Characterization of Novel Multifunctional Cationic Polymeric

School of Materials Science and Engineering, Tianjin UniVersity, Tianjin 300072, PR China, and. UniVersity of Texas Medical Branch, GalVeston, Texas 7...
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Langmuir 2008, 24, 7147-7153

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Characterization of Novel Multifunctional Cationic Polymeric Liposomes 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, and UniVersity 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 that octadecyl quaternized carboxymethyl chitosan (OQCMC) in combination with cholesterol (Chol) could form stable vesicles 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 excellent features, such as good physical and thermal stability, excellent solubility in water, and high effectiveness in drug encapsulation. Trans-activating transcriptional activator protein (TAT peptide) could be connected on the surface of cationic 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 liposomes and their ability to solublize hydrophobic materials. Using different preparation methods, OQCMC/Chol could easily be made into nanoscale particles by encapsulating both hydrophilic and hydrophobic components. We have successfully prepared polymeric liposomes encapsulating quantum dots (QDs), superparamagnetic nanoparticles, or both. Vincristine was also encapsulated in the polymeric liposomes with high drug encapsulation efficiency (90.1%). Vincristine-loaded magnetic polymeric liposomes were stable in aqueous solution and exhibited slow, steady release action over 2 weeks under physiologic pH (7.4). This allows the use of multifunctional cationic polymeric liposomes, such as those developed here from modified chitosan, in various applications such as cancer diagnosis and treatment.

Introduction Nowadays, liposomes and nanoparticles are regarded as beneficial carrier systems for drugs because of their biocompatible and biodegradable properties.1 For example, they have been used to encapsulate colchicines,2 estradiol, tretinoin,3 dithranol,4,5 and enoxacin for applications such as anticancer, antitubercular, antileishmanial, and anti-inflammatory treatments and for delivering hormonal drugs and oral vaccines.6–10 Furthermore, cationic lipids and liposomes still attract the attention of many gene therapy laboratories because of their excellent gene-transfer efficiency.11 However, liposomes also have some limitations. First, they generally show a short circulation half-life after intravenous * 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. Bull. 2001, 24, 795–799. (2) Hao, Y.; Zhao, F.; Li, N.; Yang, Y.; Li, K. Int. J. Pharm. 2002, 244, 73–80. (3) Manconi, M.; Valenti, D.; Sinico, C.; Lai, F.; Loy, G.; Fadda, A. M. Int. J. Pharm. 2003, 260, 261–272. (4) Touitou, E.; Junginger, H. E.; Weiner, N. D.; Nagai, T.; Mezei, M. J. Pharm. Sci. 1994, 83, 1189–1203. (5) Agarwal, R.; Katare, O. P.; Vyas, S. P. Int. J. Pharm. 2001, 228, 43–52. (6) Udupa, N.; Chandraprakash, K. S.; Umadevi, P.; Pillai, G. K. Drug DeV. 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, 26, 217–222. (11) Zhdanov, R. I.; Podobed, O. V.; Vlassov, V. V. Bioelectrochemistry 2002, 58, 53–64.

administration.12 Second, they are prone to adhere to each other and fuse to form larger vesicles in suspension, which may result in inclusion leakage.13 Therefore, stability is a general problem with lipid vesicles.14,15 Third, conventional phosphatidylcholine/ cholesterol (PC/Chol) liposomes do not have certain chemical groups, such as amine and carboxylic acid, so its conjugation with protein receptors is difficult. For example, trans-activating transcriptional activator (TAT) peptide must be attached to the surface of PEGylated liposomes via p-nitrophenylcarbonyl-PEGphosphatidyl ethanolamine (pNP-PEG3000-PE).16 Many attempts, such as the surface modification of liposomes,17 have been investigated to improve the properties of these liposomes. The surface modification of liposomes with several biological materials including proteins, polysaccharides, glycolipids, and water-soluble polyethylene glycol (PEG) was found to improve the circulation time of liposomes injected by decreasing the uptake of liposomes in the reticuloendothelial system (RES).12,15,18 For example, the incorporation of a lipid conjugate of PEG results in a polymeric layer around the liposome, which reduces the adhesion of plasma proteins that would otherwise cause rapid recognition of the liposomes by mononuclear (12) Bakker-Woudenberg, I. A.; Storm, G.; Woodle, M. C. J. Drug Targeting 1994, 2, 363–371. (13) Zhang, L. F.; Granick, S. Nano Lett. 2006, 6, 694–698. (14) Grit, M.; Desmidt, J. H.; Struijke, A.; Crommelin, D. J. A. Int. J. Pharm. 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– 270. (18) Ruysschaert, T.; Paquereau, L.; Winterhalter, M.; Fournier; Didier., Nano Letters. 2006, 6, 2755–2757.

