Acrylated Poly(ethylene glycol) Fabricated Drug

May 16, 2017 - The simple modified PEG nanoparticles are excellent carriers for drug delivery. As examples, the cross-linkable 1d-based drug delivery ...
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Terminal Acetylated/Acrylated Poly(ethylene glycol) Fabricated Drug Carriers: Design, Synthesis, and Biological Evaluation Jie Pang,† Fang Wu,† Chunyan Liao,† Zhongwei Gu,† and Shiyong Zhang*,†,‡ †

National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China

‡

S Supporting Information *

ABSTRACT: The simple acetylation or acrylation of poly(ethylene glycol) (PEG) terminus leads to the aggregation of PEG chains into spherical nanoparticles in water at room temperature and very low concentrations. The experiment results suggest that this aggregation happens by the variation of the local conformation of the O−CH2−CH2−O segments of PEG chains caused by the introduced acyl group, which disturbs the originally strict hydrogen bond mode between the O−CH2−CH2−O groups and the water molecules. The simple modified PEG nanoparticles are excellent carriers for drug delivery. As examples, the cross-linkable 1d-based drug delivery systems, cPEG@SN-38 and targeted cPEG@SN-38, are successfully established by their high drug loading content (18 wt %/wt) and enhanced anticancer efficacy both in vitro and in vivo while obviating the inherent toxicity of the employed chemotherapeutics. This strategy that revolves around the simple modification of the generally regarded as safe (GRAS) modules to fabricate drug carriers represents a new direction for the drug delivery systems with clinical potential.

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INTRODUCTION As classified as generally regarded as safe (GRAS) by the Food and Drug Administration (FDA), PEG, poly(ethylene glycol) or poly(ethylene glycol) monomethyl ether, has been widely used in the design of drug delivery systems.1,2 However, due to the high solubility in water, PEG itself cannot self-assemble into nanoparticles. In most applications, PEG is combined with hydrophobic moieties to form nonionic surfactants, which selfassemble into micelles to achieve the loading of anticancer drugs.1,3 Because most additional moieties themselves have no therapeutic efficacy, and they are virtually redundant substances in the body after the drug release is finished, these residual carriers may cause side effects to organs and tissues in the course of metabolism, degradation, and excretion, such as high toxicity and serious inflammation.4,5 Recently, there have been some reports directly conjugating hydrophobic drugs with PEG to construct drug delivery systems.6,7 This method appears to avoid the additional moieties, but covalent linking between the drug and PEG puts severe constraints on the structure of both components and adds considerable complexity to the production and formulation of the therapeutic package. Moreover, chemical conjugation of drugs with protecting groups can often, as was proved by many other covalent examples, dramatically change the original function of drugs.8,9 Therefore, toward the development of PEG based drug delivery systems, continuing to looking for methods to carry out the aggregation of PEG chains in the case of not using or using as little as possible an additional moiety to load drugs might be an © 2017 American Chemical Society

emerging direction to obtain nanomedicines with clinical potential. By analyzing the structure of PEG molecule, the high watersolubility of PEG is attributed to the distance between the −CH2CH2O− groups, which allows precise matching of the polymer chain into the lattice of water.10,11 By contrast, the mismatched molecules of poly(methylene glycol) and poly(propylene glycol) demonstrate low solubility in water. Further investigation shows that the water-solubility of PEG is also highly correlated with its conformation. The trans−gauche− trans conformation around the successive O−C−C−O bonds of PEG makes it soluble, while the trans−trans−trans conformation or trans−trans−gauche conformation works on the contrary.12 A typical example is the lower critical solution temperature (LCST) behavior of PEG molecules. Below the LCST, the PEG can be hydrated well by its trans−gauche−trans conformation. Once the temperature is increased above the LCST, the trans−trans−trans or trans−trans−gauche conformation becomes dominant so as to disrupt the H-bonds between PEG chains and exogenous water, and eventually cause the aggregation of the solute.13−16 Inspired by above knowledge, we hypothesize that the introduction of mismatched groups into PEG chain might tailor the local conformation of O−C−C−O and achieve the Received: March 23, 2017 Revised: May 15, 2017 Published: May 16, 2017 1956

