liver-targeting doxorubicin-conjugated polymeric prodrug with ph-triggered drug release profile

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Research ArticleReceived: 25 September 2009 Revised: 25 November 2009 Accepted: 6 February 2010 Published online in Wiley Online Library: 18 June 2010

(wileyonlinelibrary.com) DOI 10.1002/pi.2880

Liver-targeting doxorubicin-conjugatedpolymeric prodrug with pH-triggered drugrelease profileJin Huang,a,b Feng Gao,a Xiaoxin Tang,a Jiahui Yu,a∗ Daxin Wang,c

Shiyuan Liud∗ and Yaping Lie

Abstract

The aim of the research presented was to develop a potential liver-targeting prolonged-circulation polymeric prodrug ofdoxorubicin (Dox) with a pH-triggered drug release profile. In particular, linear dendritic block copolymers composed ofpolyamidoamine dendrimer (PAMAM) and poly(ethylene glycol) (PEG; number-average molecular weight of 2000 g mol−1)with or without galactose (Gal) were synthesized. Dox was coupled to the copolymers via an acid-labile hydrazone linker.These prodrugs, designated Gal-PEG-b-PAMAM-Doxn and mPEG-b-PAMAM-Doxm, showed accelerated Dox release as the pHdecreased from 8.0 to 5.6. Cytotoxicity of the prodrugs was lower than that of free Dox due to the gradual drug release nature.Compared to mPEG-b-PAMAM-Doxm, Gal-PEG-b-PAMAM-Doxn showed rather high cytotoxicity against Bel-7402, suggestive ofits galactose receptor-mediated enhanced tumor uptake. This galactose receptor-mediated liver-targeted profile was furtherconfirmed by the prolonged retention time in hepatoma tissue monitored using magnetic resonance imaging. Gal-PEG-b-PAMAM-Doxn showed better in vivo antitumor efficacy than free Dox, suggesting its great potential as a polymeric antitumorprodrug.c© 2010 Society of Chemical Industry

Keywords: Gal-PEG-b-PAMAM-Doxn; polymeric prodrug; pH-triggered drug release; doxorubicin; liver-targeting

INTRODUCTIONThe attachment of low-molecular-weight antitumor drugs tohigh-molecular-weight water-soluble polymers is a promisingstrategy for modifying biodistribution, reducing drug toxicity andthus improving the therapeutic efficacy of anticancer agents.1

Plenty of drugs, such as doxorubicin, paclitaxel, camptothecin andplatinates, conjugated with linear polymer carriers, such as poly[N-(2-hydroxypropyl) methacrylamide] copolymers,2 poly(ethyleneglycol) (PEG)3 or polyglutamate,4 have been reported. Some ofthese are presently in clinical trials.5 However, the polydispersityof the traditional linear polymers has limited their practicalapplications as drug carriers.6 Recently, increased attention hasbeen paid to dendrimers as drug carriers because of theirmonodispersity, multiple sites of attachment and controllable,well-defined size and structure.7,8 In addition, polymeric prodrugssuch as dendrimeric prodrugs have an enhanced permeabilityand retention effect that allows them to target tumor cellsmore effectively than small molecules. This passive phenomenonoccurs because of tumor vasculature hyperpermeability (allowingpolymer extravasation) and the lack of tumor tissue lymphaticdrainage which subsequently promotes the retention of polymericdrugs.9 Unfortunately, dendrimers are still associated withseveral problems, including low water solubility and highcytotoxicity, which need to be overcome for in vivo applications.10

Since PEG exhibits such properties as nonimmunogenicity,biocompatibility and improved water solubility, it has beencoupled to polymer–drug conjugates in order to resolve theseissues.11 These PEGylated conjugates show increased water

solubility, prolonged blood circulation and decreased cellularuptake.12 To enhance cellular uptake, targeting ligands can becoupled to the telechelic end of PEG. Galactose (Gal) is one of thewell-studied targeting ligands used for this strategy.13 It has beenhypothesized that polyamidoamine dendrimer (PAMAM)–drugconjugates block-modified with galactosylated PEG could reachhepatocytes via receptor-mediated active targeting due to thehigh affinity of asialo-glycoprotein (ASGP) receptor to galactosylresidues.14

