DACHPt-Loaded Unimolecular Micelles Based on Hydrophilic

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DACHPt-Loaded Unimolecular Micelles Based on Hydrophilic Dendritic Block Copolymers for Enhanced Therapy of Lung Cancer Gan Liu,†,§,∥ Hongjun Gao,‡,∥ Yixiong Zuo,†,§,∥ Xiaowei Zeng,†,§ Wei Tao,†,§ Hsiang-i Tsai,†,§ and Lin Mei*,†,§ †

The Shenzhen Key Lab of Gene and Antibody Therapy and Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, People’s Republic of China ‡ Kingfa Science & Technology Company, Ltd., Guangzhou 510663, People’s Republic of China § School of Life Sciences, Tsinghua University, Beijing 100084, People’s Republic of China S Supporting Information *

ABSTRACT: Combining sufficient stability during circulation and desirable drug release is still a great challenge for the clinical applications of nanocarriers. To satisfy this demand, we developed a novel unimolecular micelle (UM) to deliver the antitumor agent 1,2diaminocyclohexane-platinum(II) (DACHPt) for enhanced therapy of lung cancer. This DACHPt-loaded UM (UM/DACHPt) was formed through chelate complexation between DACHPt and a hydrophilic and biodegradable dendritic block copolymer poly(amidoamine)-polyglutamic acid-b-polyethylene glycol (PAM-PGlub-PEG), which was composed of generation 3 PAMAM (PAMAMG3), polyglutamic acid, and long-circulating polymer PEG. This UM/DACHPt displayed robust stability and would effectively inhibit the undesired release under physiological condition, thus exhibiting much longer in vivo half-life than diblock copolymer micelles. With significant in vitro cell cytotoxicity to A549 lung cancer cells, this UM/DACHPt demonstrated efficient antitumor efficacy on an A549 xenograft tumor model with negligible tissue cytotocxity. Therefore, this UM/DACHPt provides a promising new strategy for lung cancer therapy. KEYWORDS: unimolecular micelles, dendritic block copolymers, DACHPt, lung cancer, polyglutamic acid

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proved to be effective.24−26 However, cross-linking would reduce biodegradability and decrease the release rate of drugs, which may not be suitable for practical applications, and thus makes development of novel nanocarriers combining both stability and controlled release of value. Unimolecular micelles (UMs), fabricated from single multiarm star block copolymers, have attracted numerous attention for their excellent in vivo structural integrity as well as other advantages, such as no surrendered degradability and drug release profiles and narrow disperse dimensions.27−33 Poly(amidoamine) (PAMAM) dendrimers, the first commercialized dendrimer family,34 have been widely utilized as macroinitiors to synthesize unimolecular micelles due to their numerous tailorable terminal functional groups.35−37 For instance, aminoterminated PAMAM could directly initiate the ring-opening polymerization of the monomer γ-benzyl-L-glutamate-Ncarboxyanhydride (BLG-NCA) to obtain PAMAM- polyglutamic acid, which could be loaded with the potent antitumor

INTRODUCTION Platinum-based antitumor drugs are intensively used in clinical applications to treat ovarian, bladder, lung, head, and neck cancers.1−6 These potent antitumor agents, even the thirdgeneration platinum drug oxaliplatin, however, were greatly limited due to their low delivery efficacy, which not only decreased the antitumor efficacy but also caused severe doselimiting toxicity such as neurotoxicity and vomiting.7−11 Longcirculating nanomedicines including PEGylated liposomes and polymeric nanoparticles,12−18 which would prolong the blood circulation and improve accumulation in solid tumors through the enhanced permeability and retention (EPR) effect,19,20 are promising strategies to enhance the anticancer efficacy and decrease the side effects. Unfortunately, platinum-based PEGylated liposomes only showed modest antitumor response due to their robust stability and inefficient drug release from the carriers.21 On the other hand, conventional nanoparticles from block copolymer self-assembly suffer from insufficient stability in vivo and lead to the burst release of drugs in the bloodstream during circulation.22,23 To enhance the thermodynamic stability of the micelles, various strategies including core or shell crosslinked polymeric micelles have been widely investigated and © XXXX American Chemical Society