DOI: 10.1021/acs.biomac.7b00420 Biomacromolecules 2017, 18, 1956−1964

Article

Biomacromolecules Scheme 1. Preparation of Acetylated/Acrylated PEG Based Drug Delivery Carriers

no ZS90), with the laser of 633 nm and 4 mW at 25 °C and the collection time set to be automatic. The number-weighted mean value was obtained from triplicate samples. The fluorescence emission intensity of Nile Red at a wavelength of 575.0 nm (excited at 485.0 nm) was measured using a Hitachi F-7000 fluorescence spectrometer. Fourier-transform infrared (FTIR) spectra were recorded on a Thermo Fisher Nicolet-510P spectrometer. UV−Vis spectrometry was monitored on a Shimadzu UV-2600 instrument at 265 nm. Transmission electron microscopy (TEM) studies were carried out using a TecnaiG2F20S-TWIN instrument, operating at 120 kV. The TEM specimens were prepared by gently placing a carbon-coated copper grid on the surface of the sample. The TEM grid was then removed, stained with an aqueous solution of 2% phosphotungstic acid, dried for 0.5 h at room temperature, and then subjected to TEM observation. The mean particle diameters were determined by TEM software Digital Micrograph, and more than six particles were measured for every diameter. Human nonsmall-cell lung cancer (A549) was obtained from Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China) and used for all of cell experiments and animal experiments. The cell line was grown in Roswell Park Memorial Institute (RPMI-1640) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/ streptomycin in an incubator under 5% CO2 at 37 °C. Cell toxicity was evaluated by measuring the percentage of cell viability via the Cell Counting Kit-8 assay (CCK-8). The absorbance at 450.0 nm was then measured using a microplate reader Varioscan Flash (ThermoFisher SCIENTIFIC). The cell viability (%) was obtained according to the manufacturer’s instructions. The animals were purchased from the Institute of Laboratory Animals of Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital. All animals were left to acclimatize for a week prior to the experiments, and were performed in line with national regulations and approved by the animal experiments ethical committee. Chemicals. Unless otherwise noted, all reagents were obtained from commercial suppliers and used without further purification. Poly(ethylene glycol) monomethyl ether (Mn = 2000, 750, or 350 g/ mol) and poly(ethylene glycol) (Mn = 2000 g/mol) were purchased from Sigma-Aldrich Co. All solvents for reactions were freshly distilled prior to use. Deionized water was used in all aqueous experiments. The synthesis of compounds 1a−d and 2 is reported in the Supporting Information. Typical Preparation of the PEG Nanoparticles (PEG NPs). Compound 1c (4.0 mg, 2.0 mmol) was added into 2.5 mL of deionized water under sonication at room temperature. The resulting solution was then left to stand and the PEG NPs formed spontaneously within minutes as a clear solution. Determination of the Critical Aggregation Concentration (CAC) of Compounds 1a−d.18 A known amount of Nile Red in