∗ Correspondence to: Jiahui Yu, Institute for Advanced Interdisciplinary Research,East China Normal University, Shanghai 200062, PR China.E-mail: [email protected]

Shiyuan Liu, Department of Diagnostic Imaging, Changzheng Hospital,Shanghai 200003, P R China. E-mail: [email protected]

a Institute for Advanced Interdisciplinary Research, East China Normal University,Shanghai 200062, PR China

b College of Chemical Engineering, Wuhan University of Technology, Wuhan430070, PR China

c Subei Hospital of Jiangsu Province, Yangzhou University, Yangzhou 225001, PRChina

d Department of Diagnostic Imaging, Changzheng Hospital, Shanghai 200003,PR China

e Center for Drug Delivery System, Shanghai Institute of Materia Medica, ChineseAcademy of Sciences, Shanghai 201203, PR China

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Doxorubicin-conjugated polymeric prodrug www.soci.org

Previous reports showed that the release of drugs from thecarrier system was a prerequisite for therapeutic activity ofmost polymeric anticancer conjugates.1 Since endosomes andlysosomes show acidic pH (5.6–6.5),15 incorporation of acid-sensitive spacers between drug and carrier enables the stablecirculation of polymeric drugs in the bloodstream (pH = 7.4)and release of an active drug from the carrier in endosomes andlysosomes of cancer cells. It was expected that these pH-triggeredpolymeric prodrugs could reduce the systemic toxicity of drugs toa large extent.

From the above-mentioned considerations for an ideal poly-meric prodrug, a liver-targeting prolonged-circulation polymericprodrug of doxorubicin hydrochloride (Dox) with a pH-triggereddrug release profile was developed. Thus, Gal-PEG-b-PAMAM-Doxn blocky polymeric drugs were prepared, and characterizedusing 1H NMR and Fourier transform infrared (FTIR) spectroscopy.The cytotoxicity and acid-sensitive drug release profiles of Gal-PEG-b-PAMAM-Doxn were tested, and its liver-targeting profilewas evaluated using magnetic resonance imaging (MRI). Also, itsin vivo antitumor efficacy was evaluated by monitoring tumor sizeafter drug administration.

MATERIALS AND METHODSMaterialsPEG (number-average molecular weight Mn = 2000 g mol−1)and methoxy-poly(ethylene glycol (mPEG; Mn = 1900 g mol−1)were purchased from Fluka. PEG bis-amine (H2N-PEG-NH2)and mPEG-NH2 were synthesized according to the literature.16

Ethylenediamine, methyl acrylate lactobionic acid (Gal-COOH),dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) andthiazoyl blue tetrazolium bromide (MTT) were purchased fromSigma-Aldrich. Dox was purchased from Hisun PharmaceuticalCo. Ltd, Zhejiang, China. All other reagents and solvents were ofanalytical grade and used without further purification.

Cell line and cultureHuman hepatoma cell line Bel-7402 was supplied from theInstitute of Biochemistry and Cell Biology, Chinese Academyof Sciences. Cells were cultured in RPMI-1640, supplementedwith 10 vol% fetal bovine serum, streptomycin at 100 µg mL−1

and penicillin at 100 U mL−1. All cells were incubated at 37 ◦Cin humidified 5 vol% CO2 atmosphere. Cells were split usingtrypsin/ethylenediaminetetraacetic acid solution when almostconfluent.

Animal experimentsSix-week-old female ICR and BALB/c nude mice were receivedfrom the Shanghai Experimental Animal Center of the ChineseAcademy of Sciences. All animal experiments were performedin accordance with the National Ministry of Health recom-mendations, with induction of general anesthesia for everyprocedure. Anesthesia was induced with an intraperitonal in-jection of ketamine hydrochloride (45 mg kg−1) and xylazine(6 mg kg−1).

