- Email: [email protected]
PEGlated magnetic polymeric liposome anchored with TAT for delivery of drugs across the blood-spinal cord barrier
Biomaterials 31 (2010) 6589e6596
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
PEGlated magnetic polymeric liposome anchored with TAT for delivery of drugs across the blood-spinal cord barrier Hanjie Wang a, Shuangnan Zhang a, Zhenyu Liao a, Chunyuan Wang b, Yang Liu b, Shiqing Feng b, Xinguo Jiang c, Jin Chang a, * a
Institute of Nanobiotechnology, School of Materials Science and Engineering, Tianjin University and Tianjin Key Laboratory of Composites and Functional Materials, Tianjin 300072, PR China Department of Orthopaedics, Tianjin Medical University General Hospital, Tianjin, PR China c School of Pharmacy, Fudan University, Shanghai, 201203 PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 February 2010 Accepted 27 April 2010 Available online 4 June 2010
Due to the existence of the blood-spinal cord barrier (BSCB), many therapeutic macromolecular agents, such as drugs, protein and gene, cannot pass through this barrier to reach the site of injury, all of which restricts the treatment of spinal cord injuries (SCI). In this study, TAT-conjugated PEGlated Magnetic polymeric liposomes (TAT-PEG-MPLs) formed from PEGlated amphiphilic octadecyl quaternized carboxymethyl chitosan (PEG-OQCMC), cholesterol (Chol), superparamagnetic nanoparticles, and transactivating-transduction protein (TAT), were prepared successfully and evaluated the properties in vitro and in vivo. The result indicated that TAT-PEG-MPLs were spherical in solution, with significantly small mean diameter (83.2 nm) and excellent magnetism (magnetization saturation values of 43.5 emu/g). In vitro experiment, the uptake of PEG-MPLs with TAT by MCF-7 cells was greater than that of the PEG-MPLs without TAT. Most importantly, in vivo experiment, a low MRI signal was observed in the T2-weighted images; Histological analysis, Cryo-TEM and flame atomic absorption spectrophotometry revealed that TAT-PEG-MPLs nanoparticles significantly accumulated around the site of the SCI even inside the nerve cells. These nanoparticles may provide a promising carrier to locate to the lesion site, deliver therapeutic macromolecular agents across the BSCB and penetrate into the nerve cells for the treatment of SCI. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Chitin/chitosan Drug delivery Liposome, MRI(magnetic resonance imaging)Nanopartilce Surface modification
1. Introduction Blood-spinal cord barrier (BSCB) maintains a homeostatic environment in spinal cord (SC) and protects cord tissue by exhibiting selective permeability to plasma constituents [1]. However, due to the existence of BSCB, many therapeutic substances, such as cyclosporine A (CSA), neurotrophic factor, vascular endothelial growth factor (VEGF), excitatory amino acids (EAA), and C3, cannot pass through this barrier to reach the site of injury [2e7]. The BSCB is formed by special endothelial cells sealed with tight junctions. This unique membrane restricts the entry to the SC from the periphery. In clinical applications, high-dose therapeutic drugs are nonspecific and distributed randomly after feeding (or local injection) have been shown to be an effective treatment of spinal cord injuries (SCI). However, this high-dose therapy was accompanied with many adverse effects, such as an
* Corresponding author. Tel./fax: þ86 22 27401821. E-mail address: [email protected] (J. Chang). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.04.057
increased incidence of wound infection, low bioavailability of drugs in the setting of neural injury and an increased incidence of toxicity and side effect of drug [8]. Several approaches have been employed to circumvent this problem, including opening the tight junctions of the BSCB, the use of carrier-linked drugs and drugs embedded in nanoparticles through. While the opening of the tight junctions is so invasive that it may also allow the entry of dangerous contaminants into the setting of neural injury; the carrier-linked drugs is complicated and difficult because the therapeutic properties of the native drugs used may be altered after chemical conjugations; drugs embedded in nanoparticles, may be a better approach. Nanoparticles have recently emerged as promising carriers for drug delivery system. Over the past several decades, there has been a steady growth in the number of available nanoparticles for therapy. Among these products, polymer nanoparticles and liposomes are two dominant classes. Polymers, such as Polyethylene Glycol (PEG) [9], chitosan (CS) [10], poly(lactic-co-glycolic acid) (PLGA) [11] and so on, have been commonly used to form core-shell structure to encapsulate a variety of drugs. The advantages of liposomes include their
6590
H. Wang et al. / Biomaterials 31 (2010) 6589e6596
multilayer structure to improve the entrapment efficiency ofhydrophilic therapeutic agents, protect encapsulated drugs from external conditions and so on [12]. Recently, the TAT-PEG conjugated magnetic polymeric liposome (MPL) that combined both advantages of polymeric vesicles and liposomes, is developed in our laboratory. The NPs are formed from four materials: (1) PEGlated amphiphilic octadecyl quaternized carboxymethyl chitosan (PEG-OQCMC), forming a lamellar nanoshell, provided electrostatic and steric repulsion against particle aggregation, the ability to encapsulate high amounts of drugs, a longer circulation half-life in vivo, amino-groups for the attachment of targeting ligands such as antibodies, peptides [13]; (2) cholesterol (Chol) helped stabilize and maintain the structure of MPL; (3) superparamagnetic nanoparticles that responds to external permanent magnet with superparamagnetic characteristics, was chosen for the magnetic separation, non-invasive magnetic targeting [14] and MRI; (4) Transactivating-transduction protein (TAT), short cationic sequences with a remarkable capacity for membrane translocation [15], was employed for the delivery of the MPL across the Blood-spinal cord barrier (BSCB). Herein we report our studies on the systematic preparation and characterization of these TAT-PEG conjugated magnetic polymeric liposomes (MPLs). The properties, such as structure, morphology, size distribution, magnetic response, stability and the cellular penetration efficiency were evaluated. Further to this, the MPLs were injected through the caudal vein using a rat model of SCI and accumulation at the lesion site was evaluated through magnetic resonance imaging (MRI) tracing, histological analysis and electron microscopy. 2. Experimental method 2.1. Materials PEG-conjugated octadecyl quaternized carboxymethyl chitosan (PEG-OQCMC) and hydrophobic superparamagnetic nanoparticles, are all prepared in our lab. TAT peptide of the sequence Tyr-Gly-Arg-(Lys)2-(Arg)2-Gln-(Arg)3 was custom synthesized by Shanghai Sangon Biological Engineering Technology&Services Co. Ltd. N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and Fluorescein-5-
isothiocyanate (FITC) were purchased from Sigma-Aldrich. All other chemicals were of reagent grade and were used as received. 2.2. Synthesis and characterization of octadecyl quaternized carboxymethyl chitosan (OQCMC) and PEG-OQCMC The OQCMC was synthesized as reported in our previous work [13]. The quaternization of CMC was performed as follows. CMC (5 g) was dissolved in 100 mL of a mixture of deionized water saturated with isopropanol. QA was added slowly with different molar ratios to the glucosamine unit. An aqueous NaOH solution (42%, w/ w) was added and the mixture reacted at 80 C for 24 h with stirring. After dialysis against water performed for 4 days, the solution finally lyophilized to give OQCMC as a white powder. The monohydroxy -terminated PEG was converted to NHS-PEG according to the literature report [16]. A predetermined quantity of OQCMC was dissolved in purified water at a concentration of 5 mg/ml. NHS-PEG was dissolved in water free DMSO (50 mg/ml) solution. Subsequently, the OQCMC solution was added to the NHS-PEG solution and the mixture was stirred at room temperature for 24 h. After 24 h of stirring, the solution was purified by dialysis with a 10 000 MW cutoff membrane. This procedure was repeated at least three times and the dialyzed solution was finally freeze-dried. The 1H-NMR spectra of PEG-OQCMC was studied using a Bruker Avance 400 spectrometer (400 MHz), and D2O was used as the solvent. 2.3. Preparation and characterization of TAT-PEG conjugated magnetic polymeric liposomes (TAT-PEG-MPLs) Magnetic polymeric liposomes were prepared by reverse-phase evaporation (REV) method [17] (Fig. 2 A). PEG-OQCMC, hydrophobic superparamagnetic nanoparticles and cholesterol (weight ratio ¼ 2:0.5:1, gross mass 30 mg) were dissolved in 4 ml chloroform at room temperature to obtain the organic phase. About 6 ml deionized water was prepared to obtain aqueous phase. Then, aqueous phase was mixed with organic phase under sonication for 120 s at 100 W output. The organic solvents were evaporated on a rotary evaporator to form a gel-like MPL suspension. This MPL solution were then separated by magnet force and washed by deionized water for three times. The collected product was freeze-dried and saved. The MPLs were reacted with SPDP to yield a 2-pyridyldithiol-end group on the NPs surface. In brief, 2 ml of MPL solution (5 mg/ml) in 0.1 M phosphate buffer (pH ¼ 7.4) was mixed with 0.32 ml SPDP (2 mg) DMSO solution. The mixture was incubated at room temperature for 60 min. Low molecular weight impurities were dialyzed against PBS (0.1 M) for 5 h by dialysis bag (Mw ¼ 12 000). Then, above 2-pyridyl disulfide conjugated MPL solution was added with 1 ml TAT PBS (0.1 M) solution (5 mg/ml). The mixture was incubated overnight at 4 C to form a disulfide linkage between the surface of MPL and TAT peptide. The redundant TAT peptide was removed by magnetic separation and the obtained TAT-PEG conjugated MPLs(TAT-PEG-MPLs) solution was saved under 4 C. The FITC labeled polymeric liposomes were
Fig. 1. 1H-NMR spectrum of PEG-OQCMC.