aggregation of PEG at room temperature, thereby bringing about new drug carriers. After repeated attempts, we were pleased to find that only a simple acetyl/acrolyl group was conjugated to the end of PEG chains, the room-temperature aggregation of PEG can be realized (Scheme 1), and the critical aggregation concentration could be as low as 10−5 M. To our delight, the resulting acylated/acrylated PEG nanoparticles (NPs) feature even particle size ranging from 100 to 200 nm according to the chain length of PEG employed, which hints that these NPs are suitable to serve as drug carriers. As an example, the PEG NPs formed by cross-linkable ligand 1d were employed to load anticancer drug SN-38, an inhibitor of DNA topoisomerase I. The experiment showed that the PEG NPs owns a drug loading content of up to 18 wt %/wt, much higher than that of most reported drug carriers (typically 10 mg/mL in water), whose active metabolite is SN-38, was employed as the free drug control.34−36 As shown in Figure 7a, the tumors in all drugtreated groups demonstrate growth retardation compared to the controls (0.9% saline). The cPEG@SN-38 group gives a much higher antitumor suppression compared with the irinotecan treated group at an equivalent dose [4.26 ± 0.69 (cPEG@SN-38) versus 6.62 ± 0.76 (irinotecan) fold increase of the tumor size], demonstrating a positive therapeutic effect of SN-38-loaded nanoparticles. More attractively, the targeted cPEG@SN-38 group possesses a significantly better efficacy, which exhibited only a 2.35 ± 0.49 fold increase of the tumor size on day 20, a value 2.82-fold smaller than that from the irinotecan and 1.81-fold smaller than that from the cPEG@SN38. Simultaneous monitoring of the body weight of the administrated animals shows that less body weight shift was observed for the groups administrated by drug loaded nanoparticles compared with the free drug controls (Figure 7b), indicating better drug tolerability. At the end of the treatment, the tumors of all different groups were excised, photographed, and weighed. Proportional to the observed relative tumor volumes, the tumor sizes from mice administrated by PEG NPs were obviously smaller than those from the free irinotecan treatment group and the controls (Figure 7c). The corresponding tumor weight measurement indicates that the tumor weights in mice treated with cPEG@SN-38 and targeted cPEG@SN-38 are ∼1.44- and ∼2.97-fold lower than that treated with the irinotecan molecules, respectively (Figure 7d). Together, these findings testified to our hypothesis that the PEG nanoparticles are able to tremendously enhance the water solubility of drugs, prolong their circulation in blood compartments, and increase their accumulation in tumors via the EPR and targeting effect, thereby improving drug efficacy and minimizing the associated off-target drug effects.

high recognition capability, compound 2 with a biotin unit, which can specifically recognize cells with a high level of expression of biotin receptors (e.g., A549 cell line),29,30 is introduced into the cPEG@SN-38 by simple mixing it with 1d at the beginning of the preparation of nanoparticles (see Experiment section for details). The resulting targeted crosslinked PEG NPs (targeted cPEG@SN-38) was successfully characterized by the DLS and TEM measurement, respectively (Figures 6S and 7S). In vitro Cytotoxicity of cPEG@SN-38 and Targeted cPEG@SN-38. With the two stabilized drug delivery systems (cPEG@SN-38 and targeted cPEG@SN-38) in hand, the in vitro cytotoxicities were then tested by measuring the halfmaximal inhibitory concentration (IC50) of cell proliferation on a human lung carcinoma cancer cell line (A549) (see Experiment Section for details). As shown in Figure 6, both

Figure 6. Cell viability of free SN-38, cPEG@SN-38, and targeted cPEG@SN-38 containing 20% biotin against A549 cells after being cultured for 72 h at 37 °C with a series of concentrations. A549 cells incubated without any materials were used as the control. Data are presented as the average ± the standard deviation (n = 5).

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nanoparticles show pronounced anticancer activity. Notably, with the assistance of the targeting unit, an improved drug activity was carried out for targeted cPEG@SN-38, which gave an IC50 (0.350 μg/mL) lower than that of nontargeted cPEG@ SN-38 (0.543 μg/mL). Further control experiment discloses that the cell viability assay of nondrug loaded cross-linked PEGNPs showed negligible cytotoxicity even at a concentration of mg/mL level in A549 cells (Figure 8S), suggesting the high biocompatibility of the carriers. It should be noted that the in vitro cytotoxicity of both nanoparticles is lower than that of free SN-38 molecules (0.067 μg/mL). This variation is reasonable since SN-38 enters cells through anion-transporting,31,32 while nanoparticles are internalized by endocytosis.33 However, considering their enhanced stability and the EPR and targeting effect, the drug-loaded PEG NPs are expected to get better therapeutic outcomes in vivo. In vivo Antitumor Efficacy of cPEG@SN-38 and Targeted cPEG@SN-38. To evaluate the in vivo antitumor activity of the SN-38 loaded PEG-NPs, we chose A549 tumorbearing nude mice as an animal model. For tumor treatment, saline, irinotecan, cPEG@SN-38 and targeted cPEG@SN-38 containing equivalent drug were intravenously administered in different groups of mice. The same treatment was repeated at every 2 days after the primary injection. The tumor size of each

