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Lactoferrin-modified nanoparticles could mediate efficient gene delivery to the brain in vivo
Brain Research Bulletin 81 (2010) 600–604
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Lactoferrin-modified nanoparticles could mediate efficient gene delivery to the brain in vivo Rongqin Huang, Weilun Ke, Liang Han, Yang Liu, Kun Shao, Chen Jiang ∗ , Yuanying Pei Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
a r t i c l e
i n f o
Article history: Received 5 November 2009 Received in revised form 11 December 2009 Accepted 15 December 2009 Available online 22 December 2009 Keywords: Lactoferrin Nanoparticles Brain-targeting Gene delivery
a b s t r a c t Lactoferrin (Lf)-modified nanoparticles (NPs) have been demonstrated to mediate efficient expression of exogenous genes in the brain via intravenous administration. The brain-targeting properties of Lfmodified NPs were investigated in this study. In vivo imaging results showed that the accumulation of Lfmodified NPs was higher in the brain but lower in the other organs than that of unmodified counterparts. The results of analytical transmission electron microscopy showed that some Lf-modified NPs crossed the blood–brain barrier (BBB) and reached the neural tissues, while some remained within the BBB. Similar results were observed in the distribution of exogenous gene products. All the results demonstrated the successful delivery of Lf-modified NPs into the brain. Lf-modified NPs could be exploited as potential brain-targeting delivery systems for exogenous genes, especially for those encoding secretive proteins. © 2009 Elsevier Inc. All rights reserved.
1. Introduction The blood–brain barrier (BBB), due to the presence of tight junctions and lack of fenestrae, is usually the rate-limiting factor for the penetration of proteins, peptides or genes into the central nervous system (CNS) [2]. Fortunately, the BBB possesses specific receptormediated transport mechanisms that potentially can be exploited as a means to target drugs to the brain [19,16]. Till date, receptors discovered on the BBB mainly include transferrin (Tf) receptors, insulin receptors, epidermal growth factor receptors, insulin-like growth factor receptors, and so on [10,11,23]. Ligand-mediated brain-targeting drug delivery is one of the focuses at present. The natural ligand, Tf, and the synthetic monoclonal antibody to Tf receptors, for example, have been extensively used as braintargeting ligands for constructing drug delivery systems to the brain [10,23,15,1]. Some other ligands such as Angiopep-2 to lowdensity lipoprotein receptor-related protein-1 (LRP1) and RVG29 to GABA (B) receptor have also been exploited for conjugating brain delivery systems [18,13,22,14]. Recently, lactoferrin (Lf) has been exploited as a novel braintargeting ligand in our lab. Lf is a single-chain iron-binding glycoprotein that belongs to the Tf family. One of the advantages of Lf to be used as a brain-targeting ligand is the low plasma concentration of endogenous Lf, approximately 5 nM [21]. The plasma concentration of Lf is much lower than the Kd of Lf receptors
∗ Corresponding author. Tel.: +86 21 5198 0079; fax: +86 21 5198 0079. E-mail address: [email protected] (C. Jiang). 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.12.008
in the BBB [9], efficiently avoiding the competitive inhibition of endogenous Lf to Lf-conjugated exogenous drug delivery systems. Furthermore, the transport of Lf across the BBB monolayer model was reported to be unidirectional, from the apical to the basolateral side [4]. This unidirectional transport might result in higher accumulation of Lf-conjugated drug delivery systems in the neuron, compared to Tf-conjugated counterparts. These advantages were partly reflected in a recent study, the results of which showed that exogenous gene expression of Lf-modified NPs in brains was about 2.3-fold of that of Tf-modified NPs [8]. Similar results were obtained in the biodistribution study of Lf-modified vectors and unmodified counterparts [8]. Hu et al. also reported that an approximate 3-fold of coumarin-6 was found in mice brains carried by Lf-modified NPs compared to that carried by unmodified counterparts [6]. In this study, the brain-targeting properties of Lf-modified NPs were characterized in vivo. The brain accumulation of different NPs was evaluated in macro-level using an in vivo imaging system and in microscopic level via an analytical transmission electron microscope. The expression of exogenous genes in the brain was also studied by staining Von Willebrand in the BBB. 2. Materials and methods 2.1. Materials Polyamidoamine (PAMAM) dendrimer [generation = 5, 21.43%, w/w solution in methanol, containing 128 surface primary amino groups (MW 28,826)], was purchased from Dentritech (Midland, MI, U.S.A.). ␣-Malemidyl--Nhydroxysuccinimidyl polyethyleneglycol (NHS-PEG-MAL, MW 3400) was obtained from Nektar Therapeutics (Huntsville, AL, U.S.A.). Ethidium monoazide bromide (EMA) was purchased from Molecular Probes (Eugene, OR, U.S.A.). Copper chloro-
