Polymeric micelle composed of PLA and chitosanas a drug carrier

Yan Wu & Mingjun Li & Hongxia Gao

Received: 9 January 2008 /Accepted: 31 March 2008 / Published online: 29 April 2008# Springer Science + Business Media B.V. 2008

Abstract Water soluble chitosan (CS) oligomer was hydro-phobically modified with PLA unit. The chemical structureand physical properties of CS derivatives were confirmedby FTIR, 1HNMR, TGA and X-RD. Formation andcharacteristics of polymeric micelles of graft copolymerswere studied by fluorescence spectroscopy and dynamiclight scattering (DLS) method. To estimate the feasibility asnovel drug carriers, the copolymer micelles were preparedby the phase separation-dialysis method. Rifampin wasincorporated into polymeric micelles as a lipophilic modeldrug to investigate the drug release behavior. As PLAweight ratio increased, the micelle size and drug-loadingcontent increased, and the drug release rate decreased.

Keywords CS . PLA . Polymeric micelle . Rifampin .

Drug delivery

Introduction

Amphiphilic copolymers consisting of hydrophilic andhydrophobic segments can form micelle structures withthe hydrophobic inner core and the hydrophilic outer shellin aqueous media [1–5]. Polymeric micelles have receivedspecial attention due to their potential application andacademic interest in many interdisciplinary field [6–10].These core-shell type micelles may be used as drug delivery

vehicles for poorly water-soluble drugs, especially when themicelles are made with suitable biodegradable polymers.

PLA is a kind of biodegradable materials with lowtoxicity, excellent biocompatibility and bioabsorbability invivo. It has been widely used in biomedical applications,such as sustained drug delivery systems, implants fororthopedic devices and absorbable fibers. However, thelow hydrophilicity and high crystallinity of PLA reduce itsdegradation rate, which results in poorer soft tissuecompatibility [11].

CS has a repeated structure of (1,4)-linked 2-amino-2-deoxy-β-D-glucan. CS is already known as a biocompatible,biodegradable, and low toxic material [12, 13]. CS isconsidered to be a good candidate for the drug deliverysystem [14, 15].CS is soluble in aqueous solutions of variousacids, but CS molecules have no amphiphilic property andcannot form micelles in water. Apart from its biodegradablecharacter in physiological conditions, CS has reactive amineand hydroxyl groups, which offer possibilities of modifica-tions, graft reactions and ionic interactions. Yao [16] hasreported the synthesis and characterization of an amphotericpH sensitive biodegradable chitosan-g-(L-lactic-co-citricacid) hydrogel, and in vitro fibroblast stationery cultivationon its membrane showed that the cell growth rate was muchfaster than that on chitosan membrane.

The aim of the present work was to assess the merits ofCS-PLA polymeric micelles as drug carriers. For thepurpose, the copolymer CS-PLA was synthesized with CSoligomer and DL-lactide. The chemical structure andphysical properties of graft copolymers were characterizedand the micellar formation of the graft copolymers wasinvestigated. Finally, the lipophilic drugs rifampin, asecond-generation dihydropyridine calcium antagonist withapparent selectivity for cerebral blood vessels [17], wasselected as a model drug to incorporate into the potential of

J Polym Res (2009) 16:11–18DOI 10.1007/s10965-008-9197-z

Y. Wu (*)National Center for Nanoscience and Technology,Beijing 100080, Chinae-mail: wuy@nanoctr.cn

M. Li :H. GaoJia Mu si Hospital China,Jia Mu si 154002, China

the polymeric micelles. The drug release behavior of theCS-PLA polymeric micelles was investigated.

