Biodegradable Nanoparticles Based on Linoleic Acid andPoly(�-malic acid) Double Grafted Chitosan Derivatives as

Carriers of Anticancer Drugs

Ziming Zhao,†,‡ Miao He,†,‡ Lichen Yin,† Jiamin Bao,‡ Lili Shi,†,‡ Bingqing Wang,†,‡

Cui Tang,† and Chunhua Yin*,†,‡

State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of LifeSciences, Fudan University, Shanghai 200433, China, and Department of Biochemistry, School of Life

Sciences, Fudan University, Shanghai 200433, China

Received October 27, 2008; Revised Manuscript Received December 18, 2008

Novel chitosan derivatives carrying linoleic acid (LA) as hydrophobic moieties and poly(�-malic acid) (PMLA)as hydrophilic moieties (LA/PMLA double grafted chitosan, LMC) were synthesized. It self-assembled intonanoparticles of 190-350 nm in water, which carried negative surface charges in physiological pH. The criticalaggregation concentration of the LMC deceased with an increase in the LA content. Paclitaxel (PTX) was loadedinto the LMC nanoparticles with a high loading efficiency and the maximum loading capacity of 9.9 ( 0.4%.PTX-LMC nanoparticles exhibited a sustained release within 24 h in pH 7.4 phosphate-buffered saline (PBS),and the release rate was affected by the LA content and PMLA length. Hemolysis and acute toxicity assessmentindicated that the LMC nanoparticles were safe drug carriers for i.v. administration. Additionally, PTX-LMCshowed significantly potent tumor inhibition efficacy relative to that of TAXOL in S-180 bearing mice. Therefore,the LMC nanoparticles could be an effective and safe vehicle for systemic administration of hydrophobic drugs,especially PTX.

Introduction

Over the past few years, great effort has been made to developnovel polymeric nanoparticles as desirable drug delivery systems(DDSs) for their attractive characteristics, such as longercirculation time, targeted drug delivery, protection from enzy-matic degradation, and reduced drug toxicity or side effects.1

Research on polymeric nanoparticles has been mainly focusedon amphiphilic block copolymers2,3 and hydrophobically modi-fied water-soluble polymers,4 which can self-assemble to formhydrophobic core and hydrophilic shell structure when dissolvedin water due to the intra- or intermolecular hydrophobicinteractions. The hydrophobic domain at the same time can serveas a preservatory for hydrophobic drugs and protect them fromthe aqueous environment. As drug carriers, the safety andpotential accumulation of these polymeric nanoparticles in thebody must be taken into account. Therefore, a number ofnontoxic and biodegradable polymers, both synthetic and natural,have been utilized in formulating polymeric nanoparticlesincluding polyesters (such as poly(lactic acid) (PLA),5 poly-(lactic-co-glycolic acid) (PLGA),6 poly(L-caprolactone) (PCL)7),poly(amino acids) (such as poly(γ-glutamic acid),8 poly(L-aspartic acid),9 poly(L-lysine),10 and poly(L-ornithine)11), poly-(anhydrides),12 poly(orthoesters), and polyphosphazene deriva-tives.13 Natural polymers such as albumin,14 collagen, gliadin,15

gelatin, chitosan, pullulan,16 and Curdlan4 have also been used.In particular, chitosan has been widely applied due to its

structural and physicochemical properties. It shows satisfactorybiocompatibility, biodegradability, and low immunogenicity,17

which are beneficial attributes for an ideal drug delivery

material.18,19 However, the extended applications of chitosanare limited because of its insolubility in physiological solution(pH 7.4) and lack of amphiphilicity, which prohibits it fromforming micelles in water. Therefore, a lot of chitosan deriva-tives have been developed to be more appropriate for drugdelivery carriers. Calvo et al.20 reported the formation ofhydrophilic chitosan-polyethylene oxide nanoparticles andstudied their potential application as protein carriers. Miwa etal.21 synthesized a novel chitosan derivative with lauryl groupsattached to amino groups as the hydrophobic moieties andcarboxymethyl groups to hydroxyl groups as the hydrophilicmoieties (N-lauryl-carboxymethyl-chitosan, LCC). Kwon et al.22

prepared hydrophobically modified glycol chitosans (HGCs) bycovalent attachment of 5�-cholanic acid to glycol chitosan.Zhang et al.23 synthesized a series of novel chitosan derivativescarrying long chain alkyl groups (n ) 8, 10, 12) as hydrophobicmoieties and sulfated groups as hydrophilic moieties. Park etal.24 reported N-acetyl histidine-conjugated glycol chitosan(NAcHis-GC) self-assembled nanoparticles as a promisingsystem for intracytoplasmic delivery of drugs. Wang et al.25

synthesized a cholesterol-modified chitosan conjugate (CHCS)with succinyl linkage.