R. Huang et al. / Brain Research Bulletin 81 (2010) 600–604 phyll was supplied by Qingdao Green Source Bioengineering Co., Ltd., China. Rabbit polyclonal to Von Willebrand Factor was purchased from Abcam (Cambridge, U.K.). Rhodamine labeled goat anti-rabbit IgG was obtained from Kirkegaard & Perry Laboratories (Maryland, U.S.A.). The plasmid DNA, pEGFP-N2 (Clontech, U.S.A.) coding green fluorescent protein (GFP) and pGL2-Control Vector (Promega, U.S.A.) coding luciferase, were purified by using QIAGEN Plasmid Mega Kit (Qiagen GmbH, Germany). Lactoferrin (Lf) from bovine colostrum, and other reagents, if not specified, were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Male balb/c mice (4–5 weeks old) of 20–25 g body weight and male nude mice (4–6 weeks old) of 18–20 g body weight were supplied by the Department of Experimental Animals, Fudan University, and maintained under standard housing conditions. All animal experiments were carried out in accordance with the guidelines evaluated and approved by the ethics committee of Fudan University. 2.2. Synthesis of Lf-modified vectors and preparation of Lf-modified NPs A 1:2:1 (mol/mol/mol) conjugate of PAMAM, PEG and Lf (designated as Lfmodified vectors) was synthesized successfully and the structure was characterized, as described previously [8]. Lf-modified vectors were freshly synthesized and diluted to appropriate concentrations in distilled water. In 50 mM sodium sulfate solution, plasmid DNA solution was added to obtain specified weight ratios (10:1, PAMAM to DNA, w/w) and immediately vortexed for 30 s at room temperature. Report genes encoding GFP were used to complex with Lf-modified vectors, formulating Lfmodified NPs. PAMAM/DNA complexes were designated as unmodified NPs. Agarose gel electrophoresis was carried out to verify the complete complexation of vectors with DNA. The immuno-gold staining technique was adopted to confirm the covalent conjugation of Lf in Lf-modified NPs [7]. Freshly prepared NPs were used in the following experiments. Plasmid DNA was covalently labeled with fluorescent dye, EMA, when necessary. Fresh plasmid DNA solution (1 mg/ml 0.05 M Tris–HCl buffer, pH 8.0) was diluted with aqueous solution of EMA (0.1 mg/ml) and incubated for 30 min in dark. The complex was then exposed to UV light for 1 h, and the resulting solution was precipitated by the addition of ethanol to a final concentration of 30% (v/v). The precipitate was collected by centrifugation and further dissolved in 50 mM sodium sulfate solution to obtain EMA-labeled plasmid DNA. 2.3. In vivo imaging analysis Lf-modified and unmodified NPs loading EMA-labeled pGL2-Control Vector (10:1, PAMAM to DNA, w/w) were injected into the tail vein of nude mice at a dose of 50 g DNA/mouse. About 4 h later, the mice were anesthetized and visualized using Cri in vivo imaging system (Cri, MA, U.S.A.). Then the mice were humanely killed, following which the principle organs (including brain, heart, liver, spleen, lung and kidney) were removed and also visualized using Cri in vivo imaging system. 2.4. Tracing Lf-modified NPs in the brain Lf-modified vectors were complexed with copper chlorophyll, 1:15 (mol/mol), for 4 h at room temperature in darkness. The resulting complexes were purified by ultrafiltration through a membrane (cutoff MW = 5 kDa) to remove free copper chlorophyll. The copper chlorophyll-labeled Lf-modified vectors were used to prepare copper chlorophyll-labeled Lf-modified NPs for in vivo tracking. The copper chlorophyll-labeled Lf-modified NPs loading pGL2-Control Vector (10:1, PAMAM to DNA, w/w) were injected into the tail vein of balb/c mice at a dose of 50 g DNA/mouse. About 1 h later, the animals were humanely sacrificed. The midbrains were rapidly removed, fixed in 2.5% glutaraldehyde for 3 h, and divided to nubs of 1 mm3 . The nubs were fixed in 2.5% glutaraldehyde for another 3 h, rinsed with PBS, and further postfixed in 1% osmium tetroxide solution. Such pretreated brain nubs were dehydrated gradiently and then embedded in the epoxy resin. The dried embedded nubs were cut into ultra-thin sections (60 nm) with an ultramicrotome (LKB, Sweden). After being stained with uranyl acetate and lead citrate, the specimens were observed in an analytical transmission electron microscope (Philips, Germany). Normal mice without the administration of NPs were treated as described above and served as controls. 