H. Wang et al. / Biomaterials 31 (2010) 6589e6596 fabricated in a same way with PEG-OQMCMC replaced by FITC congjucated PEGOQMCMC. 2.4. Physicochemical characterizations of TAT-PEG-MPLs Morphology: The morphologies of different samples were observed via transmission electron microscopy (TEM). TEM observation of the sample was carried out
6591
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. Particle size and zeta potential analysis: The average particle size and size distribution were determined by quasielastic laser light scattering with a Brookhaven Zetasizer (Brookhaven Instruments Ltd., U.S.) at 25 C. About 0.2 mL of each
Fig. 2. (A) a schematic illustration shows the process of preparing TAT-PEG-MPLs by the reverse-phase evaporation method. (B) A transmission electron microscopy (TEM) image shows the nano-structure of the TAT-PEG-MPLs. (C) Pre-and post ultraviolet absorption changes of the reaction solution during the process of conjugating TAT onto the surface of PEG-MPLs. (D) The effective diameter and size distribution of TAT-PEG-MPLs measured by the particle size analyzer.
6592
H. Wang et al. / Biomaterials 31 (2010) 6589e6596 continuously as a monolayer at 37 C in a humidified atmosphere containing 5% CO2 in folate-deficient RPMI (FDRPMI) 1640 supplemented with 1% penicillin, 1% streptomycin, and 10% heat-inactivated fetal bovine serum (HIFBS). HIFBS contains endogeneous folate at concentrations sufficient for cells to grow in this medium. After 1 day of incubation, FITC-loaded samples with or without TAT were added to the growth media. The growth media were removed after 1 h,12 h and 24 h, and the cells were washed 3 times with PBS. The cells were then observed under an inverted fluorescence microscopy. 2.6. Animal model of spinal cord injury
Fig. 3. Magnetization curve of pure hydrophobic superparamagnetic nanoparticles (A) and TAT-PEG-MPLs (B) obstained by VSM. sample 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 (Brookhaven, U.S.). Zeta limits ranged from-150 to 150 V. Parameters were set as follows: strobe delay -1.00, on time 200.00 ms, and off time 1.00 ms. Magnetic Properties: Magnetic measurement of sample was performed by a vibrating sample magnetometer (VSM) from LakeShore Ltd. The samples were in the form of powder and were placed in Teflon sample holder. The magnetic measurements (hysteresis loops) were carried out in the field region of 1 T at room temperature. At 1 T, the magnetization of the samples was almost saturated. 2.5. Cell penetration tests by fluorescence microscopy The cell penetration tests of MPLs with and without TAT were performed in MCF-7 cells by labeling the samples with FITC. The cell line was cultured
The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL/100 g). The surgical area was shaved and disinfected with 70% ethanol and betadine. The spinous processes and vertebral plate were removed using a micro rongeur at T10, and the dura opened. The rat was then placed on an Impactor-2 machine, and the exposed spinal cord was struck using a 10 g 50 mm weight drop. In each case, the two hind limbs of the rat twitched involuntarily and the tail wagged, in accordance with the criteria of the SCI model. A total of 24 rats were randomly and evenly assigned to the two experimental groups. Another twelve rats were designated as treatment-free controls and were not included in the experimental groups. After surgery, the rats were placed in temperature- and humiditycontrolled incubation chambers until they awoke. They were then transferred to cages, and bladder evacuation was applied daily until the return of reflexive bladder control. 2.7. In vivo animal test Ten-week-old SD adult rats (250 g in weight)were injected with samples solution via tail vein. Animals were sacrificed at 72 h post-injection. MRI protocol: Rats were examined daily for a period of three days after injection using a 3.0 T MRI spectrometer (Signa HD 3.0 T, General Electric Company, Milwaukee, WI). Single sagittal, coronal, and transverse images were obtained by a fast gradient echo sequence for localizing subsequent T2-weighted transverse images measured with a standard turbo spin echo sequence. A “positive” MR signal was defined as a distinctive dark area in the T2-weighted images consisting of at least three contiguous pixels. Electron microscopy: Two-millimeter cross-sections of the spinal cord lesions were removed for ultra structural examination. Tissue samples were prepared for electron microscopy as follows: the spinal cord segments were removed and fixed in 2.5% glutaraldehyde for 24 h. Tissues were then post-fixed with OsO4, dehydrated in
Fig. 4. Fluorescence microscopy images of viable cells incubated with PEG-MPLs without TAT group (B) and with TAT (C).
H. Wang et al. / Biomaterials 31 (2010) 6589e6596 a graded alcohol series, and then embedded. Sections of 60e90 nm were stained with uranyl acetate and lead citrate, examined on an electron microscope (JEM 1200EX; Jeol, Tokyo, Japan), and photographed. The investigator who evaluated the sections was blinded to the group information. inverted fluorescence microscopy: Flame atomic absorption spectrophotometry: After perfusion, the length of the spinal cord collected from eight rats from each group was 4 cm. Spinal cords were maintained at 70 C with a feedback-controlled heating blanket for 48 h to facilitate dehydration. Dry weights were obtained. To eliminate the organic portions of the samples, each was treated with 1.5 mL of 65% HNO3 and 0.3 mL of 70% HClO4, digested with slow-heating until the organics dispersed totally, and then diluted with 1% HCl to 10 mL. Standards were prepared by diluting a 0.1 mg/mL Fe solution with 1% HCl. Fe concentrations were analyzed by flame atomic absorption spectrophotometry (Z-5000 atomic absorption spectrometer; Hitachi Ltd., Tokyo, Japan).
