Enhanced Intracellular Delivery and Tissue Retention ofNanoparticles by Mussel-Inspired Surface ChemistryKai Chen,†,‡ Xiaoqiu Xu,†,‡,§ Jiawei Guo,† Xuelin Zhang,† Songling Han,† Ruibing Wang,∥ Xiaohui Li,§

and Jianxiang Zhang*,†

†Department of Pharmaceutics, College of Pharmacy, and §Institute of Materia Medica, College of Pharmacy, Third Military MedicalUniversity, Chongqing 400038, China∥State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa,Macau, China

*S Supporting Information

ABSTRACT: Nanomaterials have been broadly studied for intracellular delivery ofdiverse compounds for diagnosis or therapy. Currently it remains challenging fordiscovering new biomolecules that can prominently enhance cellular internalization andtissue retention of nanoparticles (NPs). Herein we report for the first time that a mussel-inspired engineering approach may notably promote cellular uptake and tissue retentionof NPs. In this strategy, the catechol moiety is covalently anchored onto biodegradableNPs. Thus, fabricated NPs can be more effectively internalized by sensitive andmultidrug resistant tumor cells, as well as some normal cells, resulting in remarkablypotentiated in vitro activity when an antitumor drug is packaged. Moreover, the newlyengineered NPs afford increased tissue retention post local or oral delivery. Thisbiomimetic approach is promising for creating functional nanomaterials for drug delivery,vaccination, and cell therapy.

■ INTRODUCTION

Intracellular delivery of various therapeutics and contrast agentsis crucial for prevention, diagnosis, and treatment of mostdiseases. Nanomaterials have been extensively utilized to delivera plethora of bioactive compounds into cells, which include smallmolecular drugs, peptides/proteins, and nucleic acids.1−5 So far,nanoparticles (NPs) with different biophysicochemical proper-ties, such as varied size, shape, composition, surface charge, andchemistry, and biological functions have been studied for drugdelivery, gene therapy, biosensing, and molecular imaging.6−11

Biologically functionalized NPs can also be used to modulatestem cells or regulate immune cells for cell-based therapies ortissue regeneration.12,13 To fully realize these diverse applica-tions, NPs must reach their intracellular destination, whichnecessitate the capability to traverse the biological barrier of theplasma membrane. Generally, cellular uptake of NPs is mediatedby membrane-embedded receptors or by interacting with thelipid bilayer via hydrophobic and/or electrostatic interactions.14

Accordingly, different biochemical strategies have been devel-oped for rational surface modifications or coatings to increase cellbinding affinities, which are frequently implemented by physicalcoating or covalent conjugation of cell penetrating peptides,receptor ligands, antibodies, and aptamers.1,4,15,16 Althoughsome progress has been achieved based on these approaches,their in vivo applications and clinical translation remainschallenging, mainly due to synthetic cost, in vitro and in vivostability, loss or attenuation of efficiency in the complexbiological milieu, regulatory hurdles, or potential immunoge-nicity after repeated dosing.17−20 Further, exploring new

molecules that can target specific endocytic pathways is highlynecessary in certain cases such as nonviral gene delivery andvaccination. As a result, there is still unmet demand fordiscovering novel biomolecules that can effectively enhancecellular internalization of NPs for both in vitro and in vivoutilization.3,4-Dihydroxyphenylalanine is a critical component of

adhesive proteins secreted by marine mussels, and its catecholicfunctionality plays an important role in the adhesion of musselsto wet surfaces in the ocean.21,22 Materials containing thecatechol moiety afford adhesive, coating, and anchoringproperties, which can bind strongly to various inorganic andorganic surfaces.22−29 Recent studies also reveal that mussel-mimetic hydrogels with dopamine (DOPA, also has the catecholgroup) units display superior adhesive forces toward theepididymal fat pad, the external liver surfaces, the inside surfaceof blood vessels, and atherosclerotic plaques.30,31 These resultsdemonstrated that structured materials decorated with DOPAmoieties may have additionally increased absorption, binding,and adhesion capabilities to different surfaces and tissues. Thiswas largely realized by combined effects of oxidation-dependentconjugation and hydrogen-binding (H-bonding). Notably,oxidation of catechol in DOPA may produce the quinonestructure that further forms cross-linking via aryl−arylcoupling.32 Alternatively, quinones may react with amine-

Received: August 5, 2015Revised: September 28, 2015

Article

pubs.acs.org/Biomac

© XXXX American Chemical Society A DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

containing biomolecules through Michael-type addition reac-tions.33 Also, DOPA and quinones could form H-bonding withpolysaccharide moieties and amino acid residues of the cellmembrane. On the basis of these issues, herein we hypothesizethat surface engineering of NPs with DOPA may enhance theirintracellular uptake, taking advantages of the superior adhesiveand binding capability of the catechol moiety (Figure 1A). As aproof of concept, DOPA was covalently conjugated onto NPs viaa hydrophilic linker of polyethylene glycol (PEG). Thus,decorated NPs showed significantly enhanced internalizationby different cell lines, which in turn led to remarkably potentiatedefficacy of paclitaxel (PTX) payload against both sensitive andmultidrug resistant (MDR) tumor cells.

■ EXPERIMENTAL SECTIONMaterials. β-Cyclodextrin (β-CD) and lecithin (from soybean) were

purchased fromTokyoChemical Industry Co., Ltd. (Tokyo, Japan). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N -[carboxy-(polyethylene glycol)-2000] (DSPE-PEG-COOH) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) were purchased from Avanti Polar Lipids, Inc.(U.S.A.). 2-Methoxypropene (MP), dopamine hydrochloride (DOPA·HCl), and nocodazole were obtained from Sigma (St. Louis, U.S.A.).N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride(EDC·HCl) and N-hydroxysuccinimide (NHS) were purchased fromFluka (U.S.A.). Paclitaxel (PTX) was supplied by Xi’an HaoxuanBiological Technology Co., Ltd. (Xi’an, China). Pyridinium p-toluenesulfonate (PTS) was obtained from Acro Organics. Poly(lactide-co-glycolide) (PLGA, 50:50) with an intrinsic viscosity of 0.50−0.65 waspurchased from Polysciences, Inc. (U.S.A.). Penicillin, streptomycin,fetal bovine serum (FBS), and Dulbecco’s Modified Eagle’s Medium(DMEM) were purchased from HyClone (Waltham, MA, U.S.A.).RPMI1640 medium was obtained from Gibco (U.S.A.). Cy5 NHS ester

and Cy7.5 NHS ester were obtained from Lumiprobe, LLC (U.S.A.).4′,6-Diamidino-2-phenylindole (DAPI) and LysoTracker Red werepurchased from Invitrogen (U.S.A.). 1,1′-Dioctadecyl-3,3,3′,3′-tetra-methylindocarbocyanine perchlorate (Dil) was purchased fromBeyotime (China). Cytochalasin D was supplied by Cayman ChemicalCompany (U.S.A.).