2.5. Gene expression of Lf-modified NPs in the brain Lf-modified NPs loading pEGFP-N2 (10:1, PAMAM to DNA, w/w) were injected into the tail vein of balb/c mice at a dose of 50 g DNA/mouse. Two days later, the animals were humanely sacrificed. The brains were rapidly removed, fixed in cold 4% paraformaldehyde for 48 h, and then transferred to PBS containing 30% sucrose at 4 ◦ C until subsidence. Frozen coronal sections of the midbrain of 20 m thickness were cut and rinsed in PBS with a cryostat. Then the sections were incubated in 0.25% Triton X-100 for 30 min and then blocked with 2% normal goat serum (in 5% BSA) for 2 h. After that, the sections were incubated with anti-Von Willebrand Factor polyclonal antibody (1:200) overnight at 4 ◦ C, PBS rinsed, then incubated with secondary rhodamine labeled antibody for 1 h at 37 ◦ C. Finally, the sections were rinsed with PBS and mounted on slides. Digital photomicrographs were captured
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with an Olympus IX51 microscope. Sections not treated with anti-Von Willebrand Factor polyclonal antibody were used as controls.
3. Results 3.1. In vivo imaging of mice administrated with different NPs Nude mice were administered with EMA-labeled Lf-modified NPs, and unmodified NPs as control. In vivo fluorescent images were taken at 4 h after injection. As shown in Fig. 1, EMA-labeled DNA was obviously accumulated in the brain of the mouse treated with Lf-modified NPs (Fig. 1B and F), the fluorescence of which was much stronger than that of unmodified NPs (Fig. 1A and E). Furthermore, the fluorescence relatively concentrated in the central region of the brain, which might be mainly dependent on the distribution of Lf receptors. On the contrast, the accumulation of Lf-modified NPs in the liver (Fig. 1D and F) was much lower than that of unmodified NPs (Fig. 1C and E), indicating the potential function of PEGylation and Lf modification. Strong fluorescence observed in the right abdomen of mice (Fig. 1C and D) might be the autofluorescence of food. 3.2. In vivo tracking of Lf-modified NPs The specimens of brain tissues of those intravenously injected mice and normal mice were observed using an analytical transmission electron microscope. The result showed that some particles with the information of copper located at the wall of brain capillary (Fig. 2B) and some distributed within the neural tissues (Fig. 2C). The size of these compact and spherical particles was around 200 nm, concluded to be copper chlorophyll-labeled Lf-modified NPs. No similar particles were found in brain tissues of control mice (Fig. 2A). 3.3. Gene expression of Lf-modified NPs in the brain The distribution of GFP in midbrain sections of balb/c mice was observed under a fluorescent microscope, using rhodamine-labeled anti-Von Willebrand Factor polyclonal antibody to stain the brain capillaries. As shown in Fig. 3, part of green fluorescence of gene product, GFP, did not co-localized with the red fluorescence of rhodamine-labeled brain capillaries (Fig. 3A–C and G), indicating that part of Lf-modified NPs crossed the BBB and GFP could be found in neural tissues. Otherwise, part of NPs remained within the BBB and directly mediated the gene expression of Lf-modified NPs in the BBB (Fig. 3C and H). No red fluorescence was found in sections without the treatment of anti-Von Willebrand Factor polyclonal antibody (Fig. 3D), while the gene expression could still be observed (Fig. 3E and F). 4. Discussion The brain-targeting delivery of Lf-modified NPs was verified in this study. The results fully demonstrated the brain-targeting potential of Lf-modified NPs via several separate means. Several lines of evidence demonstrated the presence of specific Lf receptors in the brain [21,3,20]. Talukder et al. carried out a binding assay for Lf receptors with 125 I-Lf and revealed a Kd of 0.11 M of epithelial membranes of the choroid plexus in young calves [21]. Our previous studies also provided direct evidence of receptor-mediated endocytosis for Lf in brain capillary endothelial cells (BCECs) and membrane preparations isolated from the brain of mice [9]. The results showed that Lf receptors exhibited at least two classes of binding sites, with high or low affinity to Lf, in the BBB and brain tissues [9]. Based on these studies, Lf was
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Fig. 1. In vivo imaging of nude mice administrated with different NPs. A was the dorsal image of mice injected with unmodified NPs while C was the corresponding ventral image. B was the dorsal image of mice injected with Lf-modified NPs while D was the corresponding ventral image. E and F were main organs obtained from unmodified and Lf-modified NPs, respectively. Images were taken 4 h after treatments.