Poly(�-malic acid) (PMLA) is well-known as a water-solubleand biodegradable polymer26 that can be degraded into malicacid, an intermediate of tricarboxylic acid cycle. It also bearslateral carboxyl groups, which allow further conjugation ofbiologically active molecules. Cammas et al.27 synthesizeddegradable macromolecular micelles based on amphiphilic blockcopolymers of PMLA as hydrophilic units and poly(�-malic acidalkyl esters) as hydrophobic blocks, and their properties andstability were proven to be pH-dependent. Lee et al.28 designeda new nanoconjugate, Polycefin, where PMLA was used as thescaffold, and the drug or functional modules (such as targeting

* Corresponding author. Tel: +86-21-65643797. Fax: +86-21-55522771.E-mail address: chyin@fudan.edu.cn.

† Department of Pharmaceutical Sciences.‡ Department of Biochemistry.

Biomacromolecules 2009, 10, 565–572 565

10.1021/bm801225m CCC: $40.75 2009 American Chemical SocietyPublished on Web 01/28/2009

agent, degradation protecting module, and fluorescent agent)were conjugated to the pendant carboxyl groups of PMLA.

Although chemotherapy is one of the major cancer therapeuticmethods in clinical practice, there lies a serious problem in thatmost anticancer drugs have no specific efficacies to tumors, andnormal cells (especially bone marrow cells and endothelial cells)can also be killed. At the same time, water insolubility,biological barriers in body, and toxicity induced by high dosagealso limit the application of many anticancer drugs. NanoparticleDDSs offer major improvements in therapeutics through theirsolubilization capacity, the ability to bypass biological barriers,and sustained release. More importantly, targeted drug deliverycan be achieved by surface modification or introduction oftargeting ligands. Therefore, nanoparticle DDSs have wideapplication prospects in cancer therapy.

The aim of this study was to develop a novel polymericnanoparticle system that had good biodegradability and couldprovide modification sites on the surface for the introductionof targeting ligands. Linoleic acid (LA) and PMLA doublegrafted chitosan derivatives (LMCs) were synthesized, wherechitosan acted as the backbone, LA as the hydrophobic moiety,and PMLA as the hydrophilic moiety. It was assumed that LMCswould exhibit desirable biodegradability in that the ester oramide linkage between chitosan and LA/PMLA could be easilyhydrolyzed by lipase and amidase while the glycoside linkagecould be degraded by glycosidase. Moreover, LA is an essentialfatty acid involved in human fatty acid metabolism, and PMLAis biodegradable as mentioned above. The lateral carboxyls andresidual aminos provided abundant sites that could be modified.The physicochemical properties of LMCs were characterizedby Fourier transform infrared (FTIR) spectroscopy, 1H NMRand X-ray diffraction (XRD). LMC nanoparticles were preparedby a sonication method and were characterized by dynamic lightscattering (DLS), transmission electron microscopy (TEM),scanning electron microscopy (SEM), and fluorescence spec-troscopy. As a model hydrophobic drug, paclitaxel (PTX) wasloaded into the LMC nanoparticles; the loading capacity as wellas the loading efficiency were determined, and in vitro releaseprofiles were studied. Hemolysis and acute toxicity assessmentwere performed to evaluate the safety of LMCs. Finally, thecomparable antitumor efficacies of PTX-LMC and TAXOLwere monitored in Sarcoma180 (S-180) bearing mice.

Experimental Section

Materials. Chitosan (deacetylation degree of 85% and MW of105 000 Da) was purchased from Golden-shell Biochemical Co. Ltd.(China). LA (BR) and D,L-aspartic acid (CP) were purchased fromSinopharm Chemical Reagent Co. Ltd. (China). Trifluoroacetic acid(TFA) was supplied by Sigma (USA). All other reagents were ofanalytical grade. Kunming mice (6 weeks old, body weight 18-22 g)were obtained from the Animal Centre of Fudan University, and raisedunder normal conditions with free access to food and water. Animalexperiments were performed according to the Guiding Principles forthe Care and Use of Experiment Animals in Fudan University.

Synthesis of LMC. Benzyl malolactonate was synthesized from D,L-aspartic acid as described before.29,30 Synthesis of poly(�-benzyl malate)(PMLABz) was as follows. Lactic acid was dried under vacuum for24 h at room temperature (RT) and 0.18 g of it was added into a flaskas the initiator. TFA (0.02 g) was added as the catalyst. Benzylmalolactonate (4.12 g) was kept under N2 stream for 2 h and thentransferred under N2 to the previous flask. The polymerization wasconducted at 80 °C for 3 days. TFA was eliminated by vacuumdistillation after polymerization, and PMLABz1 (lactic acid-to-lactonemolar ratio of 1:10) was obtained. PMLABz2 (lactic acid-to-lactone

molar ratio of 1:20) was obtained when 0.09 g of lactic acid was usedfor polymerization.

Linoleic chloride was synthesized as follows. LA was dissolved indichloromethane into which 1.5 equiv of oxalyl chloride was smoothlyadded in the ice bath. The mixture was stirred at 40 °C under N2 for6 h. Excess oxalyl chloride and dichloromethane were eliminated byvacuum distillation, and linoleic chloride was obtained. PMLABz acylchloride was obtained using the same method.