Synthesis of DOPA-Conjugated DSPE-PEG. DOPA-conjugatedDSPE-PEG (DSPE-PEG-DOPA) was synthesized by coupling reaction.To this end, 100 mg of DSPE-PEG-COOH (35 μmol) was dissolvedinto 40mL PBS (pH 7.4). Then, 20mg of NHS (176 μmol) and 67.3 mgEDC·HCl (350 μmol) were added. After 12 h of activation at roomtemperature, 66.4 mg of DOPA·HCl (350 μmol) was added into thereaction mixture, followed by magnetic stirring for 24 h under theprotection of nitrogen at 4 °C. The obtained polymer was purified bydialysis for 24 h using dialysis tubing (molecular weight cutoff of 1000Da) in deionized water and then collected by freeze-drying.

Synthesis of Acetalated β-CD. Acetonation of β-CD wasperformed in the presence of excess amount of MP, using PTS as acatalyst.34 Briefly, 20 mL of MP (210 mmol) was added into 100 mL ofanhydrous DMSO containing 5 g β-CD (4.4 mmol), into which 80 mgPTS was added. After 3 h of acetalation under magnetic stirring at roomtemperature, the reaction was terminated by adding 2 mL oftriethylamine into the mixture. The acetalated product (Ac-bCD) wasprecipitated from water, collected by filtration, thoroughly washed withdeionized water, and lyophilized to a white powder.

Preparation of Various Nanoparticles. A modified nano-precipitation/self-assembly method was employed to prepare Ac-bCDNPs.35 Briefly, 50 mg Ac-bCD was dissolved in 2 mL of acetonitrile.Lecithin and DSPE-PEG at the molar ratio of 7:3 were dispersed in 0.8mL of ethanol, and then 19.2 mL of deionized water was added. Thus,obtained aqueous dispersion was heated to 65 °C for 1 h. Then, the Ac-bCD solution was added into the preheated aqueous solution dropwise(1 mL/min) under gentle stirring, followed by vortexing for 3 min. After2 h of incubation, the mixture was cooled to room temperature. Thesolidified NPs were collected by centrifugation at 16000 rpm for 10 min,

Figure 1. Engineering of high affinity nanoparticles (NPs). (A) Schematic showing enhanced intracellular uptake of DOPA-coated NPs based onacetalated β-CD (Ac-bCD): EDC, N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide; NHS, N-hydroxysuccinimide. (B) Synthesis of DOPA-conjugated DSPE-PEG (DSPE-PEG-DOPA). (C, D) TEM images (C) as well as average size and zeta-potential (D) of Ac-bCDNPs containing variouscontents of peripheral DOPA. Data are mean ± SE (n = 3).

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

B

rinsed with deionized water three times. Following similar procedures,Ac-bCD or PLGA NPs based on DSPE-PEG and DSPE-PEG-DOPA atdifferent molar ratios were fabricated. Similarly, PTX-loaded and Cy5 orCy7.5-labeled NPs were produced. The content of PTX in NPs wasquantified by high performance liquid chromatography (HPLC, LC-20A, Shimadzu), while Cy5 and Cy7.5 were quantified by UV−visspectroscopy.Measurements. 1H NMR spectra were recorded on a Bruker

BioSpin-600 spectrometer operating at 600 MHz. FT-IR spectra wereacquired on a PerkinElmer FT-IR spectrometer (100S). Dynamic lightscattering and zeta-potential measurement of NPs in aqueous solutionwas performed with a Malvern Zetasizer Nano ZS instrument at 25 °C.Transmission electron microscopy (TEM) observation was carried outon a TECNAI-10 microscope (Philips, Netherland) operating at anacceleration voltage of 80 kV.In Vitro Hydrolysis Study. In vitro hydrolysis of Ac-bCD NPs at

0% or 60% DOPA was conducted at 37 °C in PBS with pH 5 or pH 7.4.Briefly, about 0.5 mg of freshly prepared NPs was dispersed in PBS. Afterincubation for different periods of time, the transmittance at 500 nmwasmeasured.In Vitro Release Tests. In vitro release experiments were performed

in PBS at pH 5 or pH 7.4. Briefly, 0.1 mL of aqueous solution containing0.25 mg freshly prepared Ac-bCDNPs loaded with PTXwas placed intodialysis tubing, which was immersed into 30 mL of PBS and incubated at37 °C. At predetermined time points, 5.0 mL of release medium waswithdrawn, and the same volume of fresh PBS was replenished. ThePTX concentration was quantified by HPLC.Cell Culture. B16F10, HepG2, MCF-7, MDA-MB-231, MOVAS,

and RAW264.7 cells were cultured in 96-well plates at a density of 1 ×104 cells per well in 100 μL of growth medium containing 10% (v/v)FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. All cellswere incubated at 37 °C in a humidified atmosphere of 5% CO2 for 24 hbefore the addition of various NPs. B16F10, HepG2, MDA-MB-231,MOVAS, and RAW264.7 cells were cultured in RPMI 1640 medium,while MCF-7 cells were cultured in EME medium.Intracellular Uptake Study by Fluorescence Microscopy.

B16F10 and MDA-MB-231 cells were separately seeded in a 35 mmdish with 20 mm cover glasses at a density of 2 × 105 cells per well in 2mL growth medium. Cells were incubated at 37 °C with 5% CO2 for 24h. Then the culture medium was replaced by 2 mL of fresh mediumcontaining Cy5-labeled NPs and incubated for 2, 4, 8, and 12 h,respectively. Before observation, cells were stained with LysoTrackerRed (50 nM) for 1.5 h and Dil (20 μM) for 15 min. After washing withPBS, cells were counterstained with DAPI. Confocal laser scanningmicroscopy (CLSM) observation was performed on a fluorescencemicroscope (LSM780NLO, Zeiss, Germany).Quantification of Cellular Uptake by Flow Cytometry. Uptake

efficiency of different cell lines for NPs was quantified by thefluorescence activated cell sorting (FACS) technique. Specifically,B16F10, MDA-MB-231, RAW264.7, and MOVAS cells were planted in6-well plates with 5 × 105 cells per well in 2 mL of DMEM containing10% FBS (v/v) 24 h before the experiments. Generally, the treated cellswere washed three times with PBS (pH 7.4), followed by trypsinization,centrifugation (1000 rpm for 5 min), and washing with PBS (pH 7.4),and then resuspended in 200 μL of FACS solution. The cell uptake ofCy5-labeled NPs was quantified by flow cytometry (FACSVerse, BectonDickinson, U.S.A.). Approximate 10000 events were acquired persample, and the data were analyzed using FlowJo. Forward and side lightscatter gates were normally set to exclude dead cells, debris, and cellaggregates. In each study, three independent experiments wereperformed.Effects of Various Treatments on Cellular Uptake. B16F10 cells

were planted in 6-well plates with 5 × 105 cells per well in 2 mL ofDMEM containing 10% FBS (v/v) 24 h before the uptake experiment.The cells were preincubated at 4 °C for 30 min or pretreated withvarious inhibitors (nocodazole at 10 μg/mL; cytochalasin D at 5 μg/mL) for 30 min, followed by the addition of Cy5-labeled NPs. After 4 hof incubation, the cells were washed three times with PBS. Then, thecells were trypsinized, centrifuged, resuspended in 200 μL of FACS

solution, and analyzed by flow cytometry (FACSVerse, BectonDickinson, U.S.A.). Three separate experiments were performed.

Cytotoxicity Evaluation. Cells were incubated at 37 °C in ahumidified atmosphere of 5% CO2 overnight and then they were treatedwith the medium containing various concentrations of NPs with orwithout DOPA coating for 24 h. Cell viability was quantified by theMTT assay, and the experiment was repeated three times. Values of thehalf maximal inhibitory concentration (IC50) were calculated by curvefitting using Originpro 7.0.

In Vitro Antitumor Activity of PTX-Loaded Nanoparticles. Allcells were incubated under standard conditions for 24 h. Cells were thentreated with the medium containing various PTX formulations(including free PTX and PTX-loaded Ac-bCD/DSPE-PEG NPs withor without DOPA coating) at different doses of PTX. After 24 h ofincubation, the cell viability was quantified by theMTTmethod, and theIC50 values were calculated based on three separate experiments.

Study on the Intratumor Retention of Nanoparticles. Allanimal care and experimental protocols were performed in compliancewith the Animal Management Rule of the Ministry of Health of thePeople’s Republic of China (No. 55, 2001) and the guidelines for theCare and Use of Laboratory Animals of the Third Military MedicalUniversity (Chongqing, China).

Four-week-old male BALB/c athymic nude mice (16−20 g) wereacclimatized for 1 week before experimentation. To establish MCF-7human breast cancer xenografts, a suspension ofMCF-7 cells at a densityof 9 × 106 cells/mouse was subcutaneously injected in the right flank ofnude mice. When tumors reached an average volume of 100−150 mm3,tumor bearing mice were randomly assigned into three groups (n = 3).Then, intratumor injection of 50 μL of aqueous solution containingCy7.5-labeled NPs (10 mg/mL) was implemented. Real-timefluorescence imaging was performed and the fluorescence intensity atvarious time points was determined by a living imaging system (IVISSpectrum, PerkinElmer, U.S.A.). After 72 h, the mice were sacrificed,both tumors and main organs were resected for further analysis.

Retention of DOPA-Coated NPs in the Intestine. After 1 week ofacclimatization, male C57BL/6 mice (4 weeks old, 16−20 g) wererandomly assigned into three groups (n = 3). Post 12 h of fasting, 300 μLof PBS (0.01M, pH 7.4), PBS containing Cy7.5-labeled PLGANP at 0%DOPA (4 mg/mL) or PBS containing Cy7.5/PLGA NP at 60% DOPA(4 mg/mL), was separately administered by gastric gavage. At 12 and 24h post-administration, mice were sacrificed and different tissues wereresected for ex vivo fluorescence imaging by a living imaging system(IVIS Spectrum, PerkinElmer, U.S.A.).

Statistical Analysis. Statistical analysis was performed by SPSS15.0.The one-sample Kolmogorov−Smirnov test was used to determinewhether samples were in normal distribution. If samples were in normaldistribution, the one-way ANOVA test was applied for multiplecomparison of statistical analysis. If samples were not in the normaldistribution, then the Wilcoxon rank-sum test was performed. In theone-way ANOVA test, if data were homoscedastic, the LSD test wasemployed, while the Dunnett T3 test was used if data wereheteroscedastic. The p < 0.05 is considered to be statistically significant.

■ RESULTS AND DISCUSSION

Fabrication and Characterization of Nanoparticles.As aproof of concept, biodegradable NPs with peripheral PEG chainswere employed in this study, since this hydrophilic andbiocompatible surface chemistry is frequently utilized to providecolloidal stability and prolonged blood circulation for differentnanomaterials. Nevertheless, PEG coating by either noncovalentor covalent approaches can also impede intracellular uptake andsubsequent trafficking, and therefore, innovative strategies areimperative for both drug and gene delivery. DSPE-PEG was usedto fabricate NPs with PEG coating. As a hydrophobic group,DSPE may facilitate hydrophobic interactions of DSPE-PEGwith the lipophilic core composed of Ac-bCD, thus forming ahydrophilic monolayer. DOPA-conjugated DSPE-PEG (DSPE-PEG-DOPA, the molecular weight of PEG is 2 kDa) was first

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

C

synthesized by coupling reaction between carboxyl-terminatedDSPE-PEG (DSPE-PEG-COOH) and excessive DOPA, usingEDC and NHS as catalysts (Figure 1B). The product waspurified by dialysis against deionized water, and then lyophilized.

Characterization by Fourier transform infrared and 1H NMRspectroscopy showed disappearance of free carboxyl andappearance of catechol in the obtained material (FigureS1A−B), indicating successful conjugation. Calculation by the

Figure 2. Cellular uptake of Ac-bCD NPs peripherally coated with DOPA in murine B16F10 melanoma cells. (A) Internalization of different NPs byB16F10 tumor cells after incubation for various periods of time. The cell membrane was stained by Dil, while the nucleus was counterstained with DAPI.(B, C) Flow cytometric profiles (B) and quantitative results (C) showing time-dependent uptake of DOPA-coated Cy5/Ac-bCDNPs in B16F10 cells at10 μg/mL of NPs. (D)Quantification of uptake of DOPA-coated NPs by B16F10 at 20 (the left panel) and 40 μg/mL (the right panel). (E) Intracellulartrafficking of NPs in B16F10 cells. The lysosome was stained with LysoTracker Red, while the nucleus was counterstained with DAPI. Scale bars = 20μm. (F) Effects of various treatments on internalization in B16F10 tumor cells. All data are mean± SE (n = 3). For results in (C) and (D), the data at 0%DOPA were normalized to 1 at each time point; *p < 0.05, **p < 0.01, and ***p < 0.001 vs the corresponding group at 0% DOPA.

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

D

ratio of proton signals at 6.7−7.1 ppm (due to aryl protons ofDOPA) to those at 3.4−3.6 ppm (corresponding to ethylene inPEG) revealed a conjugation efficiency of 85%. On the otherhand, acetalated β-CD (Ac-bCD) was used as a modelhydrophobic carrier, since previous studies have demonstratedthat this type of materials may serve as safe and effective pH-responsive carriers for intracellular delivery of varioustherapeutics in different cell lines.36−41 Ac-bCD was synthesizedby kinetically controlled acetalation of β-CD in the presence ofexcessive amount of 2-methoxypropene.34 According to the 1HNMR spectrum (Figure S1C), the molar ratio of linear acetal tocyclic acetal was 1.65 for the synthesized Ac-bCD.Both control and DOPA-coated NPs were fabricated through

a modified nanoprecipitation/self-assembly method that is awell-established approach for processing hydrophobic materialsinto NPs of different sizes.35 It should be noted that forsimplicity, the molar percentage of DSPE-PEG-DOPA wascalculated according to the content of PEG. For NPs withoutDOPA coating, PEG was considered as 100%. Independent ofthe content of DSPE-PEG-DOPA introduced, well-definedspherical NPs could be successfully prepared, as illustrated bytypical TEM images of Ac-bCD/DSPE-PEG NPs with variedcontents of DSPE-PEG-DOPA (abbreviated as the molarpercentage of DOPA, Figure 1C). Consistent with previousresults,42 staining by phosphotungstic acid showed core−shellstructure for the obtained NPs (the last image in Figure 1C),

implying that DSPE-PEG/DSPE-PEG-DOPA was peripherallyanchored around the hydrophobic core of Ac-bCD NPs.Measurement by dynamic light scattering indicated that NPswith a mean size of 189−210 nm were manufactured when thecontent of DSPE-PEG-DOPA varied from 0 to 80% (Figure 1Dand Figure S2). As evidenced by the determined zeta-potentialvalues, surface charge of DOPA-coated NPs was slightlydecreased as compared to the control NPs without DOPA(Figure 1D). Consequently, the incorporation of DOPA had nosignificant influence on physicochemical properties of resultingNPs.

Intracellular Uptake of DOPA-Coated NPs in DifferentCell Lines. Then cellular uptake profiles of DOPA-coated Ac-bCD NPs were observed by CLSM and quantified by FACS.Using Cy5-labeled NPs, we first examined their internalization inB16F10 melanoma cells. After incubation for various periods oftime, both the time and DOPA content related cellular uptakebehaviors could be clearly observed (Figures 2A and S3). For thecontrol NPs without DOPA coating, weak green fluorescenceappeared in cells at 2 h, which was gradually intensified withprolonged incubation. By contrast, remarkably strong fluores-cence was observed when DOPA-decorated NPs at the samedose of Cy5 were used, particularly at 4 and 8 h. Furthermore,difference in the fluorescence intensity could be found when thecontent of coated DOPA was varied. Irrespective of the

Figure 3. Cellular uptake of DOPA-coated Ac-bCD NPs by MDA-MB-231 breast cancer cells. (A) Uptake of NPs in MDA-MB-231 cells afterincubation for different time periods. The cell membrane was stained by Dil, while the nucleus was counterstained with DAPI. (B) Quantification ofcellular internalization of NPs in MDA-MB-231 cells by FACS. Data at 0% DOPA were normalized to 1 at each time point. All data are mean± SE (n =3); *p < 0.05 and **p < 0.01 vs the corresponding group at 0% DOPA. (C) Intracellular trafficking of NPs in MDA-MB-231 cells. The lysosome wasstained with LysoTracker Red, while the nucleus was counterstained with DAPI. Scale bars = 20 μm.

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

E

incubation time, the maximal intensity was observed at 60%DOPA.Based on these intuitive results, cellular uptake of various NPs

by B16F10 cells was further quantified by FACS via flowcytometry. After treatment with different NPs for various times,we could observe that uptake efficiency was changed withincubation time and DOPA coating (Figure 2B). Quantitativeanalysis of repeated results indicated that internalization of NPswas gradually increased when NPs were coated with DOPAvarying from 0, 20, 40, 60, and 80% (Figure 2C). Nevertheless,whereas an initial increase in DOPA was beneficial for cellularuptake, an impairing effect was observed when the DOPAcontent was above 60%. Consistent with CLSM observation, theoptimal internalization efficiency was achieved at 60% DOPA. Inthis case, 3.3-, 4.0-, and 7.2-fold increases in mean fluorescenceintensity were reached after 2, 4, and 8 h of treatment withDOPA-decorated NPs, respectively, as compared to that ofcontrol NPs without DOPA.When the dose of Ac-bCDNPs wasincreased from 10 to 20 and 40 μg/mL, almost the similar uptakeprofiles were examined by flow cytometry (Figure 2D).Accordingly, this DOPA-facilitated cellular uptake performancewas largely independent of the NPs dosage. In other words, themaximal uptake efficiency was achieved at 60% DOPA for allexamined doses.We also found similar uptake profiles of different NPs in a

MDR human breast cancer cell line of MDA-MB-231, for whichboth CLSM observation and FACS quantification showed thesuperior performance at 60% DOPA (Figures 3A,B and S4).Besides tumor cells, DOPA was able to promote cellularinternalization in some normal cells. As exemplified by mousevascular smooth muscle (MOVAS) cells, at 60% DOPA, theinternalized NPs were considerably increased in a time-dependent manner (Figure S5A). We also found enhancedinternalization of Ac-bCD NP with 60% DOPA in RAW264.7murine macrophage cells (Figure S5B). For different cell lines itwas notable that the exact uptake kinetics was different, whichshould be attributed to their distinct phagocytosis activities.According to previous studies, DOPA-containing materialsadhere to organic substrates via H-bonding with polarcomponents and covalent coupling via reactive quinonemoieties.22,23,31 In addition, it has been found that cellularuptake of peptides, polymers, and NPs may be facilitated by H-bonding between their amino/hydroxyl groups and either sugaror protein units present in the cell membrane.43−45 In view of thepresence of amide and phenolic hydroxyl groups in DOPA-coated NPs, the combined effects of H-bonding and oxidation-dependent conjugation might account for the enhancedinternalization in various cells observed herein.

Also, CLSM observation uncovered time-dependent, sub-cellular trafficking of both DOPA-coated and control NPs. InB16F10 cells, green fluorescence was largely distributedperipherally at 2 h (Figure 2A). Staining of late endosomes/lysosomes with LysoTracker Red showed partly colocalized redand green fluorescence (yellow regions, Figure 2E), indicatingendocytosis at an early stage. After 4 h of treatment, however,intensive fluorescence near the nucleus could be observed. Thecolocalization of green and red fluorescence, as indicated bysignificant yellow areas, revealed a considerable number of NPswere transported via endosomes/lysosomes (Figure 2E). This isespecially true in the case of DOPA-free NPs. At 8 h,disseminated green fluorescence throughout the whole cyto-plasm implied endolysosomal escape of some NPs. Nevertheless,bright fluorescence appeared near the membrane after 12 h oftreatment. This might be partially related to exocytosis throughegress of NPs from late endosomes/lysosomes, as delineated bythe presence of yellow fluorescence, which was also documentedfor both smooth muscle cells and tumor cells previously;46,47

while other processes responsible for this remains elusive.Likewise, the time-dependent, endolysosomal transport wasfound in MDA-MB-231 cells (Figure 3A,C). As compared toNPs without DOPA, however, the relatively distinct accumu-lation of green fluorescence for NPs with 60% DOPA suggestedthat other cytoplasmic transportation pathways might beinvolved for DOPA-anchored NPs.We then interrogated biological processes that dominate

internalization of these NPs in B16F10 cells by perturbating theendocytosis-mediated uptake under various conditions incombination with quantification by FACS (Figure 2F). First,energy depletion was implemented by pretreatment at 4 °C.Regardless of NPs with different DOPA contents, dramaticallyreduced uptake efficiency could be found. This coincides with theprevious findings on various cells that endocytic uptake is energy-dependent.48 Intracellular uptake was also remarkably attenuatedwhen cells were pretreated with cytochalasin D, a potentinhibitor that can suppress caveolae uptake,48 suggesting thecaveolae route was also concerned in endocytosis of NPs byB16F10 cells. Likewise, decreased uptake efficiency was detectedpost-treatment with nocodazole that can inhibit microtubuledynamic instability. This effect might be related to impairedcytoplasmic trafficking along microtubules, because thedecreased cytosolic transport may lead to increased exocytosis.5

Since energy depletion and transport inhibitors did notcompletely inhibit cellular uptake of NPs, multiple pathwayssuch as caveolae-independent endocytosis, macropinocytosis, aswell as other nonidentified endocytic routes may be involved inthe internalization of NPs by B16F10 cells. It is worth noting that

Figure 4. Effect of DOPA coating on in vitro cytotoxicity of Ac-bCDNPs in B16F10 cells. (A) Viability of B16F10 cells after 6 h of incubation with Ac-bCD NPs with different contents of DOPA at 500 μg/mL. Data are mean ± SE (n = 6). (B, C) Effect of Ac-bCD NPs dose on cell viability of B16F10cells (B) and corresponding IC50 values (C) after 24 h of incubation with NPs at 0 or 80% DOPA. Data are mean ± SE (n = 3).

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

F

DOPA-coated NPs were more sensitive to all the appliedperturbations, particularly uptake and transport inhibitors, whenNPs with 60% DOPA were concerned. Consequently, at least toa certain degree, DOPA-coated NPs might be internalized andtransported in different manners as compared to DOPA-freeNPs, and this is in line with CLSM observation.Of note, coating of DOPA had no remarkable influence on the

cytotoxicity of Ac-bCD NPs. After incubation with B16F10 cellsfor 6 h, Ac-bCD NPs with DOPA varying from 20, 40, 60, and80% displayed comparable cell viability at 500 μg/mL (Figure4A). Besides, similar dose-dependent changes in viability ofB16F10 cells were detected for Ac-bCD NPs at 0 and 80%DOPA, after 24 h of treatment (Figure 4B). There was nosignificant different between the values of IC50 for Ac-bCD NPsat 0 and 80% DOPA (Figure 4C). Also, we found high cellviability for both RAW264.7 and MDA-MB-231 cells (FigureS6A,B), and the corresponding IC50 was as high as 614.1 and635.0 μg/mL (Figure S6C), respectively. Additionally, thisimplied that DOPA-coated Ac-bCD NPs did not activatemacrophages,49 in line with the fact that no acidic byproductsare produced upon hydrolysis of NPs based on acetalatedpolysaccharides.34,36,39,50

In Vitro Anticancer Activities of PTX-ContainingNanoparticles. Unambiguously, the results demonstratedenhanced intracellular uptake of NPs by peripherally decoratingwith DOPA. Accordingly, conjugation of DOPA at the terminalof PEG chains on NPs is promising to conquer the PEG dilemmaby simultaneously providing colloidal stability and maintainingeffective internalization. To examine whether the enhancedinternalization may contribute to therapeutic delivery capability,in vitro experiments were performed using PTX as a model drug.PTX-loaded Ac-bCD NPs with or without DOPA coating werealso fabricated by the modified nanoprecipitation/self-assemblymethod, resulting in PTX nanomedicines with comparative size,shape, and surface charge (Figure 5A−C). The PTX loadingcontent was 9.8 and 9.7% for NPs at 0 and 60% DOPA,respectively. Of note, similar pH-responsive hydrolysis and invitro PTX release profiles were observed for Ac-bCD NPs at 0

and 60% DOPA (Figure S7), indicating that surface coating ofDOPA had no significant effects on pH-sensitivity of resultingNPs. This may be attributed to the fact that for this type of core−shell NPs, their sensitivity is mainly determined by the corematerial.Initially, in vitro antitumor activity of PTX/Ac-bCD NPs was

evaluated in sensitive tumor cell lines of B16F10 and humanhepatocarcinoma cells (HepG2). After 24 h of incubation withvarious doses of PTX-containing NPs, cell viability wasdetermined by MTT assay. Clearly, the inhibition capabilitywas enhanced with increase in PTX doses for all formulations(Figure 5D). The pharmacological activity of PTX wassignificantly increased by packaging into Ac-bCD/DSPE-PEGNPs, consistent with our previous finding that emulsion-basedPTX nanomedicines from acetalated cyclodextrins were able topotentiate the payload efficacy.39,40 At 60% DOPA, furtherimproved PTX efficacy could be observed, as compared to thatwithout DOPA (Figure 5D). Likewise, we found additionallyenhanced antitumor activity against two MDR human breastcancer cells, that is, MCF-7 and MDA-MB-231, when PTX wasencased into NPs with 60% DOPA, in comparison to that ofDOPA-free NPs (Figure 5E).The IC50 was calculated based on the data of dose-dependent

cell viability (Table 1). For B16F10 cells, the IC50 value was 27.0,

Figure 5. Fabrication of PTX-loaded Ac-bCDNPs with or without DOPA coating and their in vitro antitumor activity. (A, B) TEM images of NPs at 0%DOPA (A) or 60% DOPA (B). (C) The average size and zeta-potential. (D, E) PTX dose-dependent viability of different tumor cells after treatmentwith NPs containing PTX. All data are mean ± SE (n = 3).

Table 1. IC50 Values of Various PTX Formulations againstBoth Sensitive and MDR Cancer Cellsa

IC50 (μg/mL)

group B16F10 HepG2 MCF-7 MDA-MB-231

PTX 27.0 ± 0.5 5.5 ± 0.3 266.0 ± 13.0 350.8 ± 10.80% DOPA 14.2 ± 1.5c 3.2 ± 0.1c 79.5 ± 2.4b 63.0 ± 1.1c

60% DOPA 8.2 ± 0.7c,e 2.2 ± 0.3c,d 28.1 ± 2.4b,f 37.5 ± 2.0c,d

aData are mean ± SE (n = 3). bp < 0.01 vs the free PTX group. cp <0.001 vs the free PTX group. dp < 0.05 vs PTX/Ac-bCD NPs with 0%DOPA. ep < 0.01 vs PTX/Ac-bCD NPs with 0% DOPA. fp < 0.001 vsPTX/Ac-bCD NPs with 0% DOPA.

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

G

14.2, and 8.2 μg/mL for free PTX, PTX/Ac-bCD NPs at 0%DOPA, and PTX/Ac-bCD NPs at 60% DOPA, respectively;while it corresponded to 5.5, 3.2, and 2.2 μg/mL in the case of

HepG2 cells. Both NPs were pharmacologically superior overfree PTX, and there was significant difference between thenanomedicines at 0 and 60% DOPA. More prominent

Figure 6. Enhanced tissue retention of DOPA-decorated NPs. (A) Representative pictures of real-time imaging in nude mice bearing human breastcancer MCF-7 xenografts post local injection of Cy7.5-labeled NPs. (B) Quantitative data of relative fluorescence intensity localized in the tumor site atdifferent time points. (C, D) Typical images (C) and quantitative results (D) of ex vivo imaging on tumors and major organs resected from micesubjected to intratumor administration. (E−H) Typical ex vivo images (E, G) and quantitative results (F, H) illustrating the distribution of Cy7.5-labeled NPs in different tissues after 12 h (E, F) or 24 h (G, H) of oral administration. Data are mean± SE (n = 3). Statistical analysis was performed bythe Wilcoxon rank-sum test; *p < 0.05 vs the NPs with 0% DOPA.

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

H

differences could be found for MDR cells. Whereas treatment byPTX/Ac-bCD NPs at 0% DOPA resulted in 3.3-fold decrease inIC50 against MCF-7 cells in comparison to free PTX, PTX/Ac-bCDNPs at 60% DOPA yielded 9.5× of reduction. As for MDA-MB-231 cells, The PTX nanomedicine at 0 and 60% DOPAafforded 5.6- and 9.4-fold decreases in the IC50 value,respectively, when compared with free PTX. Since DOPAcoating did not affect pH-sensitivity of NPs as well as releaseprofiles of PTX, the enhanced PTX activity should be largelycontributed by surface DOPA. These results strongly suggestedthat surface coating of nanomedicines with DOPA is beneficialfor their in vitro activity, primarily resulting from the enhancedintracellular delivery.Taken together, above results clearly demonstrated that

surface decoration of NPs with well-determined contents ofDOPA may remarkably enhance their cellular uptake, which inturn can potentiate the efficacy of their therapeutic payload.Besides, this type of nanosystems are promising for in vitromodulation, stimulation, or activation of different cells (such asstem/progenitor cells and immune cells) for cell therapy.Whereas this surface chemistry lacks specificity, this disadvantagecan be partly overcome by tailoring the density of DOPA orcombining with other targeting units.Enhanced Tumor Retention of DOPA-Coated Nano-

particles. Intrigued by these promising findings, we theninterrogated whether surface coating of DOPA may enhancetissue retention of NPs by the strengthened adhesion capability,which is beneficial for local drug delivery, vaccination, or tissuerepair via topical treatment. First, a tumor tissue was employed asa diseased model. For this purpose, the same dose of Cy7.5-labeled Ac-bCD NPs with or without DOPA coating wasseparately injected into tumors in nude mice with MCF-7xenografts. Real-time imaging at predetermined time points wasperformed, and representative images are illustrated in Figure 6A.It should be noted that the relatively weak intensity immediatelyafter injection was due to fluorescence quenching. For NPswithout DOPA, the fluorescence was mainly located around thetumor at 24 and 48 h (particularly at 48 h), which indicated thediffusion of NPs out of the tumor in this case, owing to the highinterstitial fluid pressure. By contrast, fluorescence washomogeneously distributed in the tumor injected with NPswith 60% DOPA. Moreover, relatively high intensity could bedetected for NPs with 60% DOPA, as compared with thosewithout DOPA, and there was significant difference at 48 h(Figure 6B). At both 24 and 48 h, the percentage of fluorescenceat tumor sites of Ac-bCD NPs at 60% DOPA was significantlyhigher than that at 0% DOPA (Figure S8A).In line with this result, ex vivo imaging and quantification of

the resected tumor tissues revealed that the fluorescenceintensity at 60% DOPA was prominently higher than that at0% DOPA (Figure 6C,D). The fluorescence intensity ratio oftumor to other main organs was 1.0 and 2.9 for Cy7.5/Ac-bCDNPs at 0 and 60% DOPA, respectively (Figure S8B).Additionally, NPs with 60% DOPA led to relatively lowdistribution in the examined major organs, especially in theheart, liver, and spleen. These results substantiated that coatingof NPs with DOPA may remarkably increase their intratumorretention after local injection while simultaneously decreasingthe undesirable distribution in other organs. This is particularlyadvantageous to enhance efficacy, yet decrease side effects forlocal drug delivery or vaccination.Subsequently, to investigate whether DOPA coating may

facilitate adhesion and retention in other tissues, we examined

tissue distribution of Cy7.5-labeled NPs with or without DOPAafter oral administration. As illustrated by ex vivo imaging(Figure 6E), only weak fluorescence could be observed in theintestine at 12 h after oral delivery in the case of Cy7.5 NP at 0%DOPA. This is consistent with the fact that orally administeredmicroparticles will be largely eliminated from the gastrointestinaltract within 12−24 h. By contrast, dramatically strongerfluorescence was found in the intestine of mice treated withCy7.5 NP at 60% DOPA. Quantitative analysis indicated asignificant difference in the intestinal fluorescence intensitybetween 0% DOPA and 60% DOPA groups (Figure 6F).Likewise, both intuitive and quantitative results suggested thatfluorescence in the intestine of the 60% DOPA group was muchmore notable at 24 h, as compared to that of Cy7.5 NP at 0%DOPA (Figure 6G,H). On the other hand, fluorescence in otherorgans such as heart, liver, spleen, lung, and kidney was slightlyincreased at 60%DOPA, while there was no significant differenceas compared to those at 0% DOPA. Since distribution in theseorgans is generally related to translocation of NPs by transcellularand paracellular pathways, these preliminary results indicatedthat DOPA coating did not affect absorption of NPs in theintestine. Nevertheless, these findings demonstrated that surfacecoating of NPs with DOPA may considerably enhance theirretention in the intestine tissue. This function is especiallyadvantageous to treat intestinal diseases (such as inflammatorybowel disease and colon cancer) or to achieve long-termmucosalimmune response for oral vaccination. The enhanced tumor andintestinal retention of NPs by DOPA coating should beattributed to increased adhesion and anchoring on theextracellular matrix and augmented intracellular uptake in localcells, although the details require further exploration.

■ CONCLUSIONSIn summary, for the first time we demonstrated herein thatbiomimetic coating of NPs with DOPA is beneficial forenhancing their intracellular delivery, potentiating cellularactivity against both sensitive and MDR tumor cells, as well asincreasing retention in tumor and intestinal tissues. Nanoma-terials engineered by this strategy may find applications inintracellular delivery of various therapeutics, local drug delivery,vaccination, and NP-mediated cell therapy.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.biomac.5b01056.

Additional results of Figures S1−8 (PDF).

■ AUTHOR INFORMATIONCorresponding Author*E-mail: jxzhang1980@gmail.com; jxzhang@tmmu.edu.cn.

Author Contributions‡These authors contributed equally (K.C. and X.Q.X.).

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis study was financially supported by the National NaturalScience Foundation of China (Nos. 81271695 and 81471774),the Research Foundation of Third Military Medical University

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

I

(2014XJY04), and also by the Program for New CenturyExcellent Talents in University (NCET-13-0703) to J.X.Z.

■ REFERENCES(1) Torchilin, V. P. Nat. Rev. Drug Discovery 2014, 13 (11), 813−827.(2) Miyata, K.; Nishiyama, N.; Kataoka, K. Chem. Soc. Rev. 2012, 41(7), 2562−2574.(3) Mitragotri, S.; Burke, P. A.; Langer, R. Nat. Rev. Drug Discovery2014, 13 (9), 655−672.(4) Bertrand, N.; Wu, J.; Xu, X. Y.; Kamaly, N.; Farokhzad, O. C. Adv.Drug Delivery Rev. 2014, 66, 2−25.(5) Sahay, G.; Alakhova, D. Y.; Kabanov, A. V. J. Controlled Release2010, 145 (3), 182−95.(6) Zhang, J. X.; Ma, P. X. Adv. Drug Delivery Rev. 2013, 65 (9), 1215−1233.(7) Mura, S.; Nicolas, J.; Couvreur, P.Nat. Mater. 2013, 12 (11), 991−1003.(8) Lobatto, M. E.; Fuster, V.; Fayad, Z. A.; Mulder, W. J. M. Nat. Rev.Drug Discovery 2011, 10 (11), 835−852.(9) Kim, B. Y. S.; Rutka, J. T.; Chan,W. C.W.N. Engl. J. Med. 2010, 363(25), 2434−2443.(10) Kabanov, A. V.; Vinogradov, S. V. Angew. Chem., Int. Ed. 2009, 48(30), 5418−5429.(11) Zhang, J. X.; Li, X. D.; Li, X. H. Prog. Polym. Sci. 2012, 37 (8),1130−1176.(12) Smith, D. M.; Simon, J. K.; Baker, J. R. Nat. Rev. Immunol. 2013,13 (8), 592−605.(13) Shi, J. J.; Votruba, A. R.; Farokhzad, O. C.; Langer, R. Nano Lett.2010, 10 (9), 3223−3230.(14) Chou, L. Y. T.; Ming, K.; Chan, W. C. W.Chem. Soc. Rev. 2011, 40(1), 233−245.(15) Petros, R. A.; DeSimone, J. M. Nat. Rev. Drug Discovery 2010, 9(8), 615−627.(16)Mascini, M.; Palchetti, I.; Tombelli, S.Angew. Chem., Int. Ed. 2012,51 (6), 1316−1332.(17) Salvati, A.; Pitek, A. S.; Monopoli, M. P.; Prapainop, K.; Bombelli,F. B.; Hristov, D. R.; Kelly, P. M.; Aberg, C.; Mahon, E.; Dawson, K. A.Nat. Nanotechnol. 2013, 8, 137−143.(18) Weissleder, R.; Kelly, K.; Sun, E. Y.; Shtatland, T.; Josephson, L.Nat. Biotechnol. 2005, 23, 1418−1423.(19) Venditto, V. J.; Szoka, F. C. Adv. Drug Delivery Rev. 2013, 65 (1),80−88.(20) Cheng, Z. L.; Zaki, A. A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas,A. Science 2012, 338 (6109), 903−910.(21) Waite, J. H.; Tanzer, M. L. Science 1981, 212 (4498), 1038−1040.(22) Lee, B. P.; Messersmith, P. B.; Israelachvili, J. N.; Waite, J. H.Annu. Rev. Mater. Res. 2011, 41, 99−132.(23) Lee, H.; Scherer, N. F.; Messersmith, P. B. Proc. Natl. Acad. Sci. U.S. A. 2006, 103 (35), 12999−3003.(24) Lee, H.; Dellatore, S. M.; Miller, W.M.; Messersmith, P. B. Science2007, 318 (5849), 426−430.(25) Lee, H.; Lee, B. P.; Messersmith, P. B. Nature 2007, 448, 338−341.(26) Lee, H.; Lee, Y.; Statz, A. R.; Rho, J.; Park, T. G.; Messersmith, P.B. Adv. Mater. 2008, 20 (9), 1619−1623.(27) Wang, J. J.; Tahir, N. M.; Kappl, M.; Tremel, W.; Metz, N.; Barz,M.; Theato, P.; Butt, H. J. Adv. Mater. 2008, 20 (20), 3872−3876.(28) Karabulut, E.; Pettersson, T.; Ankerfors, M.; Wagberg, L. ACSNano 2012, 6 (6), 4731−9.(29) Kim, J. H.; Lee, M.; Park, C. B. Angew. Chem., Int. Ed. 2014, 53(25), 6364−6368.(30) Brubaker, C. E.; Kissler, H.; Wang, L. J.; Kaufman, D. B.;Messersmith, P. B. Biomaterials 2010, 31 (3), 420−7.(31) Kastrup, C. J.; Nahrendorf, M.; Figueiredo, J. L.; Lee, H.;Kambhampati, S.; Lee, T.; Cho, S. W.; Gorbatov, R.; Iwamoto, Y.; Dang,T. T.; Dutta, P.; Yeon, J. H.; Cheng, H.; Pritchard, C. D.; Vegas, A. J.;Siegel, C. D.; MacDougall, S.; Okonkwo, M.; Thai, A.; Stone, J. R.;Coury, A. J.;Weissleder, R.; Langer, R.; Anderson, D. G. Proc. Natl. Acad.Sci. U. S. A. 2012, 109 (52), 21444−9.

(32) Burzio, L. A.; Waite, J. H. Biochemistry 2000, 39 (36), 11147−53.(33) Yu, M.; Hwang, J.; Deming, T. J. J. Am. Chem. Soc. 1999, 121 (24),5825−5826.(34) Zhang, J. X.; Jia, Y.; Li, X. D.; Hu, Y. Q.; Li, X. H. Adv. Mater. 2011,23 (27), 3035−40.(35) Zhang, L. F.; Chan, J. M.; Gu, F. X.; Rhee, J. W.; Wang, A. Z.;Radovic-Moreno, A. F.; Alexis, F.; Langer, R.; Farokhzad, O. C. ACSNano 2008, 2 (8), 1696−1702.(36) Bachelder, E. M.; Beaudette, T. T.; Broaders, K. E.; Dashe, J.;Frechet, J. M. J. J. Am. Chem. Soc. 2008, 130 (32), 10494−10495.(37) Broaders, K. E.; Cohen, J. A.; Beaudette, T. T.; Bachelder, E. M.;Frechet, J. M. J. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (14), 5497−5502.(38) Chen, H. P.; Liu, X. P.; Dou, Y.; He, B. F.; Liu, L.;Wei, Z. H.; Li, J.;Wang, C. Z.; Mao, C. D.; Zhang, J. X.; Wang, G. S. Biomaterials 2013, 34(16), 4159−72.(39) He, H. M.; Chen, S.; Zhou, J. Z.; Dou, Y.; Song, L.; Che, L.; Zhou,X.; Chen, X.; Jia, Y.; Zhang, J. X.; Li, S. H.; Li, X. H. Biomaterials 2013, 34(21), 5344−5358.(40) Liu, X. P.; Wang, G. S.; You, Z. C.; Qian, P.; Chen, H. P.; Dou, Y.;Wei, Z. H.; Chen, Y.; Mao, C. D.; Zhang, J. X. Biomaterials 2014, 35(14), 4401−16.(41) Shi, Q.; Zhang, L.; Liu, M. Y.; Zhang, X. L.; Zhang, X. J.; Xu, X. Q.;Chen, S.; Li, X. H.; Zhang, J. X. Biomaterials 2015, 67, 169−182.(42) Chan, J. M.; Zhang, L. F.; Yuet, K. P.; Liao, G.; Rhee, J. W.; Langer,R.; Farokhzad, O. C. Biomaterials 2009, 30 (8), 1627−1634.(43) Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.; Wender,P. A. J. Am. Chem. Soc. 2004, 126 (31), 9506−9507.(44) Ye, G. X.; Cao, Z. Q.; Lin, L.; Chen, D. Y.; Liu, W. G. Chin. Sci.Bull. 2008, 53 (15), 2307−2314.(45) Vasir, J. K.; Labhasetwar, V. Biomaterials 2008, 29 (31), 4244−52.(46) Panyam, J.; Labhasetwar, V. Pharm. Res. 2003, 20, 212−220.(47) Sahay, G.; Querbes, W.; Alabi, C.; Eltoukhy, A.; Sarkar, S.;Zurenko, C.; Karagiannis, E.; Love, K.; Chen, D.; Zoncu, R.; Buganim,Y.; Schroeder, A.; Langer, R.; Anderson, D. G. Nat. Biotechnol. 2013, 31(7), 653−8.(48) Khalil, I. A.; Kogure, K.; Akita, H.; Harashima, H. Pharmacol. Rev.2006, 58 (1), 32−45.(49) Sy, J. C.; Seshadri, G.; Yang, S. C.; Brown, M.; Oh, T.; Dikalov, S.;Murthy, N.; Davis, M. E. Nat. Mater. 2008, 7 (11), 863−868.(50) Zhang, D. L.; Wei, Y. L.; Chen, K.; Gong, H.; Han, S. L.; Guo, J.W.; Li, X. H.; Zhang, J. X. J. Biomed. Nanotechnol. 2015, 11 (6), 923−941.

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b01056Biomacromolecules XXXX, XXX, XXX−XXX

J