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A targeted and pH-responsive bortezomib nanomedicine in the treatment of metastatic bone tumors Mingming Wang, Xiaopan Cai, Jian Yang, Changping Wang, Lu Tong, Jianru Xiao, and Lei Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07527 • Publication Date (Web): 07 Nov 2018 Downloaded from http://pubs.acs.org on November 8, 2018

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ACS Applied Materials & Interfaces

A targeted and pH-responsive bortezomib nanomedicine in the treatment of metastatic bone tumors Mingming Wang1†, Xiaopan Cai2†, Jian Yang2†, Changping Wang1, Lu Tong1, Jianru Xiao2*, Lei Li1* 1Shanghai

Key Laboratory of Regulatory Biology, East China Normal University, Shanghai,

200241, P.R. China. 2Department

of Orthopedic Oncology, Changzheng Hospital, Shanghai, 200003, P.R. China.

†These authors contributed equally to the manuscript. Corresponding Authors *E-mail: [email protected]. (L. L.) *E-mail: [email protected]. (J. X.)

KEYWORDS: dendrimer, bortezomib, targeted drug delivery, pH-responsive, metastatic bone tumors

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ABSTRACT: Bortezomib is a boronate proteasome inhibitor widely used as an efficient anticancer drug, however, the clinical use of bortezomib is hampered by its adverse effects such as hematotoxicity and peripheral neuropathy, and low efficacy on solid tumors due to unfavorable pharmacokinetics and poor penetration in the solid tumors. In this study, we developed a RGD-targeted dendrimer conjugated with catechol and PEG groups for targeted delivery of bortezomib to metastatic bone tumors. Bortezomib was loaded on the dendrimer via a boronate-catechol linkage with pH-responsive property, which plays an essential role in the control of bortezomib loading and release. The non-targeted bortezomib nanomedicine showed minimal cytotoxicity at pH 7.4, but significantly increased anticancer activity when cRGD moieties were anchored on dendrimer surface. The ligand cRGD enabled efficient internalization of bortezomib complex by breast cancer cells such as MDA-MB-231 cells. The targeted nanomedicine efficiently depressed the progression of metastatic bone tumors and significantly inhibited the tumor-associated osteolysis in a model of bone tumors. This study provided an insight into the development of nanomedicine for metastatic bone tumors.


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INTRODUCTION Bone metastasis is a common complication of cancer, which is observed in 65-80% of patients with metastatic breast and prostate cancers.1-2 A list of devastating complications such as pathological bone fractures, pain, hypercalcemia, deregulated bone remodeling, and spinal cord and nerve compression syndromes usually occur when cancer metastasizes to bone, greatly decreasing the survival rate of cancer patients.3 Bone microenvironment provides the fertile soil for tumor growth. The engagement with bone stromal cells induces tumor cell dormancy, which causes resistance to chemo-therapy and radio-therapy in suppressing bone tumor progression.4 Bortezomib (BTZ) is the first proteasome inhibitor clinically approved for treatment of multiple myeloma and mantle cell lymphoma.5 It is also effective in the treatment of bone tumors such as giant cell tumor of bone, osteosarcoma and metastatic sarcomas through the induction of cell apoptosis, and inhibition of osteoclast recruitment and bone resorption.6-8 BTZ has extremely low half inhibitory concentrations (IC50) towards many cancer cells,9 but is also reported with significant adverse effects such as hematotoxicity, peripheral neurotoxicity and cardiotoxicity.1011

In addition, BTZ has poor aqueous solubility, unfavorable pharmacokinetics, and poor

penetration in solid tumors.12 Nanomedicine integrated with multiple functions such as targeted delivery and responsive drug release can improve the therapeutic efficacy and reduce the adverse effects of BTZ,5, 13-14 lighting up the hope of BTZ for the treatment of metastatic bone tumors. For targeted delivery of nanomedicine to bone diseases, osteoclastic bone resorption surface is usually chosen as the targeting site. In this case, bone-targeting ligands including alendronates, oligopeptides and aptamers were anchored on nanoparticle surface for bone-targeted drug delivery, gene therapy or photothermal therapy.5, 15-23 Alternatively, specific biomarkers on the