CONCLUSION

In summary, the simple acetylation or acrylation of PEG terminus was found to be an efficient strategy to tailor the local conformation of O−CH2−CH2−O segments and achieve the aggregation of PEG chains in water at room temperature and very low concentrations, thereby bringing about new drug carriers. As examples, the cross-linkable 1d-based drug delivery systems cPEG@SN-38 and targeted cPEG@SN-38 were successfully established by their enhanced anticancer efficacy both in vitro, on an A549 cancer cell line, and in vivo, on an A549 xenograft model, while suppressing the inherent toxicity of the employed chemotherapeutics. Featuring low-cost preparation, physical drug loading, high drug loading contents, robust stability, easy functionalization, and most importantly, the use of as little as possible additional moieties, the novel acetylated/acrylated polyethylene glycol fabricated drug carriers represent significant advantages over the current PEG involved drug delivery platforms. Our next aim is to further clarify the detailed mechanism of the aggregation and give birth to a general methodology of regulating the structure and property of the PEG nanoparticles to better mastery of their uses. 1962

DOI: 10.1021/acs.biomac.7b00420 Biomacromolecules 2017, 18, 1956−1964

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Biomacromolecules

Figure 7. In vivo anticancer activity of free irinotecan, cPEG@SN-38, targeted cPEG@SN-38 containing 20% biotin, and the control (saline, 0.9% NaCl) after intravenous injection through the tail vein (on days 0, 2, 4, ..., 18, and 20) of nude mice bearing A549 subcutaneous tumors. (a) Tumor growth curve over time. (b) Relative body weight change over time. (c) Representative mice bearing tumors (the first row) and excised tumors (the second row) after treatment for 20 days. (d) Excised tumor weights after treatment for 20 days. Data are presented as the average ± the standard deviation (n = 5).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00420. Synthetic procedures of chemicals, additional CAC, proposed mechanism, DLS, TEM, cell viability, NMR and MS data (PDF)

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REFERENCES

(1) Suk, J. S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L. M. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv. Drug Delivery Rev. 2016, 99, 28−51. (2) Zhang, S.; Wu, Y.; He, B.; Luo, K.; Gu, Z. Biodegradable polymeric nanoparticles based on amphiphilic principle: Construction and application in drug delivery. Sci. China: Chem. 2014, 57, 461−475. (3) He, Z.-Y.; Chu, B.-Y.; Wei, X.-W.; Li, J.; Edwards, C. K.; Song, X.R.; He, G.; Xie, Y.-M.; Wei, Y.-Q.; Qian, Z.-Y. Recent development of poly(ethylene glycol)-cholesterol conjugates as drug delivery systems. Int. J. Pharm. 2014, 469, 168−178. (4) Kahru, A.; Savolainen, K. Potential hazard of nanoparticles: From properties to biological and environmental effects. Toxicology 2010, 269, 89−91. (5) Lanone, S.; Boczkowski, J. Biomedical applications and potential health risks of nanomaterials: Molecular mechanisms. Curr. Mol. Med. 2006, 6, 651−663. (6) Fumagalli, G.; Marucci, C.; Christodoulou, M. S.; Stella, B.; Dosio, F.; Passarella, D. Self-assembly drug conjugates for anticancer treatment. Drug Discovery Today 2016, 21, 1321−1329. (7) Pasut, G.; Veronese, F. M. PEG conjugates in clinical development or use as anticancer agents: An overview. Adv. Drug Delivery Rev. 2009, 61, 1177−1188. (8) Zelikin, A. N.; Ehrhardt, C.; Healy, A. M. Materials and methods for delivery of biological drugs. Nat. Chem. 2016, 8, 997−1007. (9) Palivan, C. G.; Fischer-Onaca, O.; Delcea, M.; Itel, F.; Meier, W. Protein−polymer nanoreactors for medical applications. Chem. Soc. Rev. 2012, 41, 2800−2823.