Experimental

Materials

CS (MW=340 kDa) from Jinqiao Biochemical Co. (Hangzhou,China) with deacetylation degrees of 90%. The molecularweight was measured through the viscometric method whilethe degree of deacetylation (DD) of the chitosan wasdetermined by elemental analysis. CS oligomer (MW

6,500 Da) was prepared according the reference [18]. Themolecular weight and degree of deacetylation (DD) of thewater soluble oligomer were measured by a gel permeationchromatography (GPC) and elemental analysis, respective-ly. DL-lactide (PURAC, Holland, 99.5%) was purified twicefrom acetic ether. Dimethyl sulfoxide was distilled underreduced pressure from calcium hydride (CaH2) and storedover molecular sieves (3 Å). Rifampin was purchased fromShangdong Xinhua Pharmaceutical Co. (Shangdong, China).All other reagents were analytical grade.

Synthesis of CS-PLA graft copolymer

The polymerizations were carried out under magnetic stirringfor 12 h in dimethyl sulfoxide at 80 °C. Water soluble CSoligomer was degassed for 1 h in vacuum below 1 mmHg,and added to the reactor. A mixture of water soluble CSoligomer and DL-lactide (in the molar ratio of 6:1–40:1[DLLA/CS]) was suspended in 30 ml dimethyl sulfoxidesolution with magnetic stirring at room temperature. Themixture was kept under a vacuum below 1 mm Hg for 1 h,and then 0.5 mol triethylamine was added dropwisely via asyringe through a rubber septum. The solution was reacted at80 °C with magnetic stirring in nitrogen atmosphere. After afurther 12 h continuous stirring, the obtained polymersuspension was filtered to remove any polymer aggregation.The resulting CS-PLA precipitate obtained by pouring thesolution into ice-water was collected by filtration andthoroughly washed with distilled water, successivelyextracted with toluene in a Soxhlet’s apparatus for 48 h.Dried at 40 °C for 48 h under vacuum, a pale yellow powderwas obtained. The graft copolymers are soluble in DMSO,DMF, acetic acid and acetone.

Characterization of CS-PLA graft copolymer

Fourier-transform infrared (FTIR)

IR spectra were recorded on Fourier-transform infrared(FTIR) spectrometer (Nicolet, Magna-550). CS oligomer

and its graft copolymer were mixed with KBr and pressedto a plate for measurement.

1H NMR

1H NMRwas measured on a Bruker, DMX-500 spectrometer,1H NMR CS oligomer was dissolved in the mixed solventD2O and (CD3)2SO. The chitosan graft was dissolved in(CD3)2SO.

X-ray diffraction spectrometry

X-ray diffraction spectrometry was obtained using an XD-3A powder diffraction meter with Cu-Ka radiation in therange 5–40° (2θ) at 40 kV and 30 mA.

TGA analysis

TGA was obtained with PE, Pyrolysis-1 equipment. Thetemperature range was 30–900 °C and the heating rate is20 °C/min.

Preparation and characterization of CS-PLA graftcopolymer micelles

The formation of plain copolymer micelles was carried out bythe phase separation-dialysis method. A given amount ofcopolymer was dissolved in DMF. Then a measured amount ofcopolymer solution was added dropwisely into 10 ml distilledwater under magnetic stirring to form the micelles. The micelleswere then transferred into a dialysismembrane (cut offmolecularweight, 5,000) and were dialyzed against 3 l of deionized waterfor 48 h to remove the DMF, and the micelle was obtained. Thedialyzed solution was then analyzed or freeze-dried.

Transmission electron microscopy (TEM; Hitachi, H-600)was used to observe the morphology of the micelles. Thesample was negatively stained with 0.1% phosphotungstic acid.

The size and distribution of polymeric micelles weremeasured by DLS (Malvern Autosizer 4700). All DLSmeasurements were done with a wavelength of 532 nm at25 °C with an angle detection of 90°. The measurement foreach solution was repeated at least three times.

A solution of CS graft copolymers which containing 6×10−7 M of pyrene was placed in a square cell and thefluorescence spectrum was obtained with a fluorometer(FL-920 England). The concentrations of sample solutionwere varied from 3.02×10−5 to 1.0 mg/ml. The excitationwavelength (λex) was 336 nm.

Preparation of rifampin-loaded micelles

The rifampin-loaded polymeric micelles were prepared bythe phase separation-dialysis method as follows. 100 mg of

12 Y. Wu et al.

the CS-PLA graft copolymer and 10–30% (w/w) ofrifampin were dissolved in 6 mL of DMF, and then theorganic phase was dropped into 20 ml of water undermagnetic stirring at 25 °C to form the micelles. Themicelles were then transferred into a dialysis membrane(cut off molecular weight, 5,000) and were dialyzed against3 l of deionized water for 48 h to remove the DMF, and themicelle was obtained. Finally, the micelle suspension wasfiltered with a microfilter. The dialyzed solution was thenanalyzed or freeze-dried.

Encapsulation efficiency and drug loading capacity

The obtained micelle solution was frozen and lyophilizedby freeze dryer system to obtain a dried nanoparticleproduct. The weighted product of micelles was dissolvedand properly diluted in mixed solution of ethanol and THF(1:1, v/v). The solution was measured by UV spectropho-tometer (Perkin Elmer Lambda850 [USA]) at the wavelength of 350 nm and the weight of drug entrapped inmicelles was calculated by calibration curve. The rifampinencapsulation efficiency (AE) and the loading capacity(LC) of the nanoparticles as follows:

AE ¼ weight of rifampin inmicelles

weight of rifampin initially� 100%

LC ¼weight of rifampin inmicelles

weight of micelles� 100%

All measurements were performed in triplicate.

In vitro drug release studies of micelles

In vitro release studies, 5 ml of the rifampin-loaded CS-PLA copolymer micelle suspensions (corresponding to 10–30% [w/w] of rifampin) were placed into a dialysismembrane bag (cut off molecular weight, 5,000) and placedinto 200 ml PBS (phosphate buffered saline, 0.1 M,pH 7.4). The medium was kept at 37 °C with continuousmagnetic stirring. At selected time intervals, 4 ml ofaqueous solution was withdrawn from the release mediumand replaced with the same amount of fresh PBS. Theamount of the rifampin released from the polymericmicelles was measured with a UV spectrophotometer(Perkin Elmer Lambda850 [USA]) at 350 nm and therelease of drug was determined by a calibration curve. Allmeasurements were performed in triplicate.

Results and discussion

Synthesis and characterization of CS-PLA graft copolymers

The CS-PLA grafting copolymers were prepared byreacting of DLLA on chitosan oligomer. The synthesis ofcopolymers was carried out as shown Scheme 1.

Grafting percentage and the amount of DLLA introducedto CS oligomer increase with the molar ratio of DLLA tostructural unit of CS oligomer are shown in Table 1. Whenthe molar ratio of DLLA to CS oligomer increased from 6:1to 20:1, the grafting percentage increase from 95 to 323%;meanwhile, the molar ratio of PLA to CS oligomer in the

Table 1 Graft copolymerization of DLLA onto CS oligomer

Sample DLLA/CS molar ratio Total yield (%) Grafting percentage(%)a FDLLA/FCS oligomerb

1 6:1 35.7 95 2.182 8:1 36.3 134 3.073 11:1 42.2 198 4.544 13:1 42.7 237 5.445 15:1 45.5 272 6.246 20:1 46:1 323 7.41

a Grafting percentage %ð Þ¼ Mass of graft copolymer ðgÞ�Mass of CS oligomer ðgÞMass of CS oligomer ðgÞ � 100%

bMolar ratio in graft copolymer ¼ grafting percentage� 165:2=72

Scheme 1 Graft copolymeriza-tion of DLLA onto CSoligomer

Polymeric micelle composed of PLA and chitosan as a drug carrier 13

copolymer also rose from 2.18 to 7.41. This indicates thatthe higher the concentration of the DLLA, the higher theopportunity for the DLLA to react with CS oligomerreactive centers. Grafting percentage and molar ratio ofPLA to CS oligomer in the copolymer could approach198% and 4.54, respectively, when the molar ratio ofDLLA/aminoglucoside units is 11:1.

Compared to the IR spectrum of CS oligomer (Fig. 1),the copolymers have a new absorption peak appearingaround 1,746 cm−1, corresponding to the carbonyl group ofthe branched PLA. The methyl asymmetric deformation ofPLA appears at ~1,454 cm−1. The ~1,197 and ~1,256 cm−1

doublets observed in the copolymer are assigned to thesymmetric C–O–C stretching modes of the ester group.There are two other peaks at ~1,131 and ~1,046 cm−1

attributed to the methyl rocking and C–CH3 stretchingvibration, respectively. The increase of the amide I peak(1,668 cm−1) indicated increase of the amidation byreacting CS oligomer with DLLA. That demonstrates theformation of amide group between CS oligomer andDLLA. This evidence suggests that the DLLA can indeedreact with CS oligomer with triethylamine as catalyst.Increasing of the feed ratio of DLLA to CS oligomer madethe absorption at ~1,197 cm−1 rise, which means that themore DLLA had been grafted to CS oligomer.

The 1H-NMR spectra of the CS oligomer and graftedcopolymer of 8:1 are copmpared in Fig. 2. CS oligomershows a singlet at 3.13 (H-2) and mutiplets at 3.3–3.6 ppm(H-3, H-4, H-5, H-6) and a small singlet at 4.4 ppm (H-1)corresponding to the ring methenyl protons. The singlet at1.9 ppm is due to the survival of the N-acetylglucosamineunits of chitin [19]. Compared with CS oligomer, the 1H-NMR spectra of the graft copolymer was shown that the

signals at 4.2 and 5.1 ppm were assigned to the terminalmethenyl protons of the branched PLA and repeat units of itin the chain, respectively. The signals at 1.3 and 1.4 ppmwere attributed to the methyl protons of the PLA moietylocated at the terminal groups and the backbones [20, 21].All these results evidenced that the CS oligomer derivativescontained PLA side chains.

Physical–chemical properties of CS derivatives

Thermal properties

TGA curves for CS oligomer and grafted copolymer (11:1)are shown in Fig. 3. Compared to CS oligomer, graftcopolymer has lower thermal degradation temperatures. Afast process of weight loss appears in the TGA curvesresponse for the graft copolymer in thermal degradationranges. These results show some decrease of the thermalstability for CS-PLA graft copolymer relative to the originalCS oligomer. Introduction of substituents into polysaccharidestructures should disrupt the crystalline structure of CSoligomer, especially by the loss of the hydrogen bonding.

X-ray

X-ray diffraction profiles of CS oligomer and its graftcopolymer are show in Fig. 4. CS oligomer has tworeflection fall at 2θ=10°, 2θ=20°. The reflection fall at 2θ=10o was assigned to crystal forms I. The strongest reflectionappears at 2θ=20° which correspond to crystal forms II[22]. Compared with CS oligomer, the grafting decreasesthe intensity at both peaks. When the feed ratio reachesDLLA /CS oligomer=11:1, the graft copolymer shows onlyone broad peak at around 2θ=12°. It suggested that theability of forming hydrogen bond of CS oligomer wasdecreased after grafting. When lactide was grafted onto CSoligomer, the original crystallinity of CS oligomer wasdestroyed.

Critical micelle concentration

It is well known that amphiphilic copolymers with asuitable hydrophilic/hydrophobic balance can form amicellar structure when exposed to a selective solvent.The CS-PLA graft copolymers, consisting of hydrophilicCS oligomer and hydrophobic PLA segments, provided anopportunity to form micelles in water. The micelle behaviorof CS-PLA graft copolymer in aqueous media wasmonitored by fluorometry in the presence of pyrene as afluorescence probe. In studying the formation of micellesfrom hydrophobically modified graft coplymer in aqueoussolution, pyrene is generally used as a molecular probe, andthe variation in the ratio of intensity of first (372 nm) to

Fig. 1 IR spectra of CS oligomer and graft copolymers: (A) CSoligomer and (B) DLLA/CS oligomer=8:1, (C) DLLA/CS oligomer=11:1 (D) DLLA/CS oligomer=15:1

14 Y. Wu et al.

Fig. 3 TGA of (a) CS oligomer and (b) CS-PLA graft copolymer(DLLA/CS oligomer=11:1)

Fig. 4 X-ray diffraction profiles of CS oligomer and its graftcopolymer: (A) CS oligomer (B) DLLA/CS oligomer=8:1, (C)DLLA/CS oligomer=11:1

Fig. 2 1H-NMR spectrum ofa CS oligomer and b graftcopolymer (DLLA/CSoligomer=8:1)

Polymeric micelle composed of PLA and chitosan as a drug carrier 15

third (383 nm) vibronic peaks I372/I383, the so-calledpolarity parameter, is quite sensitive to the polarity ofmicroenvironment where pyrene is located. Thus, thechange of I372/I383, can characterize the formation ofmicelle. The change of the intensity ratio (I372/I383) isshown in Fig. 5. For CS graft copolymer, at lowerconcentrations, I372/I383 values remain nearly unchanged.Further increasing concentration, the intensity ratio start todecrease, implying the onset of micelle from grafted CSoligomer. The critical micelle concentration (cmc) isdetermined by the interception of two straight lines. Thecmc values of graft copolymers are listed in Table 2. Fromthe table, it can be seen that the cmc values of polymericamphiphiles are lower than the critical micelle concentra-tion (cmc) of low molecular weight surfactants [12],indicating the stability of micelles from graft copolymersat dilute conditions. The increasing hydrophobicity byintroduction of a large amount of hydrophobic groupsfurther reduces the cmc values (Table 2).

Morphology

Figure 6 shows the TEM image of polymeric micelles. Itcould be confirmed that polymeric micelles are spherical inshape. The size of these micelles is smaller than thatdetermined by DLS in water, presumably arising from thedry state of the TEM measurement.

Size distribution of polymeric micelles

The size and its size distribution of polymeric micelles andrifampin-loaded polymeric micelles (NP 11, NP 15, NP 20)were measured by DLS (Table 2). The size of these plainpolymeric micelles is 150–180 nm in water. The DLS datademonstrate that the micelle sizes get larger as the DLLA/CS oligomer molar ratio increase, suggesting the elongationof hydrophobic PLA side chain facilitates the growth of thehydrophobic core of polymeric micelles. It can be seenfrom Table 2 that these micelles possess a narrow unimodaldistribution (polydispersity). These results indicated that the

Fig. 5 Change of intensity ratio (I372/I383) versus the concentration ofCS-PLA graft copolymers: (○) DLLA /CS oligomer=11:1; (△)DLLA /CS oligomer=15:1; (▽) DLLA/CS oligomer=20:1

Table 2 Effect of DLLA/CS oligomer molar ratio on the properties of polymeric micelles and rifampin-loaded polymeric micelles

Sample DLLA/CS oligomer Mean diameter (nm) Polydispersity cmc×102 (mg/ml)

3 11:1 154 0.03 6.495 15:1 178 0.07 2.256 20:1 181 0.06 1.01NP11 11:1 163 0.07NP15 15:1 182 0.06NP20 20:1 210 0.06

a Rifampin content 10% (w/w)

Fig. 6 Transmission electron microscopy photographs of CS-PLAcopolymer micelles (DLLA/CS oligomer=15:1)

16 Y. Wu et al.

micelle size was dependent on the ratio of hydrophobicPLA segment to hydrophilic CS oligomer segment in thechain.

The size and size distribution of these micelles were alsomeasured after storing for 1 month. It was found that thesize and size distribution of the micelles did not change,which indicated that these micelles were fairly stable atroom temperature.

The characteristics of rifampin-loaded polymeric micellesare also shown in Table 2. The rifampin-loaded polymericmicelles showed a larger size than the plain polymericmicelles. It suggested that rifampin was incorporated into thepolymeric micelles effectively.