3. Results and discussion 3.1. Structural characterization of PEG-OQCMC From the 1H-NMR spectrum of PEG-OQCMC (Fig. 1), PEGOQCMC was successfully synthesized. The multiple peaks at d ¼ 3.5e3.7 were attributed to the protons of eCH2e groups in PEG [18], and the weak and multiple peaks at d ¼ 0.87 came from the protons of eCH3 groups on the quaternary ammonium salt. The weak and broad peak at d ¼ 4.66 was attributed to the protons of the pyranose of chitosan. The results indicated that the synthesis of PEG-OQCMC was successfully. 3.2. Formulation of TAT-conjugated PEGlated magnetic polymeric liposomes (TAT-PEG-MPLs) A schematic illustration for preparation TAT-PEG-MPL is presented in Fig. 2(A). The SPDP reagents are unique group of amineand sulfhydryl-reactive heterobifunctional cross-linkers. Amine groups exist on the surface of MPL NPs. Herein, the SPDP reagents were used to form amine-to-sulfhydryl cross-links among molecules [19]. Because TAT peptide contains sulfhydryl groups (-SH), PEG-MPL must be modified by the SPDP reagent. The modification of PEG-MPL with TAT results in displacement of a pyridine-2-thione group, which can be determined by measuring the absorbance at 343 nm. UV/Vis spectra of the reaction solutions were shown in Fig. 2(C). It can be seen that the absorbance peak at 343 nm was very clear for the solution after reaction, which approved the existence of pyridine-2-thione, all of which indicated that TAT was conjugated onto the surface of PEG-MPLs successfully. The mean
6593
diameters and polydispersity index (PDI) of TAT-PEG-MPLs were measured by dynamic light scattering (DLS) in Fig. 1(D). This data demonstrated reproducible formation of TAT-PEG-MPLs nanoparticles exhibiting significantly small mean diameter (83.2 nm) and PDI (0.193). TEM images was collected to show that these TATPEG-MPLs were spherical and nonaggregated in Fig. 2(B). TEM images showed individual, uniformly sized TAT-PEG-MPLs nanoparticles with similar diameters as calculated by DLS. 3.3. Study on the magnetic properties of TAT-PEG-MPLs The superparamagnetic colloids are promising to serve as a class of effective supports for guided delivery. In clinical applications, local injection of high-dose drugs, have been shown to be an effective treatment of spinal cord injuries (SCI). However, this high-dose therapy was accompanied with many adverse effects, such as side effects on healthy cells or conflict with other drugs in use for different diseases. Transportation of drugs to a specific site can eliminate such side effects and also reduce the dosage required. One simple way to improve targeted drug delivery is to use an external magnetic field as the guidance [20]. After biocompatible magnetic carriers containing therapeutic drugs are injected into the circulatory system, a magnetic field is applied to the targeted spinal cord to selectively collect the magnetic carriers at this site. The respond speed of biocompatible magnetic carriers to the external magnetic field is determined by the magnetization saturation values. Fig. 4 shows the hysteresis loop of TAT-PEGMPLs measured by using the vibrating sample magnetometer. It should be noted that the TAT-PEG-MPLs sample still shows high magnetization (43.5 emu/g), indicating its suitability for targeting and separation as a drug carrier. Moreover, the TAT-PEG-MPLs with homogenous dispersion show fast response to the external magnetic field due to its high magnetization and no residual magnetism is detected The result reveals that the TAT-PEG-MPLs exhibit good magnetic responsible and re-disperse properties, which suggests a potential application for magnetic targeting. 3.4. Transport of TAT-PEG-PLs into cells As seen in Fig. 3 cellular penetration of the polymeric liposomes with and without TAT was examined to demonstrate the penetration of the nanoparticles into the cells by using FITC fluorescent
Fig. 5. The magnetic resonance imaging (MRI) in vivo. (A) control group, (B) the PEG-MPL without TAT group, (C) the PEG-MPLs with TAT group.
6594
H. Wang et al. / Biomaterials 31 (2010) 6589e6596
label. The internalization of FITC conjugated samples incubated for 1 h was visualized by inverted fluorescence microscopy. After 1 h, TAT-peptide-liposomes were seen clustered in the perinuclear region with a reduced cytoplasmic distribution. Comparing with TAT-PEG-PLs at the same time point, just a little of the green fluorescence in PEG-PLs without TAT group existed in the cells. This is because the presence of the cell penetrating peptide on the surfaces of the nanoparticles promoted their cellular uptake. transactivator of transcription (Tat), having 10e27 amino acids in length, possesses multiple positive charges and penetrates by
a receptor-independent mechanism, to enhance the transport of cargoes across cell membranes and the BBB [21]. 3.5. Magnetic resonance imaging(MRI) in vivo As the TAT-PEG-MPLs used in this research possess superparamagnetic properties, the MRI signals can be reduced in T2weighted images that can be used to show the existence of magnetic NPs in CNS [22]. The accumulation and distribution of TAT-PEG-MPL in rats were examined for a period of 3 days by the
Fig. 6. The fluorescent images of the spinal cord tissue section. (A) the PEG-PLs without TAT group; (B) the PEG-PLs with TAT group.
H. Wang et al. / Biomaterials 31 (2010) 6589e6596
6595
Fig. 7. The cryo-TEM images of the spinal cord sections. the control group (A), PEG-MPLs without TAT nanoparticles (B), the TAT-PEG-MPLs group (C and D).
MR images. Signal changes in T2-weighted images before and after intravenous injection of TAT-PEG-MPL are depicted in Fig. 5. In the third day, the regions with signal drop surrounding the spinal cord injury areas increased significantly following TAT-PEG-MPLs injection and guideline by magnetic force. This phenomenon can be ascribed to the intrinsic accumulation of MPL in areas of magnetic field and sensitivity of T2-weighted sequences to iron NPs encapsulated in polymeric liposomes. Furthermore, these signal had the trend of diffusion into spinal cord parenchyma. This indicated that TAT-PEG-MPLs not only can be accumulated under the guideline of magnetic force, but also has the function of diffusion in animal parenchyma. Compared to TAT-PEG-MPLs group, the hypointense signal of PEG-MPLs without TAT was not obvious, almost same as the signal of untreated group.
but penetrate through the surrounding tissue and penetrate into the nerve cells. The conclusion was in accordance with the result got in the MCF-7 cells culture. The ability of TAT-PEG-PLs to cross the BBB and BSCB is mainly attributed to the fact of TAT, which can promoted theirpenetration by cells during a very short time.
3.6. Histological analysis To determine whether the FITC conjugated polymeric liposome with TAT can cross the BSCB, the distribution of FITC in the spinal cord sections of rats was observed 72 h after injection. FITC were unable to cross the BBB, which only existed in blood vessels [23]. The FITC-conjugated TAT-PEG-PLs nanoparticles crossed the BBB and BSCB (Fig. 6 (B)), penetrated through the surrounding tissue (Fig. 6 (B)) and penetrate into the nerve cells(Fig. 6 (C)). Moreover, the area of positive green fluorescence in TAT-PEG-PLs group was significantly more than that in PEG-PLs group without TAT. We can concluded that TAT-PEG-PLs cannot only cross the BBB and BSCB,
Fig. 8. Iron element content in the rats spinal cord Control group (A), PEG-MPLs group (B), TAT-PEG-MPLs group (C).