Received: September 19, 2016 Accepted: December 14, 2016 Published: December 14, 2016 A

DOI: 10.1021/acsami.6b11917 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic illustration of the DACHPt-loaded unimolecular micelle (UM/DACHPt) formed through the complexation between the carboxylic groups in PAM-PGlu-b-PEG and DACHPt.

Figure 2. Synthesis of the dendritic block copolymer PAM-PGlu-b-PEG.

agent 1,2-diaminocyclohexane-platinum(II) (DACHPt) through stable chelate complexation.38 Herein, we design a novel DACHPt-loaded UM (UM/ DACHPt) for enhanced therapy of lung cancer. This UM/ DACHPt was formed through chelate complexation between DACHPt and a hydrophilic and biodegradable dendritic block copolymer poly(amidoamine)- poly(glutamic acid)-b-poly(ethylene glycol) (PAM-PGlu-b-PEG), which was composed of dendrimer PAMAM-G3, DACHPt-chelator poly(glutamic acid), and long-circulating polymer PEG (Figure 1). Its size, ζ potential, drug loading content, drug encapsulation efficiency, in vitro stability, and drug release profiles were characterized. Its in vitro cellular cytotoxicity was assessed in A549 cancer cells. Pharmacokinetics study was carried out to test its half-life in vivo. Its in vivo antitumor efficiency and histopathology evaluation were further investigated on the A549 xenograft tumor model.

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EXPERIMENTAL SECTION

Materials. BLG-NCA was purchased from Kangmanlin chemicals (Nanjing, China). PAMAM-NH2-G3 (PAM-NH2, Mw = 6900 Da), CF3COOH, HBr/HAc (33% (w/v)), DACHPtCl2, and oxaliplatin were purchased from Aladdin Industrial (Shanghai, China). PEG45NH2 and PEG45-succinimidyl carboxymethyl ester (PEG45-SCM) were purchased from Ponsure Biotechnology (Shanghai, China). All the chemicals were commercially available and used as received. A549 cells were obtained from American Type Culture Collection (ATCC). Fetal bovine serum (FBS) was purchased from Lonza Walkerrsville. The Dulbecco’s modified eagle medium (DMEM) and penicillin/ streptomycin were both purchased from Invitrogen. Female Balb/C nude mice (∼18 g, 6 weeks old) were purchased from Guangdong Province Medical Animal Center and fed in a SPF (specific pathogen free) class experimental animal room. Synthesis of dendritic block copolymer PAM-PGlu-b-PEG. As shown in Figure 2, First, PAM-PBLG-NH2 was synthesized through the ring-opening polymerization. A 4 g amount of BLG-NCA in DMF was initiated by 0.24 g of the dendrimer PAM-NH2 (Supporting Information Figure S-1) to obtain dendritic copolymer PAM-PBLGB