CharacterizationFTIR spectra were recorded with a Nicolet 6700 spectrometer(USA) in the range 4000–400 cm−1. Samples were mixed with KBrand pressed into slices for measurements. 1H NMR spectra wereobtained with a Bruker Avance 500 operated at 500 MHz. UV-visible

spectra were recorded using a Yayan 1900PC spectrophotometer.MRI images were obtained with a GE Signa Twinspeed 1.5T MRI scanner. Inductively coupled plasma atomic emissionspectroscopy (ICP-AES) was carried out with a Thermo ElectronIRIS Intrepid II XSP instrument under an argon atmosphere. Opticaldensity (OD) was determined with a BIO-TEK powerwave xsmicroplate spectrophotometer. Relative molecular weights andmolecular weight distributions were measured at 30 ◦C using a gelpermeation chromatography (GPC) system (Agilent 1200 series)equipped with differential refractive index detector. A mobilephase of 0.2 mol L−1 NaNO3 and 0.01 mol L−1 NaH2PO4 (pH =7.0) at a flow rate of 1 mL min−1 was used. The injection volumewas 20 µL. PEG samples of known molecular weights with narrowmolecular weight distributions were used as standards for themolecular weight calculations.

Synthesis of Gal-PEG-NH2

Lactobionic acid (0.1778 g, 0.4966 mmol) dissolved in 20 mL ofdried dimethylformamide (DMF) was cooled to −15 ◦C undernitrogen atmosphere, followed by the addition of 0.1024 g ofDCC (0.4966 mmol). The mixture was stirred at −15 ◦C for 20 min,then at 0 ◦C for 15 min. After the addition of 0.0572 g of NHS(0.4966 mmol), the solution was maintained at 0 ◦C for 1 h, thenat room temperature for 12 h with constant stirring, followed bythe addition of 0.9146 g of H2N-PEG-NH2 (0.4573 mmol). Afterstirring at room temperature for 24 h, DMF was evaporated invacuo. The residue dissolved in 30 mL of deionized water wasthoroughly dialyzed using Spectra/Pro membrane (molecularweight cutoff (MWCO) = 1000) against deionized water atroom temperature for 6 days, and then filtered. The lyophilizedproduct was further purified by silica gel chromatography usingCHCl3/MeOH (50/1–16/1 v/v) as eluent. After removal of eluent,the vacuum-dried Gal-PEG-NH2 was obtained as an off-whitepowder in 32% yield (RF = 0.2, CHCl3/MeOH 16/1 v/v). Itschemical structure was characterized from FTIR and 1H NMRspectra.

FTIR (KBr; cm−1): 3334 (OH and NH stretching), 1710 (CONHstretching), 1631 (amino group bending), 1108 (ether bonds in PEGstretching). 1H NMR (D2O; δ, ppm): 3.49–3.63 (–OCH2), 3.07–4.48(lactobionic acid residue).17

Synthesis of Gal-PEG-b-PAMAM(G2.5)Gal-PEG-b-PAMAM(G2.5) was synthesized using Gal-PEG-NH2 asthe core via repeated Michael addition and amidation at roomtemperature according to the similar method described by Parkand co-workers.11 Its chemical structure was characterized fromFTIR and 1H NMR spectra.

FTIR (KBr; cm−1): 3328 (OH and NH stretching), 1734 (COOMestretching), 1700 (CONH stretching vibration), 1105 (ether bondsin PEG stretching). 1H NMR (CDCl3; δ, ppm): 3.49–3.63 (–OCH2),3.07–4.48 (lactobionic acid residue), 2.2–3.4 (PAMAM).

Hydrazinolysis of Gal-PEG-b-PAMAM(G2.5)Gal-PEG-b-PAMAM(G2.5) (0.9464 g, 0.2562 mmol) dissolved in30 mL of methanol was added dropwise to 12 mL of a methanolsolution of hydrazine hydrate (9.93 mL, 204.9 mmol). The mixturewas stirred at room temperature for 72 h under nitrogenatmosphere. After removal of methanol and unreacted hydrazinehydrate, the residue was recrystallized from its CH2Cl2/ether(1 : 10 v/v) solution, designated as Gal-PEG-b-PAMAM-HNNH2, andvacuum-dried to give the product in 60% yield.