6596
H. Wang et al. / Biomaterials 31 (2010) 6589e6596
3.7. Electron microscopy analysis
References
The nerve cellular penetration of the TAT-PEG-MPLs nanoparticles in vivo was studied by means of transmission electron microscopy(TEM). TEM images show cross-sectional pictures of the cells where image intensity varies according to how a beam of electrons travels through the sample. The TAT-PEG-MPLs nanoparticles were clearly visible in the nerve cells (Fig. 7 Fig. 8). In contrast, no appreciable numbers of nanoparticles were found in the PEG-MPLs without TAT group. This suggests that the TAT efficiently mediated transfer of the liposome carriers across the membrane into the cells.
[1] Pan WH, Kastin AJ. Penetration of neurotrophins and cytokines across the bloodbrain blood spinal cord barrier. Adv Drug Deliv Rev 1999;36(2e3):291e8. [2] Sharma HS, Nyberg F, Westman J, Alm P, Gordh T, Lindholm D. Brain derived neurotrophic factor and insulin like growth factor-1 attenuate upregulation of nitric oxide synthase and cell injury following trauma to the spinal cord. An immunohistochemical study in the rat. Amino Acids 1998;14(1e3):121e9. [3] McTigue DM, Horner PJ, Stokes BT, Gage FH. Neurotrophin-3 and brainderived neurotrophic factor induce oligodendrocyte proliferation and myelination of regenerating axons in the contused adult rat spinal cord. J Neurosci 1998;18(14):5354e65. [4] Hauben E, Butovsky O, Nevo U, Yoles E, Moalem G, Agranov E, et al. Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion. J Neurosci 2000;20(17):6421e30. [5] Nordal RA, Nagy A, Pintilie M, Wong CS. Hypoxia and hypoxia-inducible factor-1 target genes in central nervous system radiation injury: a role for vascular endothelial growth factor. Clin Cancer Res 2004;10 (10):3342e53. [6] Springer JE, Azbill RD, Nottingham SA, Kennedy SE. Calcineurin-mediated BAD dephosphorylation activates the caspase-3 apoptotic cascade in traumatic spinal cord injury. J Neurosci 2000;20(19):7246e51. [7] Tan EYM, Law JWS, Wang CH, Lee AYW. Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons. Pharm Res 2007;24:2297e308. [8] Weaver LC, Gris D, Saville LR, Oatway MA, Chen YH, Marsh DR, et al. Methylprednisolone causes minimal improvement after spinal cord injury in rats, contrasting with benefits of an anti-integrin treatment. J Neurotrauma 2005;22(12):1375e87. [9] Burdick JA, Anseth KS. Photoencapsulation of osteoblasts in injectable RGDmodified PEG hydrogels for bone tissue engineering. Biomaterials 2002;23 (22):4315e23. [10] Kumar M. A review of chitin and chitosan applications. React Funct Polym 2000;46(1):1e27. [11] Jain RA. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 2000;21(23): 2475e90. [12] Lian T, Ho RJY. Trends and developments in liposome drug delivery systems. J Pharm Sci 2001;90(6):667e80. [13] Liang XF, Wang HJ, Tian H, Luo H, Chang J. Syntheis, structure and properties of novel quaternized carboxymethyl chitosan with drug loading capacity. Acta Phys-Chim Sin 2008;24(2):223e9. [14] Benyettou F, Lalatonne Y, Sainte-Catherine O, Monteil M, Motte L. Superparamagnetic nanovector with anti-cancer properties: gamma Fe2O3@Zoledronate. Int J Pharm 2009;379(2):324e7. [15] Berry CC. Progress in functionalization of magnetic nanoparticles for applications in biomedicine. J Phys D Appl Phys 2009;42(22). [16] Mao SR, Shuai XT, Unger F, Wittmar M, Xie XL, Kissel T. Synthesis, characterization and cytotoxicity of poly(ethylene glycol)-graft-trimethyl chitosan block copolymers. Biomaterials 2005;26(32):6343e56. [17] Ruel-Gariepy E, Leclair G, Hildgen P, Gupta A, Leroux JC. Thermosensitive chitosan-based hydrogel containing liposomes for the delivery of hydrophilic molecules. J Control Release 2002;82(2e3):373e83. [18] Jiang HL, Kwon JT, Kim EM, Kim YK, Arote R, Jere D, et al. Galactosylated poly (ethylene glycol)-chitosan-graft-polyethylenimine as a gene carrier for hepatocyte-targeting. J Control Release 2008;131(2):150e7. [19] Liang XF, Wang HJ, Luo H, Tian H, Zhang BB, Hao LJ, et al. Characterization of novel multifunctional cationic polymeric liposomes formed from octadecyl quaternized carboxymethyl chitosan/cholesterol and drug encapsulation. Langmuir 2008;24(14):7147e53. [20] Alexiou C, Schmid RJ, Jurgons R, Kremer M, Wanner G, Bergemann C, et al. Targeting cancer cells: magnetic nanoparticles as drug carriers. Eur Biophys J 2006;35(5):446e50. [21] Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999;285 (5433):1569e72. [22] Schwartz ED. MRI and the evaluation of the blood-spinal cord barrier following injury. Am J of Neuroradiol 2005;26(7):1609e10. [23] Li LH, Guo K, Lu J, Venkatraman SS, Luo D, Ng KC, et al. Biologically active core/shell nanoparticles self-assembled from cholesterol-terminated PEG-TAT for drug delivery across the blood-brain barrier. Biomaterials 2008;29(10):1509e17. [24] Salvador A, Pascual-Marti MC, Adell JR, Requeni A, March JG. Analytical methodologies for atomic spectrometric determination of metallic oxides in UV sunscreen creams. J Pharm Biomed Anal 2000;22(2):301e6.
3.8. Flame atomic absorption spectrophotometry As seen in Fig. 8 flame atomic absorption spectrophotometry method [24] was used for quantitative analysis of TAT-PEG-MPL content in spinal cord. The results indicated that the Fe content in the spinal cord lesion sites in TAT-PEG-MPLs group was 1.563 mmol/ mg which was significantly higher than that in either PEG-MPLs group (1.322 mmol/mg) or control group(1.296 mmol/mg). The Fe content of TAT-PEG-MPL group was statistically significant compared with that of PEG-MPL without TAT group (p < 0.05) and control group (p < 0.01), while the Fe content of PEG-MPL without TAT group and control group were not statistically significant (p > 0.05). It can be concluded that TAT-conjugated method can enhance iron accumulation significantly. 4. Conclusion PEG-conjugated magnetic polymeric liposomes, which is anchored with TAT, have been successfully constructed to cross the BSCB and aggregate at the site of the lesion in an injured spinal cord. The nanoparticle formulations, exhibiting significantly small mean diameter (83.2 nm) and superparamagnetism (magnetization saturation values of 43.5 emu/g), were characterized and evaluated in vitro and in vivo. The results indicated that TAT-PEGMPLs not only can be accumulated at injury area by magnetic force, but also can be used as MRI contrast agent in rat spinal cord injury model experiment. TAT conjugation can help the PEG-MPL cross the BSCB, penetrate through the surrounding tissue and penetrate into the nerve cells. The successful transport of TAT-PEG-MPLs nanoparticles into the spinal cord may open new opportunities for the treatment of spinal cord injuries. Acknowledgments The authors gratefully acknowledge National Basic Research Program of China (973 Program) 2007CB935800, National Natural Science Foundation of China (50873076), National Natural Science Foundation of China (30876203), and Tianjin Science and Technology Program (09ZCGYSF00900). Appendix Figures with essential discrimination. Figs. 1e7 in this article have parts that are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j. biomaterials.2010.04.057.