10.1021/la703775a CCC: $40.75  2008 American Chemical Society Published on Web 06/20/2008

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

phagocytes system (MPS) phagocytes.19 Several investigators also have exploited the high affinity of chitosan for cell membranes by using chitosan derivatives as coating materials for liposomes and have reported promising results.20,21 The present study investigates the feasibility of novel cationic polymeric liposomes based on amphiphilic multifunctional octadecyl quaternized carboxymethyl chitosan (OQCMC) and cholesterol (OQCMC/Chol). The possible formation process and the chemical structure of OQCMC are shown in Figure 1. The OQCMC/Chol system is quite similar to the polymer-surfactant complexes of structures described in the literature by Kabanov et al.22 OQCMC is a new kind of chitosan derivative. The derivative has good solubility both in water and organic solvents. Here we hypothesize that polymeric liposomes formed from OQCMC/Chol may resolve most of the above problems. First, the physical and chemistry stability of the liposome can be improved by introducing carboxymethyl chitosan with a high molecular weight. Second, OQCMC has an amino group, a carboxymethyl salt group, and an octadecyl quaternized group in the same complex molecule, so targeting materials can be connected and surface modification becomes possible. Third, the OQCMC has perfectly high crystallinity compared with that of chitosan in other derivatives.23 Furthermore, OQCMC is very cheap and can easily be made into nanoparticles.

Materials and Methods Materials. Chitosan was supplied by Yuhuan Aoxing Biochemistry Co. Ltd. (Zhejiang, China) with a deacetylation degree of >99% and a molecular weight (MW) of 5 × 104. Glycidyl octadecyl dimethylammonium chloride (QA), carboxymethyl chitosan (CMC),23 oil-soluble magnetic nanoparticles (OM),24 hydrophilic magnetic nanoparticles (HM),25 and CdSe/CdS core-shell quantum dots (QDs)26 were all prepared in our laboratory. All other chemicals were reagent grade and were used as received. Preparation of OQCMC. The quaternization of CMC was conducted as follows. CMC (5 g) was dissolved in 100 mL of a (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.

mixture of deionized water saturated with isopropanol. QA was added 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 dialyzed for 4 days against water and finally lyophilized to give OQCMC as a white power.23 Polymeric Liposome Preparation. Thin-Layer EVaporation (TLE) Method. OQCMC and cholesterol (weight ratio 1/0.81, total lipids 30 mg) were dissolved in 4 mL of chloroform at room temperature. To entrap the oil-soluble magnetic nanoparticles (OM), 8 mg of OM was also dissolved in the solution. Chloroform was then evaporated with a vacuum rotary evaporator, and a thin film of polymeric liposomes was formed on the wall of a 50 mL roundbottomed flask. Then the lipid film was dispersed in deionized water and sonicated in a bath sonication unit at 30 °C for 10 min. The liposome suspensions were kept at 5 °C until further characterization was done.27 ReVerse-Phase EVaporation (REV) Method. This method allows us to obtain large unilamellar, oligolamellar, and multilamellar vesicles.28,29 OQCMC with different grafting percentages of the quaternary group and cholesterol were dissolved in 4 mL of chloroform at room temperature. Five milliliters of deionized water was mixed with this organic solvent. The weight ratio of OQCMC to 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 bathtype sonicator. Then, the solvents were evaporated on a rotary evaporator to form a gel-like, highly concentrated polymeric liposomes suspension that can be diluted with a suitable aqueous buffer solution. The liposome suspension was kept under vacuum for at least 3 h to remove trace amounts of the organic solvent. To remove the largest particles and obtain a more homogeneous polymeric liposome population, the liposome suspension can be extruded through cellulose acetate membrances of 0.45 µm pore size in a Millipore filtration cell. Polymeric liposomes encapsulating OM were obtained by adding OM to organic solvent by the TLE method. Magnetic polymeric liposomes encapsulating vincristine were obtained by adding HM and vincristine to the aqueous phase by the REV method. Polymeric liposomes encapsulating QDs were obtained by adding QDs to an organic solvent by the REV method. QD-tagged magnetic polymeric liposomes were obtained by adding QDs and HM to the organic phase and the aqueous phase, respectively. Physicochemical Characterizations of Cationic Polymeric Liposomes X-ray diffraction patterns of different sample fractions were measured with a Rigaku D/max 2500v/pc with a Cu °C source operating at 40 kV and 50 mA at 20 °C. The relative intensity was (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.

