characterization of novel multifunctional cationic polymeric liposomes formed from octadecyl...

Characterization of Novel Multifunctional Cationic PolymericLiposomes Formed from Octadecyl Quaternized Carboxymethyl

Chitosan/Cholesterol and Drug Encapsulation

Xiao F. Liang,† Han J. Wang,† Hao Luo,† Hui Tian,† Bing B. Zhang,† Li J. Hao,†

Jon I. Teng,‡ and Jin Chang*,†

School of Materials Science and Engineering, Tianjin UniVersity, Tianjin 300072, PR China, andUniVersity of Texas Medical Branch, GalVeston, Texas 77551

ReceiVed December 2, 2007. ReVised Manuscript ReceiVed May 4, 2008

The design and construction of effective delivery vectors for drugs is very important. We have discovered thatoctadecyl quaternized carboxymethyl chitosan (OQCMC) in combination with cholesterol (Chol) could form stablevesicles with structure similar to that of conventional liposomes prepared from phosphatidylcholine/cholesterol (PC/Chol). Compared to conventional liposomes, our polymeric liposomes formed by OQCMC/Chol have many excellentfeatures, such as good physical and thermal stability, excellent solubility in water, and high effectiveness in drugencapsulation. Trans-activating transcriptional activator protein (TAT peptide) could be connected on the surface ofcationic polymeric liposomes by using cross-linking reagent N-hydroxysuccinimidyl-3-(2-pyridyldithio) propionate(SPDP). Also, oil-soluble magnetic nanoparticles were used to verify the bilayer structure of the polymeric liposomesand their ability to solublize hydrophobic materials. Using different preparation methods, OQCMC/Chol could easilybe made into nanoscale particles by encapsulating both hydrophilic and hydrophobic components. We have successfullyprepared polymeric liposomes encapsulating quantum dots (QDs), superparamagnetic nanoparticles, or both. Vincristinewas also encapsulated in the polymeric liposomes with high drug encapsulation efficiency (90.1%). Vincristine-loadedmagnetic polymeric liposomes were stable in aqueous solution and exhibited slow, steady release action over 2 weeksunder physiologic pH (7.4). This allows the use of multifunctional cationic polymeric liposomes, such as thosedeveloped here from modified chitosan, in various applications such as cancer diagnosis and treatment.

Introduction

Nowadays, liposomes and nanoparticles are regarded asbeneficial carrier systems for drugs because of their biocompatibleand biodegradable properties.1 For example, they have been usedto encapsulate colchicines,2 estradiol, tretinoin,3 dithranol,4,5 andenoxacin for applications such as anticancer, antitubercular,antileishmanial, and anti-inflammatory treatments and fordelivering hormonal drugs and oral vaccines.6–10 Furthermore,cationic lipids and liposomes still attract the attention of manygene therapy laboratories because of their excellent gene-transferefficiency.11

However, liposomes also have some limitations. First, theygenerally show a short circulation half-life after intravenous

administration.12 Second, they are prone to adhere to each otherand fuse to form larger vesicles in suspension, which may resultin inclusion leakage.13 Therefore, stability is a general problemwith lipid vesicles.14,15 Third, conventional phosphatidylcholine/cholesterol (PC/Chol) liposomes do not have certain chemicalgroups, such as amine and carboxylic acid, so its conjugationwith protein receptors is difficult. For example, trans-activatingtranscriptional activator (TAT) peptide must be attached to thesurface of PEGylated liposomes via p-nitrophenylcarbonyl-PEG-phosphatidyl ethanolamine (pNP-PEG3000-PE).16

Many attempts, such as the surface modification of liposomes,17

have been investigated to improve the properties of theseliposomes. The surface modification of liposomes with severalbiological materials including proteins, polysaccharides, gly-colipids, and water-soluble polyethylene glycol (PEG) was foundto improve the circulation time of liposomes injected by decreasingthe uptake of liposomes in the reticuloendothelial system(RES).12,15,18 For example, the incorporation of a lipid conjugateof PEG results in a polymeric layer around the liposome, whichreduces the adhesion of plasma proteins that would otherwisecause rapid recognition of the liposomes by mononuclear

* To whom correspondence should be addressed. Tel:+86-022-27401821.Fax: +86-022-27401821. E-mail: [email protected].

† Tianjin University.‡ University of Texas Medical Branch.(1) Takeuchi, H.; Kojima, H.; Yamamoto, H.; Kawashima, Y. Biol. Pharm.