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
PEGlated magnetic polymeric liposome anchored with TAT for delivery of drugs across the blood-spinal cord barrier Hanjie Wang a, Shuangnan Zhang a, Zhenyu Liao a, Chunyuan Wang b, Yang Liu b, Shiqing Feng b, Xinguo Jiang c, Jin Chang a, * a
Institute of Nanobiotechnology, School of Materials Science and Engineering, Tianjin University and Tianjin Key Laboratory of Composites and Functional Materials, Tianjin 300072, PR China Department of Orthopaedics, Tianjin Medical University General Hospital, Tianjin, PR China c School of Pharmacy, Fudan University, Shanghai, 201203 PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 February 2010 Accepted 27 April 2010 Available online 4 June 2010
Due to the existence of the blood-spinal cord barrier (BSCB), many therapeutic macromolecular agents, such as drugs, protein and gene, cannot pass through this barrier to reach the site of injury, all of which restricts the treatment of spinal cord injuries (SCI). In this study, TAT-conjugated PEGlated Magnetic polymeric liposomes (TAT-PEG-MPLs) formed from PEGlated amphiphilic octadecyl quaternized carboxymethyl chitosan (PEG-OQCMC), cholesterol (Chol), superparamagnetic nanoparticles, and transactivating-transduction protein (TAT), were prepared successfully and evaluated the properties in vitro and in vivo. The result indicated that TAT-PEG-MPLs were spherical in solution, with significantly small mean diameter (83.2 nm) and excellent magnetism (magnetization saturation values of 43.5 emu/g). In vitro experiment, the uptake of PEG-MPLs with TAT by MCF-7 cells was greater than that of the PEG-MPLs without TAT. Most importantly, in vivo experiment, a low MRI signal was observed in the T2-weighted images; Histological analysis, Cryo-TEM and flame atomic absorption spectrophotometry revealed that TAT-PEG-MPLs nanoparticles significantly accumulated around the site of the SCI even inside the nerve cells. These nanoparticles may provide a promising carrier to locate to the lesion site, deliver therapeutic macromolecular agents across the BSCB and penetrate into the nerve cells for the treatment of SCI. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Chitin/chitosan Drug delivery Liposome, MRI(magnetic resonance imaging)Nanopartilce Surface modification
1. Introduction Blood-spinal cord barrier (BSCB) maintains a homeostatic environment in spinal cord (SC) and protects cord tissue by exhibiting selective permeability to plasma constituents [1]. However, due to the existence of BSCB, many therapeutic substances, such as cyclosporine A (CSA), neurotrophic factor, vascular endothelial growth factor (VEGF), excitatory amino acids (EAA), and C3, cannot pass through this barrier to reach the site of injury [2e7]. The BSCB is formed by special endothelial cells sealed with tight junctions. This unique membrane restricts the entry to the SC from the periphery. In clinical applications, high-dose therapeutic drugs are nonspecific and distributed randomly after feeding (or local injection) have been shown to be an effective treatment of spinal cord injuries (SCI). However, this high-dose therapy was accompanied with many adverse effects, such as an
* Corresponding author. Tel./fax: þ86 22 27401821. E-mail address: [email protected] (J. Chang). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.04.057
increased incidence of wound infection, low bioavailability of drugs in the setting of neural injury and an increased incidence of toxicity and side effect of drug [8]. Several approaches have been employed to circumvent this problem, including opening the tight junctions of the BSCB, the use of carrier-linked drugs and drugs embedded in nanoparticles through. While the opening of the tight junctions is so invasive that it may also allow the entry of dangerous contaminants into the setting of neural injury; the carrier-linked drugs is complicated and difficult because the therapeutic properties of the native drugs used may be altered after chemical conjugations; drugs embedded in nanoparticles, may be a better approach. Nanoparticles have recently emerged as promising carriers for drug delivery system. Over the past several decades, there has been a steady growth in the number of available nanoparticles for therapy. Among these products, polymer nanoparticles and liposomes are two dominant classes. Polymers, such as Polyethylene Glycol (PEG) [9], chitosan (CS) [10], poly(lactic-co-glycolic acid) (PLGA) [11] and so on, have been commonly used to form core-shell structure to encapsulate a variety of drugs. The advantages of liposomes include their
6590
H. Wang et al. / Biomaterials 31 (2010) 6589e6596
multilayer structure to improve the entrapment efficiency ofhydrophilic therapeutic agents, protect encapsulated drugs from external conditions and so on [12]. Recently, the TAT-PEG conjugated magnetic polymeric liposome (MPL) that combined both advantages of polymeric vesicles and liposomes, is developed in our laboratory. The NPs are formed from four materials: (1) PEGlated amphiphilic octadecyl quaternized carboxymethyl chitosan (PEG-OQCMC), forming a lamellar nanoshell, provided electrostatic and steric repulsion against particle aggregation, the ability to encapsulate high amounts of drugs, a longer circulation half-life in vivo, amino-groups for the attachment of targeting ligands such as antibodies, peptides [13]; (2) cholesterol (Chol) helped stabilize and maintain the structure of MPL; (3) superparamagnetic nanoparticles that responds to external permanent magnet with superparamagnetic characteristics, was chosen for the magnetic separation, non-invasive magnetic targeting [14] and MRI; (4) Transactivating-transduction protein (TAT), short cationic sequences with a remarkable capacity for membrane translocation [15], was employed for the delivery of the MPL across the Blood-spinal cord barrier (BSCB). Herein we report our studies on the systematic preparation and characterization of these TAT-PEG conjugated magnetic polymeric liposomes (MPLs). The properties, such as structure, morphology, size distribution, magnetic response, stability and the cellular penetration efficiency were evaluated. Further to this, the MPLs were injected through the caudal vein using a rat model of SCI and accumulation at the lesion site was evaluated through magnetic resonance imaging (MRI) tracing, histological analysis and electron microscopy. 2. Experimental method 2.1. Materials PEG-conjugated octadecyl quaternized carboxymethyl chitosan (PEG-OQCMC) and hydrophobic superparamagnetic nanoparticles, are all prepared in our lab. TAT peptide of the sequence Tyr-Gly-Arg-(Lys)2-(Arg)2-Gln-(Arg)3 was custom synthesized by Shanghai Sangon Biological Engineering Technology&Services Co. Ltd. N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and Fluorescein-5-
isothiocyanate (FITC) were purchased from Sigma-Aldrich. All other chemicals were of reagent grade and were used as received. 2.2. Synthesis and characterization of octadecyl quaternized carboxymethyl chitosan (OQCMC) and PEG-OQCMC The OQCMC was synthesized as reported in our previous work [13]. The quaternization of CMC was performed as follows. CMC (5 g) was dissolved in 100 mL of a mixture of deionized water saturated with isopropanol. QA was added slowly with different molar ratios to the glucosamine unit. An aqueous NaOH solution (42%, w/ w) was added and the mixture reacted at 80 C for 24 h with stirring. After dialysis against water performed for 4 days, the solution finally lyophilized to give OQCMC as a white powder. The monohydroxy -terminated PEG was converted to NHS-PEG according to the literature report [16]. A predetermined quantity of OQCMC was dissolved in purified water at a concentration of 5 mg/ml. NHS-PEG was dissolved in water free DMSO (50 mg/ml) solution. Subsequently, the OQCMC solution was added to the NHS-PEG solution and the mixture was stirred at room temperature for 24 h. After 24 h of stirring, the solution was purified by dialysis with a 10 000 MW cutoff membrane. This procedure was repeated at least three times and the dialyzed solution was finally freeze-dried. The 1H-NMR spectra of PEG-OQCMC was studied using a Bruker Avance 400 spectrometer (400 MHz), and D2O was used as the solvent. 2.3. Preparation and characterization of TAT-PEG conjugated magnetic polymeric liposomes (TAT-PEG-MPLs) Magnetic polymeric liposomes were prepared by reverse-phase evaporation (REV) method [17] (Fig. 2 A). PEG-OQCMC, hydrophobic superparamagnetic nanoparticles and cholesterol (weight ratio ¼ 2:0.5:1, gross mass 30 mg) were dissolved in 4 ml chloroform at room temperature to obtain the organic phase. About 6 ml deionized water was prepared to obtain aqueous phase. Then, aqueous phase was mixed with organic phase under sonication for 120 s at 100 W output. The organic solvents were evaporated on a rotary evaporator to form a gel-like MPL suspension. This MPL solution were then separated by magnet force and washed by deionized water for three times. The collected product was freeze-dried and saved. The MPLs were reacted with SPDP to yield a 2-pyridyldithiol-end group on the NPs surface. In brief, 2 ml of MPL solution (5 mg/ml) in 0.1 M phosphate buffer (pH ¼ 7.4) was mixed with 0.32 ml SPDP (2 mg) DMSO solution. The mixture was incubated at room temperature for 60 min. Low molecular weight impurities were dialyzed against PBS (0.1 M) for 5 h by dialysis bag (Mw ¼ 12 000). Then, above 2-pyridyl disulfide conjugated MPL solution was added with 1 ml TAT PBS (0.1 M) solution (5 mg/ml). The mixture was incubated overnight at 4 C to form a disulfide linkage between the surface of MPL and TAT peptide. The redundant TAT peptide was removed by magnetic separation and the obtained TAT-PEG conjugated MPLs(TAT-PEG-MPLs) solution was saved under 4 C. The FITC labeled polymeric liposomes were
Fig. 1. 1H-NMR spectrum of PEG-OQCMC.