Drug loading into polymeric micelles

Table 3 demonstrates that the entrapment efficiency anddrug loading of CS-PLA micelles. The entrapment efficien-cy and drug loading depended mainly on the copolymercomposition ratio of PLA to CS oligomer. The drug-loadingcontent in micelles increased from 6.8% to 10.4% withincreasing the molar ratio of DLLA /CS oligomer from 11to 20. This result could be explained by the rifampin havinga hydrophobic character. Therefore, the higher the PLAcontent in copolymer, the more easily the drug wasentrapped in micelles. It suggested that the elongation ofhydrophobic PLA side chain facilitates the compatible ofthe hydrophobic core of polymeric micelles and hydrophobicdrug rifampin.

In vitro release behavior of drug-loaded micelles

Figure 7 shows release profiles of rifampin from CS-PLAmicelles with various DLLA/CS oligomer molar ratios. Theresults were illustrated by plotting the relative releasepercentages of rifampin based on loading amount versustime. For all polymeric micelles, rifampin release bothshowed an initial burst release and after rifampin releaseprofiles displayed a sustained fashion. An initial burstrelease, a significant amount of rifampin was release within10 h, 35.6% for CS-PLA graft copolymer micelles. Afterthe initial burst, rifampin release profiles displayed asustained release. The amount of cumulated rifampinrelease over 5 days was 85.1% for CS-PLA polymeric

micelles (DLLA/CS oligomer 11:1, rifampin content 20%).This sustained release could result from diffusion ofrifampin into the polymer wall and the drug throughpolymer wall as well as the erosion of the polymers.

The release of a drug from the polymer micelles is rathercomplicated process. It can be affected by many factorssuch as the polymer degradation, molecular weight,crystallinity, the binding affinity between the polymer andthe drug, and so on [23]. In the study, the drug release ratemight be mainly determined by the diffusion of the drugthrough the polymer matrix. The initial burst might beattributed to the rapid release of drugs in the microchannelsprobably existing in micelles [24, 25]. Rifampin, because ofits lipophilic character, was physically entrapped in thehydrophobic core of a micelle. Accordingly, the in vitrorelease behaviors of a lipophilic compound from thesepolymeric micellar systems are largely affected by its innercore with hydrophobic properties [23]. Therefore, as thePLA content of a copolymer increased, the hydrophobicsegments in a copolymer increased, resulting in the increaseof the binding affinity between rifampin and PLA.Consequently, in this micelle system, the drug release ratewas inversely proportional to the PLA content of acopolymer. Another reason for the fast release rate of CS-PLA (DLLA/CS oligomer=11:1) was their small particlesize with relatively high surface area.

Table 3 Drug loading efficiency, drug entrapment efficiency and micelle yield of rifampin-loaded copolymeric micellesa

Sample DLLA/CS oligomer Entrapment efficiency (%) Drug loading (%) Micelle yield (%)

3 11:1 57.1 6.8 54.55 15:1 62.7 8.1 68.76 20:1 71.3 10.4 84.2

a The mass of rifampin used was 20% (w/w) in relation to polymer mass.

Fig. 7 Release profiles of rifampin from CS-PLA polymeric micelles

Polymeric micelle composed of PLA and chitosan as a drug carrier 17

Conclusion

Amphiphilic CS-PLA graft copolymers which can form thepolymeric micelles were prepared. The graft copolymerswith controlled structure were obtained by adjusting themolar ratio of the DLLA to CS glucose unit. The rifampin-loaded polymeric micelles were prepared by the phaseseparation-dialysis method. The preliminary investigationsfor the novel micelle system have shown that thecomposition of the copolymer makes a large influence onthe micelle size and size distribution, and drug releasebehavior. Control of the micelle size, drug-loading content,and drug release behavior can be achieved by optimizingthe DLLA/CS oligomer ratio of the copolymer. Theseresults showed that CS-PLA polymeric micelles could bepromising as a new drug delivery system for lipophilicdrugs.

Acknowledgements This work was supported by the NationalNatural Science Foundation of China (Foundation no.: 90406024)and the National High Technology Research and DevelopmentProgram of China (863) (no.: 2006AA03Z321)

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