AUTHOR INFORMATION

Corresponding Author

*Phone: +86-28-85411109. Fax: +86-28-85411109. E-mail: [email protected]. ORCID

Zhongwei Gu: 0000-0003-1547-6880 Shiyong Zhang: 0000-0001-7633-358X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21372170, 51673130), the Excellent Young Foundation of Sichuan Province (2014JQ0032, 2016JQ0028), and the Applied Basic Research Project of Sichuan Province (2015JY0279). 1963

DOI: 10.1021/acs.biomac.7b00420 Biomacromolecules 2017, 18, 1956−1964

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Biomacromolecules (10) Goutev, N.; Ohno, K.; Matsuura, H. Raman spectroscopic study on the conformation of 1,2-dimethoxyethane in the liquid phase and in aqueous solutions. J. Phys. Chem. A 2000, 104 (40), 9226−9232. (11) Begum, R.; Matsuura, H. Conformational properties of short poly(oxyethylene) chains in water studied by IR spectroscopy. J. Chem. Soc., Faraday Trans. 1997, 93 (21), 3839−3848. (12) Wada, R.; Fujimoto, K.; Kato, M. Why is poly(oxyethylene) soluble in water? Evidence from the thermodynamic profile of the conformational equilibria of 1,2-dimethoxyethane and dimethoxymethane revealed by raman spectroscopy. J. Phys. Chem. B 2014, 118 (42), 12223−12231. (13) Wei, P.; Cook, T. R.; Yan, X.; Huang, F.; Stang, P. J. A discrete amphiphilic organoplatinum(II) metallacycle with tunable lower critical solution temperature behavior. J. Am. Chem. Soc. 2014, 136, 15497−15500. (14) Jeong, B.; Kim, S. W.; Bae, Y. H. Thermosensitive sol−gel reversible hydrogels. Adv. Drug Delivery Rev. 2012, 64, 154−162. (15) Li, W.; Zhang, A.; Chen, Y.; Feldman, K.; Wu, H.; Schlüter, A. D. Low toxic, thermoresponsive dendrimers based on oligoethylene glycols with sharp and fully reversible phase transitions. Chem. Commun. 2008, 45, 5948−5950. (16) Lee, E.; Jeong, Y.-H.; Kim, J.-K.; Lee, M. Controlled selfassembly of asymmetric dumbbell-shaped rod amphiphiles: Transition from toroids to planar nets. Macromolecules 2007, 40, 8355−8360. (17) Cai, K.; He, X.; Song, Z.; Yin, Q.; Zhang, Y.; Uckun, F. M.; Jiang, C.; Cheng, J. Dimeric drug polymeric nanoparticles with exceptionally high drug loading and quantitative loading efficiency. J. Am. Chem. Soc. 2015, 137, 3458−3461. (18) Shen, Y. Q.; Jin, E.; Zhang, B.; Murphy, C. J.; Sui, M. H.; Zhao, J.; Wang, J. Q.; Tang, J. B.; Fan, M.; Van Kirk, E.; Murdoch, W. J. Prodrugs forming high drug loading multifunctional nanocapsules for intracellular cancer drug delivery. J. Am. Chem. Soc. 2010, 132, 4259− 4265. (19) Benson, S. P.; Pleiss, J. Self-assembly nanostructures of triglyceride−water interfaces determine functional conformations of candida antarctica lipase B. Langmuir 2017, 33, 3151−3159. (20) Guo, M.; Rong, W. T.; Hou, J.; Wang, D. F.; Lu, Y.; Wang, Y.; Yu, S. Q.; Xu, Q. Mechanisms of chitosan-coated poly (lactic-coglycolic acid) nanoparticles for improving oral absorption of 7-ethyl10-hydroxycamptothecin. Nanotechnology 2013, 24, 245101. (21) Bjorling, M.; Karlstroem, G.; Linse, P. Conformational adaption of poly (ethylene oxide). A 13C NMR study. J. Phys. Chem. 1991, 95, 6706−6709. (22) Yanagida, S.; Takahashi, K.; Okahara, M. Metal-ion complexation of noncyclic poly(oxyethylene) derivatives. III. Complexation in aprotic solvent and isolation of their solid complexes. Bull. Chem. Soc. Jpn. 1978, 51, 3111−3120. (23) Dahal, U. R.; Dormidontova, E. E. Spontaneous insertion, helix formation, and hydration of polyethylene oxide in carbon nanotubes. Phys. Rev. Lett. 2016, 117, 027801. (24) Tasaki, K. Poly(oxyethylene)−water interactions: A molecular dynamics study. J. Am. Chem. Soc. 1996, 118, 8459−8469. (25) Heymann, B.; Grubmüller, H. Elastic properties of poly (ethylene-glycol) studied by molecular dynamics stretching simulations. Chem. Phys. Lett. 1999, 307, 425−432. (26) Shephard, J. J.; Bremer, P. J.; McQuillan, A. J. Structure and conformation in mixtures of methyl-terminated poly(ethylene oxide) and water. Principal component analysis and band fitting of infrared absorptions. J. Phys. Chem. B 2009, 113, 14229−14238. (27) Gradishar, W. J.; Tjulandin, S.; Davidson, N.; Shaw, H.; Desai, N.; Bhar, P.; Hawkins, M.; O’Shaughnessy, J. Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil−based paclitaxel in women with breast cancer. J. Clin. Oncol. 2005, 23, 7794−7803. (28) Uster, P. S.; Working, P. K.; Vaage, J. Pegylated liposomal doxorubicin (DOXIL®, CAELYX®) distribution in tumour models observed with confocal laser scanning microscopy. Int. J. Pharm. 1998, 162, 77−86.