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Ind. Pharm. 1993, 19, 1331–1342.(7) Parthasarathi, G.; Udupa, N.; Umadevi, P.; Pillai, G. K. J. Drug Targeting

1994, 2, 173–182.(8) Uchegbu, I. F.; Double, J. A.; Turton, J. A.; Florence, A. T. Pharm. Res.

1995, 12, 1019–1024.(9) Williams, D. M.; Carter, K. C.; Baillie, A. J. J. Drug Targeting 1995, 3,

1–7.(10) Ruckmani, K.; Jayakar, B.; Ghosal, S. K. Drug DeV. Ind. Pharm. 2000,

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1989, 50, 1–6.(15) Graff, A.; Winterhalter, M.; Meier, W. Langmuir 2001, 17, 919–923.(16) Torchilin, V. P.; Levchenko, T. S. Curr. Protein Pept. Sci. 2003, 4, 133–

140.(17) Oku, N.; Namba, Y. Crit. ReV. Ther. Drug Carrier Syst. 1994, 11, 231–

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7147Langmuir 2008, 24, 7147-7153

10.1021/la703775a CCC: $40.75 2008 American Chemical SocietyPublished on Web 06/20/2008

phagocytes system (MPS) phagocytes.19 Several investigatorsalso have exploited the high affinity of chitosan for cell membranesby using chitosan derivatives as coating materials for liposomesand have reported promising results.20,21

The present study investigates the feasibility of novel cationicpolymeric liposomes based on amphiphilic multifunctionaloctadecyl quaternized carboxymethyl chitosan (OQCMC) andcholesterol (OQCMC/Chol). The possible formation process andthe chemical structure of OQCMC are shown in Figure 1. TheOQCMC/Chol system is quite similar to the polymer-surfactantcomplexes of structures described in the literature by Kabanovet al.22 OQCMC is a new kind of chitosan derivative. Thederivative has good solubility both in water and organic solvents.Here we hypothesize that polymeric liposomes formed fromOQCMC/Chol may resolve most of the above problems. First,the physical and chemistry stability of the liposome can beimproved by introducing carboxymethyl chitosan with a highmolecular weight. Second, OQCMC has an amino group, acarboxymethyl salt group, and an octadecyl quaternized groupin the same complex molecule, so targeting materials can beconnected and surface modification becomes possible. Third,the OQCMC has perfectly high crystallinity compared with thatof chitosan in other derivatives.23 Furthermore, OQCMC is verycheap and can easily be made into nanoparticles.

Materials and MethodsMaterials. Chitosan was supplied by Yuhuan Aoxing Biochem-

istry Co. Ltd. (Zhejiang, China) with a deacetylation degree of>99% and a molecular weight (MW) of 5 × 104. Glycidyl octadecyldimethylammonium chloride (QA), carboxymethyl chitosan(CMC),23 oil-soluble magnetic nanoparticles (OM),24 hydrophilicmagnetic nanoparticles (HM),25 and CdSe/CdS core-shell quantumdots (QDs)26 were all prepared in our laboratory. All other chemicalswere reagent grade and were used as received.

Preparation of OQCMC. The quaternization of CMC wasconducted as follows. CMC (5 g) was dissolved in 100 mL of a

mixture of deionized water saturated with isopropanol. QA wasadded slowly with different molar ratios to the glucosamine unit.The mixture was trickled with an aqueous NaOH solution (42%,w/w) and reacted at 80 °C for 24 h with stirring before being dialyzedfor 4 days against water and finally lyophilized to give OQCMC asa white power.23

Polymeric Liposome Preparation. Thin-Layer EVaporation(TLE) Method. OQCMC and cholesterol (weight ratio 1/0.81, totallipids 30 mg) were dissolved in 4 mL of chloroform at roomtemperature. To entrap the oil-soluble magnetic nanoparticles (OM),8 mg of OM was also dissolved in the solution. Chloroform was thenevaporated with a vacuum rotary evaporator, and a thin film ofpolymeric liposomes was formed on the wall of a 50 mL round-bottomed flask. Then the lipid film was dispersed in deionized waterand sonicated in a bath sonication unit at 30 °C for 10 min. Theliposome suspensions were kept at 5 °C until further characterizationwas done.27

