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Acta Biomaterialia 30 (2016) 144–154
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Acta Biomaterialia
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Docetaxel (DTX)-loaded polydopamine-modified TPGS-PLAnanoparticles as a targeted drug delivery system for the treatment ofliver cancer
http://dx.doi.org/10.1016/j.actbio.2015.11.0311742-7061/� 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
⇑ Corresponding authors.E-mail addresses: [email protected] (X. Zeng), [email protected]
ghua.edu.cn (L. Mei).
Dunwan Zhu a, Wei Tao b, Hongling Zhang b,c, Gan Liu b, Teng Wang b, Linhua Zhang a,Xiaowei Zeng b,⇑, Lin Mei b,⇑a Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials, Tianjin 300192, PR Chinab The Shenzhen Key Lab of Gene and Antibody Therapy, and Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR Chinac Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China
a r t i c l e i n f o
Article history:Received 24 June 2015Received in revised form 13 September2015Accepted 16 November 2015Available online 18 November 2015
Keywords:Cancer nanotechnologyDopamine polymerizationSurface modificationGalactosylated nanoparticlesLiver targeting
a b s t r a c t
Polydopamine-based surface modification is a simple way to functionalize polymeric nanoparticle (NP)surfaces with ligands and/or additional polymeric layers. In this work, we developed DTX-loadedformulations using polydopamine-modified NPs synthesized using D-a-tocopherol polyethylene glycol1000 succinate-poly(lactide) (pD-TPGS-PLA/NPs). To target liver cancer cells, galactosamine was conju-gated on the prepared NPs (Gal-pD-TPGS-PLA/NPs) to enhance the delivery of DTX via ligand-mediatedendocytosis. The size and morphology of pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs changed obviouslycompared with TPGS-PLA/NPs. In vitro studies showed that TPGS-PLA/NPs, pD-TPGS-PLA/NPs andGal-pD-TPGS-PLA/NPs had similar release profiles of DTX. Both confocal laser scanning microscopy andflow cytometric results showed that coumarin 6-loaded Gal-pD-TPGS-PLA/NPs had the highest cellularuptake efficiency in liver cancer cell line HepG2. Moreover, DTX-loaded Gal-pD-TPGS-PLA/NPs inhibitedthe growth of HepG2 cells more potently than TPGS-PLA/NPs, pD-TPGS-PLA/NPs, and a clinicallyavailable DTX formulation (Taxotere�). The in vivo biodistribution experiments show that theGal-pD-TPGS-PLA/NPs are specifically targeted to the tumor. Furthermore, the in vivo anti-tumor effectsstudy showed that injecting DTX-loaded Gal-pD-TPGS-PLA/NPs reduced the tumor size most significantlyon hepatoma-bearing nude mice. These results suggest that Gal-pD-TPGS-PLA/NPs prepared in the studyspecifically interacted with the hepatocellular carcinoma cells through ligand–receptor recognition andthey may be used as a potentially eligible drug delivery system targeting liver cancers.
Statement of Significance
Polydopamine-based surface modification is a simple way to functionalize polymeric nanoparticle sur-faces with ligands and/or additional polymeric layers. In this work, we developed docetaxel (DTX)-loaded formulations using polydopamine-modified NPs synthesized from D-a-tocopherol polyethyleneglycol 1000 succinate-poly(lactide) (pD-TPGS-PLA/NPs). To target liver cancer cells, galactosamine wasconjugated on the prepared NPs (Gal-pD-TPGS-PLA/NPs) to enhance the delivery of DTX via ligand-mediated endocytosis. Both confocal laser scanning microscopy and flow cytometric results showed thatcoumarin 6-loaded Gal-pD-TPGS-PLA/NPs had the highest cellular uptake efficiency for liver cancer cellline HepG2. The in vivo biodistribution experiments show that the Gal-pD-TPGS-PLA/NPs are specificallytargeted to the tumor. Furthermore, the in vivo anti-tumor effects study showed that injecting DTX-loaded Gal-pD-TPGS-PLA/NPs reduced the tumor size most significantly on hepatoma-bearing nude mice.These results suggest that Gal-pD-TPGS-PLA/NPs prepared in the study specifically interacted with thehepatocellular carcinoma cells through ligand–receptor recognition and they could be used as a poten-tially eligible drug delivery system targeting liver cancers.
� 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
D. Zhu et al. / Acta Biomaterialia 30 (2016) 144–154 145
1. Introduction
Liver cancer is one of the most prevalent cancers and hasbecome the 3rd leading cause of cancer death after lung cancerand stomach cancer around the world [1]. Up to now, liver canceris mainly clinically treated by chemotherapy besides surgery.However, most anticancer drugs have high toxicity and low speci-ficity, leading to systemic toxicity and severe side effects. Poly-meric nanoparticles (NPs) have been employed as promisingcarriers of anticancer drugs, functioning as sustained, controlledand targeted drug delivery systems to improve the therapeuticeffects and to reduce the side effects on normal organs [2–6]. Inthe existing systems, short interfering RNAs (siRNAs) deliveredby liposomes has get promising results and has been the first suc-cessful clinical trial against liver cancer [7,8]. Poly(lactide) (PLA) isone of the most often used FDA-approved biodegradable polymersin drug delivery [9,10]. TPGS is a water-soluble derivative of natu-ral vitamin E, which is generally recognized as an excellent emul-sifier due to its bulky structure and large surface area. Moreover,co-administration of TPGS can enhance cytotoxicity, suppress P-glycoprotein-mediated multi-drug resistance, and increase the oralbioavailability of anticancer drugs [11,12]. TPGS-PLA copolymerscan greatly improve drug encapsulation efficiency (EE), acceleratedrug release, and enhance the cellular uptake of drug-loaded NPsto meet therapeutic needs [13,14]. The preparation techniques ofpolymeric nanoparticles include nanoprecipitation, dialysis, sol-vent evaporation and multiple emulsions [14,15].