DOI: 10.1021/acsami.6b11917 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces NH2. The degree of polymerization of PBLG in PAM-PBLG-NH2 was verified by 1H NMR spectroscopy (Varian UNITY-plus 400 M nuclear magnetic resonance spectrometer; solvent, CDCl3). Then 20 mL of trifluoroacetic acid dissolving PAM-PBLG-NH2 (2 g) was added in 2 mL of hydrogen bromide (HB; 33% in acetic acid) and stirred for 1 h at room temperature. Then the solution was neutralized by sodium hydroxide (NaOH) and dialyzed against distilled water (DI water) using a dialysis membrane with molecular weight cutoff (MWCO) of 3.5 kDa. The aqueous solution of purified product was lyophilized to obtain PAM-PGlu-NH2. Then PAM-PGlu-NH2 was reacted with excess PEG-SCM in DI water at room temperature for 2 h to obtain the dendritic block copolymer PAM-PGlu-b-PEG. The unreacted PEG and impurities were dialyzed out of the dialysis membrane with MWCO of 3.5 kDa. PAM-PGlu-b-PEG was obtained after lyophilization. The diblock copolymer PEG-b-PGlu was synthesized according to our previous work (Figure S-2).39 Preparation of DACHPt-Loaded Micelles. DACHPt-loaded micelles were prepared similar to the procedure outline in ref 38. Briefly, PAM-PGlu-b-PEG and PEG-b-PGlu polymer solutions were separately mixed with the DACHPt aqueous solution (Molar ratio Glu/DACHPt = 3/1) and reacted for 72 h. The formed micelles were purified by ultrafiltration using Centricon Plus-20 centrifugal filter units (MWCO, 50 kDa; Millipore, MA, USA). Characterization of DACHPt-Loaded Micelles. The size distribution and ζ potential of DACHPt-loaded micelles were measured using a Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., Malvern, U.K.). Before measurement, the freshly prepared micelles were diluted as needed. All measurements were carried out at 25 °C. The data were obtained as the average of three measurements. The morphologies of the micelles were then observed by transmission electron microscopy (TEM; Tecnai G2 20, FEI Co., Hillsboro, OR, USA). A 1 μL aliquot of the sample solution was placed on the resulting grids. The sample was stained by depositing a drop of mixture of 50% ethanol and 1% sodium phosphotungstate (pH 7.0) onto the surface of the sample-loaded grid and dried in the air. To measure the drug loading content (LC) and drug encapsulation efficiency (EE) of the DACHPt-loaded micelles, a predefined amount of micelles was dissolved in 1 mL of water. The Pt concentration in the micelles was quantified by inductively coupled plasma mass spectrometry (ICP-MS; Xseries II, Thermo Scientific, Waltham, MA, USA). The LC and EE of DACHPt-loaded micelles were calculated according to

LC/% =

amout of DACHPt in the micelles × 100 amount of the micelles

EE/% =

amount of DACHPt in the micelles × 100 amount of feeding DACHPt

from orbital cavity was collected, heparinized, and centrifuged (14000 rpm, 10 min, 4 °C) to obtain the plasma.40 After being heated in nitric acid, the plasma samples were decomposed and their Pt contents were quantified by ICP-MS. The plasma concentration was fitted as a biphasic behavior using the equation as follows: C p = A1e−kdt + A 2 e−ket + A3 where Cp is the concentration of plasma Pt and kd and ke are respectively the rate constants for the distribution and elimination processes. In Vivo Antitumor Efficacy. After being randomly divided into four groups (n = 5), the mice were built with a human A549 xenograft tumor model by injection of 1.5 × 106 A549 cells (150 μL) subcutaneous at the right axilla of each mouse. Before initiating treatment, tumors were observed frequently and allowed to grow to ∼50 mm3 in volume. Mice were injected intravenously four times via tail vein on days 0, 4, 8, and 12 with saline, oxaliplatin, and DACHPtloaded micelles (6 mg/kg on a Pt basis). The antitumor efficacy was determined in accordance with the tumor volume (V), which was calculated similarly to that of our previous work.32 The body weights of the mice were simultaneously measured to evaluate the systemic toxicity. Histopathology Evaluation. Optical microscopy was used to assess the histopathology evaluation of tissues after treatments with hematoxylin and eosin (H&E).40 On the 20th day, after the animals were sacrificed, the hearts, lungs, livers, spleens, kidneys, and tumors of the mice were collected and dehydrated in PBS with 10% formaldehyde overnight. Subsequently the tissues were embedded with paraffin followed by being cut into 5 μm slices. After staining with H&E, the tissues were observed under optical microscopy and the photographs were taken by a Nikon camera fitted on a Nikon Eclipse 600 microscope (Melville, NY, USA). Statistical Analysis. All of the experiments were carried out at least three times. The data are expressed as mean ± SD unless noted otherwise and analyzed for significance using Student’s t-test.32 Probability value (P) < 0.05 indicates a statistically significant value: *, P < 0.05; **, P < 0.01.

