group was weaker than that of the GalcoPBA groups, indicating less capture by the RES. A rather high fluorescence intensity was observed in the tumors, and the ZnPc-loaded PBA/Gal micelles exhibited a much stronger fluorescence intensity than did the GalcoPBA groups, indicating the achievement of better tumor accumulation. For quantitative analysis, the ZnPc content in the liver and tumor was examined by homogenization. As shown in Figure 5D, the ZnPc content of PBA/Gal micelles in the liver (1.09%ID) was approximately 54% lower than that of GalcoPBA micelles (2.39%ID). Cy5.5labeled PBA/Gal micelles also showed lower accumulation in the liver than did Cy5.5-labeled


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GalcoPBA micelles (Figure 4B). Considering the results from blood clearance and fluorescence imaging, we speculated that the irreversible-shielding GalcoPBA micelles re-entered the bloodstream with the targeting ligands exposed on the surface of the micelles, leading to immune recognition and RES capture. This caused their higher content in the liver. Significantly, the reversible-shielding PBA/Gal micelles could mutually shield the dual ligands once leaked out of the tumor site, thus decreasing recognition and capture by the RES. The tumor-targeting capability is the most important factor. The tumor accumulation of Cy5.5-labeled PBA/Gal micelles was higher than Cy5.5-labeled GalcoPBA (Figure 4B), and the ZnPc content of ZnPcloaded PBA/Gal micelles in the tumors (1.34%ID) was 40% higher than that of ZnPc-loaded GalcoPBA micelles (0.96%ID) (Figure 5D), demonstrating the better accumulation of the reversible-shielding system. Figure S12 and Figure S16 shows that the tumor accumulation and liver capture of Cy5.5-labeled PBAcoGal micelles and ZnPc-loaded PBAcoGal micelles were similar to those of Cy5.5-labeled GalcoPBA micelles and ZnPc-loaded GalcoPBA micelles, respectively. Moreover, the tumor-bearing mice with glucose infusion exhibited higher tumor accumulation of ZnPc than did those without glucose infusion (Figure 5D), again demonstrating key roles of tumor acidification and pHe-dependent cellular uptake in vivo. Taken together, these results suggested that the pH-responsive dual-ligand reversible shielding strategy holds better abilities to decrease RES capture, prolong blood circulation and facilitate tumor accumulation than do irreversible ligand shielding systems.


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Figure 5. (A) In vivo fluorescence imaging of the tumor-bearing mice at 6, 12, 24, 36, and 48 h after intravenous injection of ZnPc-loaded PBA/Gal micelles, GalcoPBA micelles, and PBA/Gal micelles perturbated by glucose infusion. (B) The quantitative analysis of the average radiance of the tumor site at different time points. (C) Ex vivo fluorescence imaging of a liver and tumor excised from sacrificed mice. (D) ZnPc content in the liver and tumor. In vivo photodynamic tumor inhibition in H22 tumor-bearing mice receiving intravenous injections of different formulations: (E) tumor volume curves and (F) tumor weights obtained on day 14. Data are presented as means with standard deviations (n = 3) (mean  SD).  p < 0.05,  p < 0.01 and  p < 0.001.


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Encouraged by the decreased RES capture and improved tumor accumulation, we investigated whether the dual-ligand reversible ligand shielding strategy can realize more effective PDT in vivo. The irreversible GalcoPBA micelles were utilized as controls. As displayed in Figure 5E, the groups receiving treatment with PBS (laser) and PBA/Gal without laser irradiation group showed remarkable tumor volume increases, indicating that laser irradiation alone wouldn’t inhibit tumor growth. Although the tumor growth rate of the free ZnPc (laser) group was slightly slower than that of the PBS group, the tumor volume was still approximately 9.8 times the initial tumor volume since the nonspecific accumulation of free ZnPc in the tumor was very limited. In contrast, the ZnPc-loaded micelles showed better tumor inhibition upon 660 nm laser irradiation due to the passive and active targeting effects. Importantly, the group treated with PBA/Gal (laser) showed the most efficacious antitumor action, which was better than that from GalcoPBA (laser), indicating the effectiveness and superiority of the dual-ligand reversible shielding strategy in cancer therapy. This was further confirmed by the average weights of the excised tumors (Figure 5F and Figure S17). Specifically, the tumor inhibition rate of the PBA/Gal (laser) group was as much as 90.7%, whereas that of the GalcoPBA (laser) group was 80.4%. The ligand reversible shielding system exhibited a tumor inhibition rate more than 10% higher than that of the irreversible ligand shielding system. Taking the results of the pharmacokinetics and tumor targeting capability studies into consideration, the improved accumulation in the tumor site accounts for the better antitumor efficacy of the reversible shielding strategy. The irreversible PBAcoGal micelles had an antitumor efficacy comparable to that of the GalcoPBA micelles (Figure S18). Taken together, these results validated the practicality of the dual-ligand reversible shielding strategy in cancer therapy.


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In summary, we proposed a new reversible shielding strategy based on the pH-responsive mutual shielding of dual ligands. Due to the formation of a more stable complex between PBA and Gal at pH 7.4, the exposed ligands were reduced, and the cellular uptake of the dual-ligand micelles was effectively suppressed in a physiological environment. Upon reaching the acidic tumor site at pH 6.8, the borate formed by PBA and Gal became unstable, and the unbound PBA and Gal regained their targeting abilities toward Me-SA and ASGPR, leading to an efficient increase in cellular uptake. Compared with the irreversible ligand shielding strategy, this dualligand mutual shielding and re-shieldable targeting system exhibited prolonged blood circulation, reduced RES capture and better tumor accumulation. Furthermore, ZnPc-encapsulated reversible shielding micelles significantly improved the tumor inhibition efficacy compared with irreversible micelles, confirming the effectiveness and superiority of the dual-ligand mutual shielding strategy in vivo. We believe that this dual-ligand mutual shielding strategy will provide inspiration for the design of re-shieldable theranostic nanoparticles. ASSOCIATED CONTENT Detailed experimental materials and methods, additional 1H NMR spectra, gel permeation chromatography traces, cellular uptake analysis, standard curve, pharmacokinetic profile study, stability and release profile, tumor imaging and accumulation of control group (PDF) AUTHOR INFORMATION Corresponding Author *Email: [email protected]. ORCID Zhi Yuan: 0000-0002-5847-2823


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Funding Sources This work was supported by the National Natural Science Foundation of China (Grant 51433004, 51773096), Natural Science Foundation of Tianjin (Grant 17JCZDJC33500), and PCSIRT (Grant IRT1257). ACKNOWLEDGMENT This study is dedicated to 100th anniversary of Nankai University. This work was supported by the National Natural Science Foundation of China (Grant 51433004, 51773096), Natural Science Foundation of Tianjin (Grant 17JCZDJC33500), and PCSIRT (Grant IRT1257). We appreciate Prof. Qiang Wu at Nankai University for help with the characterization of materials and Dr. Chuangnian Zhang, from the Chinese Academy of Medical Sciences & Peking Union Medical College Institute of Biomedical Engineering, for help with polymer synthesis. ABBREVIATIONS PBA, phenylboronic acid; Gal, galactose; DLC, drug loading content; PEG, poly(ethylene glycol); PCL, poly(ε-caprolactone); GPC, gel permeation chromatography. REFERENCES (1) Srinivasarao, M.; Low, P. S. Chem. Rev. 2017, 117 (19), 12133–12164. (2) Zhong, Y.; Meng, F.; Deng, C.; Zhong, Z. Biomacromolecules 2014, 15 (6), 1955–1969. (3) Chen, W.; Zhou, S.; Ge, L.; Wu, W.; Jiang, X. Biomacromolecules 2018, 19 (6), 1732– 1745. (4) Cabral, H.; Miyata, K.; Osada, K.; Kataoka, K. Chem. Rev. 2018, 118 (14), 6844–6892.


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68x43mm (600 x 600 DPI)


Reversible Shielding between Dual Ligands for Enhanced Tumor

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