Synthesis of LMC was as follows. Chitosan (1.67 g) was dissolvedin 20 mL of MeSO3H into which 2.99 g of linoleic chloride (0.9 equiv/sugar unit of chitosan) was added. After stirring at RT for 2 h,PMLABz1 acyl chloride (2.15 g) was added. The mixture was stirredfor 4 h at RT before 30 g of crushed ice was added to stop the acylationreaction. The acidic mixture was stirred at RT for 0.5 h and dialyzed(molecular weight cutoff (MWCO) 3600 Da) for 24 h to remove mostof the acid, followed by adjusting the pH to 7.4 with NaHCO3. Themixture was then filtered to remove insoluble matter and dialyzed(MWCO 3600 Da) again for more than 3 days. Finally the pH ofdialysis solution was adjusted to 6.0 and allowed to precipitate for 10min at 4 °C. After filtration, the precipitate was washed twice withwater and twice with acetone and dried in vacuum, and LMC1 wasobtained.

LMC2 was obtained when 1.99 g of linoleic chloride (0.6 equiv/sugar unit of chitosan) was used. LMC3 was obtained when 0.99 g oflinoleic chloride (0.3 equiv/sugar unit of chitosan) was used. WhenPMLABz1 acyl chloride was replaced by PMLABz2 acyl chloride,LMC4, 5, and 6 were obtained using the same method as LMC1, 2,and 3, respectively (Figure 1).

Characterization of LMC. 1H NMR measurements were performedon an AVANCE DMX 500 spectrometer (Bruker, Germany). Chitosanwas dissolved in the mixed solvent of D2O and F3CCOOD, and chitosanderivatives were dissolved in the mixed solvent of DMSO-d6 andF3CCOOD. The internal reference was tetramethylsilane (TMS).

IR spectra were recorded on a Nexus 470 FTIR spectrometer(Nicolet, USA) in KBr discs.

XRD spectra were obtained using a D/max-γB multicrystal diffrac-tion meter (Rigaku, Japan) with Cu KR radiation in the range of 5-40°(2θ) at 40 kV and 60 mA.

Preparation of LMC Nanoparticles. Nanoparticles were preparedby a sonication method. LMCs (10 mg) were dissolved in 10 mL ofwater, which was then sonicated using a probe-type sonifier (JY92-R,Scientz Biotechnology, China) at 100 W for 10 min in an ice bath.The pulse was turned off for 2 s with the interval of 5 s to avoid increasein temperature. The resulting solution was filtered through a 0.8 µmmembrane, and a LMC nanoparticle solution was obtained.

DLS Measurement. The particle size and zeta potential of thenanoparticles were determined by DLS (Nicomp380/ZLS, SantaBarbara, CA). The particle size measurements were performed at awavelength of 635 nm and a scattering angle of 90° at 23 °C. The zetapotential measurements were monitored at a wavelength of 635 nmand a scattering angle of 14° at 23 °C. Sample concentration was keptat 1.0 mg/mL.

Morphology. The morphological examination of LMC nanoparticleswas conducted by TEM (H-600A, Hitachi, Japan). The samples wereplaced on copper grids and dehydrated for TEM observation.

The morphology of LMC nanoparticles was also observed by SEM(XL30, Philips-FEI, Netherlands) at 20 kV after dehydration, desic-cation, fixing, and gold spraying.

Fluorescence Spectroscopy. The critical aggregation concentrations(CACs) of the LMC nanoparticles were determined by fluorescencespectroscopy (Cary Eclipse, Varian, USA) with pyrene as a hydrophobicprobe. A pyrene solution (6.0 × 10-4 M) was prepared in acetone andstored at 4 °C until use. The pyrene solution was added to 5 mLvolumetric flasks and evaporated to dryness under nitrogen. Then 5mL of LMC solutions with various concentrations (1 × 10-4 to 0.1mg/mL) were added, which were then sonicated for 1 h beforemeasurement of steady-state fluorescence spectra. Fluorescence excita-

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tion spectra were recorded at the emission wavelength (λem) of 395nm and excitation and emission bandwidths at 5 nm.

Drug Loading and In Vitro Release. LMC (50 mg) was dissolvedin 10 mL of 0.15 M phosphate-buffered saline (PBS) (pH 7.4), and 1mL of PTX solution in ethanol (10 mg/mL) was added. The mixturewas then sonicated using a probe-type sonifier at 100 W for 10 min inan ice bath. To remove ethanol, the solution was dialyzed (MWCO3600 Da) against 0.15 M PBS (pH 7.4) overnight, and the nanoparticlesolution was filtered through a 0.8 µm membrane to remove insolublePTX.