ReVerse-Phase EVaporation (REV) Method. This method allowsus to obtain large unilamellar, oligolamellar, and multilamellarvesicles.28,29 OQCMC with different grafting percentages of thequaternary group and cholesterol were dissolved in 4 mL ofchloroform at room temperature. Five milliliters of deionized waterwas mixed with this organic solvent. The weight ratio of OQCMCto cholesterol changed from 1/0 to 1/1.96 (total weight 30 mg).Oil-soluble substances and water-soluble substances (vincristine)can be added to the organic phase and aqueous phase before mixing,respectively. The mixture was sonicated for 10 min using a bath-type sonicator. Then, the solvents were evaporated on a rotaryevaporator to form a gel-like, highly concentrated polymericliposomes suspension that can be diluted with a suitable aqueousbuffer solution. The liposome suspension was kept under vacuumfor at least 3 h to remove trace amounts of the organic solvent.

To remove the largest particles and obtain a more homogeneouspolymeric liposome population, the liposome suspension can beextruded through cellulose acetate membrances of 0.45 µm poresize in a Millipore filtration cell.

Polymeric liposomes encapsulating OM were obtained by addingOM to organic solvent by the TLE method. Magnetic polymericliposomes encapsulating vincristine were obtained by adding HMand vincristine to the aqueous phase by the REV method. Polymericliposomes encapsulating QDs were obtained by adding QDs to anorganic solvent by the REV method. QD-tagged magnetic polymericliposomes were obtained by adding QDs and HM to the organicphase and the aqueous phase, respectively.

Physicochemical Characterizations of Cationic PolymericLiposomes X-ray diffraction patterns of different sample fractionswere measured with a Rigaku D/max 2500v/pc with a Cu °C sourceoperating at 40 kV and 50 mA at 20 °C. The relative intensity was

(19) Josbert, M. M.; Peter, B.; Leo, W. T. D. B.; Tom, D. V.; Cor, S.; Christien,O.; Marca, H. M. W.; Daan, J. A. C.; Gert, S.; Wim, E. H. Bioconjugate Chem.2003, 14, 1156–1164.

(20) Otake, K.; Shimomura, T.; Goto, T.; Imura, T.; Furuya, T.; Yoda, S.;Takebayashi, Y.; Sakai, H.; Abe, M. Langmuir 2006, 22, 4054–4059.

(21) Guo, J.; Ping, Q.; Jiang, G.; Huang, L.; Tonga, Y. Int. J. Pharm. 2003,260, 167–173.

(22) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A.J. Am. Chem. Soc. 1998, 120, 9941–9942.

(23) Liang, X. F.; Wang, H. J.; Tian, H.; Luo, H.; Chang, J. Acta Phys. Chim.Sin. 2008, 24, 223–229.

(24) Sun, S. H.; Zeng, H. J. Am. Chem. Soc. 2002, 124, 8204–8205.(25) Mornet, S.; Portier, J.; Duguet, E. J. Magn. Magn. Mater. 2005, 293,

127–134.(26) Peng, Z. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 183–184.

(27) Evonne, M. R.; David, R. K.; Janelle, L. F.; Mare, C.; Diane, B. L.; Gregg,B. F. J. Am. Chem. Soc. 2007, 129, 4961–4972.

(28) Souza, E. F. D.; Teschke, O. ReV. AdV. Mater. Sci. 2003, 5, 34–40.(29) Magin, R. L.; Meisman, M. R. Chem. Phys. Lipids 1984, 34, 245–256.

Figure 1. Chemical structures of OQCMC and possible formation of cationic polymeric liposomes from OQCMC and cholesterol when hydratedin aqueous solution (i.e., via the film dispersion method).