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RESULTS AND DISCUSSION

Synthesis and Characterization of Dendritic Copolymer PAM-PGlu-b-PEG. The chemical structure of the dendritic copolymer PAM-PGlu-b-PEG was demonstrated by 1 H NMR. First, the representative spectrum of PAM-PBLGNH2 was shown in Figure 3A; peak a represents the signal of the methylene protons of PAMAM; and peak b−e represent the signals of -CHCH2CH2C(O)-, -CHCH2CH2C(O)-, -C(O)CH(CH2)NH-, and C6H5CH2- of PBLG. The degree of polymerization of PBLG in each PAM-PBLG-NH2 was calculated to be 384 by comparing the peak areas of a, c, and d with e. Because each PAMAM-G3 contains 32 primary amines, the average number of PBLG units initiated by each amine should be 12. Then after acid hydrolysis, results the vanishment of the 1H NMR signal corresponding to the benzyl group (Figure 3B, peak e) and the identical DP between PGlu and PBLG segments both indicate complete deprotection of BLG without breakage of the PGlu backbones.39 Then as shown in Figure 3C, peak e represented the methylene of PEG (-OCH2CH2-: δ = 3.7 ppm), demonstrating the successful grafting of PEG45 onto the dendritic copolymer. The number of PEG units grafted onto each copolymer was quantified to be 30 by comparing the proton ratios of the methylene units of PEG (peak e) with the methyne units of PGlu (peak b), confirming that almost all the PGlu blocks were grafted with PEG, and the dendritic block copolymer PAM-PGlu-b-PEG was successfully synthesized.

In Vitro Drug Release Study. The stability of UM/DACHPt in cell culture media including 10% FBS at 25 °C was evaluated by DLS. The DACHPt release profile of the micelles was monitored by the dialysis method. First, 1 mL of DACHPt-loaded micelles was placed into the dialysis bag (MWCO, 3.5 kDa) and immersed in 30 mL of cell culture media including 10% FBS (pH 7.4 and 5.5) at 37 °C. The media outside were taken out at defined periods, and the concentrations of Pt were measured by ICP-MS. MTT Assay. The 50% growth inhibitory concentrations (IC50) of free oxaliplatin, M/DACHPt, and UM/DACHPt in the A549 cell lines were measured by the MTT assay. After incubated in a 96-well culture plate (104 cells/well) for 24 h, A549 cells were then exposed to 10, 20, 40, 100, and 200 μM oxaliplatin or DACHPt-loaded micelles (on a platinum basis) for another 24, 48, and 72 h. At each point of time, The MTT solution was added, and cell viability was measured in a BioRad 680 microplate reader by formazan absorbance at 490 nm. Pharmacokinetics Study. To analyze the in vivo plasma clearance of DACHPt-loaded micelles, mice (n = 3) were intravenously injected with oxaliplatin, M/DACHPt, and UM/DACHPt at 100 μg/mouse on a Pt basis. At defined time periods (1, 4, 8, 12, 24, and 48 h), blood C