The PTX-loaded LMC nanoparticle solution was loaded onto thesilica column (50-100 mesh) and eluted with 0.15 M PBS (pH 7.4).The fraction of LMC nanoparticles was collected, and its volume (V1)was measured. Then, the column was eluted in gradient mode withethanol/water to collect free PTX fraction, and its volume (V2) wasmeasured. The whole elution process was monitored with a UV detector(UV-WXJ 9388 spectrophotometer). Methanol (4.5 mL) was added to0.5 mL of the nanoparticles fraction, which was sonicated for 5 minand centrifuged at 12 000 rotations/min for 10 min. The concentration(C1) of PTX in the supernatant was determined by high-performanceliquid chromatography (HPLC). The mobile phase was acetonitrile/water (46/54, v/v); the flow rate was 1.0 mL/min, and the detectionwavelength was 227 nm. The column was a Hypersil C18 column (5µm, 150 mm × 4.6 mm, Yilite, China). The fraction of free PTX wastreated in the same way, and the concentration (C2) was determined.

The drug loading capacity (LC) and loading efficiency (LE) weredefined as follows:

LC(%, w ⁄ w)) [(C1 × V1 × 10) ⁄ (C1 × V1 × 10+Mass of LMC)] × 100%

LE(%, w ⁄ w)) [(C1 × V1) ⁄ (C1 × V1 +C2 × V2)] × 100%

In vitro release of PTX from the LMC nanoparticles was investigatedin PBS (0.15 M, pH 7.4) containing 0.1% (w/v) Tween 80. Briefly,

0.5 mL of PTX-loaded LMC nanoparticle solution was added to 99.5mL of the release medium, which was incubated at 37 °C and 100rotations/min. At predetermined time intervals, 1 mL of the samplewas taken out and centrifuged at 12 000 rotations/min. The amount ofPTX in the supernatant was determined by HPLC, and the cumulativereleased amount was calculated.

Hemolysis. Erythrocytes were isolated from the whole blood of ahealthy New Zealand rabbit, washed three times with 0.15 M PBS (pH7.4), and resuspended to obtain a concentration of 2% (v/v). PBS wasadded into 0.1 mL, 0.2 mL, and 0.3 mL nanoparticle suspensions (10mg/mL) to obtain a final volume of 2.5 mL, into which 2.5 mL of theerythrocyte suspension was added. Samples were incubated at 37 °Cfor 1 h and centrifuged at 3000 rotations/min for 10 min. Erythrocytesincubated with 0.15 M PBS and water served as negative and positivecontrols, respectively. Nanoparticle solution without erythrocytes wasused as the blank to compensate the turbidity of free LMC nanoparticles.The hemolysis ratio (HR) was determined spectrophotometrically at541 nm and calculated from the following equation:

HR(%)) (ODsample -ODnegative control) × 100 % ⁄ (ODpositive control -ODnegative control)

Acute Toxicity. Mice were fasted for 12 h and randomly grouped(five males and five females in each group). LMC was dissolved insaline and administered via i.v. at a dose of 625 mg/kg body weightonce or twice a day. Dominant signs of toxicity and mortality weremonitored for the subsequent two weeks. At the end of the observationalperiod, the animals were sacrificed and a thorough autopsy was carriedout.

Antitumor Efficacy of PTX-Loaded LMC Nanoparticles. S-180bearing mice were obtained by subcutaneous inoculation of S-180 cellsat the axillary region of male mice. When the tumor volume reached50-200 mm3, mice were grouped and administered with saline(control), PTX injection at 6 mg/kg, PTX-loaded LMC nanoparticles

Figure 1. Reaction scheme of LMC derivatives.

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at 6 mg/kg, and PTX-loaded LMC nanoparticles at 3 mg/kg. Drugadministration was performed three times via tail vein injection with 2days spaced between each administration. Tumor size and body weightwere recorded every other day.

The long diameter (L(t), mm) and the short diameter (W(t), mm) ofthe tumor were measured with a vernier caliper, and the change ofbody weight of each mouse was recorded every other day. The tumorvolume (V(t))was calculated using the following equation:

V(t)) L(t) × W(t)2 ⁄ 2

The mice were sacrificed on the third day after the last administration,and the tumors were excised and weighed. The tumor inhibition ratio(TIR) was calculated according to the following equation:

TIR(%)) (1-WT ⁄ WC) × 100%

where WT and WC refer to the tumor weight of the test and controlgroups, respectively.

Results and Discussion

Synthesis of LMC. Lactic acid was selected as the initiatorbecause it was a hydroxy acid that could initiate the ring-openingpolymerization of lactone (benzyl malolactonate) and thus formPMLABz with lactic acid as its carboxyl terminal. Chitosan wasa macromolecule with steric hindrance, so it was difficult forpure PMLABz to be conjugated onto chitosan because of thehigh steric hindrance of its lateral benzyl. Comparatively, lacticacid without side chains was suitable for conjugation ontochitosan via the carboxyl terminal group. On the other hand,the length of PMLABz could be controlled by adjusting themolar ratio of lactic acid to lactone.