7148 Langmuir, Vol. 24, No. 14, 2008 Liang et al.

recorded in the scattering range (2θ) of 3-40°. Samples of polymericliposomes (OQCMC/Chol) were prepared by a lyophilized lipidsuspension to obtain a freeze-dried mixture. The physical mixtureof OQCMC/Chol was prepared as follows: OQCMC/Chol (weightratio 1/0.81) was mixed with chloroform, and then the chloroformwas evaporated to obtain the dried mixture directly. The thermalproperties and the phase-transition temperature of polymericliposomes were characterized with a differential scanning calorimeter(Diamond DSC, Perkin-Elmer instrument). Each dried sample wasweighed in an aluminum pan and heated from 0 to 120 °C at ascanning rate of 10 °C/min. The morphologies of different cationicpolymeric liposomes were observed via transmission electronmicroscopy (TEM). TEM observation of the liposomes was carriedout at an operating voltage of 200 kV with a JEOL-100CXII (Japan)in bright-field mode and by electron diffraction. Dilute suspensionsof polymeric liposomes in water were dropped onto a carbon-coatedcopper grid by negatively staining with 2% phosphotungstic acidand then air dried. Samples of cationic polymeric liposomesencapsulating magnetic nanoparticles were prepared similarly withoutstaining. The average particle size and size distribution weredetermined by quasielastic laser light scattering with a MalvernZetasizer (Malvern Instruments Ltd., U.K.) at 25 °C. About 0.2 mLof each polymeric liposomes suspension was diluted with 2.5 mLof water immediately after preparation. Each experiment was repeatedthree times. The zeta potential was measured by using a ZetasizerS (Malvern, U.K.). Zeta limits ranged from-150 to 150 V. Strobingparameters were set as follows: strobe delay -1.00, on time 200.00ms, and off time 1.00 ms.

Vincristine Release from the Nanoparticles in Vitro Unen-capsulated vincristine was removed from the magnetic polymericliposomes by magnetic separation or dialysis. The amount ofvincristine was determined by UV spectrophotometry at 298 nm.About 2 mg of dried magnetic polymeric liposomes was dissolvedin 5 mL of chloroform to destroy the liposome structure, and then3 mL of deionized water was added to the chloroform to extract thereleasing drug by the extraction method. This process was repeatedfive times. The vincristine encapsulation efficiency (EE) andvincristine loading efficiency (LE) of the process were calculatedfrom

EE) A-BA

× 100 (1)

LE) CD

× 100 (2)

where A is the total amount of vincristine, B is the amount ofunencapsulated vincristine, C is the weight of vincristine in thevesicle, and D is the weight of the vesicle.

The in vitro release profiles of vincristine from polymericliposomes were determined as follows: about 2 mL of the vincristine-loaded polymeric liposomes suspension was placed in a dialysis bagwith 10 mL of Tris-HCl (pH 7.4) buffer solutions in test tubes andincubated at 37( 0.5 °C with stirring. At appropriate time intervals,buffer solutions outside the dialysis bag were taken out of the testtube to determine the amount of vincristine released from the vesiclesby UV, and 10 mL of fresh medium was added. All release testswere run in triplicate, and the mean values were reported.

Results and Discussion

Formation Process of Polymeric Liposomes In our work,a series of OQCMC were successfully prepared. As a newamphiphilic derivative of chitosan with high molar mass (>1 ×104), OQCMC exhibited excellent solubility both in water andorganic solvents such as chloroform and toluene. OQCMC alsohad high crystallinity compared with chitosan and other deriva-tives. The chemical structure of OQCMC is shown in Figure 1.OQCMC has amine groups, carboxymethyl salt groups, octadecylquaternized groups, and hydroxyl groups.23 Its molecular structurewas similar to that of PC to some extent, but compared with PC,

it exhibits excellent solubility in water. The hydrophilic propertiesand lipophilic properties within the molecule are balanced verywell, and various functions associated with biomembranes inliposomes, such as aggregation, fusion, and selective permeability,are all dependent on this balance.

The process of vesicle preparation from OQCMC andcholesterol by the film dispersion method is shown in Figure 1.After the evaporation of chloroform, the OQCMC membranecould be formed spontaneously. The transformation of thepolymeric vesicle membrane structure may be similar to that ofthe phospholipids membrane. The size of the vesicles remainedstable for >60 days and was almost the same as that after 1 hwhen the system temperature increased to 50 °C and thendecreased to the original temperature. While the system tem-perature increases, the thermal motion and the layer interspacingalso increased. Because most of the observed liposomes wereaggregated vesicles, this aggregated polymeric structure had asubstantially larger stability than the single-vesicle structure andconsequently a larger resistance in maintaining its shape andfunction as a carrier of cosmetics, food additives, and drugs.This observation also had some important consequences in theliposomes’ selective permeability when they were used ascarriers.30

Structural Characterization of Polymeric Liposomes Theeffects of different preparation methods on the formation ofpolymeric liposomes are discussed. The size and shape of thepolymeric vesicles can be directly observed by TEM. Figure 2shows the TEM images of the polymeric nanoparticles preparedby thin-layer evaporation (TLE) and reverse-phase evaporation(REV), respectively, at a weight ratio of 1/0.81(OQCMC/Chol).The vesicles were different sizes. They were, in fact, differenttypes, including multilamellar vesicles (MLV), large unilamellarvesicles (LUVs), and small unilamellar vesicles (SUVs). Fromthe average results of 50 vesicles and under the same experimentalconditions, the sizes of the vesicles made from the TLE methodwere slightly larger than that from the REV method (Figure 2).Also, this result could be seen from the particle size analyzer.Under the same condition, the mean particle size of polymericliposomes by the TLE method is 172.5(2.1 nm, which comparedwith 108.5 ( 0.5 nm by the REV method.

The TLE and REV methods both allow for the formation ofmultilamellar vesicles. Basically, a mixture of OQCMC and lipidcompounds can be dissolved in an organic solvent (chloroform)or a mixture of two organic solvents (chloroform-methanol).Other hydrophobic components (e.g., drugs) could be cosolu-bilized with the liposome-forming materials. This polymeric lipidfilm was then hydrated with an aqueous solution buffered to the

(30) Teschke, O. Langmuir 2002, 18, 6513–6520.(31) Bouwstra, J. A.; Van Hal, D. A.; Hofland, H. E. J.; Junginger, H. E.

Colloids Surf., A. 1997, 123-124, 71–80.

Figure 2. Transmission electron micrograph (TEM) images of cationicpolymeric liposomes (a) Prepared by the thin-layer evaporation method(TLE) and (b) prepared by reverse-phase evaporation (REV).

Characterization of Cationic Polymeric Liposomes Langmuir, Vol. 24, No. 14, 2008 7149

desired pH value and any hydrophilic component that should beentrapped within vesicles (e.g., water-soluble drugs) was solu-bilized. Also, hydrophobic components could be cosolubilizedin organic solvent, and the hydrophilic component could bedissolved in an aqueous solution. This method represents a goodapproach to increasing the amount of drug entrapped withinvesicles.

The effects of the weight ratios of OQCMC/Chol and thedegree of substitution (DS) of the quaternary group in car-boxymethyl chitosan on the formation of polymeric liposomesare also discussed. Cholesterol (C27H45OH) is a cell membraneconstituent. It modulates membrane fluidity, elasticity, andpermeability by closing the gaps created by imperfect packingof other lipid species when proteins are embedded in themembrane. From an analysis of particle size, the size of thevesicles made from OQCMC only was larger than that frommixed samples of OQCMC and cholesterol with a weight ratiobelow 1:0.81 (Figure 3a). Furthermore, cholesterol can enablethe formation of vesicles and reduced aggregation and providedgreater stability.31 With the increase in cholesterol content fromzero to 0.81, the mean sizes of polymeric liposomes decreased,but when the cholesterol content was more than the content ofOQCMC, the mean size of the polymeric liposomes increaseddrastically.

From Figure 3b, the mean size of the polymeric liposomesfirst decreased and then increased with the DS of the quaternarygroup increasing. However, they are all very stable, and thesmallest mean size is about 60.4 ( 0.2 nm from particle sizeanalysis when the DS of the quaternary group is about 73.2%.The zeta potential of cationic polymeric liposomes increasedfrom +26.32 to +42.17 mV as the DS of the quaternary groupincreased. This finding suggests that cationic polymeric liposomeswith different charges can be prepared by controlling the DS ofthe quaternary group.

Figure 4 shows the particle size distributions based on theintensity of polymeric liposomes prepared by the REV method.The mean particle size of polymeric liposomes was about 74.1( 0.1 nm. The polymeric liposomes size measured by parti-cle size analyzer was bigger than those visualized by TEM. Thepolydispersity index is used to describe the spread in particlediameters produced in a sample of particles. In particle sizeanalysis system, the normalized variable is usually referred toas the polydispersity index. As the index approaches zero, thesize range produced becomes narrower. And the polydispersityindex of the polymeric liposomes was 0.224.

Stability of Polymeric Liposomes. X-ray diffractograms ofOQCMC, polymeric liposomes, and the OQCMC/Chol physical

mixture are shown in Figure 5. It could be seen that there weresome differences in peak height, width, and position among them.Compared with chitosan, which showed a relatively broad peakat 2θ ) 20°,23 OQCMC had a narrow peak at 2θ ) 21.4° anda new narrow peak at 2θ ) 5° that are indicative of a bilayeredlamellar structure.32 Sharp, strong peaks of OQCMC confirmed

(32) Grant, J.; Tomba, J. P.; Lee, H.; Allen, C. J. Appl. Polym. Sci. 2007, 103,3453–3460.

Figure 3. (a) Effect of the weight ratio of OQCMC to cholesterol on the mean size of nanoparticles. (b) Effect of DS of the quaternary group onthe mean size and zeta potential of nanoparticles.

Figure 4. Particle size distribution based intensity of cationic polymericliposomes prepared by OQCMC and cholesterol. (The molar ratio ofOQCMC/Chol is 1:0.81, and the DS of OQCMC is 73.2%.)

Figure 5. XRD patterns of (1) OQCMC, (2) polymeric liposomes, and(3) the OQCMC/Chol physical mixture. (The weight ratio of OQCMC/Chol is 1:0.81, and the DS of OQCMC is 73.2%.)


that the polymeric lipid is a highly crystalline material. However,the narrow peak at 2θ ) 21.4° disappeared when OQCMC andcholesterol were blended together in chloroform. Also, polymericliposomes and the OQCMC/Chol physical mixture both had arelatively broad peak at 2θ ) 18°. The absence or change inseveral peaks in the diffraction pattern for the blends is anindication that interactions between OQCMC and cholesterolare operative. In addition, polymeric liposomes of OQCMC/Chol after hydration with a relatively narrow peak at 2θ ) 21.4°should be better organized into lamellar-like structures thancomponents of the OQCMC/Chol physical mixture.

The differential scanning calorimetry (DSC) traces forcholesterol, QA, OQCMC, OQCMC/Chol physical mixtures,and polymeric liposomes are shown in Figure 6. The phase-transition temperature was defined as the temperature requiredto induce a change in the lipid physical state from the orderedgel phase (hydrocarbon chains are fully extended and closelypacked) to the disordered liquid-crystalline phase (hydrocarbonchains are randomly oriented and fluidic). Compared withcholesterol, the DSC traces for QA and OQCMC exhibited a

single narrow peak characteristic of the main gel to liquid-crystalline phase-transition (Tm) temperature. The positions ofthese peaks, at Tm(1) ) 40.14 °C, Tm(2) ) 46.98 °C, and Tm(3) )52.55 °C, representing the melting temperatures of cholesterol,QA, and OQCMC, increased slightly. As the molecular weightincreased from QA to OQCMC, molecular interactions becamestronger, requiring more energy to disrupt the ordered packing;thus the phase-transition temperature increased.

Note that there was no peak for OQCMC/Chol (1:0.81 (wt/wt)) physical mixtures and polymeric liposomes. It showed thatthe blending of OQCMC with cholesterol altered the gel to liquid-crystalline transition of OQCMC. This huge reduction in enthalpyto zero is likely caused by the strong interactions betweenamphiphilic OQCMC and cholesterol.33 As with conventionalliposomes, the addition of cholesterol to OQCMC membranesalso acts to broaden the transition of the lipid with the enthalpyof the transition reaching zero. These curves clearly indicatedthat at temperature of <115 °C the main transition peak did notcome up, owing to the formation of the stable lipid bilayer inthe gel state of the chains. Interestingly, strong interaction andrearranging of the cholesterol and OQCMC molecules had takenplace when they were mixed in chloroform. Thus, cationicpolymeric liposomes formed from OQCMC had good thermalstabilization with changing temperature. The DSC study furthersupported the results obtained by XRD.

Physical Structure of Polymeric Liposomes and TheirApplications To compare OQCMC/Chol mixtures with con-ventional PC/Chol liposomes, oil-soluble magnetic nanoparticles(OM) were selected as kind of hydrophobic component and werecosolubilized in the bilayer of polymeric liposomes. In this work,the dehydration-rehydration method was used. After the thinfilm containing OM was prepared, the aqueous phase wasintroduced. Figure 7a-c shows TEM images of magnetic cationicpolymeric liposomes encapsulating OM. The images displayeddistinct patterns of adhered cationic polymeric liposomes: singleand multiple lamellar vesicles and flat supported bilayers. As aresult of the OQCMC/Chol liposome formation process, inter-mediate-sized unilamellar vesicles (Figure 7a), large unilamellarvesicles (Figure 7b), and multilamellar vesicles (Figure 7c) wereformed and encapsulated OM, which could be clearly seen in the

(33) Zhu, A. P.; Fang, N.; Chan-Park, M. B.; Chan, V. Biomaterials 2005, 26,6873–6879.

Figure 6. DSC traces of the thermal transformation of (1) cholesterol,(2) QA, (3) OQCMC, (4) the OQCMC/Chol physical mixture, and (5)polymeric liposomes. (The weight ratio of OQCMC/Chol is 1:0.81; theDS of OQCMC is 73.2%.)

Figure 7. TEM images of magnetic cationic polymeric liposomesencapsulating OM. (a) Intermediate-sized unilamellar vesicles (IUV).(b) Large unilamellar vesicles (LUV). (c) Multilamellar vesicles (MLV).(c) Schematic drawing of cationic multilamellar polymeric vesicles thatmay encapsulate materials with various properties.

Figure 8. TEM images of (a) a TAT peptide-bearing cationic poly-meric liposomes; (b) a QD-tagged cationic polymeric liposome; (c)magnetic cationic polymeric liposomes encapsulating hydrophilicmagnetic nanoparticles (HM); and (d) QD-tagged magnetic cationicpolymeric liposomes encapsulating HM.


lipid bilayer. The lamellar structure with OM could also be seenin Figure 7c. The physical structure of this cationic polymericliposome was very similar to that of conventional liposomes(PC/Chol). They were bilayer structures made by OQCMC/Chol.It was a microscopic vesicle consisting of an aqueous coreenclosed in one or more quaternized carboxymethyl chitosanlayers that could be used to deliver vaccines, drugs, enzymes,or other substances to target cells or organs. Figure 7d shows aschematic drawing of cationic multilamellar polymeric vesicles(three films). DNA, hydrophobic components, hydrophiliccomponents and targeting materials can be carried simultaneouslyby cationic polymeric liposomes.

However, in light of the short half-life of conventional PC/Chol liposomes, several researchers have introduced a protectivecoating by designing liposomes that are nonreactive or poly-morphous.34 It is possible that cationic polymeric liposomes witha OQCMC shell also help prevent liposomes from sticking toeach other and to blood cells or vascular walls, and monoclonalantibodies covalently bonded to this small unilamellar polymericliposomes could be easily accomplished by using quaternizedcarboxymethyl chitosan, which was substituted by the hetero-bifunctional cross-linking reagent N-hydroxysuccinimidyl-3-(2-pyridyldithio) propionate (SPDP).35 In our work, TAT peptide-bearing cationic polymeric liposome had also been prepared usingcross-linking reagent SPDP. Figure 8a shows the TEM imagesof these TAT peptide-bearing cationic polymeric liposome inPBS (pH 7.4). They were sphere-shaped and larger than polymericliposome without connecting TAT peptide. The whole systemwas stable within an aqueous medium of PBS with a transparentappearance.

Furthermore, polymeric liposomes were used to encapsulatehydrophobic quantum dots (QDs) and hydrophilic magneticnanoparticles (HM). A TEM image of QD-tagged polymericliposome is shown in Figure 8b. These polymeric liposome-encapsulated QDs had high fluorescent intensity and colloidalstability and could be prepared easily by the REV method.Fluorescence self-quenching of QDs inside polymeric liposomeswas not observed. The average size of these nanoparticles wasabout 60.4 nm in aqueous solution with a polydispersity indexof 0.24. TEM image of magnetic polymeric liposomes containing

HM made by the TLE method is shown in Figure 8c. Withineach magnetic polymeric liposome sphere, nanosized Fe3O4

crystals on the order of 10 nm are randomly embedded in thepolymer matrix. This revealed that the magnetic cationicpolymeric liposomes particles were dispersed independently withan average diameter of about 15 nm in aqueous solution andwere very stable for about 2 weeks. The saturation magnetizationvalue of these superparamagnetic magnetic polymeric liposomeswas 28 emu/g at 300 K (data not shown).36 A TEM image ofa QD-tagged magnetic cationic polymeric liposome containingHM is shown in Figure 8d. The polymeric liposomes encap-sulating magnetic nanoparticles and QDs were superparamagneticand luminescent. They also had a small size of about 20 nm andwere smaller than QD-tagged polymeric liposomes withoutmagnetic nanoparticles. Similar to conventional liposomes, therewere two primary mechanisms for polymeric liposomes (OQCMC/Chol), such as encapsulation (formation of polymeric liposomespassively entrapping water-soluble materials in the interlamellarspaces) and partitioning (formation of polymeric liposomespassively entrapping organic-soluble materials in the intrabilayerspaces). The analysis of this mechanism also applied to drugswith different hydrophilities.