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recorded in the scattering range (2θ) of 3-40°. Samples of polymeric liposomes (OQCMC/Chol) were prepared by a lyophilized lipid suspension to obtain a freeze-dried mixture. The physical mixture of OQCMC/Chol was prepared as follows: OQCMC/Chol (weight ratio 1/0.81) was mixed with chloroform, and then the chloroform was evaporated to obtain the dried mixture directly. The thermal properties and the phase-transition temperature of polymeric liposomes were characterized with a differential scanning calorimeter (Diamond DSC, Perkin-Elmer instrument). Each dried sample was weighed in an aluminum pan and heated from 0 to 120 °C at a scanning rate of 10 °C/min. The morphologies of different cationic polymeric liposomes were observed via transmission electron microscopy (TEM). TEM observation of the liposomes was carried out at an operating voltage of 200 kV with a JEOL-100CXII (Japan) in bright-field mode and by electron diffraction. Dilute suspensions of polymeric liposomes in water were dropped onto a carbon-coated copper grid by negatively staining with 2% phosphotungstic acid and then air dried. Samples of cationic polymeric liposomes encapsulating magnetic nanoparticles were prepared similarly without staining. The average particle size and size distribution were determined by quasielastic laser light scattering with a Malvern Zetasizer (Malvern Instruments Ltd., U.K.) at 25 °C. About 0.2 mL of each polymeric liposomes suspension was diluted with 2.5 mL of water immediately after preparation. Each experiment was repeated three times. The zeta potential was measured by using a Zetasizer S (Malvern, U.K.). Zeta limits ranged from -150 to 150 V. Strobing parameters were set as follows: strobe delay -1.00, on time 200.00 ms, and off time 1.00 ms. Vincristine Release from the Nanoparticles in Vitro Unencapsulated vincristine was removed from the magnetic polymeric liposomes by magnetic separation or dialysis. The amount of vincristine was determined by UV spectrophotometry at 298 nm. About 2 mg of dried magnetic polymeric liposomes was dissolved in 5 mL of chloroform to destroy the liposome structure, and then 3 mL of deionized water was added to the chloroform to extract the releasing drug by the extraction method. This process was repeated five times. The vincristine encapsulation efficiency (EE) and vincristine loading efficiency (LE) of the process were calculated from

A-B × 100 A C LE ) × 100 D

EE )

(1) (2)

where A is the total amount of vincristine, B is the amount of unencapsulated vincristine, C is the weight of vincristine in the vesicle, and D is the weight of the vesicle. The in vitro release profiles of vincristine from polymeric liposomes were determined as follows: about 2 mL of the vincristineloaded polymeric liposomes suspension was placed in a dialysis bag with 10 mL of Tris-HCl (pH 7.4) buffer solutions in test tubes and incubated at 37 ( 0.5 °C with stirring. At appropriate time intervals, buffer solutions outside the dialysis bag were taken out of the test tube to determine the amount of vincristine released from the vesicles by UV, and 10 mL of fresh medium was added. All release tests were 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 new amphiphilic derivative of chitosan with high molar mass (>1 × 104), OQCMC exhibited excellent solubility both in water and organic solvents such as chloroform and toluene. OQCMC also had high crystallinity compared with chitosan and other derivatives. The chemical structure of OQCMC is shown in Figure 1. OQCMC has amine groups, carboxymethyl salt groups, octadecyl quaternized groups, and hydroxyl groups.23 Its molecular structure was similar to that of PC to some extent, but compared with PC,

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

it exhibits excellent solubility in water. The hydrophilic properties and lipophilic properties within the molecule are balanced very well, and various functions associated with biomembranes in liposomes, such as aggregation, fusion, and selective permeability, are all dependent on this balance. The process of vesicle preparation from OQCMC and cholesterol by the film dispersion method is shown in Figure 1. After the evaporation of chloroform, the OQCMC membrane could be formed spontaneously. The transformation of the polymeric vesicle membrane structure may be similar to that of the phospholipids membrane. The size of the vesicles remained stable for >60 days and was almost the same as that after 1 h when the system temperature increased to 50 °C and then decreased to the original temperature. While the system temperature increases, the thermal motion and the layer interspacing also increased. Because most of the observed liposomes were aggregated vesicles, this aggregated polymeric structure had a substantially larger stability than the single-vesicle structure and consequently a larger resistance in maintaining its shape and function as a carrier of cosmetics, food additives, and drugs. This observation also had some important consequences in the liposomes’ selective permeability when they were used as carriers.30 Structural Characterization of Polymeric Liposomes The effects of different preparation methods on the formation of polymeric liposomes are discussed. The size and shape of the polymeric vesicles can be directly observed by TEM. Figure 2 shows the TEM images of the polymeric nanoparticles prepared by 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, different types, including multilamellar vesicles (MLV), large unilamellar vesicles (LUVs), and small unilamellar vesicles (SUVs). From the average results of 50 vesicles and under the same experimental conditions, the sizes of the vesicles made from the TLE method were 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 polymeric liposomes by the TLE method is 172.5 ( 2.1 nm, which compared with 108.5 ( 0.5 nm by the REV method. The TLE and REV methods both allow for the formation of multilamellar vesicles. Basically, a mixture of OQCMC and lipid compounds 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 cosolubilized with the liposome-forming materials. This polymeric lipid film 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.

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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 on the mean size and zeta potential of nanoparticles.

desired pH value and any hydrophilic component that should be entrapped within vesicles (e.g., water-soluble drugs) was solubilized. Also, hydrophobic components could be cosolubilized in organic solvent, and the hydrophilic component could be dissolved in an aqueous solution. This method represents a good approach to increasing the amount of drug entrapped within vesicles. The effects of the weight ratios of OQCMC/Chol and the degree of substitution (DS) of the quaternary group in carboxymethyl chitosan on the formation of polymeric liposomes are also discussed. Cholesterol (C27H45OH) is a cell membrane constituent. It modulates membrane fluidity, elasticity, and permeability by closing the gaps created by imperfect packing of other lipid species when proteins are embedded in the membrane. From an analysis of particle size, the size of the vesicles made from OQCMC only was larger than that from mixed samples of OQCMC and cholesterol with a weight ratio below 1:0.81 (Figure 3a). Furthermore, cholesterol can enable the formation of vesicles and reduced aggregation and provided greater stability.31 With the increase in cholesterol content from zero to 0.81, the mean sizes of polymeric liposomes decreased, but when the cholesterol content was more than the content of OQCMC, the mean size of the polymeric liposomes increased drastically. From Figure 3b, the mean size of the polymeric liposomes first decreased and then increased with the DS of the quaternary group increasing. However, they are all very stable, and the smallest mean size is about 60.4 ( 0.2 nm from particle size analysis when the DS of the quaternary group is about 73.2%. The zeta potential of cationic polymeric liposomes increased from +26.32 to +42.17 mV as the DS of the quaternary group increased. This finding suggests that cationic polymeric liposomes with different charges can be prepared by controlling the DS of the quaternary group. Figure 4 shows the particle size distributions based on the intensity 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 particle size analyzer was bigger than those visualized by TEM. The polydispersity index is used to describe the spread in particle diameters produced in a sample of particles. In particle size analysis system, the normalized variable is usually referred to as the polydispersity index. As the index approaches zero, the size range produced becomes narrower. And the polydispersity index of the polymeric liposomes was 0.224. Stability of Polymeric Liposomes. X-ray diffractograms of OQCMC, polymeric liposomes, and the OQCMC/Chol physical

Figure 4. Particle size distribution based intensity of cationic polymeric liposomes prepared by OQCMC and cholesterol. (The molar ratio of OQCMC/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%.)

mixture are shown in Figure 5. It could be seen that there were some differences in peak height, width, and position among them. Compared with chitosan, which showed a relatively broad peak at 2θ ) 20°,23 OQCMC had a narrow peak at 2θ ) 21.4° and a new narrow peak at 2θ ) 5° that are indicative of a bilayered lamellar 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.

Characterization of Cationic Polymeric Liposomes

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; the DS of OQCMC is 73.2%.)

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

that the polymeric lipid is a highly crystalline material. However, the narrow peak at 2θ ) 21.4° disappeared when OQCMC and cholesterol were blended together in chloroform. Also, polymeric liposomes and the OQCMC/Chol physical mixture both had a relatively broad peak at 2θ ) 18°. The absence or change in several peaks in the diffraction pattern for the blends is an indication that interactions between OQCMC and cholesterol are 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 than components of the OQCMC/Chol physical mixture. The differential scanning calorimetry (DSC) traces for cholesterol, QA, OQCMC, OQCMC/Chol physical mixtures, and polymeric liposomes are shown in Figure 6. The phasetransition temperature was defined as the temperature required to induce a change in the lipid physical state from the ordered gel phase (hydrocarbon chains are fully extended and closely packed) to the disordered liquid-crystalline phase (hydrocarbon chains are randomly oriented and fluidic). Compared with cholesterol, the DSC traces for QA and OQCMC exhibited a

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

single narrow peak characteristic of the main gel to liquidcrystalline phase-transition (Tm) temperature. The positions of these 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 weight increased from QA to OQCMC, molecular interactions became stronger, 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 that the blending of OQCMC with cholesterol altered the gel to liquidcrystalline transition of OQCMC. This huge reduction in enthalpy to zero is likely caused by the strong interactions between amphiphilic OQCMC and cholesterol.33 As with conventional liposomes, the addition of cholesterol to OQCMC membranes also acts to broaden the transition of the lipid with the enthalpy of the transition reaching zero. These curves clearly indicated that at temperature of