Linoleic chloride was added prior to PMLABz acyl chloridebecause the latter was immiscible with MeSO3H and the reactionwould be uneven. The feeding sequence resulted in a homo-geneous and complete reaction. MeSO3H acted as a solvent andcatalyst in the acylation of chitosan. After ice was added, thebenzyl groups could be hydrolyzed directly by the strong acidof MeSO3H.

Compared with other amphiphilic chitosan derivatives re-ported, such as LCC21 or N-octyl-O-sulfate chitosan (OCS),23

LMC had longer alkyl chains and longer hydrophilic chains. Itwas reported that the solubility of hydrophobic compounds wasincreased with an increase in the carbon length in hydrophobicmoieties, so the long chains of LA might be helpful for theencapsulation of PTX.31 The long PMLA chains might form ahydration layer and reduce the absorption of plasma opsoninsand thus might decrease the clearance of the reticuloendothelialsystem (RES). In addition, the lateral carboxyls of PMLAprovided modification sites for conjugation of bioactive mol-ecules, and the jointed substances on the outer surface wouldbe easily recognized.

Characterization of LMC. FTIR spectra of chitosan and itsderivatives are shown in Figure 2. New peaks at 2924, 2853,1467 cm-1 were attributed to the long alkyl chain of LA, andthe peak at 1733 cm-1 was assigned to the carboxyl group ofPMLA. Peaks at 1173 and 1088 cm-1 showed the formation ofan ester bond. There was a slight peak at 1650 cm-1 in LMC1representing the amide linkage, which was not detected in LMC2or LMC3. Such results suggested that LA and PMLA werelinked to chitosan mainly by ester linkage, and when the C-6position was saturated they could also be linked to NH2 viaamide bond. This accorded with previous investigations thatC-6 OH was more active than C-3 OH, and the positivelycharged NH2 was inactive for acylation reaction.32

In the 1H NMR spectrum of chitosan (Figure 3), the peak at1.9 ppm was assigned to the NHAc, while peaks at 2.9-3.8ppm were attributed to the H-2,3,4,5,6 and OH. In comparison,LMC1 showed the peak at 0.8 ppm, which was assigned to themethyl hydrogen. The peaks at 1.1-1.6 ppm were attributed tothe methene hydrogen of LA. The peak at 4.5 ppm was assignedto the CH of the terminal lactic acid residue, and the peaks at4.7 ppm represented the CH of PMLA. Such results evidencedboth LA and PMLA in the modified chitosan.

The degree of substitution (DS) of LA in LMC was calculatedby comparing the ratio of linoleic methyl protons (δ ) 0.8 ppm)to sugar protons (δ ) 2.9-3.8 ppm), which was defined as the

Figure 2. IR spectra of chitosan (a), LMC3 (b), LMC2 (c), and LMC1(d).

Figure 3. 1H NMR spectra of chitosan (a) and LMC1 (b).

568 Biomacromolecules, Vol. 10, No. 3, 2009 Zhao et al.

number of LA groups per 100 anhydroglucose units of chitosan.The DS of PMLA was calculated by comparing the ratio ofmethylene protons of PMLA (δ ) 4.5 ppm) to sugar protons(δ ) 2.9-3.8 ppm), which was defined as the number of PMLAgroups per 100 anhydroglucose units of chitosan. The resultswere shown in Table 1.

XRD graphs of chitosan and LMC derivatives were shownin Figure 4. Chitosan showed two sharp diffraction peaks at 2θ) 11° and 20°, and a small shoulder peak at 2θ ) 22°. Thereflection fall at 2θ ) 11° was assigned to crystal form I, whichwas orthorhombic and represented hydrated crystal structure ofchitosan. The strongest reflection at 2θ ) 20° corresponded tocrystal form II, which was also orthorhombic.33 LMC1, LMC2and LMC3 showed only one broad peak at around 2θ ) 20°,and the peak intensity decreased, which suggested that thehydrogen bond was decreased after chemical modification, andthe crystalline structure of LMC appeared to be amorphous. Themolecules of amorphous LMC were in a disorder state, and thelong chains of them were easy to twist together, which wasbeneficial to encapsulate drugs and form nanoparticles.

Morphology of LMC Nanoparticles. The morphology ofthe LMC nanoparticles as examined by TEM and SEM areshown in Figure 5. The nanoparticles appeared spherical inshape.