H. Wang et al. / Biomaterials 31 (2010) 6589e6596 fabricated in a same way with PEG-OQMCMC replaced by FITC congjucated PEGOQMCMC. 2.4. Physicochemical characterizations of TAT-PEG-MPLs Morphology: The morphologies of different samples were observed via transmission electron microscopy (TEM). TEM observation of the sample was carried out
6591
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. Particle size and zeta potential analysis: The average particle size and size distribution were determined by quasielastic laser light scattering with a Brookhaven Zetasizer (Brookhaven Instruments Ltd., U.S.) at 25 C. About 0.2 mL of each
Fig. 2. (A) a schematic illustration shows the process of preparing TAT-PEG-MPLs by the reverse-phase evaporation method. (B) A transmission electron microscopy (TEM) image shows the nano-structure of the TAT-PEG-MPLs. (C) Pre-and post ultraviolet absorption changes of the reaction solution during the process of conjugating TAT onto the surface of PEG-MPLs. (D) The effective diameter and size distribution of TAT-PEG-MPLs measured by the particle size analyzer.
6592
H. Wang et al. / Biomaterials 31 (2010) 6589e6596 continuously as a monolayer at 37 C in a humidified atmosphere containing 5% CO2 in folate-deficient RPMI (FDRPMI) 1640 supplemented with 1% penicillin, 1% streptomycin, and 10% heat-inactivated fetal bovine serum (HIFBS). HIFBS contains endogeneous folate at concentrations sufficient for cells to grow in this medium. After 1 day of incubation, FITC-loaded samples with or without TAT were added to the growth media. The growth media were removed after 1 h,12 h and 24 h, and the cells were washed 3 times with PBS. The cells were then observed under an inverted fluorescence microscopy. 2.6. Animal model of spinal cord injury
Fig. 3. Magnetization curve of pure hydrophobic superparamagnetic nanoparticles (A) and TAT-PEG-MPLs (B) obstained by VSM. sample 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 (Brookhaven, U.S.). Zeta limits ranged from-150 to 150 V. Parameters were set as follows: strobe delay -1.00, on time 200.00 ms, and off time 1.00 ms. Magnetic Properties: Magnetic measurement of sample was performed by a vibrating sample magnetometer (VSM) from LakeShore Ltd. The samples were in the form of powder and were placed in Teflon sample holder. The magnetic measurements (hysteresis loops) were carried out in the field region of 1 T at room temperature. At 1 T, the magnetization of the samples was almost saturated. 2.5. Cell penetration tests by fluorescence microscopy The cell penetration tests of MPLs with and without TAT were performed in MCF-7 cells by labeling the samples with FITC. The cell line was cultured
The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL/100 g). The surgical area was shaved and disinfected with 70% ethanol and betadine. The spinous processes and vertebral plate were removed using a micro rongeur at T10, and the dura opened. The rat was then placed on an Impactor-2 machine, and the exposed spinal cord was struck using a 10 g 50 mm weight drop. In each case, the two hind limbs of the rat twitched involuntarily and the tail wagged, in accordance with the criteria of the SCI model. A total of 24 rats were randomly and evenly assigned to the two experimental groups. Another twelve rats were designated as treatment-free controls and were not included in the experimental groups. After surgery, the rats were placed in temperature- and humiditycontrolled incubation chambers until they awoke. They were then transferred to cages, and bladder evacuation was applied daily until the return of reflexive bladder control. 2.7. In vivo animal test Ten-week-old SD adult rats (250 g in weight)were injected with samples solution via tail vein. Animals were sacrificed at 72 h post-injection. MRI protocol: Rats were examined daily for a period of three days after injection using a 3.0 T MRI spectrometer (Signa HD 3.0 T, General Electric Company, Milwaukee, WI). Single sagittal, coronal, and transverse images were obtained by a fast gradient echo sequence for localizing subsequent T2-weighted transverse images measured with a standard turbo spin echo sequence. A “positive” MR signal was defined as a distinctive dark area in the T2-weighted images consisting of at least three contiguous pixels. Electron microscopy: Two-millimeter cross-sections of the spinal cord lesions were removed for ultra structural examination. Tissue samples were prepared for electron microscopy as follows: the spinal cord segments were removed and fixed in 2.5% glutaraldehyde for 24 h. Tissues were then post-fixed with OsO4, dehydrated in
Fig. 4. Fluorescence microscopy images of viable cells incubated with PEG-MPLs without TAT group (B) and with TAT (C).
H. Wang et al. / Biomaterials 31 (2010) 6589e6596 a graded alcohol series, and then embedded. Sections of 60e90 nm were stained with uranyl acetate and lead citrate, examined on an electron microscope (JEM 1200EX; Jeol, Tokyo, Japan), and photographed. The investigator who evaluated the sections was blinded to the group information. inverted fluorescence microscopy: Flame atomic absorption spectrophotometry: After perfusion, the length of the spinal cord collected from eight rats from each group was 4 cm. Spinal cords were maintained at 70 C with a feedback-controlled heating blanket for 48 h to facilitate dehydration. Dry weights were obtained. To eliminate the organic portions of the samples, each was treated with 1.5 mL of 65% HNO3 and 0.3 mL of 70% HClO4, digested with slow-heating until the organics dispersed totally, and then diluted with 1% HCl to 10 mL. Standards were prepared by diluting a 0.1 mg/mL Fe solution with 1% HCl. Fe concentrations were analyzed by flame atomic absorption spectrophotometry (Z-5000 atomic absorption spectrometer; Hitachi Ltd., Tokyo, Japan).