Fig. 2. Observation of Lf-modified NPs using analytical transmission electron micrographs. The midbrain tissues of balb/c mice without any treatment (A) and intravenously injected with copper chlorophyll-labeled Lf-modified NPs (B and C) were shown. The NPs were indicated by black arrows in B and C. Bar = 200 nm.
R. Huang et al. / Brain Research Bulletin 81 (2010) 600–604
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Fig. 3. Localization of gene expression of Lf-modified NPs in the midbrain using a fluorescent microscope. Brain sections were immunostained with (A) or without (D) rhodamine-labeled anti-Von Willebrand Factor polyclonal antibody. B and E were images of GFP expression. C was the merged picture of A and B, while F was that of D and E. The circles indicated GFP expressed in neural cells, and the quadrangles in white indicated the brain capillaries without gene expression (C). Green: GFP. Red: rhodamine. Bar = 100 m. G and H were magnified insets (bar = 20 m) of quadrangles in orange in C. The arrow in H indicated the GFP expression in the BBB (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
exploited as a novel ligand for designing brain-targeting drug delivery systems and exhibited great brain delivery effects (Fig. 1) [8,6]. Detailed brain delivery and localization of Lf-modified NPs was also demonstrated in this study. In fact, the distribution of Lf receptors in the brain differs in some pathological conditions. In parkinsonian patients, Lf receptors have been reported to be increased on neurons and microvessels and more pronounced in those regions of the midbrain where the loss of dopaminergic neurons is severe (PD) [3,5]. On the other hand, almost all the current drug delivery systems would remain in part within the BBB via intravenous administrations. Although the transport of Lf across the BCECs monolayer was reported as unidirectional, from the apical to the basolateral side [4], this phenomenon still exists. Lf-modified NPs were found to locate at both the BBB and neural tissues (Fig. 2), and similar result was observed in the distribution of gene products (Fig. 3). Enhanced brain delivery of therapeutic genes might at the same time increase their accumulation within the BBB, which was not the final target. Therefore, therapeutic genes encoding secreted proteins such as the glial cell line-derived neurotrophic factor (GDNF) are preferred. GDNF has been reported to functionalize in nerve cells in a paracrine and autocrine manner [24,12]. It will meet the therapeutic demand in whatever kind of cells that express these neurotrophic factors, because they will finally secrete out for their function. Moreover, GDNF have been in the forefront of PD treatments [17]. In conclusion, Lf-modified NPs might be one of the most potential delivery systems for gene therapy of PD.