DOI: 10.1021/acsami.6b11917 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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hydrodynamic diameter with greatly extended PEG chains. On the other hand, the M/DACHPt self-assembled from the diblock copolymer PEG-b-PGlu and DACHPt through the complexation between the carboxylic groups of poly(glutamic acid) and DACHPt was also monodispersed with the hydrodynamic diameter as ∼60.5 nm (Table 1). In Vitro Stability and Drug Release Profiles. After the fabrication of UM/DACHPt, its in vitro stability was investigated. As expected, the size of UM/DACHPt remained stable after incubation in cell culture media including 10% FBS for 48 h (Figure 5A). The DACHPt loading content of UM/DACHPt was measured to be 25.4% by ICP-MS (Table 1). Then the in vitro DACHPt release from UM/DACHPt and M/DACHPt in cell culture media at pH 7.4 and 5.5 were monitored, as the previous study reported that the micelles complexed from DACHPt and carboxyl group would release DACHPt under chloride ion-containing and acidic conditions.38 As shown in Figure 5B, DACHPt was obviously released from M/DACHPt at pH 7.4 with the released amounts being ∼50% at 96 h, whereas its release was accelerated at pH 5.5 with the released amounts as >80% at 96 h. These results are consistent with other reports,40 confirming the burst release of M/DACHPt under physiological conditions. In stark contrast, the released amounts of DACHPt from UM/DACHPt were much less than that of M/DACHPt at pH 7.4, which was only 20% at 96 h. This result proves that the UM/DACHPt would effectively inhibit the burst release under physiological conditions and maybe hold longer circulation time in vivo. Moreover, the DACHPt was also rapidly released from the UM/DACHPt at pH 5.5, anticipating its fast release in tumor. In Vitro Cytotoxicity. To verify the in vitro cytotoxicity of UM/DACHPt, the MTT assay in A549 cells was carried out. M/DACHPt and oxaliplatin with the same DACHPt concentrations and DACHPt-free UM with the same polymer concentration were used as the controls. The UM/DACHPt and control groups with varying DACHPt concentrations were added into the cells for 24, 48, and 72 h incubation. As shown in Figure 6A, after 24 h incubation, oxaliplatin exhibited obvious cell cytotoxicity, whereas the cytotoxicities of M/ DACHPt and UM/DACHPt were much weaker. The IC50 of oxaliplatin was calculated to be 98.8 μM while those of M/ DACHPt and UM/DACHPt were calculated to be 214.0 and 361.9 μM (Table 2). The weaker cytotoxicity of UM/DACHPt compared with M/DACHPt would mainly be attributed to the less DACHPt release in the beginning, which is consistent with the in vitro release profiles, while, after 72 h incubation, the cytotoxicity of UM/DACHPt significantly increased (Figure

Figure 3. 1H NMR spectra of PAM-PBLG384-NH2 in CDCl3 (A), PAM-PGlu384-NH2 in D2O (B), and PAM-PGlu384-b-(PEG45)30 in D2O (C).

Similarly, the diblock copolymer PEG-b-PGlu was characterized by 1H NMR spectroscopy. As shown in Figure S-3, the number of PGlu units was calculated to be 18, approximately identical with that of each arm of PAM-PGlu-b-PEG. Therefore, PEG-b-PGlu could be used as the control group of PAM-PGlu-b-PEG. Fabrication and Characterization of DACHPt-Loaded Micelles. Because of its completely hydrophilic structure, the dendritic block copolymer PAM-PGlu-b-PEG would form a unimolecular micelle in water with the hydrodynamic diameter as 59.4 ± 2.1 nm (Figure 4A). After complexation of PAMPGlu-b-PEG and DACHPt, the Pt-loaded micelle UM/ DACHPt was obtained. This UM/DACHPt was narrow, monodispersed with the hydrodynamic diameter as ∼54.2 ± 2.9 nm (Figure 4A). The size of UM/DACHPt was smaller than non-Pt-loaded UM, demonstrating that the UM/DACHPt was still a unimolecular micelle without aggregation of micelles after the complexation of UM and DACHPt. Meanwhile, its slightly decreased size should be attributed to the shrink of PGlu layer after complexation. TEM image showed that the UM/DACHPt was also monodispersed with diameter as 20− 40 nm (Figure 4B), which is consistent with that of DLS. Compared to the size of UM/DACHPt measured by DLS, that measured by TEM was relatively smaller because the TEM measures the size of dried micelles while the DLS measures the