3. Results and discussion 3.1. Structural characterization of PEG-OQCMC From the 1H-NMR spectrum of PEG-OQCMC (Fig. 1), PEGOQCMC was successfully synthesized. The multiple peaks at d ¼ 3.5e3.7 were attributed to the protons of eCH2e groups in PEG [18], and the weak and multiple peaks at d ¼ 0.87 came from the protons of eCH3 groups on the quaternary ammonium salt. The weak and broad peak at d ¼ 4.66 was attributed to the protons of the pyranose of chitosan. The results indicated that the synthesis of PEG-OQCMC was successfully. 3.2. Formulation of TAT-conjugated PEGlated magnetic polymeric liposomes (TAT-PEG-MPLs) A schematic illustration for preparation TAT-PEG-MPL is presented in Fig. 2(A). The SPDP reagents are unique group of amineand sulfhydryl-reactive heterobifunctional cross-linkers. Amine groups exist on the surface of MPL NPs. Herein, the SPDP reagents were used to form amine-to-sulfhydryl cross-links among molecules [19]. Because TAT peptide contains sulfhydryl groups (-SH), PEG-MPL must be modified by the SPDP reagent. The modification of PEG-MPL with TAT results in displacement of a pyridine-2-thione group, which can be determined by measuring the absorbance at 343 nm. UV/Vis spectra of the reaction solutions were shown in Fig. 2(C). It can be seen that the absorbance peak at 343 nm was very clear for the solution after reaction, which approved the existence of pyridine-2-thione, all of which indicated that TAT was conjugated onto the surface of PEG-MPLs successfully. The mean
6593
diameters and polydispersity index (PDI) of TAT-PEG-MPLs were measured by dynamic light scattering (DLS) in Fig. 1(D). This data demonstrated reproducible formation of TAT-PEG-MPLs nanoparticles exhibiting significantly small mean diameter (83.2 nm) and PDI (0.193). TEM images was collected to show that these TATPEG-MPLs were spherical and nonaggregated in Fig. 2(B). TEM images showed individual, uniformly sized TAT-PEG-MPLs nanoparticles with similar diameters as calculated by DLS. 3.3. Study on the magnetic properties of TAT-PEG-MPLs The superparamagnetic colloids are promising to serve as a class of effective supports for guided delivery. In clinical applications, local injection of high-dose drugs, have been shown to be an effective treatment of spinal cord injuries (SCI). However, this high-dose therapy was accompanied with many adverse effects, such as side effects on healthy cells or conflict with other drugs in use for different diseases. Transportation of drugs to a specific site can eliminate such side effects and also reduce the dosage required. One simple way to improve targeted drug delivery is to use an external magnetic field as the guidance [20]. After biocompatible magnetic carriers containing therapeutic drugs are injected into the circulatory system, a magnetic field is applied to the targeted spinal cord to selectively collect the magnetic carriers at this site. The respond speed of biocompatible magnetic carriers to the external magnetic field is determined by the magnetization saturation values. Fig. 4 shows the hysteresis loop of TAT-PEGMPLs measured by using the vibrating sample magnetometer. It should be noted that the TAT-PEG-MPLs sample still shows high magnetization (43.5 emu/g), indicating its suitability for targeting and separation as a drug carrier. Moreover, the TAT-PEG-MPLs with homogenous dispersion show fast response to the external magnetic field due to its high magnetization and no residual magnetism is detected The result reveals that the TAT-PEG-MPLs exhibit good magnetic responsible and re-disperse properties, which suggests a potential application for magnetic targeting. 3.4. Transport of TAT-PEG-PLs into cells As seen in Fig. 3 cellular penetration of the polymeric liposomes with and without TAT was examined to demonstrate the penetration of the nanoparticles into the cells by using FITC fluorescent
Fig. 5. The magnetic resonance imaging (MRI) in vivo. (A) control group, (B) the PEG-MPL without TAT group, (C) the PEG-MPLs with TAT group.
6594
H. Wang et al. / Biomaterials 31 (2010) 6589e6596
label. The internalization of FITC conjugated samples incubated for 1 h was visualized by inverted fluorescence microscopy. After 1 h, TAT-peptide-liposomes were seen clustered in the perinuclear region with a reduced cytoplasmic distribution. Comparing with TAT-PEG-PLs at the same time point, just a little of the green fluorescence in PEG-PLs without TAT group existed in the cells. This is because the presence of the cell penetrating peptide on the surfaces of the nanoparticles promoted their cellular uptake. transactivator of transcription (Tat), having 10e27 amino acids in length, possesses multiple positive charges and penetrates by
a receptor-independent mechanism, to enhance the transport of cargoes across cell membranes and the BBB [21]. 3.5. Magnetic resonance imaging(MRI) in vivo As the TAT-PEG-MPLs used in this research possess superparamagnetic properties, the MRI signals can be reduced in T2weighted images that can be used to show the existence of magnetic NPs in CNS [22]. The accumulation and distribution of TAT-PEG-MPL in rats were examined for a period of 3 days by the
Fig. 6. The fluorescent images of the spinal cord tissue section. (A) the PEG-PLs without TAT group; (B) the PEG-PLs with TAT group.
H. Wang et al. / Biomaterials 31 (2010) 6589e6596
6595
Fig. 7. The cryo-TEM images of the spinal cord sections. the control group (A), PEG-MPLs without TAT nanoparticles (B), the TAT-PEG-MPLs group (C and D).
MR images. Signal changes in T2-weighted images before and after intravenous injection of TAT-PEG-MPL are depicted in Fig. 5. In the third day, the regions with signal drop surrounding the spinal cord injury areas increased significantly following TAT-PEG-MPLs injection and guideline by magnetic force. This phenomenon can be ascribed to the intrinsic accumulation of MPL in areas of magnetic field and sensitivity of T2-weighted sequences to iron NPs encapsulated in polymeric liposomes. Furthermore, these signal had the trend of diffusion into spinal cord parenchyma. This indicated that TAT-PEG-MPLs not only can be accumulated under the guideline of magnetic force, but also has the function of diffusion in animal parenchyma. Compared to TAT-PEG-MPLs group, the hypointense signal of PEG-MPLs without TAT was not obvious, almost same as the signal of untreated group.
but penetrate through the surrounding tissue and penetrate into the nerve cells. The conclusion was in accordance with the result got in the MCF-7 cells culture. The ability of TAT-PEG-PLs to cross the BBB and BSCB is mainly attributed to the fact of TAT, which can promoted theirpenetration by cells during a very short time.
3.6. Histological analysis To determine whether the FITC conjugated polymeric liposome with TAT can cross the BSCB, the distribution of FITC in the spinal cord sections of rats was observed 72 h after injection. FITC were unable to cross the BBB, which only existed in blood vessels [23]. The FITC-conjugated TAT-PEG-PLs nanoparticles crossed the BBB and BSCB (Fig. 6 (B)), penetrated through the surrounding tissue (Fig. 6 (B)) and penetrate into the nerve cells(Fig. 6 (C)). Moreover, the area of positive green fluorescence in TAT-PEG-PLs group was significantly more than that in PEG-PLs group without TAT. We can concluded that TAT-PEG-PLs cannot only cross the BBB and BSCB,
Fig. 8. Iron element content in the rats spinal cord Control group (A), PEG-MPLs group (B), TAT-PEG-MPLs group (C).
6596
H. Wang et al. / Biomaterials 31 (2010) 6589e6596
3.7. Electron microscopy analysis
References
The nerve cellular penetration of the TAT-PEG-MPLs nanoparticles in vivo was studied by means of transmission electron microscopy(TEM). TEM images show cross-sectional pictures of the cells where image intensity varies according to how a beam of electrons travels through the sample. The TAT-PEG-MPLs nanoparticles were clearly visible in the nerve cells (Fig. 7 Fig. 8). In contrast, no appreciable numbers of nanoparticles were found in the PEG-MPLs without TAT group. This suggests that the TAT efficiently mediated transfer of the liposome carriers across the membrane into the cells.
[1] Pan WH, Kastin AJ. Penetration of neurotrophins and cytokines across the bloodbrain blood spinal cord barrier. Adv Drug Deliv Rev 1999;36(2e3):291e8. [2] Sharma HS, Nyberg F, Westman J, Alm P, Gordh T, Lindholm D. Brain derived neurotrophic factor and insulin like growth factor-1 attenuate upregulation of nitric oxide synthase and cell injury following trauma to the spinal cord. An immunohistochemical study in the rat. Amino Acids 1998;14(1e3):121e9. [3] McTigue DM, Horner PJ, Stokes BT, Gage FH. Neurotrophin-3 and brainderived neurotrophic factor induce oligodendrocyte proliferation and myelination of regenerating axons in the contused adult rat spinal cord. J Neurosci 1998;18(14):5354e65. [4] Hauben E, Butovsky O, Nevo U, Yoles E, Moalem G, Agranov E, et al. Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion. J Neurosci 2000;20(17):6421e30. [5] Nordal RA, Nagy A, Pintilie M, Wong CS. Hypoxia and hypoxia-inducible factor-1 target genes in central nervous system radiation injury: a role for vascular endothelial growth factor. Clin Cancer Res 2004;10 (10):3342e53. [6] Springer JE, Azbill RD, Nottingham SA, Kennedy SE. Calcineurin-mediated BAD dephosphorylation activates the caspase-3 apoptotic cascade in traumatic spinal cord injury. J Neurosci 2000;20(19):7246e51. [7] Tan EYM, Law JWS, Wang CH, Lee AYW. Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons. Pharm Res 2007;24:2297e308. [8] Weaver LC, Gris D, Saville LR, Oatway MA, Chen YH, Marsh DR, et al. Methylprednisolone causes minimal improvement after spinal cord injury in rats, contrasting with benefits of an anti-integrin treatment. J Neurotrauma 2005;22(12):1375e87. [9] Burdick JA, Anseth KS. Photoencapsulation of osteoblasts in injectable RGDmodified PEG hydrogels for bone tissue engineering. Biomaterials 2002;23 (22):4315e23. [10] Kumar M. A review of chitin and chitosan applications. React Funct Polym 2000;46(1):1e27. [11] Jain RA. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 2000;21(23): 2475e90. [12] Lian T, Ho RJY. Trends and developments in liposome drug delivery systems. J Pharm Sci 2001;90(6):667e80. [13] Liang XF, Wang HJ, Tian H, Luo H, Chang J. Syntheis, structure and properties of novel quaternized carboxymethyl chitosan with drug loading capacity. Acta Phys-Chim Sin 2008;24(2):223e9. [14] Benyettou F, Lalatonne Y, Sainte-Catherine O, Monteil M, Motte L. Superparamagnetic nanovector with anti-cancer properties: gamma Fe2O3@Zoledronate. Int J Pharm 2009;379(2):324e7. [15] Berry CC. Progress in functionalization of magnetic nanoparticles for applications in biomedicine. J Phys D Appl Phys 2009;42(22). [16] Mao SR, Shuai XT, Unger F, Wittmar M, Xie XL, Kissel T. Synthesis, characterization and cytotoxicity of poly(ethylene glycol)-graft-trimethyl chitosan block copolymers. Biomaterials 2005;26(32):6343e56. [17] Ruel-Gariepy E, Leclair G, Hildgen P, Gupta A, Leroux JC. Thermosensitive chitosan-based hydrogel containing liposomes for the delivery of hydrophilic molecules. J Control Release 2002;82(2e3):373e83. [18] Jiang HL, Kwon JT, Kim EM, Kim YK, Arote R, Jere D, et al. Galactosylated poly (ethylene glycol)-chitosan-graft-polyethylenimine as a gene carrier for hepatocyte-targeting. J Control Release 2008;131(2):150e7. [19] Liang XF, Wang HJ, Luo H, Tian H, Zhang BB, Hao LJ, et al. Characterization of novel multifunctional cationic polymeric liposomes formed from octadecyl quaternized carboxymethyl chitosan/cholesterol and drug encapsulation. Langmuir 2008;24(14):7147e53. [20] Alexiou C, Schmid RJ, Jurgons R, Kremer M, Wanner G, Bergemann C, et al. Targeting cancer cells: magnetic nanoparticles as drug carriers. Eur Biophys J 2006;35(5):446e50. [21] Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999;285 (5433):1569e72. [22] Schwartz ED. MRI and the evaluation of the blood-spinal cord barrier following injury. Am J of Neuroradiol 2005;26(7):1609e10. [23] Li LH, Guo K, Lu J, Venkatraman SS, Luo D, Ng KC, et al. Biologically active core/shell nanoparticles self-assembled from cholesterol-terminated PEG-TAT for drug delivery across the blood-brain barrier. Biomaterials 2008;29(10):1509e17. [24] Salvador A, Pascual-Marti MC, Adell JR, Requeni A, March JG. Analytical methodologies for atomic spectrometric determination of metallic oxides in UV sunscreen creams. J Pharm Biomed Anal 2000;22(2):301e6.
3.8. Flame atomic absorption spectrophotometry As seen in Fig. 8 flame atomic absorption spectrophotometry method [24] was used for quantitative analysis of TAT-PEG-MPL content in spinal cord. The results indicated that the Fe content in the spinal cord lesion sites in TAT-PEG-MPLs group was 1.563 mmol/ mg which was significantly higher than that in either PEG-MPLs group (1.322 mmol/mg) or control group(1.296 mmol/mg). The Fe content of TAT-PEG-MPL group was statistically significant compared with that of PEG-MPL without TAT group (p < 0.05) and control group (p < 0.01), while the Fe content of PEG-MPL without TAT group and control group were not statistically significant (p > 0.05). It can be concluded that TAT-conjugated method can enhance iron accumulation significantly. 4. Conclusion PEG-conjugated magnetic polymeric liposomes, which is anchored with TAT, have been successfully constructed to cross the BSCB and aggregate at the site of the lesion in an injured spinal cord. The nanoparticle formulations, exhibiting significantly small mean diameter (83.2 nm) and superparamagnetism (magnetization saturation values of 43.5 emu/g), were characterized and evaluated in vitro and in vivo. The results indicated that TAT-PEGMPLs not only can be accumulated at injury area by magnetic force, but also can be used as MRI contrast agent in rat spinal cord injury model experiment. TAT conjugation can help the PEG-MPL cross the BSCB, penetrate through the surrounding tissue and penetrate into the nerve cells. The successful transport of TAT-PEG-MPLs nanoparticles into the spinal cord may open new opportunities for the treatment of spinal cord injuries. Acknowledgments The authors gratefully acknowledge National Basic Research Program of China (973 Program) 2007CB935800, National Natural Science Foundation of China (50873076), National Natural Science Foundation of China (30876203), and Tianjin Science and Technology Program (09ZCGYSF00900). Appendix Figures with essential discrimination. Figs. 1e7 in this article have parts that are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j. biomaterials.2010.04.057.