Release Profile of Vincristine Sulfate from PolymericLiposomes The cationic polymeric liposomes can be used indrug delivery, and the preliminary rearch showed that OQCMChad a lower cytotoxic effect than polyethylenimine (PEI) asrevealed by the MTT test. Here, we demonstrate such utilityusing vincristine as a model for water-soluble drugs. Figure 9depicts the release profiles of vincristine from PC/Chol liposomes,cationic polymeric liposomes, and magnetic cationic polymericliposomes in pH 7.4 Tris-HCl buffer at 37 ( 0.5 °C. There wasno distinct difference between two samples of cationic polymericliposomes and magnetic cationic polymeric liposomes. Allliposome groups were better than controls (drug only) and adrug/ferrofluid physical mixture. A biphasic release pattern hadbeen observed with all of these samples. With the first phaselasting about 11 h, 57.2, 35.7 and 34.1% of loaded vincristinewas released from PC/Chol liposomes, cationic polymericliposomes, and magnetic polymeric liposomes, respectively. Thiswas considered to be a burst effect, and it had been attributedto the unbound excess of vincristine on the surface of the lipidbilayer. Then, all three samples gave a slow drug release afterthe initial burst, but compared with PC/Chol liposomes, polymericliposomes had a relatively slow burst release and a longer release

(34) Silvander, M.; Bergstrand, N.; Edwards, K. Chem. Phys. Lipids. 2003,126, 77–83.

(35) Barbet, J.; Machy, P.; Leserman, L. D. J. Supramol. Struct. Cell Biochem.1981, 16, 243–258.

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Figure 9. In vitro release of vincristine in Tris-HCl (pH 7.4) at 37 (0.5 °C.

Figure 10. Photographic images of the liposomal dispersions afterpreparation and release in Tris-HCl (pH 7.4) after about 1 month at 37( 0.5 °C: (a) PC/Chol liposome and (b) OQCMC/Chol polymericliposome.


time because of their high molar mass. Polymeric liposomes alsohad higher drug loading and encapsulation efficiency. Forcomparison, the vincristine encapsulation efficiencies of OQCMC/chol and PC/Chol were 90.1 and 81.8%, respectively.

Figure 10 shows photographic images of PC/Chol andOQCMC/Chol liposome dispersions after preparation and releasein Tris-HCl (pH 7.4) after about one month at 37 ( 0.5 °C. Itcan be seen from the Figure that PC/Chol and OQCMC/Cholliposome dispersions were both very homogeneous and stableafter preparation. However, 1 month after the release of vincristine,precipitates and large particles could be found in PC/Cholliposomal dispersions, and the dispersions of OQCMC/Chol hadnot changed a lot. Our results clearly demonstrate that OQCMC/Chol polymeric liposomes are more stable than PC/Cholliposomes in Tris-HCl buffer solution.

Conclusions

We have developed new cationic polymeric liposomes fromoctadecyl quaternized carboxymethyl chitosan and cholesterol.The physical structure of these cationic polymeric liposomeswas similar to that of conventional PC/Chol liposomes. Micro-spheres or nanospheres of multilamellar vesicles (MLV), largeunilamellar vesicles (LUVs), or small unilamellar vesicles (SUVs)with a lipid bilayer could be formed. Oil-soluble magnetic

nanoparticles or other hydrophobic components could besolubilized in the bilayer of polymeric liposomes, and vincristineas a hydrophilic molecule could also be entrapped in its aqueouscore. These polymeric liposomes exhibited high thermal stability,good solubility in water, high drug encapsulation efficiency ofvincristine, and a long duration of controlled release. Thenanospheres could be modified to be multifunctional, which wouldallow them to serve as potentially useful materials for biomedicalapplications such as nucleic acid extraction, cancer diagnosisand treatment, biosensors, and drug delivery.

Acknowledgment. We gratefully acknowledge the NationalNatural Science Foundation of China (50373033), Key ProjectFoundation from Tianjin Science and Technology Committee(no. 05YFJZJC01001), and Tianjin International CooperationFoundation (05YFGHHZ20070).

Supporting Information Available: Particle size distributionof polymeric liposomes by the TLE and REV methods. Zeta potentialof polymeric liposomes by the REV method. UV/vis and fluorescencespectra analysis of QD-tagged polymeric liposomes. Photographic imageof QDs and QD-tagged polymeric liposomes. This material is availablefree of charge via the Internet at http://pubs.acs.org.

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characterization of novel multifunctional cationic polymeric liposomes formed from octadecyl...

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