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FTIR (KBr; cm−1): 3428 (OH and NH stretching), 1654 (CONHand CONH–NH2 stretching), 1559 (CONH–NH2 bending), 1104(ether bonds in PEG stretching). 1H NMR (D2O; δ, ppm): 7.85–8.25(CH2CONHNH2), 3.49–3.63 (–OCH2), 3.07–4.48 (lactobionic acidresidue), 2.2–3.4 (PAMAM).

Synthesis of Gal-PEG-b-PAMAM-Doxn prodrugGal-PEG-b-PAMAM-HNNH2 (0.4478 g, 0.1181 mmol) and Dox(0.6569 g, 1.1326 mmol) were separately dissolved in 15 mL ofmethanol. Their solutions were mixed, and maintained at roomtemperature with constant stirring for 7 days under nitrogen at-mosphere. After removal of methanol, the aqueous solution ofGal-PEG-b-PAMAM-Doxn was purified using gel chromatographywith a Sephadex-G50 column using pure water as eluent, andlyophilized to give the product in 50% yield. The molar ratioof Dox to Gal-PEG-b-PAMAM-HNNH2 calculated from UV analysis(λmax = 495 nm) was 5.7 : 1.

FTIR (KBr; cm−1): 3428 (OH and NH stretching), 1654 (CONH andCONH–NH2 stretching), 1559 (CONH–NH2 bending), 1104 (etherbonds in PEG stretching). 1H NMR (CD3OD; δ, ppm): 3.49–3.63(–OCH2), 3.07–4.48 (lactobionic acid residue), 2.2–3.4 (PAMAM),1.3, 5.47, 7.62, 7.89 (Dox).18

The mPEG-b-PAMAM-Doxm prodrug was also synthesizedaccording to a similar procedure.

Preparation of Gal-PEG-b-PAMAM(G3.0)-COOHGal-PEG-b-PAMAM(G3.0) (0.3064 g, 0.0644 mmol), dimethy-laminopyridine (0.4 mg, 0.0032 mmol) and succinic anhydride(0.2576 g, 2.576 mmol) were separately dissolved in 25 mL ofCH2Cl2. Their solutions were mixed, and maintained at roomtemperature with constant stirring for 2 days under nitrogenatmosphere. After removal of CH2Cl2, the aqueous solution ofGal-PEG-b-PAMAM(G3.0)-COOH was thoroughly dialyzed using aSpectra/Pro membrane (MWCO = 1000) against deionized waterfor 3 days. The lyophilized product, designated as Gal-PEG-b-PAMAM(G3.0)-COOH, was obtained in 70% yield.

FTIR (KBr; cm−1): 3422 (OH and NH stretching), 1664 (COOHstretching), 1108 (ether bonds in PEG stretching). 1H NMR (CD3OD;δ, ppm): 3.54–3.66 (–OCH2), 3.01–4.74 (lactobionic acid residue),2.2–3.4 (PAMAM), 2.45–2.51 (CH2COOH).

Preparation of Gal-PEG-b-PAMAM(G3.0)–Gd complexesIn a typical procedure, gadolinium chloride hexahydrate (GdCl3·6H2O; 0.0524 g, 0.14 mmol) was dissolved in 2 mL of double-distilled water, and the pH of the solution was adjusted to5–7 with 0.1 mol L−1 hydrochloric acid. Gal-PEG-b-PAMAM(G3.0)-COOH (0.1337 g, 0.0282 mmol) dissolved in 2 mL of double-distilled water was added dropwise to the solution of GdCl3·6H2O. The mixture was gently stirred at 37 ◦C for 48 h in thedark. Gal-PEG-b-PAMAM(G3.0)–Gd complexes were purified usinggel chromatography with a Sephadex-G25 column to removefree Gd3+. The lyophilized product was obtained in 68% yieldand stored at −4 ◦C. The mass percentage content of Gd(III) inGal-PEG-b-PAMAM(G3.0)–Gd was measured using ICP-AES. mPEG-b-PAMAM(G3.0)–Gd was also synthesized according to a similarprocedure.