(29) Kumar, R.; Han, J.; Lim, H.-J.; Ren, W. X.; Lim, J.-Y.; Kim, J.-H.; Kim, J. S. Mitochondrial induced and self-monitored intrinsic apoptosis by antitumor theranostic prodrug: In vivo imaging and precise cancer treatment. J. Am. Chem. Soc. 2014, 136, 17836−17843. (30) Heo, D. N.; Yang, D. H.; Moon, H.-J.; Lee, J. B.; Bae, M. S.; Lee, S. C.; Lee, W. J.; Sun, I.-C.; Kwon, II. K. Gold nanoparticles surfacefunctionalized with paclitaxel drug and biotin receptor as theranostic agents for cancer therapy. Biomaterials 2012, 33, 856−866. (31) Nozawa, T.; Minami, H.; Sugiura, S.; Tsuji, A.; Tamai, I. Role of organic anion transporter OATP1B1 (OATP-C) in hepatic uptake of irinotecan and its active metabolite, 7-ethyl-10-hydroxycamptothecin: in vitro evidence and effect of single nucleotide polymorphisms. Drug Metab. Dispos. 2005, 33, 434−439. (32) Wang, H.; Xu, K.; Wang, B.; Liu, J.; Wang, X.; Xing, M.; Huang, P.; Guo, Z.; Xu, L. Microcystin-LR induces a wide variety of biochemical changes in the A549 human non-small cell lung cancer cell line: Roles for protein phosphatase 2A and its substrates. Environ. Toxicol. 2017, 32, 1065−1078. (33) Mahmoudi, M.; Azadmanesh, K.; Shokrgozar, M. A.; Journeay, W. S.; Laurent, S. Effect of nanoparticles on the cell life cycle. Chem. Rev. 2011, 111, 3407−3432. (34) Bala, V.; Rao, S.; Prestidge, C. A. Facilitating gastrointestinal solubilisation and enhanced oral absorption of SN38 using a molecularly complexed silica-lipid hybrid delivery system. Eur. J. Pharm. Biopharm. 2016, 105, 32−39. (35) Wang, H.; Xie, H.; Wang, J.; Wu, J.; Ma, X.; Li, L.; Wei, X.; Ling, Q.; Song, P.; Zhou, L.; Xu, X.; Zheng, S. Self-assembling prodrugs by precise programming of molecular structures that contribute distinct stability, pharmacokinetics, and antitumor efficacy. Adv. Funct. Mater. 2015, 25, 4956−4965. (36) Peng, C.-L.; Lai, P.-S.; Lin, F.-H.; Wu, S. Y.-H.; Shieh, M.-J. Dual chemotherapy and photodynamic therapy in an HT-29 human colon cancer xenograft model using SN-38-loaded chlorin-core star block copolymer micelles. Biomaterials 2009, 30, 3614−3625.

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DOI: 10.1021/acs.biomac.7b00420 Biomacromolecules 2017, 18, 1956−1964