Targeted drug delivery, which can carry drugs to specific organsor tissues, has been highlighted in cancer nanotechnology [16–19].Introducing various targeting ligands, such as antibodies, peptides,nucleic acids, and small molecules, has significantly improved thespecificity to deliver drugs within the tumor cells via endocytosismechanisms [20–24]. Targeted drug delivery also significantlydecreases toxic side effects compared to traditional chemotherapy.Hepatic carcinoma cells are known to recognize galactose- and N-acetylgalactosamine-terminated glycoproteins through asialogly-coprotein (ASGP) receptors located on their surfaces [25], and ASGPreceptor-mediated targeted therapy has been generally recognizedas one of the most effective targeted drug delivery systems intreating hepatocellular carcinoma [26–30].
NPs are typically modified with cell-interactive ligands to pro-mote cell binding and uptake abilities. However, due to the lackof reactive functional groups, the NP surface is commonly activatedby reactive linkers, coupling agents or prefunctionalization, fol-lowed by complicated and inefficient purification processes toremove catalysts and excess reactants [31–34]. Dopamine poly-merization has been used to introduce a reactive chemical groupon the surface of NPs [35,36]. In weak alkaline conditions (�pH8–8.5), dopamine catechol is oxidized to quinone which reactswith other catechols and/or quinones to form polymerized dopa-mine, finally giving a water-insoluble polymer film on solid sur-faces. Functional ligands possess nucleophilic functional groupssuch as amine and thiol that can be incorporated into the surfacelayer via Michael addition and/or Schiff base reactions [37–39].Being both simple and versatile, this method has been widely usedto functionalize various types of substrates since its discovery in2007 [35–37].
DTX, as one of the most active anticancer drugs for solid tumors[40,41], is a mitotic inhibitor for cell cycle progression by inducingtubulin polymerization and by arresting cells in the G2-M phase[42]. The aim of the study was to develop DTX-loaded formulationsfor the treatment of liver cancers. To this end, we conjugatedgalactosamine as a target moiety on the polydopamine layerintroduced to the surface of TPGS-PLA/NPs. The particle size
distribution, zeta potential, surface morphology, drug loading con-tent (LC), encapsulation efficiency (EE), drug release profile, cellu-lar uptake efficiency and cytotoxicity against HepG2 cells wereinvestigated in vitro. Additionally, the in vivo biodistribution andanti-tumor effects of the prepared NPs on hepatoma-bearing nudemice were also evaluated.
2. Materials and methods
2.1. Materials
Dopamine hydrochloride, galactosamine hydrochloride, cou-marin 6, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and IR-780 were purchased from Sigma–Aldrich(St. Louis, MO, USA). DTX (purity: 99.9%) was bought from Shang-hai Jinhe Bio-Technology Co., Ltd. (Shanghai, China). CopolymerTPGS-PLA was synthesized as described previously [43]. Humanhepatoma cell line HepG2 and human nasopharyngeal carcinomacell line CNE-2 were get from American Type Culture Collection(ATCC).
2.2. Preparation of DTX- or coumarin 6-loaded TPGS-PLA/NPs
DTX-loaded TPGS-PLA NPs were synthesized as described previ-ously [44]. Briefly, 10 mg DTX and 100 mg TPGS-PLA copolymerswere dissolved in 8 ml of acetone. Then the solution was dropwiseadded into 100 ml of 0.03% (w/v) TPGS aqueous solution using a1 ml injector under stirring. After stirring overnight at room tem-perature to remove acetone, the particles were collected by beingcentrifuged at 20,000 rpm for 20 min and then washed three timesin 10 ml of deionized water to remove TPGS emulsifier and unen-capsulated drug. The precipitated NPs were prepared for poly-dopamine coating. Fluorescent coumarin 6-loaded NPs werefabricated by the same protocol, only with DTX replaced by 2 mgcoumarin 6.
2.3. Prime-coating with polydopamine
Polydopamine-coated NPs (pD-TPGS-PLA) were synthesized byincubating TPGS-PLA NPs in 0.5 mg/mL dopamine hydrochloridedissolved in a 10 mM Tris buffer (pH 8.5) for 3 h at room temper-ature with stirring. The suspensions turned dark when dopaminehydrochloride was added, indicating dopamine was successfullypolymerized. The obtained pD-TPGS-PLA/NPs were centrifugedand lyophilized for galactosamine conjugation.
2.4. Conjugation of galactosamine to pD-TPGS-PLA/NPs
The functional ligand galactosamine was bound to the surfaceof the pD-TPGS-PLA/NPs via the Michael addition reaction [35].Briefly, pD-TPGS-PLA/NPs were resuspended in Tris buffer(10 mM, pH 8.5) containing galactosamine. The final concentra-tions of NPs and ligand were 1 and 2 mg/mL, respectively. After2 h of incubation at room temperature with stirring, the resultingNPs (designated as Gal-pD-TPGS-PLA/NPs) were centrifuged,washed three times with deionized water and then lyophilized.Dopamine polymerization includes brief incubation of the pre-formed NPs in a weak alkaline solution of dopamine, followed bysecondary incubation with amine- or thiol-terminated functionalligands in aqueous solution via the Michael addition reaction.Using this method, we functionalized pD-TPGS-PLA NPs withgalactosamine as the targeting ligand. The surface modification of
Fig. 1. Schematic representation of the preparation technique for DTX-loaded Gal-pD-TPGS-PLA/NPs.
146 D. Zhu et al. / Acta Biomaterialia 30 (2016) 144–154
polymeric NPs using dopamine polymerization and galactosamineis schematized in Fig. 1.
2.5. Particle size and zeta potential
The particles (about 2 mg) were suspended in deionized waterby sonication before measurement. The mean size, sizedistribution and zeta potential of DTX-loaded TPGS-PLA/NPs,pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs were measured by aMalvern Mastersizer 2000 particle size analyzer (Zetasizer NanoZS90, Malvern Instruments Ltd, UK). The data were the averagesof three measurements.
2.6. Surface morphology
The morphology of the NPs was examined by field emissionscanning electron microscopy (FESEM) using a JEOL JSM-6700Fsystem operated at a 3.0 kV accelerating voltage. To preparesamples for FESEM, the particles were fixed on the stub by adouble-sided sticky tape and then coated with platinum layer byJFC-1300 automatic fine platinum coater (JEOL, Tokyo, Japan) for40 s. Transmission electron microscopy (TEM, Tecnai G2 20, FEICompany, Hillsboro, Oregon, USA) was also used to study theNPs. Sample was dropped onto a carbon-coated-on lacey supportfilm that was allowed to dry before characterization.
2.7. Fourier transform infrared (FTIR) spectroscopy and X-rayphotoelectron spectroscopy (XPS) analysis
The changes of the chemical composition in the surfaces of NPsafter modification with polydopamine and targeted ligandgalactosamine were analyzed by FTIR spectrometer (Nicolet, USA)with a pellet of powdered KBr and adsorbent in the range of500–4000 cm�1. XPS (Kratos Ltd., UK) was obtained with an AXISHis spectrometer using a monochromatic Al Ka X-ray source(1486.6 eV photons, 150 W). Binding energy values were refer-enced to the Fermi edge, and charge correction was performedsetting the C 1s peak at 284.8 eV.
2.8. Drug loading content (LC) and encapsulation efficiency (EE)
Drug LC and EE of DTX-loaded NPs were determined by HPLC(LC 1200, Agilent Technologies, Santa Clara, CA) using previouslypublished methods [45]. Briefly, 5 mg NPs were dissolved in 1 mlof dichloromethane (DCM) under vigorous vortexing. This solutionwas transferred to 5 ml of mobile phase consisting of acetonitrileand deionized water (50:50, v/v). A nitrogen stream wasintroduced to evaporate DCM for about 15 min, and then a clearsolution was obtained for HPLC analysis. A reverse-phase C-18 col-umn (150 � 4.6 mm, 5 lm, C18, Agilent Technologies, CA, USA)was used. The flow rate of mobile phase was set at 1.0 ml/min.The column effluent was detected at 227 nm with a UV/VIS detec-tor. The drug LC and EE of DTX-loaded NPs were calculated by the
following equations respectively. Measurements were carried outthree times for each batch.
LC ð%Þ ¼ Weight of DTX in the NPsWeight of NPs
� 100%
EE ð%Þ ¼ Weight of DTX in the NPsWeight of the feeding DTX
� 100%
2.9. Differential scanning calorimetry
The physical status of DTX inside NPs was investigated by dif-ferential scanning calorimetry (DSC 822e, Mettler Toledo, Greifen-see, Switzerland). The samples were purged with dry nitrogen at aflow rate of 20 ml/min. The temperature was raised at a rate of10 �C/min.
2.10. In vitro drug release
Accurately weighed lyophilized DTX-loaded NPs (5 mg) weredispersed in 1 ml of release medium (pH 7.4 phosphate buffer solu-tion containing 0.1% w/v Tween 80) to form a suspension. Tween80 was used to increase the solubility of DTX in the buffer and toavoid the binding of DTX to the tube wall. The suspension wastransferred into a dialysis membrane bag (MWCO = 3500, ShanghaiSangon Biotechnology Co. China) which was immersed in 15 ml ofPBS release medium in a centrifuge tube. The tube was transferredinto an orbital water bath and shaken at 200 rpm at 37 �C. Therelease buffer outside the dialysis bag was replaced with fresh buf-fer every day for 14 days and subjected to HPLC analysis. Thecumulative release of drug from DTX-loaded NPs was plottedagainst time.
2.11. Cellular uptake of fluorescent NPs
HepG2 cells were cultured in a chambered cover glass system inDulbecco’s Modified Eagle’s Medium (DMEM) supplemented with10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/mlstreptomycin in 5% CO2 at 37 �C. Coumarin 6-loaded NPs were usedfor the observation and analysis of cellular uptake. After beingincubated with 25 lg/ml coumarin 6-loaded NPs at 37 �C for 1 or2 h, the cells were rinsed three times with cold PBS and then fixedwith cold methanol for 20 min. The nuclei were counterstainedwith DAPI for 10 min, and then the stained cells were washed threetimes with PBS to remove free DAPI. The cell monolayer wasobserved by confocal laser scanning microscope (CLSM) (Olympus,Japan) with the following channels: a blue channel excited at340 nm and a green channel excited at 485 nm.
For flow cytometric (FCM) assay, the HepG2 cells wereincubated with 100 lg/ml coumarin 6-loaded TPGS-PLA/NPs,pD-TPGS-PLA/NPs or Gal-pD-TPGS-PLA/NPs at 37 �C for 1 h,respectively. The cells were collected and washed with PBS, andthe intracellular fluorescence of coumarin 6 was detected by FCM
D. Zhu et al. / Acta Biomaterialia 30 (2016) 144–154 147
after being excited at 488 nm. Fluorescence emissions at 530 nmfrom 10,000 cells were collected, amplified and scaled to generatea single parameter histogram.
For quantitative analysis, HepG2 cells (initial density: 2 � 104
cells per well) were plated in 96-well black plates and incubatedovernight. Then the cells were equilibrated with HBSS at 37 �Cfor 1 h, and incubated for 2 h with 50, 100 and 500 lg/ml coumarin6-loaded NPs respectively. After incubation, the medium wasremoved and the wells were washed three times with cold PBS.An aliquot of 50 ll 0.5% Triton X-100 in 0.2 N NaOHwas then intro-duced into each well to lyse the cells. The fluorescence intensitywas measured with a microplate reader (GENios, Tecan, Switzer-land) with excitation wavelength at 430 nm and emission wave-length at 485 nm. Cell uptake efficiency was expressed as thepercentage of cell-associated fluorescence versus fluorescence ofthe feed solution.
2.12. Cell viability
HepG2 cells were seeded in 96-well plates at a density of1 � 104 cells/well and incubated overnight. Then the cells wereincubated with drug-free TPGS-PLA/NPs, pD-TPGS-PLA/NPs, Gal-pD-TPGS-PLA/NPs, commercial Taxotere�, DTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs or Gal-pD-TPGS-PLA/NPs suspension at0.25, 2.5, 12.5 and 25 lg/ml equivalent DTX concentrations for24 and 48 h. At the predetermined time, the formulations werereplaced with DMEM containing MTT (5 mg/ml) and the cells werethen incubated for additional 4 h. MTT was aspirated off and DMSOwas added to dissolve the formazan crystals. Absorbance was mea-sured at 570 nm using a microplate reader (Bio-Rad Model 680,UK). Untreated cells were taken as control with 100% viability.The drug concentration at which the growth of 50% cells was inhib-ited (IC50) in comparison with that of the control sample was cal-culated by curve fitting of the cell viability data.
2.13. Animals and tumor model
All the protocols for animal experiments were approved by theAdministrative Committee on Animal Research in the GraduateSchool at Shenzhen, Tsinghua University. Female BALB/c nude mice(15–20 g) aged 4–5 weeks old were purchased from the Institute ofLaboratory Animal Sciences, Chinese Academy of Medical Science.About 0.1 ml of HepG2 cells in PBS were implanted into the subcu-taneous (s.c.) space of right flank region of the BALB/c nude mice ata dosage of 2 � 106 cells/mouse. After inoculation, the s.c. tumorgrowth in each mouse was closely observed. The tumor volumewas calculated by 4p/3 � (length/2) � (width/2)2.
2.14. In vivo imaging and biodistribution analysis
IR-780 was used to replace DTX in the formulation. Excess dyemolecules and other impurities produced during pD coating andGal conjugation were removed by centrifugation filtration through100 kDa MWCO Amicon filters (Millipore, Billerica, MA, USA) andwashing with water 3 times [46]. When the tumor volumesreached about 100–150 mm3, the mice were divided into freeIR-780, non-coated TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPsgroups. HepG2 tumor-bearing nude mice were intravenouslyinjected with 100 lL of free IR-780 (0.7 mg/kg), non-coatedTPGS-PLA/NPs or Gal-pD-TPGS-PLA/NPs (0.7 mg/kg IR-780 equiva-lent for nanoparticles) solutions and then imaged using a MaestroIn Vivo Imaging System (CRI Inc., USA). Images were taken at 0.5, 6and 24 h after injection using the Maestro system. The nude micewere sacrificed by cervical vertebra dislocation at 24 h after injec-tion. Then the organs including heart, liver, spleen, lung, kidneyand tumor were collected and analyzed using the imaging system.
The excitation wavelength of IR-780 is 704 nm and emission spec-trum is 740–950 nm. Dye accumulation and retention in organswas evaluated by calculating contrast index (CI) values usinginstrument software.
2.15. In vivo therapeutic efficacy study
When the tumor volumes reached about 50 mm3, the micewere randomly divided into five groups (n = 5), which wereintraperitoneally injected with saline as a control, or DTX-loadedTPGS-PLA/NPs, pD-TPGS-PLA/NPs, Gal-pD-TPGS-PLA/NPs or Tax-otere�, respectively, at 10 mg/kg DTX doses on days 0, 4, 8 and12. The animals were closely observed for clinical signs and behav-iors, and tumor volumes were measured with a vernier caliperevery other day. The mice were sacrificed by cervical decapitationafter 14 days of treatment, and the final tumor weights were mea-sured and also utilized to evaluate the anti-tumor activity.
2.16. Statistical methodology
All the experiments were repeated at least three times unlessotherwise stated. The results are expressed as mean ± SD, and thestatistical significance of all the results was determined by the Stu-dent’s t-test. P < 0.05 was considered to be statistically significant.
3. Results and discussion
3.1. Characteristics of NPs
In a weak alkaline condition, dopamine is able to undergooxidative polymerization in the presence of oxygen as an oxidant.During the polymerization of dopamine, a tightly adherent pDlayer is created on the surface of TPGS-PLA/NPs which areimmersed in the dopamine solution [37,47]. After 3 h reaction withdopamine at room temperature, all NP suspensions turned dark,implying dopamine polymerization was happened. The dark pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs powders could beobtained after lyophilized (Supplementary data, Fig. S1). The sizeand size distribution of the NPs were measured by dynamic lightscattering (DLS) and the data were listed in Table 1. The particlesizes of pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs were largerthan TPGS-PLA/NPs due to the coating of polydopamine ontoNPs. The zeta potential of TPGS-PLA/NPs is �16.4 ± 3.7, the nega-tive surface charge of the NPs may be ascribed to the presence ofcarboxylate on the surface of PLA segment [45]. After surface mod-ification with polydopamine, the zeta potential of pD-TPGS-PLA/NPs was also negative. This could be attributed to the poly-dopamine coating layer has a negative charge because of thedeprotonation of phenolic hydroxyl groups at neutral pH [48,49].The zeta potential of Gal-pD-TPGS-PLA/NPs was not significantlydifferent from pD-TPGS-PLA/NPs, suggesting that the ligand galac-tosamine conjugated to pD-TPGS-PLA/NPs was not substantial.Similar results were reported by Park and co-workers [35]. Thedrug LC and loading efficiency of Gal-pD-TPGS-PLA/NPs were a lit-tle lower than those of TPGS-PLA/NPs and pD-TPGS-PLA/NPsbecause galactosamine was conjugated to the NPs after beingloaded with DTX, during which time a few drug molecules werelost.
FESEM and TEM images of the prepared NPs are shown in Fig. 2.TPGS-PLA/NPs were spherical and smooth-surfaced. As shown inFig. 2A (FESEM images), we cannot see the significant morphologychange after the TPGS-PLA/NPs were surface modified with poly-dopamine and targeting ligand galactosamine. However, inFig. 2B (TEM images), a layer on the periphery of TPGS-PLA/NPscould be clearly observed after surface modification with poly-
Table 1Characterization of DTX-loaded nanoparticles. (PDI = polydispersity index, ZP = zeta potential, LC = loading content, EE = encapsulation efficiency, n = 3.)
Samples Size (nm) PDI ZP (mV) LC (%) EE (%)
TPGS-PLA 126.5 ± 7.2 0.142 �16.4 ± 3.7 8.23 ± 0.14 89.68 ± 1.56pD-TPGS-PLA 205.2 ± 8.3 0.137 �14.5 ± 2.5 8.07 ± 0.21 89.25 ± 2.37Gal-pD-TPGS-PLA 209.4 ± 5.1 0.145 �13.7 ± 2.1 7.69 ± 0.19 88.47 ± 1.95
Fig. 2. (A) FESEM images of DTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs; local images in the box are shown in the lower panel. (B) TEM images ofDTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs.
148 D. Zhu et al. / Acta Biomaterialia 30 (2016) 144–154
dopamine and ligand galactosamine, and the size of modified NPswas larger than bare TPGS-PLA/NPs. Furthermore, the observationof TEM images showed that the collected NPs free of extraneousparticles in size and shape, indicating the absence of polydopaminegrains, indicating under the reaction conditions of this study, thepolydopamine grains were absent [35,50]. The results demon-strated that the coating of polydopamine to TPGS-PLA/NPs surfacewas successful. The average size which was evaluated from TEMwas significantly smaller than that obtained from DLS experiment.This difference could be ascribed to a tendency of shrink and col-lapse while the NPs were in dry state. This result was in good con-sistency with the report of Wang et al. [51].
Fig. 3. DSC thermograms of pure DTX, and DTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs.
3.2. FTIR and XPS analysis
FTIR analysis was employed to characterize the surface chemi-cal group composition of NPs. As shown in Fig. S2, several newabsorption signals appeared after surface modification with poly-dopamine layer. The peaks at 1600 and 1530 cm�1 were assignedto the overlap of the C@C resonance vibrations in the aromatic ringand the NAH bending vibrations, respectively. The broad absor-bance between 3390 and 3150 cm�1 corresponded to the stretch-ing vibrations of NAH/OAH. The strong stretching vibration peakat 1750 cm�1 indicated the existence of ester groups on NPs. The
results indicated the incorporation of the polydopamine layer onthe surface of the TPGS-PLA/NPs after modification. Similar resultswere reported by Jiang and co-workers [47]. The FTIR spectra ofpD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs were almost the
Fig. 4. In vitro drug release profiles of DTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPsand Gal-pD-TPGS-PLA/NPs.
Fig. 5. Cellular uptake of coumarin 6-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-incubation with coumarin 6-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGSloaded NPs (*P < 0.05, **P < 0.01, ***P < 0.001). (C) FCM histograms for coumarin 6-loaded
D. Zhu et al. / Acta Biomaterialia 30 (2016) 144–154 149
same, likely because of the small quantity of conjugated ligandgalactosamine. As proof, XPS also provides information with higherdetection sensitivity to determine the surface chemical composi-tion. The appearance N1s spectrum of XPS at the binding energyof �400 eV on the surface of pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs (but bare NPs did not) further verified the presenceof a polydopamine layer onto the precursor NPs after modification(Supplementary data, Fig. S3A) [52]. The coating of polydopamineon the TPGS-PLA/NPs and the functionalized NPs with targetingligand galactosamine were also confirmed by narrow XPS scanfor C1s peaks. As shown in Fig. S3B, after polydopamine modifica-tion of the TPGS-PLA/NPs, a large proportion of the peak C@O(288.8 eV) was shielded. Meanwhile, the peak C@O was virtuallyeliminated after functionalization with ligand galactosamine. Fur-thermore, the intensity of peak CAO/CAN (286.0 eV) in Gal-pD-TPGS-PLA/NPs was larger than pD-TPGS-PLA/NPs, indicating theligand galactosamine was conjugated to pD-TPGS-PLA/NPs viathe Michael addition reaction [53,54]. Taken together, the FTIRand XPS analysis suggested that the experiments of polydopamine
pD-TPGS-PLA/NPs in HepG2 cells. (A) CLSM images of HepG2 cells after 1 h or 2 h-PLA/NPs at 37 �C. Scale bars, 20 lm. (B) Cellular uptake efficiency of coumarin 6-NPs on HepG2 cells after 1 h incubation.
Fig. 6. Viability of HepG2 cells detected by MTT assays. Viability of HepG2 cells cultured with DTX-loaded NPs in comparison with that of Taxotere� at the same DTX dose anddrug-free NPs with the same amount of NPs for 24 h (A) and 48 h (B) (*P < 0.05, **P < 0.01, ***P < 0.001).
Table 2IC50 values of Taxotere�, and DTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs on HepG2 cells after 24 h and 48 h incubation.
Incubation time (h) IC50 (lg/ml)
Taxotere TPGS-PLA/NPs pD-TPGS-PLA/NPs Gal-pD-TPGS-PLA/NPs
24 25.25 ± 1.23 25.66 ± 1.54 23.34 ± 1.47 10.19 ± 0.9248 12.26 ± 0.82 3.29 ± 0.56 3.47 ± 0.78 0.56 ± 0.06
150 D. Zhu et al. / Acta Biomaterialia 30 (2016) 144–154
coating to bare NPs and galactosamine grafting to polydopamine-modified NPs surface were successful.
3.3. Physical state of DTX in NPs
The physical state of DTX in TPGS-PLA, pD-TPGS-PLA and Gal-pD-TPGS-PLA NPs was investigated by DSC which reflected drugrelease from the systems both in vitro and in vivo. Fig. 3 showsthe DSC thermograms of pure DTX powder, and DTX-loadedTPGS-PLA, pD-TPGS-PLA and Gal-pD-TPGS-PLA NPs. The endother-mic melting peak of pure DTX appeared at 173 �C. However, whenDTX was loaded into TPGS-PLA, pD-TPGS-PLA and Gal-pD-TPGS-PLA NPs, no melting peak is detected, which indicated the absenceof crystalline DTX in the NP formulations. Thus, DTX was in anamorphous, disordered crystalline phase of a molecular dispersionor a solid solution state after being encapsulated by NPs [45].
3.4. In vitro drug release
The in vitro drug release patterns of DTX-loaded TPGS-PLA/NPs,pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs for 14 days areshown in Fig. 4. All the NPs exhibited a burst release of DTX atthe initial stage, i.e. about 30% of the encapsulated drug wasreleased in the first 2 days. After 14 days, 55.07%, 54.33% and53.86% of drugs were released from TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs, respectively. Hence, the threetypes of NPs had similar release profiles of DTX (P > 0.05).
3.5. Cellular uptake of NPs
To assess the extent of internalization of the prepared NPs intoHepG2 cells, DTX was replaced by a fluorescent probe coumarin 6and observed by CLSM [55]. Fig. 5A displays the CLSM images ofcells after 1 and 2 h of incubation with a suspension of 25 lg/mlcoumarin 6-loaded NPs in DMEM. The intensity of fluorescenceincreased significantly in HepG2 cells incubated with Gal-pD-TPGS-PLA/NPs for 1 h compared to those of the cells incubatedwith TPGS-PLA/NPs and pD-TPGS-PLA/NPs. Galactosamine on thesurface of Gal-pD-TPGS-PLA/NPs is essential for targeted drugdelivery by retaining the liver-targeting activity. To investigatethe role of Gal in the cellular uptake of Gal-pD-TPGS-PLA/NPs, weperformed a receptor competition experiment by selecting galac-tosamine as the competitive reagent. When galactosamine andGal-pD-TPGS-PLA/NPs were added to wells at the same time, thefluorescence was significantly quenched (Fig. 5A). To further inves-tigate the liver-targeting specificity of Gal-pD-TPGS-PLA/NPs, CNE-2 cells were used as control. Little fluorescence was observed inCNE-2 cells incubated with the galactosamine-conjugated NPs(Fig. 5A). Additionally, the intensity of fluorescence in HepG2 cellsincubated with the NPs with or without galactosamine conjugatedincreased as incubation proceeded (Fig. 5A) (i.e. from 1 h to 2 h).Therefore, the galactosylated NPs specifically interacted withHepG2 cells through ligand-receptor (ASGP) recognition.
To further investigate the cellular uptake efficiency of NPs,HepG2 cells were treated with 50, 100 and 500 lg/ml coumarin6-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/
Fig. 7. In vivo imaging and biodistribution analysis of nude mice bearing HepG2 tumors after tail vein injection of free IR-780, IR-780 loaded TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs. (A) Time-lapse NIR fluorescence images of nude mice. The tumors are circled with dotted line. (B) NIR fluorescence intensity of tumors was quantified at indicatedtime points. (C) NIR fluorescence images of major organs and tumors after injection of free IR-780, IR-780 loaded TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs at 24 h. (D)Semiquantitative biodistribution of free IR-780, IR-780 loaded TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs in nude mice at 24 h. The data are shown as mean ± SD (n = 3,*P < 0.05, **P < 0.01).
D. Zhu et al. / Acta Biomaterialia 30 (2016) 144–154 151
NPs and incubated for 2 h. As shown in Fig. 5B, the cellular uptakeefficiency decreases with increasing concentration of NPs. The cel-lular uptake efficiency of Gal-pD-TPGS-PLA/NPs was significantlyhigher than that of TPGS-PLA/NPs and pD-TPGS-PLA/NPs at allthree concentrations.
The cellular uptake of coumarin 6-loaded NPs was also con-firmed by FCM assay. As shown in Fig. 5C, the fluorescence inten-sity of cells treated with coumarin 6-loaded Gal-pD-TPGS-PLA/NPs exceeds that of cells treated with coumarin 6-loaded TPGS-PLA/NPs or pD-TPGS-PLA/NPs.
3.6. Effect of NPs on cell viability
To evaluate the cytotoxicity of DTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs, MTT assay was per-formed using HepG2 cells. The results were compared with thatof a clinically available DTX formulation Taxotere�, and the cyto-toxicity of drug-free NPs was also investigated to exclude anynon-specific effect. HepG2 cells were treated with drug-free orDTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs suspensions and Taxotere� at 0.25, 2.5, 12.5 and 25 lg/ml equivalent DTX concentrations for 24 (Fig. 6A) and 48 h(Fig. 6B). The growth of cells was inhibited more significantly byGal-pD-TPGS-PLA/NPs than by pD-TPGS-PLA/NPs, TPGS-PLA/NPsand Taxotere�, and drug-free NPs did not obviously affect cell cyto-toxicity. This indicated that the synthesized NPs were biocompat-ible and non-toxic to tissues and cells.
The IC50 value, which quantitatively evaluates the in vitro ther-apeutic effects of a pharmaceutical formulation, is defined as the
drug inhibitory concentration needed to cause 50% tumor cell mor-tality in a designated period [56]. Table 2 lists the IC50 values forHepG2 cells after 24 and 48 h of incubation with DTX formulationTaxotere�, and drug-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs andGal-pD-TPGS-PLA/NPs. The IC50 values of Taxotere� and DTX-loaded TPGS-PLA/NPs, and pD-TPGS-PLA/NPs were almost thesame after 24 h, while those of Gal-pD-TPGS-PLA/NPs were lower.However, after 48 h, the IC50 values of all of the drug-loaded NPformulations were significantly lower than those of Taxotere�,with the value of DTX-loaded Gal-pD-TPGS-PLA/NPs being mini-mal. These results suggest that DTX-loaded NPs had betterin vitro therapeutic effects on HepG2 cells, and Gal-pD-TPGS-PLA/NPs were superior to the non-functionalized NPs as a drug carrierfor targeted chemotherapy of liver cancer.
3.7. In vivo imaging and biodistribution of Gal-pD-TPGS-PLA/NPs
The above results have confirmed that Gal-pD-TPGS-PLA/NPscan specifically bind to ASGPR on HepG2 cells (Fig. 5). We thenestablished a HCC xenograft mouse model using HepG2 cells, andthen assessed the in vivo delivery of Gal-pD-TPGS-PLA/NPs afterintravenous injections using in vivo imaging technique. Thebiodistribution of free IR-780, IR-780 loaded TPGS-PLA/NPs andGal-pD-TPGS-PLA/NPs were assessed. As shown in Fig. 7A and B,the fluorescence signals of free IR-780 showed whole body distri-bution with a small number of the IR-780 loaded TPGS-PLA/NPsand Gal-pD-TPGS-PLA/NPs located in the lung and tumor tissueat 0.5 h after injection. At 6 h post-injection, a strong signal wasdetected in the tumor tissue for free IR-780, IR-780 loaded
Fig. 8. Anti-tumor efficacy of DTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs, and Taxotere� and saline on the BALB/c nude mice bearing HepG2cells xenograft. (A) Tumor growth curve period. (B) Percentage change of animal weight curve of the BALB/c nude mice bearing HepG2 cells xenograft after intraperitoneallyinjected with DTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs or Gal-pD-TPGS-PLA/NPs, or Taxotere� or saline. (C) Images of tumors in each group removed from the sacrificedmice at the end of study. (D) Tumor weights for each group after removal from the sacrificed mice at the end of the study. 1, saline; 2, Taxotere�; 3, DTX-loaded TPGS-PLA/NPs; 4, DTX-loaded pD-TPGS-PLA/NPs; 5, DTX-loaded Gal-pD-TPGS-PLA/NPs (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001).
152 D. Zhu et al. / Acta Biomaterialia 30 (2016) 144–154
TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs. The tumor signal inten-sity of IR-780 loaded Gal-pD-TPGS-PLA/NPs was stronger than thatof the IR-780 loaded TPGS-PLA/NPs and free IR-780. Signal inten-sity in lung and liver for the latter groups was still quite high. How-ever, after 24 h post-injection, the signal intensity was observedmuch stronger in tumor tissue than that in lung and liver for allthree groups. It thus could be deduced that Gal-pD-TPGS-PLA/NPs has liver- and hepatoma-targeting capability due to its highaffinity for ASGPR that is often overexpressed by hepatocytes andhepatoma cells. It was very exciting that Gal-pD-TPGS-PLA/NPsexhibited the most remarkable hepatoma-targeting. After adminis-tration, Gal-pD-TPGS-PLA/NPs began to accumulate in the tumor at0.5 h, and further analysis of major tissues at 24 h showed that thefluorescent signal in the tumor was obviously stronger than in theliver (Fig. 7C and D). We believe this distinct hepatoma targetingproperty is a result of Gal-pD-TPGS-PLA/NPs being a specific ligandof ASGPR and that these NPs can selectively accumulate in thetumor via specific ligand/receptor binding.
3.8. In vivo anti-tumor effects
To investigate the in vivo anti-tumor effects of DTX formulatedin Gal-pD-TPGS-PLA/NPs, BALB/c nude mice were subcutaneously(s.c.) inoculated into the right flank with 2 � 106 HepG2 cells in100 ll of PBS. When the tumor volumes reached about 50 mm3,the mice were randomized into 5 groups (n = 5) and treated withDTX-loaded TPGS-PLA/NPs, pD-TPGS-PLA/NPs or Gal-pD-TPGS-PLA/NPs or Taxotere�, at 10 mg/kg DTX dose or saline on days0, 4, 8 and 12 through intraperitoneal injection. Tumor sizes ofthe mice were recorded every 2 days until the 14th day. Aftertwo weeks of treatment, all the mice were sacrificed and thetumors were separated from the bodies. Both DTX-loaded
nanoformulations and Taxotere� significantly inhibited tumorgrowth compared with saline control (Fig. 8A). Being consistentwith the in vitro cytotoxicity data, the group treated withDTX-loaded Gal-pD-TPGS-PLA/NPs showed the lowest growth rateamong the five groups (Fig. 8A). Notably, all mice experiencedslight weight losses on the day of drug administration, and thebody weights were restored after four days (Fig. 8B). The tumorimages and weights of each group are shown in Fig. 8C and D.Evidently, DTX-loaded Gal-pD-TPGS-PLA/NPs worked significantlybetter than Taxotere� and non-functionalized NPs in suppressingtumors, suggesting that they treated liver tumors in the estab-lished animal model more effectively.
4. Conclusions
In conclusion, a new galactosamine-conjugated polydopaminemodified copolymer (Gal-pD-TPGS-PLA/NPs) was synthesized asa carrier for liver tumor-targeted drug delivery. By using the faciledopamine polymerization method, TPGS-PLA/NPs were functional-ized through galactosamine conjugation. The size of Gal-pD-TPGS-PLA/NPs is about 200 nm. Dopamine polymerization and galac-tosamine conjugation barely changed the characteristics of zetapotential and drug release profile of NPs. In vitro cellular uptakeand cytotoxicity assay demonstrated that Gal-pD-TPGS-PLA/NPstargets HepG2 cells via ASGP receptor-mediated recognition andsignificantly inhibits cell proliferation. In addition, Gal-pD-TPGS-PLA/NPs showed a strong hepatoma-targeting property at animallevel. Furthermore, DTX-loaded Gal-pD-TPGS-PLA/NPs reducedtumor size more evidently in vivo than Taxotere, DTX-loadedTPGS-PLA/NPs or pD-TPGS-PLA/NPs, or saline. Therefore, the pre-pared Gal-pD-TPGS-PLA/NPs could potentially qualify as a drugdelivery system targeting liver cancers or other liver diseases.
D. Zhu et al. / Acta Biomaterialia 30 (2016) 144–154 153
Disclosure
The authors report no conflicts of interest in this work.
Acknowledgments
This work was supported by the National Natural Science Foun-dation of China (Nos. 31270019, 51203085, 81571793), GuangdongNatural Science Funds for Distinguished Young Scholar (No.2014A030306036), Natural Science Foundation of GuangdongProvince (No. 2015A030313848), Scientific and TechnologicalInnovation Bureau of Nanshan District (No. KC2014JSCX0023A),Science, Technology & Innovation Commission of Shenzhen Munic-ipality (Nos. JCYJ20150430163009479, JCYJ20150529164918738,CYZZ 20130320110255352, JCYJ20140718171607436) and TianjinMunicipal Natural Science Foundation (No. 15JCZDJC38300).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.actbio.2015.11.031.
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