Size and Zeta Potential of LMC Nanoparticles. Particlesize of LMC nanoparticles demonstrated a unimodal distribution(figure not shown). The hydrodynamic diameter and thepolydispersity index of the nanoparticles are illustrated in Table1. The size of LMC nanoparticles was affected by the DS ofLA and the length of PMLA. At a certain PMLA chain length,the particle size decreased with the increase of DS (LA), possibly

due to the increasing hydrophobic interactions between LAmoieties, which made the hydrophobic core more compact.When the DS (LA) was fixed, the particle size increased withthe length of PMLA, which was possibly due to an increase ofthe extending hydrophilic shell. Long-circulating nanoparticlesare able to penetrate into tumor tissues, accumulate, and releasethe therapeutic drug locally at tumor sites because of the“enhanced permeability and retention” (EPR) effect. Particlesize is a key factor that affects the blood circulation half-life ofnanoparticles. As a rough approximation, nanoparticles withdiameters larger than 200 nm, providing that they are rigidstructure, are readily scavenged by macrophages and the RES.For very small particles (<50 nm), they can easily pass throughthe leaky capillary wall in the tumor but can also be easilypushed out from the tumor into the blood.34,35 Generally,nanoparticles that have a mean diameter of 50-200 nm areappropriate for drug delivery into tumors. Therefore, LMC1 andLMC4 with smaller particle size might be more suitable asdelivery carriers for anticancer drugs.

The zeta potential of LMC nanoparticles is also shown inTable 1. The negative charge confirmed the presence of ionizedcarboxyl groups at the nanoparticle surface. In the case ofLMC1, the zeta potential of -17.1 ( 0.7 mV suggested that itmight have good dispersion stability.

When the pH of the nanoparticle solution changed from 3.0to 7.4, the size of the nanoparticles increased obviously.However, as pH further increased from 7.4 to 9.0, no appreciableincrease of particle size was detected (Figure 6). This wasprobably because the carboxyls of PMLA were in the protonatedform at pH 3.0 and the PMLA chains were folded. When pHincreased to 7.4, the carboxyls of PMLA were negativelycharged, and the electrostatic repulsion allowed extension ofthe PMLA chains. Since the ionization of carboxyl was completeat pH 7.4, further increase in the pH value showed negligibleeffect on particle size. The change in the particle size influencedby deprotonation of PMLA was remarkable on particles havinglow PMLA substitution. It was because the molar ratio of LAto malic acid was fixed to keep the equilibrium of hydrophobicand hydrophilic moieties of LMC. Therefore, a longer lengthof PMLA related to a lower substitution degree. When thecarboxyls were deprotonated, the extension of PMLA was moreobvious in long chains. So the size of the LMC nanoparticle

Table 1. Particle Size, Zeta Potential, and DS of LMCNanoparticles (n ) 3)

samplemean diameter

(nm)azeta potential

(mV)DS

(LA)bDS

(PMLA)b

LMC1 190.0 ( 69.7 (0.14) -17.1 ( 0.7 72.9 2.9LMC2 223.3 ( 66.1 (0.09) -10.3 ( 4.0 47.0 3.6LMC3 240.7 ( 185.6 (0.59) -16.9 ( 2.5 22.5 3.2LMC4 207.9 ( 78.4 (0.14) -13.6 ( 4.6 67.1 1.8LMC5 266.2 ( 116.6 (0.19) -10.0 ( 3.6 41.1 1.5LMC6 328.8 ( 127.6 (0.15) -10.7 ( 2.6 24.9 1.5

a Values in parentheses represent the polydispersity index. b Determinedby 1H NMR.

Figure 4. XRD patterns of chitosan (a), LMC1 (b), LMC2 (c), andLMC3 (d).

Figure 5. TEM (a) and SEM (b) images of LMC1 nanoparticles.

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that had lower PMLA substitution but longer chains changedremarkably with pH.

Figure 7 showed that zeta potential of LMC1 nanoparticleswas sensitive toward pH. When pH was lower than 6.0, thenanoparticles showed positive surface charges, and the zetapotential was between 20 mV and 30 mV. When pH was higherthan 7.0, the nanoparticles were negatively charged, and thezeta potential was between -15 mV and -30 mV. LMCpossessed both carboxyls and aminos, the pKa values of whichwere about 5.0 and 6.5, respectively. When the pH was belowtheir pKa, the protonation led to the decrease of negative chargesof carboxyls and increase of positive charges of aminos, andthe LMC nanoparticles appeared to be positively charged. Incomparison, when the pH was above their pKa, the deprotonationled to the increase of negative charges of carboxyls and decreaseof positive charges of aminos, and the nanoparticles werenegatively charged. When the pH was between 6.0 and 7.0, theLMC nanoparticles were nearly uncharged because the negativecharge and positive charge were neutralized.

Since the pH of the lysosome was below 5.0, surface chargereversal from negative to positive would occur when LMCnanoparticles were uptaken and transported to the endolysosome,which was proposed as the mechanism for the endolysosomalescape of the nanoparticles. The cationic nanoparticles interactedwith vesicular membranes and led to localized destabilizationof the membrane and the escape of nanoparticles into cytoplas-mic compartment.36 Therefore, the surface charge property ofLMC nanoparticles was beneficial for the endolysosomal escape.

Fluorescence Spectroscopy. Nanoparticle formation in aque-ous media was investigated using a fluorescence probe. Thefluorescence excitation spectra of the LMC1 nanoparticles atvarious concentrations in the presence of 6.0 × 10-7 M pyreneare shown in Figure 8a. At low concentrations, there were

negligible changes in both the total fluorescence intensity andthe shift of the (0,0) band at 334 nm. However, as the LMC1concentration increased, a notable increase in the total fluores-cence intensity and a red shift of the (0,0) band was observed,indicating that the probe was transferred from the aqueous phaseto the nonpolar environment of the nanoparticles interior. TheCAC value was determined from the crossover point as shownin Figure 8b. The CAC values of the LMC1, 2, and 3nanoparticles were 2.5 × 10-3, 3.5 × 10-3, and 4.5 × 10-3

mg/mL, respectively. The increase of LA moieties improvedthe stability of the nanoparticles by stronger hydrophobicinteractions and thus reduced the CAC values.

PTX Loading and Release Test. The study on the CAC ofthe LMC nanoparticles using pyrene as a fluorescence probesuggested that hydrophobic molecules could be incorporatedinto the inner core of the nanoparticles. This property wasextended to a hydrophobic anticancer drug, PTX.

The PTX loading efficiency and loading capacity of LMCnanoparticles are shown in Table 2. Loading efficiencies of allLMC nanoparticles were higher than 70%, which suggested thatthe sonication method was effective for PTX loading into theinner core of nanoparticles. As compared with LMC1, LMC2and LMC3 with less hydrophobic moieties showed lowerloading capacity. The same trend was observed among LMC4,LMC5, and LMC6. The increased drug loading capacity of LMCnanoparticles with the DS of LA might be attributed to thehydrophobic interactions between LA moieties and hydrophobicdrugs.

Figure 6. The particle size of LMC nanoparticles at pH 3.0, 7.4, and9.0.

Figure 7. Zeta potentials of LMC1 nanoparticles at different pH values(mean ( SD, n ) 3).

Figure 8. (a) Excitation spectra of pyrene (6.0 × 10-7 M) in water inthe presence of LMC1 with various concentrations (emission wave-length of 395 nm). (b) Plot of intensity ratio (I337/I334) from excitationspectra vs log C of LMC1 nanoparticles.

570 Biomacromolecules, Vol. 10, No. 3, 2009 Zhao et al.

The total amount of PTX released from LMC1, LMC2,LMC3, and LMC4 within 4 h was 49.5%, 60.7%, 63.5%, and47.0%, respectively (Figure 9). A slower release rate of PTXwith increased DS of LA was also attributed to the hydrophobicinteractions between LA moieties and PTX. This finding wasconsistent with the results of other researchers.4,37 Na et al.4

reported that an increased DS of hydrophobic moieties and drugloading capacity of self-assembled nanoparticles resulted in aslower release of all-trans retinoic acid (ATRA). Jeong et al.37

also announced that the release rate of clonazepam at higherdrug loading was slower than that at lower drug loading. Alonger PMLA chain could also slow the release rate of PTX,which suggested that the thicker hydrophilic shell would restrictthe dissolution and diffusion of the hydrophobic drug into theouter aqueous medium. Therefore, in vitro drug release kineticscould be controlled by the DS of hydrophobic LA and the lengthof hydrophilic PMLA chains.

Hemolysis. Since LMC could form self-assembled nanopar-ticles, it might have surfactant activity, suggesting that mem-brane damage such as hemolysis might occur following i.v.administration. The blood compatibility of LMC nanoparticlesin vivo can be predicted by investigating the hemolysis ratio invitro. Hemolysis ratios of LMC1 nanoparticles at differentconcentrations (0.1, 0.2, and 0.3 mL) were 1.5 ( 0.6%, 2.3 (0.6%, and 4.5 ( 0.7%, respectively. In other studies, thehemolysis of N-acyl chitosan modified with butanoyl groups,hexanoyl groups, and benzoyl groups was compared with thatof unmodified chitosan. Unmodified chitosan nanoparticlesshowed a negligible hemolysis (less than 2%) while the N-acylchitosan nanoparticles revealed a relatively higher hemolysisratio, which increased with the length of alkyl groups. Suchresult suggested that hemolysis might be due to the longhydrophobic acyl groups on the surface.38 In this research, thelong LA chains were in the core and covered with hydrophilicPMLA chains, so their damage to the red blood cells (RBCs)was negligible. The hemolytic ratio was lower than 5%, whichindicated its safety in blood-contacting applications and suit-ability for i.v. administration.

Acute Toxicity. In the acute toxicity study, no signs oftoxicity were observed when mice were i.v. administered withLMC1 nanoparticles at a dose of 625 mg/kg, and all the animalssurvived during the observational period. When the dose reached1250 mg/kg, treatment-related toxic signs were observed includ-ing prostration and respiratory distress, which gradually disap-peared after 24 h. However, no animal death was observed andno macroscopic changes were detected in major organs. Themaximum tolerated dose (MTD) of LMC1 via i.v. administrationwas determined to be above 1250 mg/kg in mice. Such resultsindicated that LMC1 nanoparticles had minimal acute toxicity.