Figure 4. (A) Size distribution of the UM and UM/DACHPt micelles and (B) TEM image of the UM/DACHPt micelles. D

DOI: 10.1021/acsami.6b11917 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Table 1. Characterization of DACHPt-Loaded Micellesa

a

micelles

Dh (nm)

PDI

ZP (mV)

LC (%)

EE (%)

UM/DACHPt M/DACHPt

54.2 ± 2.9 60.5 ± 3.2

0.12 0.15

−20.3 ± 2.1 −23.8 ± 2.6

25.4 ± 1.2 24.3 ± 1.3

70 ± 3.2 68 ± 3.0

PDI = polydispersity index; ZP = ζ potential; LC = loading content; EE = encapsulation efficiency.

Figure 5. (A) DACHPt-loaded micelles incubated in media containing 10% FBS. They maintained their sizes for 48 h. (B) Accumulative release of the DACHPt-loaded micelles in media containing 10% FBS with different pH. Each datum was obtained as the average of three measurements.

Figure 6. Cell viability of A549 cells incubated with the UM/DACHPt compared with that of oxaliplatin and M/DACHPt at the same oxaliplatin dose and that of the drug-free UM with the same polymer concentrations: (A) 24, (B) 48, and (C) 72 h.

Table 2. IC50 Values of Oxaliplatin, M/DACHPt, and UM/ DACHPt on A549 Cells Following 24, 48, and 72 h Incubation IC50 (μM) time (h)

oxaliplatin

M/DACHPt

UM/DACHPt

24 48 72

98.8 ± 8.4 16.1 ± 1.1 12.2 ± 0.91

214.0 ± 2.0 34.1 ± 2.8 9.3 ± 0.82

361.9 ± 3.2 30.2 ± 2.6 8.0 ± 0.78

6C) and its IC50 decreased to 8 μM. This is even slightly stronger than that of free oxaliplatin (12.2 μM), demonstrating the sustained release of DACHPt from UM/DACHPt with the increase of incubation time and acidity of environment. Moreover, the DACHPt-free UM had no obvious effect on the cell viability, indicating its biocompatibility and further potential clinical practice. Pharmacokinetics Study. To study the in vivo pharmacokinetics of UM/DACHPt, the plasma Pt concentrations were monitored over time after the intravenous injection of UM/ DACHPt. The Pt concentrations were quantified by ICP-MS and fitting to the two-compartment pharmacokinetic model. As shown in Figure 7, the Pt concentration of the free oxaliplatin group decreased instantly after injection, with only less than 10% left after 4 h. In contrast, M/DACHPt significantly

Figure 7. Plasma Pt concentrations in mice after intravenous injection of oxaliplatin, M/DACHPt, and UM/DACHPt as a function of time postinjection.

extended the blood residence time of DACHPt and increased its concentration in the blood compartment, remaining detectable even at 24 h after injection, whereas compared to that of M/DACHPt, the plasma Pt concentrations and its residence times in mice injected with UM/DACHPt increased further, with obvious levels remaining even at 48 h after E

DOI: 10.1021/acsami.6b11917 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 8. Effect of the DACHPt formats on antitumor efficacy (A) and changes of body weight (B) of A549 xenograft-bearing nude mice. Images (C) and weights (D) of the tumors resected from each group of the sacrificed mice on the last day.

Figure 9. H&E stained tumor, heart, lung, liver, spleen, and kidney slices from mice on the 20th day after treatments of saline, oxaliplatin, and DACHPt-loaded micelles.