Drug release experimentRelease of Dox from the polymeric prodrugs dissolved in buffersolution with various pH values (5.6, 7.4 and 8.0) was performed

using a dialysis method.19 An amount of 90 mL of buffer solutionof corresponding pH was used as a dialysis medium at 37 ± 0.5 ◦Cunder constant stirring. Briefly, Gal-PEG-b-PAMAM-Doxn prodrug(31.8 mg) was dissolved in 10 mL of buffer solution. The samplesolution was charged into a dialysis tube (MWCO = 1000).Then the dialysis tube was immersed in the dialysis mediumof corresponding pH. At certain time intervals, 1 mL aliquots ofthe dialysis medium were withdrawn, and the same volume offresh medium was added. The sample solution was analyzed usingUV-visible spectroscopy at 495 nm.20 Each experiment was carriedout in triplicate. Means and corresponding standard deviations(mean ± SD) were recorded.

Evaluation of cytotoxicity using the MTT methodCytotoxicity of Gal-PEG-b-PAMAM-Doxn, mPEG-b-PAMAM-Doxm

and Dox to inhibit cells growth was determined by evaluationof the viability of human hepatoma cell line Bel-7402 using theMTT method. Cells were seeded in 96-well plates at an initialdensity of 104 cells per well in 200 µL of growth medium andincubated for 18–20 h to reach 80% confluency at the time oftreatment. Growth medium was replaced with 100 µL of freshserum-free media containing various amounts of drug (containingDox at 2, 8, 20 and 40 µg mL−1). Cells were incubated for 24 h.The culture medium was then replaced by 100 µL of MTT solution(0.5 mg mL−1). After further incubation for 4 h in an incubator,100 µL of dimethylsulfoxide was added to each well to replace theculture medium and dissolve the insoluble formazan-containingcrystals. OD was measured at 570 nm using an automatic BIO-TEKmicroplate reader (Powerwave XS, USA), and the cell viability wascalculated from the following equation:

Cell viability (%) = ODsample

ODcontrol× 100 (1)

where ODsample is the OD value from a well treated with sample andODcontrol that from a well treated with phosphate buffered saline(PBS) buffer only. Each experiment was carried out in triplicate.Means and corresponding standard deviations (mean ± SD) wererecorded.

Evaluation of cytotoxicity from acridine orange (AO) stainingThe Bel-7402 cells were plated on collagen-coated glass coverslipsin 6-well plates at an initial density of 3.0 × 105 cells per well in5 mL of growth medium and incubated for 18–20 h to reach 80%confluency at the time of treatment. Growth medium was replacedwith 5 mL of fresh serum-free media containing 0.3 mg mL of Dox.After incubation for 1 h, the cells were washed with PBS, and thenincubated with fresh medium containing serum for an additional12 h. Cells were washed twice with PBS and immobilized for 30 minby adding 2 mL of 95% alcohol. After immobilization, cells werestained with 2 mL of AO (100 µg mL−1) for 15 min, washed withPBS and treated with 2 mL of CaCl2 solution for 45 s. The coverslipswere washed with PBS, and imaged under dark-field conditionswith a Motic AE31 microscope equipped with a cooled-frame CCDcamera. Green fluorescence was excited at 490 nm and emissionwas collected at 520 nm. All images were obtained at the samemagnification.

Approximate evaluation of liver-targeting profile using MRITo evaluate approximately the liver-targeting profile of galactosy-lated PEG-b-PAMAM, MRI experiment was carried out with female

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Figure 1. Synthesis route to Gal-PEG-b-PAMAM-Doxn prodrug.

ICR mice, which were divided into two groups for two contrastagents (Gal-PEG-b-PAMAM–Gd, mPEG-b-PAMAM–Gd). Mice foreach contrast agent were also divided into seven groups (threemice each) for testing at various time points after injections. Thecontrast agents with Gd concentrations of 15 mmol L−1 were in-jected at a dose of 1 mmol Gd kg−1 into anesthetized mice via atail vein.

MRI was performed using a GE Signa Twinspeed 1.5 T MRIscanner with a knee coil. The study was performed in thetransverse plane with a T1-weighted spinecho sequence (TR/TE,400 ms/10 ms; section thickness, 3 mm; field of view, 8 cm2; matrix,128 × 128; number of signals acquired, 4) and a total duration of6 min 32 s.

To assess the liver-targeting profile of the contrast agents, theregion of interest (ROI) was set on liver. Signal intensity in liver(SIliver) was measured at various time points. In order to avoidpossible nonlinearly varying factors that could have influencedthe SI measurements, a cylindrical oil recipient was simultaneouslyimaged as an external standard (SIoil), and SIliver values were dividedby SIoil to obtain a relative signal intensity (RI) value:21

RI = SIliver

SIoil(2)

Thus, the enhancement (ENH) values were calculated from theresults of Eqn (2) as follows:

ENH (%) = RI − RI0RI0

× 100 (3)

where RI0 is the mean RI value for liver in the six control micewithout contrast injection.

Animal model and antitumor activity of Gal-PEG-b-PAMAM-Doxn

The in vivo antitumor efficacy of Gal-PEG-b-PAMAM-Doxn wastested using human hepatoma cells (Bel-7402) xenografted intoBALB/c nude mice. Briefly, tumors were established by injecting107 Bel-7402 tumor cells subcutaneously at the right axillary flankof 4- to 6-week-old female BALB/c nude mice. When the tumorsreached an average volume of 500 mm3, mice were divided intothree groups of eight mice each. Treatments were initiated andthis day was designated as day 0. Multiple doses of PBS or Gal-PEG-b-PAMAM-Doxn were injected via tail vein administration every2 days. A total dose of Gal-PEG-b-PAMAM-Doxn was equivalentto 30 mg Dox kg−1. The free Dox was injected at the maximumtolerated dose of 12 mg Dox kg−1. Controls were treated with PBS


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alone. The tumor sizes of the mice were measured every 2 days.Tumor volume was estimated using the following equation:22

V = 3.14ab2

6(4)

where a and b are major and minor axes of the tumor measuredwith a caliper.

Statistical data analysisStatistical data analysis was performed using the Student’s t-testwith p < 0.05 as the level of significance.

RESULTS AND DISCUSSIONSynthesis and characterization of polymeric prodrug and MRIcontrast agentTo synthesize Gal-PEG-b-PAMAM(G2.5), Gal-PEG-NH2 was initiallysynthesized using the direct coupling reaction between lacto-bionic acid and H2N-PEG-NH2 with DCC/NHS as activator (Fig. 1).After purification by dialysis and silica gel chromatography, thepurified Gal-PEG-NH2 was obtained as an off-white powder in32% yield. The low yield is due to the formation of Gal-PEG-Galand the absorption of Gal-PEG-NH2 in the silica gel. Gal-PEG-NH2

easily dissolves in anhydrous methanol. Its chemical structure wasconfirmed from FTIR and 1H NMR spectra (Fig. 2(a)). The molarratio of lactobionic acid residue to PEG in Gal-PEG-NH2 calculatedfrom the integral area ratio of acetal peak at 4.47–4.49 ppm (Hf)to the peak at 3.49–3.63 ppm (–OCH2) in the 1H NMR spectrum is1.1 : 1, very similar to the theoretical value of 1 : 1, indicating thatone primary amino group per H2N-PEG-NH2 molecule participatesin the coupling reaction. Subsequently, Gal-PEG-b-PAMAM(G2.5)dendrimer was synthesized using Gal-PEG-NH2 as the core viarepeated Michael addition and amidation. The chemical structureof Gal-PEG-b-PAMAM(G2.5) was also confirmed from FTIR and 1HNMR spectra (Fig. 2(b)). Its weight-average molecular weight (Mw)measured using GPC s about 3690 g mol−1, with a narrow poly-dispersity index of 1.18, very similar to the theoretical value of3730 g mol−1, indicating its structural integrity.

After hydrazinolysis, Gal-PEG-b-PAMAM-HNNH2 was obtained.The disappearance of the FTIR spectral peak at 1734 cm−1 assignedto ester bonds clearly indicates that the ester groups in Gal-PEG-b-PAMAM(G2.5) had been fully hydrazinolyzed by hydrazine hydrate.

To preserve the biological activity of Dox, Gal-PEG-b-PAMAM-Doxn prodrug was synthesized by the direct coupling reactionbetween Gal-PEG-b-PAMAM-HNNH2 and Dox via hydrazone bondswithout heating for one week under nitrogen atmosphere.The resultant prodrug was purified using Sephadex-G50 gelchromatography. The purified Gal-PEG-b-PAMAM-Doxn easilydissolves in water. Its structure is confirmed by the presence ofthe characteristic peaks at 1.3, 5.47, 7.62 and 7.89 ppm assigned toDox in its 1H NMR spectrum (Fig. 2(d)).14 The molar ratio of Dox toGal-PEG-b-PAMAM-HNNH2 calculated from the 1H NMR spectrumis 5.5 : 1, similar to that obtained from UV analysis (5.7 : 1), andlower than the theoretic value of 8 : 1. It is thought that the sterichindrance results in the lower drug loading of Dox.

To evaluate the liver-targeting profile using MRI, Gal-PEG-b-PAMAM(G3.0) and mPEG-b-PAMAM(G3.0) were modified withsuccinic anhydride. Gd(III) was conjugated with their carboxylicgroups. The contents of Gd(III) in Gal-PEG-b-PAMAM(G3.0)–Gd andmPEG-b-PAMAM(G3.0)–Gd measured using ICP-AES are 21.5 and23.4 wt%, respectively.

Figure 2. 1H NMR spectra of (a) Gal-PEG-NH2, (b) Gal-PEG-b-PAMAM,(c) Gal-PEG-b-PAMAM-HNNH2 and (d) Gal-PEG-b-PAMAM-Doxn.

Figure 3. In vitro release profiles of Dox from Gal-PEG-b-PAMAM-Doxnprodrug in buffer solutions of various pH at 37 ◦C (mean ± SD, n = 3):(a) pH = 5.6; (b) pH = 7.4; (c) pH = 8.0.

Release profiles of Dox from Gal-PEG-b-PAMAM-Doxn prodrugTo investigate the pH-triggered drug release profile of the Gal-PEG-b-PAMAM-Doxn prodrug, a controlled release of drug experimentwas performed in buffer solution with pH = 5.6, 7.4 and 8.0. Asshown in Fig. 3, only 14 and 32% of Dox release is observed in mediaof pH=8.0 and 7.4, respectively, after incubation for 30 h. However,97% of Dox release is reached at pH = 5.6 in about 15 h, three timeshigher than that at pH = 7.4. It is thought that the pH-triggereddrug release profile of the Gal-PEG-b-PAMAM-Doxn prodrug results


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Figure 4. Cell viability of mPEG-b-PAMAM-Doxm, Gal-PEG-b-PAMAM-Doxnand Dox against Bel-7402 determined using the MTT method (mean ± SD,n = 3; ∗p < 0.05).

from the acid-sensitive degradation of hydrazone linker betweenDox and Gal-PEG-b-PAMAM carrier. This pH-triggered drug releaseprofile enables the stable circulation of the polymeric drug in thebloodstream (pH = 7.4), while the active drug is released from thecarrier in endosomes and lysosomes (pH = 5.6–6.5) of cancer cells,which results in the reduction of systemic toxicity of the drug to alarge extent.

Cytotoxicity of polymeric prodrugThe in vitro cytotoxicity of Gal-PEG-b-PAMAM-Doxn, mPEG-b-PAMAM-Doxm and Dox was evaluated with Bel-7402 using theMTT assay. Representative concentration–growth inhibition dataare shown in Fig. 4. Although both the polymeric prodrugs and Doxinhibit cell growth in a dose-dependent manner, the former areconsistently less toxic than Dox, especially at higher dose. The cellviability of Bel-7402 is only 11% when treated with 40 µg mL−1 of

Dox, while a cell viability above 60% is observed when treated withcorresponding Dox concentration of polymeric drug. It is thoughtthat the decreased cytotoxicity of the polymeric prodrugs resultsfrom their gradual drug release profiles.

Interestingly, at the same concentration, Gal-PEG-b-PAMAM-Doxn shows higher cytotoxocity than mPEG-b-PAMAM-Doxm. Itis thought that receptor-mediated cell uptake occurs due to thepresence of ASGP receptor in Bel-7402 and galactose residue in Gal-PEG-b-PAMAM-Doxn,23 which results in the increased intracellulardrug concentration.

The cytotoxicity of Gal-PEG-b-PAMAM-Doxn, mPEG-b-PAMAM-Doxm and Dox was further checked by the observation of greenfluorescence of treated cells after AO staining. As shown in Fig. 5, incomparison with the control, less green fluorescence is observedwhen cells are incubated with the drugs, suggestive of the toxicityof the drugs. In addition, the cytotoxicity in the order mPEG-b-PAMAM-Doxm < Gal-PEG-b-PAMAM-Doxn < Dox can also beobserved from the green fluorescence images, consistent with theMTT assay results.

Approximate evaluation of liver-targeting profile using MRITo avoid radiochemical pollution and scarification of a largenumber of animals, contrast-enhanced MRI, a noninvasive andreal-time method in vivo, was selected to evaluate approximatelythe liver-targeting profile of Gal-PEG-b-PAMAM. Liver was set as theROI. As shown in Fig. 6, although an obvious signal enhancementin liver is observed after injection of Gal-PEG-b-PAMAM–Gd andmPEG-b-PAMAM–Gd contrast agents, the former shows bettersignal enhancement. The maximum liver ENH of both contrastagents occurs 6 h after injection. After that, mPEG-b-PAMAM–Gdshows a rapid decrease of ENH between 6 and 120 h, while agradual decrease of ENH for Gal-PEG-b-PAMAM–Gd is observedfor the same interval. It is thought that the high affinity ofASGP receptor at the liver surface to galactosyl residues in Gal-PEG-b-PAMAM–Gd results in the gradual decrease of ENH forGal-PEG-b-PAMAM–Gd, suggesting its active targeting potentialto liver.

Figure 5. Cytotoxicity of (a) control, (b) mPEG-b-PAMAM-Doxm, (c) Gal-PEG-b-PAMAM-Doxn and (d) Dox against Bel-7402 using AO staining.


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Figure 6. Signal enhancement of (a) Gal-PEG-b-PAMAM–Gd and(b) mPEG-b-PAMAM–Gd in mouse liver (mean ± SD, n = 3).

Figure 7. In vivo antitumor efficacy of (a) PBS, (b) Dox and (c) Gal-PEG-b-PAMAM-Doxn (mean ± SD, n = 8).

Evaluation of antitumor activity of Gal-PEG-b-PAMAM-Doxn invivoThe in vivo antitumor efficacy of Gal-PEG-b-PAMAM-Doxn wasevaluated by monitoring tumor size after drug administration.As shown in Fig. 7, the size of the tumor in the control groupincreases significantly with time, indicating that PBS has noeffect on preventing tumor growth. In contrast, tumor growthis efficiently inhibited in the mice treated with Gal-PEG-b-PAMAM-Doxn, suggesting a rather good antitumor activity. In the case offree Dox, antitumor activity is also observed up to the tenth day,after which tumor size shows an increasing tendency.

CONCLUSIONSA polymeric prodrug of linear dendritic-structured Gal-PEG-b-PAMAM-Doxn was synthesized, and characterized using 1H NMRand FTIR spectroscopy. This prodrug shows a pH-triggered drugrelease profile, liver-targeting potential, lower cytotoxicity andbetter in vivo antitumor efficacy in comparison with free Dox,indicative of its great potential as highly efficient polymericprodrug.

ACKNOWLEDGEMENTSThe research work was supported by the Shanghai Municipal-ity Commission for Special Project of Nanometer Science andTechnology (0852nm03700, 0952nm05300), Shanghai Municipal-ity Commission for Non-governmental International CorporationProject (09540709000), International Corporation Project of Shang-hai Municipality Commission (10410710000), the 973 Projects ofChinese Ministry of Science and Technology (2007CB936104 and2009CB930300), and Self-Determined and Innovative ResearchFunds of WUT.

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liver-targeting doxorubicin-conjugated polymeric prodrug with ph-triggered drug release profile

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