In vivo Antitumor Efficacy. With regard to the change inthe tumor volume and tumor weight, it was evident that PTX-LMC1 and PTX injection treatments effectively suppressed thetumor growth (Table 3 and Figure 10); e.g., 8 days after i.v.injection, tumor volumes of the mice treated with PTX-LMC1and PTX-injection were significantly smaller than those treatedwith saline (p < 0.05). The TIR of PTX-LMC1 (6 mg/kg), PTX-LMC1 (3 mg/kg), and PTX injection (6 mg/kg) were 47.5%,30.6% and 31.6%, respectively. PTX-LMC1 was more effectivethan PTX injection (p < 0.05) with respect to tumor suppressionat the same dose. Such results could be attributed to multiple

Table 2. Loading Capacity and Loading Efficiency of LMCNanoparticles for PTX (n ) 3)

sample loading capacity (wt %) loading efficiency (wt %)

LMC1 9.5 ( 0.3 81.2 ( 0.5LMC2 6.3 ( 0.4 84.3 ( 3.8LMC3 5.6 ( 0.3 74.0 ( 3.8LMC4 9.9 ( 0.4 89.4 ( 0.3LMC5 6.5 ( 0.4 85.7 ( 1.0LMC6 5.9 ( 0.5 81.7 ( 4.3

Figure 9. In vitro drug release profiles of PTX from LMC1, LMC2,LMC3, and LMC4 nanoparticles in pH 7.4 PBS containing 0.1%Tween 80 (w/v) at 37 °C (mean ( SD, n ) 3).

Table 3. In Vivo Antitumor Effect of PTX-LMC1 and PTX Injectionin S-180 Bearing Mice

body weight (g)a

formulationdose

(mg/kg)before

administrationafter

administrationtumor

weight (g)a,b

PTX-LMC 3 23.3 ( 1.1 26.8 ( 2.2 1.36 ( 0.28*6 23.8 ( 1.0 26.7 ( 2.7 1.03 ( 0.32***

PTX injection 6 24.1 ( 1.1 26.9 ( 2.1 1.34 ( 0.38*saline 24.0 ( 0.8 27.7 ( 1.7 1.96 ( 0.60

a Data represent mean value ( SD, n ) 10. b Statistically significantdifference from control: *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 10. (a) Tumor growth curve of the S-180 bearing micefollowing i.v. injection of distinct PTX formulations. (b) Tumor dissectedfrom mice 8 days after drug administration (mean ( SD, n ) 10).Statistically significant difference from control: *p < 0.05, **p < 0.01,and ***p < 0.001.

LMC Nanoparticles as Anticancer Drug Carriers Biomacromolecules, Vol. 10, No. 3, 2009 571

mechanisms. First, the hydrophilic PLMA on the surface ofLMC nanoparticles might decrease the uptake by the RES andprolong the circulation time of PTX-LMC1 in the blood ascompared to the rapid elimination of free PTX, which couldtherefore increase the tumor selectivity of the nanoparticles.33

Second, PTX-LMC1 could allow a slow release of the encap-sulated PTX, thus providing sustained drug effect in the targetsite. Lastly, PTX encapsulated in nanoparticles could avoid rapidenzymatic/hydrolytic degradation, which was an advantage overPTX injection.

Conclusion

Biodegradable amphiphilic graft copolymers of LMC couldbe obtained through modification of chitosan with LA andPMLA. It could form self-assembled polymeric nanoparticlesin water under physiological conditions that were 190-350 nmin size and negatively charged. DS of LA, PMLA length, andpH showed significant effect on the particle size. Additionally,zeta potentials of the LMC nanoparticles were sensitive towardpH in that it changed from positive to negative when pH wasconverted from acid to slightly basic. The CAC of LMCnanoparticles in water were 2.5 × 10-3, 3.5 × 10-3, 4.5 × 10-3

mg/mL for LMC1, LMC2, and LMC3, respectively. Loadingefficiencies and capacities of LMC nanoparticles for PTX werealso influenced by DS of LA, achieving the maximum loadingefficiency of more than 80% and maximum loading capacityof 9.9%. PTX release from LMC nanoparticles showed an initialburst release followed by a slowly sustained release phase, andthe release rate could be controlled by the LA content andPMLA length. Hemolysis ratios of LMC nanoparticles werebelow 5%, and the MTD via i.v. administration was above 1250mg/kg, which suggested its desired safety. PTX-LMC was potentin antitumor effect than PTX injection at the same dose of 6mg/kg. The results of particle size, surface charge, releaseprofiles, and antitumor efficacy showed that LMC nanoparticlesmight deliver drugs to the tumor and get topical release. Inaddition, LMC derivatives had abundant carboxyls and aminoson the surface which allowed the introduction of biologicallyactive molecules. It would be formulated for targeted deliveryto specific tissues or tumors by adjusting the particle size andsurface charge or conjugating different ligands. These propertiescould lead to development of LMC as a promising DDS forantitumor therapy.

Acknowledgment. The authors are thankful for the financialsupport from National Natural Science Foundation of China (No.30873204).

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