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metastatic bone tumors cells could be considered as the target.24-28 Integrin αvβ3 was overexpressed on various tumor cells such as breast cancer cells, and it promotes tumor growth and progression, angiogenesis and metastasis.29 Tripeptide Arg-Gly-Asp (RGD) can specifically recognize integrin αvβ3, and is widely used as a targeting ligand for cancer therapy. 30-33 In this study, we reported a cyclic RGD (cRGD)-targeted polyamidoamine (PAMAM) dendrimer modified with catechol and poly (ethylene glycol) (PEG) ligands for BTZ delivery in the treatment of metastatic bone tumors. Dendrimers with unique properties such as monodisperse, well-defined molecular weight and high density of surface functionality were used as the polymeric scaffold to synthesize the nanomedicine.34-36 Dendrimers are monomolecular micelles, which are distinct from traditional micelles assembled by amphiphilic polymers, and have been widely used as scaffolds for drug and gene delivery.37 Here, the PAMAM dendrimer was successively modified with bifunctional PEG chains for improved blood circulation time, cRGD for targeting of metastatic bone tumors, and catechol groups for BTZ loading via the boronate-catechol linkage.12,

38-40

The catechol-boronate linkage in the

nanomedicine has pH-responsive property and allows “off-on” BTZ release triggered by tumor extracellular acidity. These properties together enable the stability of nanomedicine in normal tissues, targeted delivery of nanomedicine to tumor sites, and rapid release of BTZ in acidic microenvironments. As demonstrated by in vitro and in vivo studies, the designed BTZ nanomedicine showed high efficacy in the inhibition of tumor progression and bone tumorassociated osteolysis. EXPERIMENTAL SECTION Materials.


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Ethylenediamine-cored and amine-terminated generation 5 (G5) PAMAM dendrimer (molecular weight 28826 Da) was purchased from Dendritech (Midland, MI). PEG (N-hydroxysuccinimide 5-pentanoate) ether N’-(3-maleimidopropionyl) aminoethane (NHS-PEG-MAL, molecular weight 2057 Da) was purchased from JenKem Tech. (Beijing, China). The anticancer drug BTZ was

supplied

by

Yeexin

(Shanghai).

N,N’-dicyclohexylcarbodiimide

(DCC),

N-

hydroxysuccinimide (NHS), 3,4-dihydroxyphenylacetic acid (DPA) were supplied by Aladdin Biotech. (Shanghai, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltrazolium bromide (MTT) was purchased from Sangon (Shanghai, China). The chemicals methanol, ethanol, triethylamine, dimethyl sulfoxide, and ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate were purchased from Sinopharm Chemical Reagent (Shanghai, China). G5 dendrimer dissolved in distilled water was lyophilized as a light yellow colored gel before use. Synthesis and characterization of cRGD-targeted dendrimer with DPA and PEG groups. G5 PAMAM dendrimer (100 mg, 3.5 μmol) and NHS-PEG-MAL (122 mg, 59.3 μmol) were dissolved in 1 mL dimethyl sulfoxide and 4 mL PBS buffer, respectively. The solutions were mixed together (pH 7.2), and stirred for 24 h under room temperature. The product was intensively dialyzed against distilled water (molecular weight cut off 3500 Da), lyophilized to obtain PEGylated G5 dendrimer (G5-PEG-MAL), and further characterized by NMR spectroscopy (700 MHz, Varian) to calculate average number of modified PEG-MAL. For cRGD-modified G5-PEG-MAL, the peptide cRGD-SH (9.2 mg, 16.0 μmol) was dissolved in 2 mL EDTA buffer (3 wt%, pH 7.2). Then the solution was mixed with G5-PEG-MAL (106 mg, 2.0 μmol), and stirred for 24 h at room temperature. The product was intensively dialyzed against PBS buffer and distilled water (molecular weight cut off 3500 Da), and lyophilized to


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obtain G5-PEG-cRGD. The product was characterized by NMR to calculate the average number of cRGD on G5 PAMAM dendrimer. For DPA-modified G5-PEG-cRGD, DPA (14.6 mg, 86.8 μmol) was activated by DCC (23.3 mg, 113 μmol) and NHS (12 mg, 104 μmol) in 2 mL dehydrated dimethyl sulfoxide for six hours. Triethylamine was added into the reaction mixture as an acid-binding agent. G5-PEGcRGD (105 mg, 1.93 μmol) in 2 mL dehydrated dimethyl sulfoxide was then added slowly into DPA, and the reaction solution was kept stirring at room temperature for seven days. The crude product was intensively dialyzed (molecular weight cut off 3500 Da), and lyophilized to obtain DPA-G5-PEG-cRGD. The average number of DPA modified on each G5 dendrimer was characterized by 1H NMR (700 MHz, Varian). DPA-G5-PEG without cRGD modification was synthesized as described above and used as the non-targeted control material. Fabrication of BTZ-loaded DPA-G5-PEG-cRGD. The anticancer drug BTZ was dissolved in dimethyl sulfoxide, and the polymer DPA-G5-PEGcRGD was in deionized water as stock solutions. The drug was then mixed with the polymer at BTZ/polymer molar ratios of 10:1 and 20:1, respectively. Drug-loaded polymers were kept at 37 oC

for two hours prior to further experiments. To reveal the role of cRGD targeting, BTZ-loaded

DPA-G5-PEG complexes without cRGD (BTZ/polymer molar ratio of 10:1 and 20:1) were prepared as negative controls. The BTZ/polymer complexes (10:1) prepared at pH 7.4 or 5.0 in D2O (containing 0.5% d6-dimethyl sulfoxide to dissolve BTZ during complex formation) were characterized by 1H NMR (700 MHz, Varian). BTZ release from the DPA-G5-PEG-cRGD/BTZ complex.


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The drug release experiments were conducted as described below. BTZ-loaded DPA-G5-PEGcRGD (3.9 mmol BTZ, BTZ to polymer molar ratio of 10:1) was prepared as described above and the free BTZ in the complex solution was removed by quickly dialyzed against PBS buffer for 2 h. After that, the purified complex in the dialysis bag was placed in a cup containing 50 mL buffer solutions (pH 7.4, 6.5 or 5.0, respectively). The outer phase solution was kept stirring during the release experiments, and 100 μL of the outer phase solution was collected at different time. Drug concentrations in the samples were measured by HPLC (Agilent1200 equipped with a C18 column). The mobile solvent was methanol/deionized water at a volume ratio of 7:3. The flow rate is 1.0 mL/min. 10 µL of the collected sample was injected and the flowed sample was detected at 260 nm. Three independent experiments were repeated in drug release experiments. The size and zeta potential of DPA-G5-PEG, DPA-G5-PEG-cRGD, DPA-G5-PEG/BTZ and DPA-G5-PEG-cRGD/BTZ complexes were measured by dynamic light scattering (DLS) at 25 oC

(Malvern Zetasizer Nano ZS 90, UK).

Cell culture and BTZ cytotoxicity assay. MDA-MB-231 cells (human breast cancer cells, ATCC) were cultured in Minimal Essential Medium (MEM). The cells were cultured in 96-well plates at 37 oC for 24 h before cytotoxicity assay. The cells (around 104 cells per well) were incubated with BTZ, BTZ-loaded DPA-G5PEG-cRGD or DPA-G5-PEG complexes (pH 7.4) for 48 h, followed by the removal of culture media, washing with fresh PBS for three times, and addition with MTT containing media. After incubation for two hours, dimethyl sulfoxide was used to dissolve the produced purple formazan by living cells. Absorbances of the samples were measured at 490 nm by a microplate reader (MQX200R). The absorbances were used to reveal the sample toxicity according to the value of non-treated cells. Five independent experiments were repeated in the MTT assay.


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Cellular uptake of DPA-G5-PEG-cRGD and DPA-G5-PEG. The polymers DPA-G5-PEG-cRGD and DPA-G5-PEG were labelled with a blue fluorescent dye 1-pyreneboronic acid (PBA) via the same catechol-boronate linkage. The polymers were incubated with PBA for 2 h at 37 oC before cellular uptake assay. MDA-MB-231 cells were seeded in a 24-well plate overnight before incubated with PBA-labelled polymers (100 nM) at 37 oC

for 6 h. After that, the media in each well were removed, and the cells were washed with PBS

for three times. The washed cells were then observed by the fluorescence microscope (Olympus, Japan). Non-treated cells or the ones incubated with PBA in the absence of polymers were negative controls. Establishment of metastatic bone tumor model. MDA-MB-231 cells that stably expressing luciferase (MDA-MB-231-luc) were used to establish the bone tumor model. BALB/c nude mice (five weeks, female, 20~22 g) were purchased from the Animal Center in East China Normal University (ECNU). All the animal experiments were carried out according to the NIH guidelines for care and use of laboratory animals, and approved by the ethics committee of ECNU. Generally, MDA-MB-231-luc (~105 cells) in 20 μL PBS buffer were injected into the right tibia of each animal via a percutaneous approach. Two weeks after the injection, the mice were intravenously injected with D-luciferin, and the bioluminescence images of bone tumors in the tibia of each animal were observed by in vivo imaging system (Xenogen IVIS-200, Caliper Life Sciences, Hopkinton). The mice with bone tumors were randomly separated to four groups (five animals in each group). In vivo therapeutic efficacy of the BTZ nanomedicine.


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The mice bearing bone tumors were injected with PBS buffer, BTZ, BTZ/DPA-G5-PEG-cRGD (RGD+) and BTZ/DPA-G5-PEG (RGD-) complexes, respectively via tail vein injection (200 μL, 0.5 mg BTZ per kilogram animal, BTZ/polymer molar ratio is 10:1). The injections were repeated every 2 d with 5 injections in total. The body weight of the animals was recorded every day. Two days after the final injection, bioluminescence images of bone tumors were obtained by the in vivo imaging system. X-ray and micro-CT images on the tibias with bone tumor were taken to detect the bone destruction in the mice. The excised bone tumors in each group were weighted. The in vivo data were analyzed by student’s t-test, one tailed. Pharmacokinetic behavior and biodistribution of the BTZ nanomedicne. DPA-G5-PEG-cRGD and DPA-G5-PEG were labeled with an NIR fluorescent dye Cy5.5. The BTZ-loaded complexes were prepared as described above and intravenously injected into BALB/c mice without tumor. The doses of polymers and BTZ were equal to those used in therapeutic experiments. Then, blood was collected at scheduled time post injection, and 200 μL plasma was obtained by centrifugation at 3000 rpm. The plasma was diluted with PBS, and fluorescence intensity of the sample was tested by a fluorescence spectrophotometer (Excitation wavelength 700 nm, and Emission wavelength 720 nm). The amount of Cy5.5-labeled polymer in the blood was calculated according to a standard curve. For biodistribution studies, the mice bearing MDA-MB-231 tumors were administrated with Cy5.5-labeled BTZ/DPA-G5-PEG-cRGD and BTZ/DPA-G5-PEG complexes. The doses of polymers and BTZ were equal to those used in therapeutic experiments. The mice were sacrificed at 6 h, 12 h or 24 h post-injection. The tumors and major organs were harvested, and


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fluorescence image of the washed tumors and tissues were imaged by an in vivo imaging system (IVIS, Lumina-II, Caliper Life Sciences). For TUNEL staining, the tumor tissues harvested from mice in the four groups were fixed in 4% formalin solution for 12 h. The tumor tissues were then embedded in paraffin and sectioned. The sections were incubated with proteinase K, TUNEL reaction mixture and Hoechest 33342 according to the protocols of in situ apoptosis detection kit (Roche, Mannheim Germany). The sections were observed by using a fluorescence microscope. Blood toxicity assay and H&E staining Healthy BALB/c nude mice were treated with PBS, free BTZ, BTZ/DPA-G5-PEG-cRGD and BTZ/DPA-G5-PEG complexes. The doses of BTZ and polymer were equal to those in therapeutic experiments, and a total number of five injections were administrated as described above. The mice were sacrificed, and the blood were collected. The blood toxicity was measured by a hematology analyzer (HEMAVET-950, Drew Scientific Inc.). The harvested heart, liver, spleen, kidneys, and lung tissues were fixed in 4% formalin solution. The tissues were embedded in paraffin blocks, sectioned into slices, and stained with hematoxylin and eosin, respectively. The tissue sections were then observed by an optical microscope.

RESULTS AND DISCUSSION Preparation of DPA-G5-PEG-cRGD and the BTZ complex. The cRGD-targeted and pH-responsive polymeric carrier for BTZ delivery was synthesized by facile chemistry shown in Figure 1. To prolong the blood circulation of G5 PAMAM dendrimer,


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the polymer was first grafted with NHS-PEG-MAL, and the introduced MAL moieties on dendrimer surface were conjugated with cRGD-SH via thiol-maleimide addition reaction. cRGD was used to improve the internalization of polymeric nanocarriers by breast cancer cells MDAMB-231 overexpressing integrin αvβ3.41-42 DPA molecules for BTZ loading were coupled to dendrimer surface by condensation reaction between carboxyl on DPA and primary amine on G5 dendrimer.9 The synthesized polymers were characterized by 1H NMR (Figure 2). Characteristic peaks of methylene protons for PEG and G5 PAMAM, vinyl protons for MAL, and aromatic protons for cRGD and DPA were observed. According to the NMR spectra, the average numbers of PEG, cRGD and DPA molecules conjugated on each G5 dendrimer were calculated to be 12, 2 and 30, respectively.

Figure 1. Synthesis of cRGD-targeted and pH-responsive BTZ nanomedicine.


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Figure 2. The 1H NMR spectra of G5-PEG-MAL (a), G5-PEG-cRGD (b), DPA-G5-PEG-cRGD (c), and DPA-G5-PEG (d) in D2O. The proton assignments for vinyl protons on MAL, aromatic protons on cRGD and DPA, and methylene protons on G5 dendrimer were shown.

BTZ molecules were loaded on DPA-G5-PEG-cRGD via the pH-responsive catechol-boronate linkage. The BTZ/dendrimer molar ratio was 10:1. We measured the size and zeta-potential of DPA-G5-PEG, DPA-G5-PEG-cRGD and BTZ-loaded nanoparticles by DLS. As shown in Figure S1, the size of DPA-G5-PEG was about 17 nm in aqueous solution. The RGD-modified polymer DPA-G5-PEG-RGD showed a slightly increased hydrodynamic size. After BTZ loading, both the DPA-G5-PEG/BTZ and DPA-G5-PEG-RGD/BTZ nanoparticles showed a hydrodynamic size around 80 nm and the nanoparticles were nearly neutrally charged,


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suggesting the self-assembly of BTZ-loaded polymer in aqueous solution. The complex was also characterized by 1H NMR. As shown in Figure 3, free BTZ was observed with sharp peaks such as the methyl protons H11, H12, the pyrazine protons H1-3, and the phenyl protons H4-8 (Figure 3a). After complexation with DPA on the dendrimer at pH 7.4, all the peaks for BTZ became broaden (Figure 3b), suggesting the reduced molecular motion for BTZ and the formation of DPA-G5-PEG-cRGD/BTZ complex 43. When the pH was tailored to 5.0, sharp proton peaks for BTZ were almost recovered, which is due to the acid-labile property of catechol-boronate linkage in the complex.44 This result enabled the rapid release of BTZ molecules from the polymer matrix at acidic microenvironments. To confirm the pH-responsive drug release behaviors of DPA-G5-PEG-cRGD/BTZ complex, we investigated the BTZ release kinetics from the polymer using an equilibrium dialysis method. As shown in Figure 4a, BTZ showed extremely slow release from the polymer at pH 7.4. Less than 5% of BTZ molecules were released at 12 h, suggesting good stability of the complex at physiological condition. However, the release rate of BTZ was significantly promoted at pH 5.0, which is usually known as the lysosomal pH.45 Nearly 57% of the drugs were detected in the outer phase at 6 h under pH 5.0. Warburg effect is a well-known phenomenon in most malignant tumors.46 Increased glycolysis and reduced oxidative phosphorylation in tumor cells produce excess lactic acid in tumor tissues.47 Therefore, most solid tumors showed an acidic environment with pH ranging from 6.5 to 6.8.48 To determine if BTZ in the complex could be released when accumulated in the tumor extracellular environment, we also conducted the in vitro drug release study at pH 6.5 to mimic the tumor extracellular acidity. As shown in Figure 4a, the weak acidity at pH 6.5 also significantly triggered the release of BTZ from DPA-G5-PEG-cRGD. 33% of the drugs were


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released at 6 h under this pH condition. This property is beneficial for the targeted delivery of anticancer drugs in tumor microenvironments.

Figure 3. The 1H NMR of free BTZ (a) and BTZ nanomedicine prepared at BTZ to DPA-G5PEG-cRGD molar ratio of 10:1 and pH 7.4 (b), and the same nanomedicine prepared at pH 5.0 (c) in D2O. The proton assignments for BTZ, cRGD, DPA and G5 dendrimer protons were shown.

Cellular uptake and anticancer activity of the DPA-G5-PEG-cRGD/BTZ complex. The integrin αvβ3 were reported to overexpress on breast cancers such as MDA-MB-231 cells, 49 and the biomarker is associated with tumor growth, invasion and metastasis. To confirm the targeting ability of the synthesized polymer containing cRGD towards breast cancer cells, we


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first used PBA to label the polymer via the same catechol-boronate linkage. PBA is a blue fluorescent dye with a boric acid group.50 As shown in Figure 4b, free PBA treated cells exhibited extremely low internalization. The MDA-MB-231 cells treated with the DPA-G5PEG-cRGD/PBA complex showed significant blue fluorescence signals. In comparison, cells treated with the non-targeted control complex exhibited minimal uptake, suggesting the essential role of cRGD in targeted delivery of PBA into the breast cancer cells. We further tested the toxicity of BTZ and the complexes with DPA-G5-PEG-cRGD or DPAG5-PEG on MDA-MB-231 cells. The complexes were prepared at a BTZ to polymer molar ratio of 10:1. As shown in Figure 4c, free BTZ showed high anticancer activity against MDA-MB-231 cells with an IC50 value around 12.9 nM, after complexation with DPA-G5-PEG, the drug showed minimal toxicity on the cells at concentrations up to 2000 nM, this is due to stable binding of BTZ to the DPA ligands on polymer. The non-targeted polymer DPA-G5-PEG had a neutral and hydrophilic surface and showed minimal cellular uptake as shown in Figure 4b. In addition, the linkage between BTZ and DPA in the complex is stable in the culture media (pH 7.4), and minimal drugs were released (Figure 4a). Therefore, the DPA-G5-PEG/BTZ complex caused low toxicity towards MDA-MB-231 cells. For cRGD-targeted nanoparticle, the targeted nanomedicine showed significantly higher toxicity on the breast cancer cells in comparison with the non-targeted one (Figure 4c). The IC50 of the targeted complex is 370.6 nM. Since both polymers without BTZ were non-toxic on the MDA-MB-231 cells at concentrations up to 2000 nM. The increased toxicity of cRGD-targeted BTZ nanomedicine is due to the improved internalization of BTZ complex by the cancer cells via the RGD/αvβ3 recognition. The anticancer activity of the internalized complex could be turned on by lysosomal pH as shown in Figure 4a. As a result, the DPA-G5-PEG-cRGD/BTZ complex showed potent anticancer activity towards


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MDA-MB-231 cells, and the activity is attributed to both cRGD-mediated internalization and acidity-triggered BTZ release. Similar results were observed when the complexes were prepared at a BTZ to polymer molar ratio of 20:1 (Figure 4d). The activity of prepared DPA-G5-PEGcRGD/BTZ complex is not as high as free BTZ, which can be explained by the sustained release of BTZ from the nanoparticle and the high activity of BTZ itself on the cancer cells. The targeting and pH-responsive properties of the BTZ nanomedicine are beneficial for increasing the therapeutic outcome and reducing adverse effects of BTZ during cancer therapy.

Figure 4. BTZ release kinetics from the targeted nanoformulation at different pH conditions (a). Uptake of PBA, PBA-loaded DPA-G5-PEG-cRGD and DPA-G5-PEG complexes by the breast cancer cell MDA-MB-231 for six hours (b). Viability of MDA-MB-231 incubated with free BTZ


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and BTZ complexes prepared at BTZ and polymer molar ratio of 10:1 (c) and 20:1 (d), respectively for 48 h. In vivo therapeutic efficacy of the DPA-G5-PEG-cRGD/BTZ complex on metastatic bone tumors. We investigated the pharmacokinetic behaviors of DPA-G5-PEG/BTZ and DPA-G5-PEGRGD/BTZ nanoparticles in vivo. Both polymers were labeled with an NIR dye Cy5.5 and then loaded with BTZ at a BTZ to polymer molar ratio of 10:1. As shown in Figure S2, both the DPA-G5-PEG/BTZ and DPA-G5-PEG-RGD/BTZ nanoparticles showed a relative long blood circulating time, which is probably due to the presence of PEG chains on the nanoparticles. The biodistributions of non-targeted and targeted nanoparticles labeled with Cy5.5 in the main tissues as well as intratibial tumors were measured by IVIS. As shown in Figure S3, the targeted nanoparticles DPA-G5-PEG-RGD/BTZ showed a higher tumor accumulation than the nontargeted control at 6 h, 12 h and 24 h, which is due to the specific molecular recognition between RGD and the integrin αvβ3 overexpressed on breast cancer cells such as MDA-MB-231 cells. We further evaluated the anticancer activity of the prepared BTZ nanomedicine in a bone tumor model (Figure 5a). MDA-MB-231 cells stably expressing firefly luciferase gene was injected into the tibia of nude mice to establish the animal model. Two weeks after the injection, the mice were administrated with D-luciferin, and examined by in vivo imaging system. The animals with luminescence in the tibia were divided into four groups, and treated with PBS, free BTZ, DPA-G5-PEG/BTZ (RGD-), and DPA-G5-PEG-cRGD/BTZ (RGD+), respectively via intravenous injection. As shown in Figure 5b, the animals in the PBS group showed much stronger luminescence intensities after treatment than those before the treatment, suggesting


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rapid progression of the bone tumors during the therapeutic period. In comparison, the animals treated with the DPA-G5-PEG-cRGD/BTZ complex showed the lowest luminescence intensities among the four groups (Figure 5c), which indicated the effectiveness of the targeted BTZ nanomedicine in tumor inhibition. The cRGD targeted BTZ nanomedicine is more effective than the non-targeted BTZ formulation. After the treatment, the tumors in each animal were weighted. The animals treated with targeted BTZ nanomedicine had the smallest tumors among the groups (Figure 5d). TUNEL staining results in Figure 5e suggest that the targeted BTZ nanomedicine caused a much higher level of apoptosis in the tumors than the control groups. We also evaluated the blood toxicity in healthy mice receiving PBS, BTZ, DPA-G5-PEG/BTZ and DPA-G5-PEGRGD/BTZ nanoparticles, respectively. The doses and injection schedules were equal to those in the in vivo therapeutic experiments. As shown in Figure S4, BTZ caused serious blood toxicity after intravenous injection, the levels of WBC, LYMPH#, BSO# and PLT were significantly inhibited in the mice receiving free BTZ. However, the mice treated with non-targeted or targeted BTZ nanomedicine showed limited blood toxicity due to the efficient binding of BTZ in the nanoparticles during blood circulation. The main organs and tissues including heart, kidney, liver, lung and spleen in the mice treated with targeted and non-targeted BTZ nanoparticles were similar to those in the mice treated with PBS only (Figure S5), suggesting the low toxicity of the nanoparticles. In addition, no obvious decrease in body weight of the mice was observed during the therapeutic period (Figure 5f), suggesting the low systemic toxicity of the nanoformulation. Free BTZ also did not cause the decrease of body weight during period, and this is due to the relatively low BTZ dose (0.5 mg/kg) administrated in this study.


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Figure 5. Treatment of MDA-MB-231 bone tumors via intravenous injection of cRGDtargeted BTZ nanomedicine (a). Bioluminescence images of MDA-MB-231-luc tumorbearing mice before and after the treatment with PBS, free BTZ, non-targeted BTZ nanomedicine (RGD-), and cRGD-targeted BTZ nanomedicine (RGD+), respectively (b). Relative luminescence intensity of mice in the four groups (c). The luminescence intensity of mice in the cRGD-targeted BTZ group was set as 1. Weights of the excised tumors after treatment are shown in (d). Apoptosis of tumor cells analyzed by a TUNEL assay. The cell nuclei were stained by Hoechst 33342. Magnification, ×20. (Scale bar, 50μm). (e). Body weights of animals during the therapeutic period (f). *p < 0.05 and **p < 0.01 analyzed by Student’s t-test, one tailed.


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Figure 6. X-ray images of tibias in the mice administrated with PBS, BTZ, non-targeted BTZ (RGD-), and cRGD-targeted BTZ (RGD+) nanomedicine, respectively (a). 3D micro-CT reconstruction of the mice tibias in the four groups after treatment (b). The bone volume (mm3, c) and trabecular number (mm-1, d) of tibias at the tumor site after


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treatment according to the micro-CT analysis. *p < 0.05 analyzed by Student’s t-test, one tailed. It is reported that the crosstalk between bone cancer cells and osteoblasts will disturb the bone homeostasis, and thus the growth of bone tumors is usually associated with osteolysis. We therefore measured the tibias with bone tumors by X-ray and micro-CT after the treatment. As shown in Figure 6a and 6b, both X-ray and micro-CT images revealed that the tibias in the RGD+ group had the lowest bone destruction compared to those in the control groups. We further quantitatively evaluated the bone volume and the trabecular number (Tb.N) in the tibia of each mouse. As shown in Figure 6c and 6d, both the bone volume and the trabecular number in the RGD+ group were much higher than those in the control groups, and the results were in accordance with the X-ray and micro-CT images. These results confirmed that the DPA-G5PEG-cRGD/BTZ formulation can inhibit the growth of bone tumors and tumor-associated osteolysis.

CONCLUSIONS In summary, we synthesized a cRGD-targeted and pH-responsive polymer for BTZ delivery. The targeted BTZ nanomedicine was stable at physiological condition, efficiently internalized by breast cancer cells, released the BTZ cargo triggered by endolysosomal acidity, and effectively killed the cancer cells. In vivo therapeutic results revealed that the targeted BTZ nanomedicine successfully inhibited the growth of bone tumors and osteolysis in a metastatic bone tumor model. The results provided a promising polymeric carrier for the targeted delivery of BTZ in the treatment of bone tumors.


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AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. (L. L.) *E-mail: [email protected]. (J. X.)

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Size and zeta-potential of RGD-, RGD+, RGD- BTZ and RGD+ BTZ nanoparticles. Pharmacokinetic behaviors of DPA-G5-PEG/BTZ and DPA-G5-PEG-RGD/BTZ nanoparticles in vivo. The biodistribution of DPA-G5-PEG/BTZ and DPA-G5-PEG-RGD/BTZ nanoparticles in the main tissues and intratibial tumors. Histological examination of the H&E sections of major organs.

Acknowledgements We would like to thank the financial supports form the National Key Research and Development Program of China (2016YFC0902100) and the National Natural Science Foundation of China (81871470, 81672883). REFERENCES (1) Weilbaecher, K. N.; Guise, T. A.; McCauley, L. K. Cancer to bone: a fatal attraction. Nat Rev Cancer 2011, 11 (6), 411-25. (2) Wang, C.; Cai, X.; Zhang, J.; Wang, X.; Wang, Y.; Ge, H.; Yan, W.; Huang, Q.; Xiao, J.; Zhang, Q.; Cheng, Y. Trifolium-like Platinum Nanoparticle-Mediated Photothermal Therapy Inhibits Tumor Growth and Osteolysis in a Bone Metastasis Model. Small 2015, 11 (17), 20806,. (3) Gartrell, B. A.; Saad, F. Managing bone metastases and reducing skeletal related events in prostate cancer. Nat Rev Clin Oncol 2014, 11 (6), 335-45. (4) Meads, M. B.; Hazlehurst, L. A.; Dalton, W. S. The bone marrow microenvironment as a tumor sanctuary and contributor to drug resistance. Clin Cancer Res 2008, 14 (9), 2519-26.


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