treatment, as shown in Figure 8A, the tumor volume of the saline-treated, oxaliplatin-treated, and M/DACHPt-treated groups increased to 396.5 ± 42.5, 277.3 ± 32.8, and 177.3 ± 19.6 mm3, respectively, whereas the tumor volume of the UM/ DACHPt-treated group only increased to 115.2 ± 29.5 mm3. Obviously, the UM/DACHPt formulation outperforms much more than the oxaliplatin and M/DACHPt in inhibition of the tumor growth, consistent with the in vitro drug release profiles and in vivo pharmacokinetics. At the same time, the UM/ DACHPt-treated mice did not exhibit significant weight loss during the study, similar to the three other controls (Figure 8B). On the 20th day, the tumors were resected following

treatment. The half-lives of M/DACHPt and UM/DACHPt were calculated to be 7.1 and 13.2 h, respectively. This result is consistent with the more robust structure of UM/DACHPt, anticipating that the UM/DACHPt would increase accumulation in tumor through EPR effect and reduce the system toxicity.41,42 In Vivo Antitumor Efficacy. Inspired by the above positive results, the in vivo antitumor efficacy of UM/DACHPt was evaluated last. A549 tumor xenograft-bearing mice were treated with saline, oxaliplatin, M/DACHPt, and UM/DACHPt every 4 days for continuous four times from when their tumor volumes became ∼50 mm3. Twenty days after the first F

DOI: 10.1021/acsami.6b11917 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (Grant No. 31270019), the China Postdoctoral Science Foundation (Grant No. 2015M580109), the Guangdong Natural Science Funds for Distinguished Young Scholar (Grant No. 2014A030306036), the Natural Science Foundation of Guangdong Province (Grant No. 2016A030310023), Guangdong Special Support Program, Science and Technology Planning Project of Guangdong Province (Grant No. 2016A020217001), Science, Technology & Innovation Commission of Shenzhen Municipality (Grant Nos. JCYJ20150529164918738 and JCYJ20150430163009479). We thank Lingyan Luo for her kind assistance in the animal studies.

killing of all the mice. Panels C and D of Figure 8 clearly show the morphology and average weight of the tumors in all of the groups, directly confirming the efficient antitumor efficacy of UM/DACHPt. Histopathology Evaluation. To assess the necrotic degree of the tumor and organs, the mice were sacrificed followed by collecting the tumor and major organs. The tissue slices were stained by hematoxylin and eosin (H&E) and observed using optical microscopy. In normal tissues, hematoxylin would dye the cell nuclei as indigo, while eosin would dye the cell cytoplasm and extracellular matrix as pink. However, necrotic cells could hardly show clear cell morphology. As Figure 9 showed, the tumor from all of the Pt-treated groups displayed obvious necrosis. In particular, more severe necrosis could be observed from DACHPt-loaded micelles-treated groups, which is consistent with the in vivo antitumor results. On the other hand, H&E staining results of major organs containing heart, lung, liver, spleen, and kidney showed that free oxaliplatin did not cause significant damage mainly due to the much reduced toxicity compared to cisplatin.40 Furthermore, injection of DACHPt-loaded micelles also had negligible side effects to the mice, thus greatly broadening the therapeutic dose of the platinum drugs.

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CONCLUSIONS The unimolecular micelle UM/DACHPt based on the hydrophilic and biodegradable dendritic block copolymer PAM-PGlu-b-PEG was developed for delivery of the potent anticancer drug DACHPt. Compared to the block copolymer micelles, this unimolecular micelle exhibited more robust stability, more desirable drug release profile, and longer in vivo half-life. Moreover, it demonstrated significant in vitro cytotoxicity and further efficient in vivo antitumor efficacy with negligible side effects. Considering that maintaining stability during the circulation is a precondition for further in vivo applications of micelles, this stable platform brings this goal closer and provides firm foundation for further multifunctionalization, such as grafting active targeting ligands and combination therapy. ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at on the The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b11917. Chemical structure of PAMAM dendrimer, synthesis of block copolymer PEG-b-PGlu, preparation of DACHPt aqueous solution, antitumor efficacy measurements, and 1 H NMR spectra of polymers (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Lin Mei: 0000-0001-6503-5149 Author Contributions ∥

REFERENCES

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G.L., H.G., and Y.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest. G

DOI: 10.1021/acsami.6b11917 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.6b11917 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX