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ORIGINAL CONTRIBUTION pH-responsive release of paclitaxel from hydrazone-containing biodegradable micelles Peilan Qi 1 & Yongqiang Bu 1 & Jing Xu 1 & Benkai Qin 1 & Shujuan Luan 1 & Shiyong Song 1 Received: 8 August 2016 /Revised: 16 October 2016 /Accepted: 17 October 2016 /Published online: 5 November 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract Many tumor cells have acidic microenvironment that can be exploited for the design of pH-responsive drug delivery systems. In this work, well-defined pH-sensitive and biodegradable polymeric micelles were prepared and evaluate as carrier of paclitaxel (PTX). A diblock copolymer constituting of a poly(ethylene glycol) (PEG) and a polycaprolactone (PCL) segment linked by a pH-sensitive hydrazone bond (Hyd), which was denoted as mPEG-Hyd- PCL, was synthesized. The copolymer was assembled to mi- celles with mean diameters about 100 nm. The mean diame- ters and size distribution of the hydrazone-containing micelles increased obviously in mildly acidic environments while kept unchanged in the neutral. No significant change in size was found on polymeric micelles without hydrazone (mPEG- PCL). PTX was loaded into micelles, and the anticancer drug released from mPEG-Hyd-PCL micelles was promoted by the increased acidity. In vitro cytotoxicity study showed that the PTX-loaded mPEG-Hyd-PCL micelles exhibited significantly enhanced cytotoxicity against HepG2 cells compared to the non-sensitive mPEG-PCL micelles. These results suggest that hydrazone-containing copolymer micelles with pH sensitivity and biodegradability show excellent potential as carriers of anticancer drugs. Keywords Biodegradable copolymer . pH-sensitive . Polymeric micelle . Controlled release Introduction Polymeric micelles have shown great potential in hydrophobic anticancer drug delivery [13], while protecting them during circulation, to maximize the therapeutic efficacy and minimize side effects. Some micelle anticancer drug formulations, e.g., NK911®, SP1049C®, NK105®, and Genexol-PM®, have advanced to clinical trials [ 4, 5]. Their nano-size and prolonged circulating time facilitate the passive accumulation around tumor tissues via enhanced permeability and retention (EPR) effect. There are also more and more functionalized micelles that were designed with active targeting abilities to minimize systemic toxicity of therapeutic agents [6]. It should be noted that the effectiveness of a micelle drug delivery sys- tem is determined by the control of not only where the payload should be delivered but also when the payload should be re- leased; e.g., drugs should be released quickly as soon as the drug delivery system arrives targeting sites. To this end, stimulus-responsive micelle carriers have recently attracted much attention because of their capability to release incorpo- rated therapeutic agents in a responsive manner in accordance with the signals stemming from disease-associated microen- vironment changes. Physical and chemical stimuli such as pH [79], temperature [10], and reduction agents [11, 12] were explored to trigger controlled drug release from the micelles, determining when the drugs are to be released. Among the many triggered release systems that are being evaluated, pH-sensitive drug delivery systems are particularly interesting, because the acidity of many pathological sites have been well characterized. A pH gradient exists between normal tissues and tumor which has acidic microenvironment Electronic supplementary material The online version of this article (doi:10.1007/s00396-016-3968-6) contains supplementary material, which is available to authorized users. * Shiyong Song [email protected] 1 Present address: Institute of Pharmacy, Henan University, North Jinming Rd, Kaifeng 475004, China Colloid Polym Sci (2017) 295:112 DOI 10.1007/s00396-016-3968-6

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  • ORIGINAL CONTRIBUTION

    pH-responsive release of paclitaxel from hydrazone-containingbiodegradable micelles

    Peilan Qi1 & Yongqiang Bu1 & Jing Xu1 & Benkai Qin1 & Shujuan Luan1 & Shiyong Song1

    Received: 8 August 2016 /Revised: 16 October 2016 /Accepted: 17 October 2016 /Published online: 5 November 2016# Springer-Verlag Berlin Heidelberg 2016

    Abstract Many tumor cells have acidic microenvironmentthat can be exploited for the design of pH-responsive drugdelivery systems. In this work, well-defined pH-sensitiveand biodegradable polymeric micelles were prepared andevaluate as carrier of paclitaxel (PTX). A diblock copolymerconstituting of a poly(ethylene glycol) (PEG) and apolycaprolactone (PCL) segment linked by a pH-sensitivehydrazone bond (Hyd), which was denoted as mPEG-Hyd-PCL, was synthesized. The copolymer was assembled to mi-celles with mean diameters about 100 nm. The mean diame-ters and size distribution of the hydrazone-containing micellesincreased obviously in mildly acidic environments while keptunchanged in the neutral. No significant change in size wasfound on polymeric micelles without hydrazone (mPEG-PCL). PTX was loaded into micelles, and the anticancer drugreleased frommPEG-Hyd-PCL micelles was promoted by theincreased acidity. In vitro cytotoxicity study showed that thePTX-loaded mPEG-Hyd-PCLmicelles exhibited significantlyenhanced cytotoxicity against HepG2 cells compared to thenon-sensitive mPEG-PCL micelles. These results suggest thathydrazone-containing copolymer micelles with pH sensitivityand biodegradability show excellent potential as carriers ofanticancer drugs.

    Keywords Biodegradable copolymer . pH-sensitive .

    Polymeric micelle . Controlled release

    Introduction

    Polymeric micelles have shown great potential in hydrophobicanticancer drug delivery [1–3], while protecting them duringcirculation, tomaximize the therapeutic efficacy andminimizeside effects. Some micelle anticancer drug formulations, e.g.,NK911®, SP1049C®, NK105®, and Genexol-PM®, haveadvanced to clinical trials [4, 5]. Their nano-size andprolonged circulating time facilitate the passive accumulationaround tumor tissues via enhanced permeability and retention(EPR) effect. There are also more and more functionalizedmicelles that were designed with active targeting abilities tominimize systemic toxicity of therapeutic agents [6]. It shouldbe noted that the effectiveness of a micelle drug delivery sys-tem is determined by the control of not only where the payloadshould be delivered but also when the payload should be re-leased; e.g., drugs should be released quickly as soon as thedrug delivery system arrives targeting sites. To this end,stimulus-responsive micelle carriers have recently attractedmuch attention because of their capability to release incorpo-rated therapeutic agents in a responsive manner in accordancewith the signals stemming from disease-associated microen-vironment changes. Physical and chemical stimuli such as pH[7–9], temperature [10], and reduction agents [11, 12] wereexplored to trigger controlled drug release from the micelles,determining when the drugs are to be released.

    Among the many triggered release systems that are beingevaluated, pH-sensitive drug delivery systems are particularlyinteresting, because the acidity of many pathological siteshave been well characterized. A pH gradient exists betweennormal tissues and tumor which has acidic microenvironment

    Electronic supplementary material The online version of this article(doi:10.1007/s00396-016-3968-6) contains supplementary material,which is available to authorized users.

    * Shiyong [email protected]

    1 Present address: Institute of Pharmacy, Henan University, NorthJinming Rd, Kaifeng 475004, China

    Colloid Polym Sci (2017) 295:1–12DOI 10.1007/s00396-016-3968-6

    http://dx.doi.org/10.1007/s00396-016-3968-6http://crossmark.crossref.org/dialog/?doi=10.1007/s00396-016-3968-6&domain=pdf

  • [13]. The extracellular pH values in cancerous tissues are low-er (5.7–7.0) than the normal blood pH of 7.4. pH gradients canalso be found between the extracellular environment and in-tracellular compartments such as endosomes and lysosomes(pH 4.5–6.5). As a result, the active anticancer drug could bereleased effectively in mild acidic medium of the extracellularspace of the tumor or in the acidic environment of endosomesor lysosomes following cellular uptake.

    Polymer-drug conjugate systems with acid-sensitive link-ages between therapeutic molecule and macromolecules havebeen prepared firstly. Ulbrich [7, 14] and Kataoka [15] syn-thesized block copolymers conjugated with doxorubicin(DOX) via acid labile hydrazone bond, which released conju-gated drug in an acidic intracellular compartment upon cleav-age of hydrazone bonds. Jing and his co-workers [16] havedeveloped micelles based hydrazone and amide linkage forDOX and found higher pH sensitivity of hydrazone-containing co-polymer. As acid labile bond, the hydrazonebond formed between DOX and polymer hydrazines contain-ing spacer has been most often studied. Other acid-sensitivebonds have been seldom used. It is obvious that the conjuga-tion approach requires appropriate bonding sites on both poly-mer and a drug molecule to form acid labile bond, while otheranticancer drugs (such as paclitaxel (PTX), camptothecin(CPT), and gemcitabine) cannot form a hydrazone bond di-rectly as DOX.

    An alternative way to form a pH-sensitive drug deliverysystem is to physically entrap drugs into the hydrophobic coreof pH-responsive polymeric micelles. The pH-sensitive bondis a part of the copolymer, side chain or backbone. In this way,sufficient structural change in the copolymer triggers boostdrug release upon the cleavage of pH-sensitive bond in anacidic environment. Zhong and his co-workers [17] synthe-s i z ed po ly ( e t hy l ene g lyco l ) - b l ock -po ly (2 , 4 , 6 -trimethoxybenzylidene-pentaerythritolcarbonate) (PEG-PTMBPEC) block copolymer and formulated pH-responsivebiodegradable micelles and polymersomes. It was shown thatthe pH-dependent release of PTX originated from the hydro-lysis of pendant acetal bonds on hydrophobic PTMBPECblocks under endosomal pH condition. Yang and his co-workers [18] constructed a pH-sensitive block polymerPEG-b-C18. The hydrophilic block (PEG) and hydrophobicblock (stearic acid) were connected by a Schiff base bond. Theacid labile Schiff base bond cleaved under acidic condition,which resulted in disassociation of micelle and consequentprompted drug release. It is obvious that the pH-responsivedrug delivery system based on cleave-disassociate-release(CDR) mechanism is applicably limited not only to DOXbut also to PTX and CPT.

    The profile of pH-triggered drug release is mainly deter-mined by the sensitivity of acid labile bonds. The bondsshould hydrolyze quickly at mild acidic environments andkeep stable at a pH of 7.4. Acid labile linkages such as

    hydrazone [7, 14–16], acetal [19, 20], orthoester [21, 22],citraconic amide [23], and Schiff base bonds [18] were report-ed. They appeared in either side pendant chains or backboneof the copolymers, acting either as drug-polymer linkages ordiblock linkages. Among them, hydrazone bonds were mostlyexplored for their easy synthesis, good stability, and moderatesensitivity. Fu [24] was the first researcher who designeddiblock polymer with hydrazone linkage. The copolymersshowed pH-dependent degradation but not used as anticancerdrug carriers. The controllable degradation and good compat-ibility of the copolymers were approved. Georgiadou [25] andhis co-workers designed diblock polymer with hydrazonelinkage. The copolymers showed pH-dependent degradationbut not used as anticancer drug carriers.

    PTX has demonstrated significant effect against a widerange of tumors. However, some vital issues including poorwater solubility, low bioavailability, and emergence of drugresistance largely limited the application of PTX. The clinical-ly used PTX formulation is a mixture of cremophor EL (alsocalled polyoxyethylenated castor oil; cremorphor EL is a kindof non-ionic surfactant, often used as a solubilizing agent ofinsoluble drug use) and ethanol, which often leads to signifi-cant side effects [26]. Due to the hydrophobic environment ofthe core of micelles, water-insoluble drugs can easily be sol-ubilized and thus loaded for delivery at the required targets[27]. So, PTX has been formulated into nano-sized micellesand found enhanced antitumor efficiency and minimized sideeffects. To realize temporally and spatially controlled drugrelease, PTX has been seldom involved in a pH-triggered drugdelivery system [28].

    In this work, pH-sensitive copolymer was designed withhydrazone bond as the acid labile linkage. Poly(ethylene gly-col) was used for hydrophilic shell-forming block connectedwith hydrophobic core-forming block, biodegradable poly(ε-caprolactone). The linkage of the two blocks was hydrazonebond with adjacent conjugate group, which was added to im-prove its chemical stability in air atmosphere. Micelles basedon the copolymer were formed and PTX was loaded. Perfectdrug delivery system with the characteristics of intelligence,stealthiness, and biodegradability is expected, and cleave-disassociate-release mechanism is hypothesized.

    Experimental section

    Materials

    Methyl poly(ethylene glycol) (mPEG; Mn = 5000) was pur-chased from Sigma-Aldrich and was dried for 24 h in a vac-uum oven at 50 °C before use. ε-Caprolactone (99 %,Aladdin) was dried with calcium hydride by stirring for 24 hand distilled under reduced pressure before use. Stannousoc tanoa te (Sn(Oct ) 2 ; 95 %, Sigma-Aldr ich) , 4 -

    2 Colloid Polym Sci (2017) 295:1–12

  • carboxybenzaldehyde (98 %, Shanghai Darui), N,N′-dicyclohexylcarbo-diimide (DCC; 98 % Shanghai Darui), 4-dimethylaminopyridine (DMAP; 98 % Shanghai Darui),methyl 4-hydroxybenzoate (98 %, Shanghai Darui), and hy-drazine hydrate aqueous solution (80 %, Tianjin Kemiou)were used as received. All organic solvents were analyticalreagents and used as received, except that toluene was driedwith sodium method to get anhydrous toluene.

    Preparation of hydrazone-containing block copolymermPEG-Hyd-PCL

    Synthesis of aldehyde capped methyl poly(ethylene glycol)Methyl poly(ethylene glycol) (mPEG-CHO) was synthesizedaccording to the reported procedure with some modifications[18]. Briefly, mPEG (10 g) dissolved in dichloromethane(DCM) (150 mL) reacted with 4-carboxybenzaldehyde inthe presence of DCC and DMAP for 24 h at room tempera-ture. Then, the solution was filtered and the filtrate was con-centrated by rotary evaporation to remove DCM. The rawproduct was dissolved in isopropanol and recrystallized atbelow 5 °C in a refrigerator. Resulted solid was washed withcold isopropanol and ethyl ether subsequently. mPEG-CHOyellow powder was obtained with a yield of 91.1 %, 1H NMR(400MHz, CDCl3), δ10.10 (Ar-CHO), 3.60 (–OCH2CH2O–),4.50 (–COOCCHO–), 3.80 (–COOCHC–), 3.37 (CH3O–),and 8.20 and 7.9 (aromatic protons).

    Synthesis of 4-hydroxybenzoylhydrazine According to ref-erence [29], methyl 4-hydroxybenzoate (9.2 g) and 17 mL ofhydrazine hydrate (80 %) solution were mixed with smallamount of ethanol to get a clear solution. The mixture wasrefluxed for 48 h and then filtered. Washing it with ethanoltwo times yielded a white solid, 4-hydroxybenzoylhydrazine,which was dried in a vacuum oven for 24 h, 1H NMR(400 MHz, CDCl3), δ9.49 (Ar-OH), 7.60 and 6.70 (aromaticprotons), 9.90 (Ar-CONH–), and 4.36 (–N-NH2).

    Synthesis of hydrazone-containing macroinitiator mPEG-Hyd-phenol mPEG-CHO (1 g) reac ted wi th 4-hydroxybenzoichydrazine (0.15 g) in DMF (10 mL) at60 °C for 24 h. mPEG-Hyd-phenol was isolated by precipita-tion from cold ethyl ether and dried at 40 °C in vacuo, 1HNMR (400 MHz, CDCl3), δ8.40 (Ar-CH = N), 11.81 (Ar-CONH-N=), 10.16 (Ar-OH), and 3.60 (–OCH2CH2O–).

    Synthesis of mPEG-Hyd-PCL block copolymer by ring-opening polymerization Under a nitrogen atmosphere, thereis certain amount of mPEG-Hyd-phenol and ε-caprolactone inthe presence of stannous octanoate (100 μL) in anhydroustoluene. Polymers with different molecular weights were syn-thesized by varying the ratio between macroinitiator andmonomer. Typically, 0.304 g (0.057 mmol) mPEG-Hyd-

    phenol and 1.14 g (10.00 mmol) ε-caprolactone were placedin 20 mL anhydrous toluene. The mixture was stirred at100 °C in the presence of stannous octanoate (100 μL) for24 h. Then, chloroform was added to dissolve all the solid.The block copolymer was isolated by precipitation from coldethyl ether and washed at least five times with cold ethyl ether.It was dried at 40 °C in vacuo. Block copolymers mPEG-Hyd-PCL with a target molecular weight of 25,000 were obtainedwith a yield of 59 %, 1H NMR (400 MHz, CDCl3), δ3.60 (–OCH2CH2O–); δ8.40 (Ar-CH = N); 2.30, 1.60, 1.37, and 4.05(protons on poly(ε-caprolactone) part); and 6.91, 7.89, and8.09 (aromatic protons). Polymers of different molecularweights were listed in Table 1.

    Synthesis of mPEG-PCL block copolymer by ring-opening polymerization mPEG-PCL copolymers withouthydrazone linkage were also prepared as pH non-responsivecounterpart. The procedure was the same as the mPEG-Hyd-PCL block copolymer except using mPEG as initiator, 1HNMR (400 MHz, CDCl3), δ1.38, 1.64, 2.30, and 4.06 (pro-tons on poly(ε-caprolactone) part) and 3.64 (–OCH2CH2O–).

    Characterization

    Fourier transformed infrared spectroscopy (FTIR) wasperformed using an AVATAR360 (Nicolet, USA) spec-trometer. 1H NMR spectra were recorded on anAVANCE 400 spectrometer (Brucker, Switzerland) oper-ating at 400 MHz using deuterated chloroform or deuter-ated dimethyl sulfoxide as solvents. Chemical shift wascalibrated against residual solvent signals. The molecularweight and polydispersity of the copolymers were deter-mined by a Damn Eos (Wyatt, USA) gel permeationchromatograph (GPC) instrument equipped withPhenogel 10E6A column and a OPTILAB rEXrefractive-index detector. Measurements were performedusing tetrahydrofuran (THF) as the eluent at a flow rateof 1.0 mL/min at 30 °C and a series of narrow polysty-rene standards for the calibration of the columns. Thesize of the micelles was determined by dynamic lightscattering (DLS) at 25 °C using a Zetasizer Nano-ZS90(Malvern Instruments, UK) equipped with a 633-nm He–Ne laser. The micelle suspension was filtered through a0.22-μm syringe filter before measurements. The amountof PTX was determined by high-performance liquidchromatography (HPLC) (Shimadzu LC-20AT, Japan)with UV detection at 227 nm using a mixture of aceto-nitrile and water (v/v = 55/45) as a mobile phase and aBDS HYPERSIL C18 (4.6 × 250 mm, 5 μm) column.Transmission electron microscopy (TEM) was performedusing a JEM-100CX II TEM. The samples were preparedby dropping 10 μL of micelle dispersion on the coppergrid and dried in air.

    Colloid Polym Sci (2017) 295:1–12 3

  • Micelle formation and pH-triggered change of micelle size

    Micelles were prepared using a simple solvent evapora-tion method. mPEG-Hyd-PCL or mPEG-PCL (20 mg)was dissolved in acetone (1 mL). Under stirring, thesolution was added into 20 mL pure water by dropwise.The resulting suspension was stirred at room tempera-ture for 24 h. The resulting micelles were filteredthrough 0.22-μm syringe filter, and the size was deter-mined by DLS. Three 10-mL aliquots were taken fromfreshly prepared micelle dispersions, and the pH of mi-celle dispersions was adjusted to pH 5.0 and pH 4.0using acetate buffer or maintained at pH 7.4 using phos-phate buffer. The sizes were measured on DLS after 24-h incubation at 37 °C with shaking.

    Critical micelle concentration measurement

    Critical micelle concentration (CMC) was determinedusing pyrene as a fluorescence probe. Ten vessels wereadded 0.5 mL stock solution of pyrene in acetone(6.0 × 10−6 mol/L), respectively. They were left in thedark and dried in air. Then, 5-mL micelle dispersionwith different polymer concentration was added into

    each vessel. The concentration varied from 1.0 × 10−10

    to 0.1 mg/mL, and the pyrene concentration was fixedat 0.55 μM. Fluorescence spectra were recorded usingan F-7000 (Hitachi, Japan) fluorescence spectrometerand an excitation wavelength of 335 nm. Fluorescenceemissions at 375 and 386 nm were monitored. TheCMC was estimated at the cross-point when extrapolat-ing the intensity ratio I375/I386 at low- and high-concentration regions.

    Encapsulation and release of PTX

    PTX-loaded micelles were prepared by a solvent evaporationmethod. Typically, copolymers (20 mg) and PTX (2 mg) weredissolved in 1 mL acetone. Under magnetic stirring, the solu-tion was added by dropwise into 40 mL pure water at roomtemperature. PTX-loaded micelles were formed after evapo-ration of acetone. The dispersion was filtered through0.22-μm syringe filter to remove undissolved PTX, thensealed, and stored in refrigerator. To determine drug-loadingcontent (DLC), 1 mL of PTX-loaded micelle dispersion wasfreeze-dried, the residue was dissolved in acetonitrile, and theamount of PTX was determined by HPLC. DLC was deter-mined according to the following formula:

    DLC wt%ð Þ ¼ weight of loaded drug=total weight of loaded drug and polymerð Þ � 100%

    The in vitro release of PTX from micelles was investigatedat 37 °C under three conditions. Three aliquots (2 mL) ofPTX-loaded micelle suspension were put into three dialysisbag with a molecular weight cutoff (MWCO) of 8000–14,000,respectively. The bags were sealed and immersed in the fol-lowing three 20-mL different buffers: acetate buffer (0.01 M,pH 4), acetate buffer (0.01 M, pH 5), and PBS (0.01 M, pH7.4). There were 0.5 wt% Tween 80 in all the buffers. Atdesired time intervals, 2 mL of the release medium was takenout and replenished with an equal volume of fresh medium.

    The concentration of PTXwas determined byHPLCmeasure-ments. Cumulative release was calculated according to fol-lowing formula:

    Er ¼Ve

    Xn−1

    1

    Ci þ V0Cnmdrug

    In this equation, Er is the cumulative release of PTX (%),Veis the volume to be taken every time (mL), V0 is the volume of

    Table 1 Synthesis of mPEG-Hyd-PCL Polymer (Mi/Mm)

    a Yields (%) Mnb (kg/mol) Mnc (kg/mol) PDId CMCe (mg/L)

    mPEG-Hyd-PCL25K 1:175 59 28.2 15.9 1.840 0.603

    mPEG-Hyd-PCL35K 1:260 67 36.2 19.3 1.791 0.288

    mPEG-Hyd-PCL45K 1:350 68 45.2 24.6 1.683 0.191

    a Feed ratios in mole between initiator and monomerb Calculated from 1H NMR spectrac GPC resultsd PDI polydispersity indexe Critical micelle concentration determined using pyrene as a fluorescent probe

    4 Colloid Polym Sci (2017) 295:1–12

  • medium (mL), Ci is the concentration when certain volume isto be taken (μg/mL), mdrug is the total mass of PTX containedin the release system (μg), and n is the sampling time.

    In vitro toxicity evaluation

    The cytotoxicity of the empty micelle, PTX-loaded mi-celles, and free PTX were evaluated by MTT assay usingHepG2 and HeLa cells. Cells were seeded onto a 96-wellplate at a density of 5 × 103 cells per well in 100 μL of1640 medium containing 10 % FBS or Dulbecco’s mod-ified Eagle medium (DMEM) containing 10 % FBS andincubated for 24 h (37 °C, 5 % CO2). The medium in

    each well was then replaced by 100 μL of new 1640medium or DMEM (containing 10 % FBS) containingvarious concentrations of micelles, PTX-loaded micelles,or free PTX. The tests were conducted in replicates offour for each concentration. Each sample was performedin quintuplicate and the samples were calculated withcells for 24 and 48 h, and the viability of cells wasmeasured using the methylthiazoletetrazolium method.In brief, 100 μL methylthiazoletetrazolium solutions(0.5 mg/mL in culture medium) were added to each well.The cells were incubated for 4 h, and then, 100 μL ofDMSO was added to dissolve the resulting purple crys-tals. The formazan crystals were dissolved in dimethyl

    H3CO

    O

    H113

    CHO

    +

    DCC/DMAP

    H3CO

    O

    113

    O

    CHO

    CH2CH2

    OCH3

    HO

    O

    NHNH2

    HO

    O

    HO

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    +

    H3CO

    O

    113

    O

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    C NNH

    O

    OH

    H3CO

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    113

    O

    H

    C NNH

    O

    O

    O

    H

    O

    n

    N2H4.H

    2O

    O

    O

    DMF

    Scheme 1 Synthesis pathway ofpH-sensitive mPEG-Hyd-PCLblock copolymer

    Colloid Polym Sci (2017) 295:1–12 5

  • sulfoxide, and the absorbance correlatable with the num-ber of viable cells was measured using a thermo micro-plate reader at 570 nm. Cells cultured in DMEM mediumor 1640 medium containing 10 % FBS (without micelles)were used as controls. Cell viability (%) was calculatedby the following equation [30]:

    Cell viability %ð Þ ¼ Asample=Acontrol� �� 100

    where Asample and Acontrol denote the absorbance of thesample well and control well, respectively. Data are pre-sented as average SD ± (n = 3).

    Result and discussion

    Synthesis of hydrazone-containing block copolymers

    Placing acid labile hydrazone bond onto backbone but not sidechain of a copolymer is the main point of the current work.The hydrazone-containing diblock polymer was not preparedby direct coupling of polymers with reacting end groups.Preparation was started with hydrazone-containingmacroinitiator and followed by a ring-opening polymeriza-tion. The synthesis of the diblock copolymer is illustrated inScheme 1.

    As shown in Scheme 1, mPEG-CHO was firstly formedby esterification of mPEG with 4-carboxybenzaldehyde.

    Then, 4-hydroxybenzoichydrazide was synthesized byhydrazinolysis of methyl 4-hydroxybenzoate, which bearhydrazine group (–NH2HN2). Hydrazone containingmacroinitiator was obtained by coupling reaction betweenmPEG-CHO and 4 - hyd r oxyben zo i c hyd r a z i d e .Subsequently, hydrazone containing the copolymermPEG-Hyd-PCL was synthesized via ring-opening poly-merization with the initiator which has a benzyl hydroxylgroup. The 1H NMR spectra of mPEG-CHO (Fig. 1a) showsignal characteristic of δ10.10 (Ar-CHO), 3.60 (–OCH2CH2O–), 4.50 (–COOCCHO–), 3.80 (–COOCHC–),3.37 (CH3O–), and 8.20 and 7.9 (aromatic protons).Through the above analysis, we can confirm the successof the synthesis of mPEG-CHO. 1H NMR showed thatthe macroinitiator mPEG-Hyd-phenol was successfullysynthesized, as revealed by presence of signal δ8.40attributable to hydrazone protons (Ar-CH = N)(Fig. 1b). The 1H NMR spectra of mPEG-Hyd-PCL(Fig. 1c) show clearly signal characteristic of PEG(δ3.60) and poly(ε-caprolactone) (δ1.37, 1.60, 2.30,4.05). And most importantly, characteristic signals forproton on hydrazone bond (δ8.40) and aromatic protons(δ6.91, 7.89, 8.09) were found on the spectra. mPEG-PCL which has no pH-sensitive linkage was synthesizedas control.

    The molecular weights of synthesized polymers aregiven in Table 1. mPEG-Hyd-PCL25K, mPEG-Hyd-PCL35K, and mPEG-Hyd-PCL45K were designed basedon feed ratios (1:175, 1:126, and 1:350, respectively)

    Fig. 1 The 1H NMR spectra ofcopolymer mPEG-CHO (a),mPEG-Hyd-phenol (b), andmPEG-Hyd-PCL (c)

    6 Colloid Polym Sci (2017) 295:1–12

  • between initiator and monomer, and the yields were 59,67, and 68 %, respectively. Their hydrophilic segmenthad the same molecular weight (Mn = 5267 g/mol),while their hydrophobic block varied in molecularweight. Their real molecular weights were calculatedbased on 1H NMR. The integral ratio between reso-nances at δ4.05 (one of methylene protons on PCL)and 3.60 (methoxy proton of PEG) was used to calcu-

    late. It was found that the values based on 1H NMRspectra were almost consistent with the theoretical ones.Additionally, GPC measurements revealed a unimodaldistribution with Mn of 15.9, 19.3, and 24.6 kg/mol,respectively (polystyrene standards) and polydispersityindices (PDIs) of 1.840, 1.791, and 1.683 (Fig. S1 inSupplementary Materials). So, well-defined mPEG-Hyd-PCL block copolymer was successfully synthesized.

    Formation and pH-triggered size change of micelles

    The amphiphilic mPEG-Hyd-PCL polymers can be self-assembled into micelles in aqueous solution by a solventevaporation method. The particle size of the micelles wascharacterized by DLS. As shown in Fig. 2a and Table 2,the micelles have diameters ranged from 105 to 121 nmand increase with their molecular weight. The CMCs ofthe polymeric micelles were 6.03 × 10−4, 2.88 × 10−4, and1.91 × 10−4 mg/mL for mPEG-Hyd-PCL (25 K), mPEG-Hyd-PCL (35 k), and mPEG-Hyd-PCLA (45 k), respec-tively, determined by fluorescence measurements usingpyrene as a probe (Fig. S2 in Supplementary Materials).The CMC values were calculated from the intensity ratioof bands at 375 and 386 (I375/I386) (Fig. S3 inSupplementary Materials). It was found that the CMC ofthe polymers decreased from mPEG-Hyd-PCL25K tomPEG-Hyd-PCL45K, which originated from the in-creased hydrophobic interaction of micelle core. The mi-celles had a relatively low CMC, indicating the excellentstability to keep their construct under the in vivo dilutedconditions, which will be critical for the efficient deliveryof drugs to tumors.

    The average particle size of the drug-loaded micelleswas smaller than that of the blank micelles (Fig. 2b andTable 2). The incorporation of PTX into the core ofmicelles may enhance the van der Waals force of thehydrophobic segments of the micelles, resulting in amore compact structure and a decrease of particle size[31]. As is known to all, nanoparticles below 200 nmcan accumulate in tumor tissue via the enhanced perme-ability and retention (EPR) effect [32, 33]; therefore,mPEG-Hyd-PCL micelle drug delivery system below100 nm in size would effectively reach lesion sites

    100010010

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    mPEG-Hyd-PCL25K-PTX

    mPEG-Hyd-PCL35K-PTX

    mPEG-Hyd-PCL45K-PTX

    a

    b

    Fig. 2 The particle size of different molecular weight of mPEG-Hyd-PCLblock copolymer micelles (a) and PTX-loaded polymeric micelles (b)

    Table 2 Average sizes of blankmicelles and drug-loaded micelles Blank micelles Size (d nm) PDI PTX-loaded micelles Size (d nm) PDI

    mPEG-Hyd-PCL25K 105 0.077 mPEG-Hyd-PCL25K-PTX 86 0.072

    mPEG-Hyd-PCL35K 110 0.099 mPEG-Hyd-PCL35K-PTX 96 0.018

    mPEG-Hyd-PCL45K 121 0.104 mPEG-Hyd-PCL45K-PTX 102 0.088

    Colloid Polym Sci (2017) 295:1–12 7

  • and achieve the goal of pH-controlled drug delivery. Asshown in Fig. 3, the morphology of PTX-loaded mPEG-Hyd-PCL35K polymer micelles was observed by TEM.The drug-loaded micelles have a spherical core-shellstructure, and the particle size of the micelles is about100 nm, which is consistent with the result of DLSmeasurement. And, the drug-loading content of mPEG-Hyd-PCL35K was about 2.7 %.

    The evolution of micelle sizes responsive to pH wasmonitored by DLS measurements. The size changes ofmicelles with or without hydrazone bonds in response todifferent pH were illustrated in Fig. 4. As shown inFig. 4a, the size distribution of hydrazone-containingmPEG-Hyd-PCL35K micelles underwent obvious changesin pH 4.0, pH 5.0 conditions, and the multiple peaksappeared; there was obvious turbidity and sedimentationwhile keeping stable in pH 7.4. The appearance ofmulti-scale nanoparticles was resulted from the

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    Fig. 3 TEM images of PTX-loaded mPEG-Hyd-PCL35K polymermicelles

    8 Colloid Polym Sci (2017) 295:1–12

  • decomposition of the pH-sensitive micelles. The aggrega-tion of PCL blocks which are insoluble in water makesthe larger particles of 1000 nm above. In contrast, mPEG-PCL35K micelles without hydrazone bonds kept un-changed under all pH conditions (Fig. 4b). The pH-sensitive micelle drug delivery system will keep stableand protect their payload from being cleared in bloodcirculation. When they accumulate around acidic tumortissue via EPR effect or internalized by tumor cell, boostrelease of drugs will occur to enhance the therapeuticeffect.

    pH-controlled release of PTX

    The pH-trigged in vitro drug release behaviors were studied atpHs 4.0, 5.0, and 7.4. As shown in Fig. 5, PTX released fromPTX-loaded mPEG-Hyd-PCL35K micelles at physiological

    pH was about ca. 40 % in 24 h. The release rate was signifi-cantly accelerated at pH 5.0 and 4.0, with accumulated releaseabove 80 % in 24 h, respectively. The system showed higherdrug release rate at acidic environment than at physiologicalmedium, that is because acid labile characteristic of hydrazonebond endowed the polymeric mPEG-Hyd-PCL35K micelleswith pH-controlled drug release profile. To compare the re-lease curves (Fig. 5a, b), it can be found that PTX-loadedmPEG-PCL35K polymeric micelles without hydrazone bond,at a pH of 4, 5, and 7.4, all showed a consistent trend ofrelease, and under different pH conditions, all the cumulativerelease was about 45 % in 24 h; there was no pH-dependentrelease profile. Consequently, the molecular structure of thepolymer-containing hydrazone bond can affect the drugin vitro release behavior of micelles. As is well known, theextracellular pH in tumors (pH 5.7–6.8) is lower than thephysiological conditions (pH 7.4) [34]; therefore, the pH-

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    Colloid Polym Sci (2017) 295:1–12 9

  • responsive release system might be applied in anticancertherapies.

    Cytotoxicity of the drug-loaded micelles

    The toxicities of the blank micelles were tested in HeLa andHepG2 cells using a MTT assay. The cell viabilities of HeLaand HepG2 cells were above 90% for both blank mPEG-PCLand mPEG-Hyd-PCL micelles, following 24- and 48-h incu-bation (Fig. 6),which means that the blank micelles were re-markably non-toxic and biocompatible up to a concentrationof 100 μg/mL.

    To demonstrate the effect of hydrazone linkages ofmPEG-Hyd-PCL35K on antitumor capability, in vitro cy-totoxicities of free PTX, mPEG-PCL35K, and mPEG-Hyd-PCL35K micelles at a series of equivalent concen-trations of PTX were evaluated against HeLa and HepG2

    cell lines. As shown in Figs. 7 and 8, all the formula-tions showed a dose-dependent cell proliferation inhibi-tion behavior, and prolonging incubation time from 24 to48 h led to more death of tumor cells. Free PTX showedhigher in vitro toxicity to each cancerous cell, comparedto the other two micelle formulations. While, pH-sensi-tive, PTX-loaded mPEG-Hyd-PCL35K micelles weremore toxic than PTX-loaded mPEG-PCL35K. Superiorcell-killing capability of PTX-loaded mPEG-Hyd-PCL35K micelles may be due to the fact that entry ofpH-sensitive micelles through endocytosis and drug re-lease into the cytoplasm triggered by endosome pH arequick and efficient processes [21].

    Additionally, a significant difference in proliferationinhibition between HeLa and HepG2 cells incubatedwith the same formulation was found. The liver carci-noma cell line HepG2 demonstrated less sensitivity topaclitaxel than the HeLa cell line. The relative resis-tance to paclitaxel of the HepG2 cells when comparedto the cancerous HeLa cells likely results from the me-tabolism of PTX by liver cells to less toxic compounds[35, 36].

    Conclusions

    In summary, a new type of pH-sensitive biodegradablemPEG-Hyd-PCL block copolymer was synthesized viar ing -open ing po lymer iza t i on in i t i a t ed f rom ahydrazone-containing macroinitiator. The resulted copol-ymer was composed of hydrophilic PEG with fixedlength and hydrophobic PCL with different lengths,which could be self-assembled into micelles withuniformed size and narrow size distribution. The con-taining hydrazone provided the formed micelles withpH-responsive properties of degradation into pieces un-der acidic conditions. PTX was used as a model drugand was effec t ive ly loaded into the micel les .Copolymers without hydrazone bonds were also pre-pared as control. In vitro drug release results showedthat PTX-loaded mPEG-Hyd-PCL presented a more rap-id and complete drug release in the intracellular lyso-some environment (pH 5.0). The results of in vitro cellassay revealed that the micelles were non-toxic andgood biocompatibility. PTX-loaded mPEG-Hyd-PCL mi-celles possessed higher antitumor activity to kill theHeLa cells in comparison with PTX-loaded mPEG-PCL micelles. Although further study in vivo is neces-sary to determine the potential of the pH-sensitive mi-celles as drug delivery vehicle, the initial results dem-onstrate they are promising for drug-controlled releasein tumor therapy.

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    10 Colloid Polym Sci (2017) 295:1–12

  • Acknowledgments This work was supported by the National NaturalScience Foundation of China (NSFC 51375142), Research Fund forExcellent Young College Teachers of Henan Province, and a key projectfunded by the Education Department of Henan Province.

    Compliance with ethical standards

    Conflict of interest The authors declare that they have no conflict ofinterest.

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    pH-responsive release of paclitaxel from hydrazone-containing biodegradable micellesAbstractIntroductionExperimental sectionMaterialsPreparation of hydrazone-containing block copolymer mPEG-Hyd-PCLCharacterizationMicelle formation and pH-triggered change of micelle sizeCritical micelle concentration measurementEncapsulation and release of PTXInvitro toxicity evaluation

    Result and discussionSynthesis of hydrazone-containing block copolymersFormation and pH-triggered size change of micellespH-controlled release of PTXCytotoxicity of the drug-loaded micelles

    ConclusionsReferences