Acknowledgments This work was supported by the grants from National Basic Research Program (2007CB935802) of China (973 Program), and National Natural Science Foundation of China (30901861 and 30973652). References [1] R.J. Boado, Y. Zhang, Y. Wang, W.M. Pardridge, Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood–brain barrier delivery in the mouse, Biotechnol. Bioeng. 102 (2009) 1251– 1258. [2] M. Demeule, J.C. Currie, Y. Bertrand, C. Ché, T. Nguyen, A. Régina, R. Gabathuler, J.P. Castaigne, R. Béliveau, Involvement of the low-density lipoprotein receptorrelated protein in the transcytosis of the brain delivery vector angiopep-2, J. Neurochem. 106 (2008) 1534–1544. [3] B.A. Faucheux, N. Nillesse, P. Damier, G. Spik, A. Mouatt-Prigent, A. Pierce, B. Leveugle, N. Kubis, J.J. Hauw, Y. Agid, Expression of lactoferrin receptors is increased in the mesencephalon of patients with Parkinson disease, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 9603–9607. [4] C. Fillebeen, L. Descamps, M.P. Dehouck, L. Fenart, M. Benaissa, G. Spik, R. Cecchelli, A. Pierce, Receptor-mediated transcytosis of lactoferrin through the blood–brain barrier, J. Biol. Chem. 274 (1999) 7011–7017. [5] A.J. Grau, V. Willig, W. Fogel, E. Werle, Assessment of plasma lactoferrin in Parkinson’s disease, Mov. Disord. 16 (2001) 131–134. [6] K. Hu, J. Li, Y. Shen, W. Lu, X. Gao, Q. Zhang, X. Jiang, Lactoferrin-conjugated PEG-PLA nanoparticles with improved brain delivery: in vitro and in vivo evaluations, J. Control. Release 134 (2009) 55–61. [7] R.Q. Huang, W.L. Ke, L. Han, Y. Liu, K. Shao, L.Y. Ye, J.N. Lou, C. Jiang, Y.Y. Pei, Brain-targeting mechanisms of lactoferrin-modified DNA-loaded nanoparticles, J. Cereb. Blood Flow Metab. (2009) (on line).
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[16] M.M. Patel, B.R. Goyal, S.V. Bhadada, J.S. Bhatt, A.F. Amin, Getting into the brain: approaches to enhance brain drug delivery, CNS Drugs 23 (2009) 35–58. [17] S. Ramaswamy, K.E. Soderstrom, J.H. Kordower, Trophic factors therapy in Parkinson’s disease, Prog. Brain Res. 175 (2009) 201–216. [18] A. Régina, M. Demeule, C. Ché, I. Lavallée, J. Poirier, R. Gabathuler, R. Béliveau, J.P. Castaigne, Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector angiopep-2, Br. J. Pharmacol. 155 (2008) 185–197. [19] J. Rip, G.J. Schenk, A.G. de Boer, Differential receptor-mediated drug targeting to the diseased brain, Expert Opin. Drug Deliv. 6 (2009) 227–237. [20] Y.A. Suzuki, B. Lonnerdal, Baculovirus expression of mouse lactoferrin receptor and tissue distribution in the mouse, Biometals 17 (2004) 301–309. [21] M.J. Talukder, T. Takeuchi, E. Harada, Receptor-mediated transport of lactoferrin into the cerebrospinal fluid via plasma in young calves, J. Vet. Med. Sci. 65 (2003) 957–964. [22] F.C. Thomas, K. Taskar, V. Rudraraju, S. Goda, H.R. Thorsheim, J.A. Gaasch, R.K. Mittapalli, D. Palmieri, P.S. Steeg, P.R. Lockman, Q.R. Smith, Uptake of ANG1005, a novel paclitaxel derivative, through the blood–brain barrier into brain and experimental brain metastases of breast cancer, Pharm. Res. (2009) (on line). [23] K. Ulbrich, T. Hekmatara, E. Herbert, J. Kreuter, Transferrin- and transferrinreceptor-antibody-modified nanoparticles enable drug delivery across the blood–brain barrier (BBB), Eur. J. Pharm. Biopharm. 71 (2009) 251– 256. [24] E. Woodhall, A.K. West, M.I. Chuah, Cultured olfactory ensheathing cells express nerve growth factor, brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor and their receptors, Brain Res. Mol. Brain Res. 88 (2001) 203–213.
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Lactoferrin-modified nanoparticles could mediate efficient gene delivery to the brain in vivo Rongqin Huang, Weilun Ke, Liang Han, Yang Liu, Kun Shao, Chen Jiang ∗ , Yuanying Pei Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
a r t i c l e
i n f o
Article history: Received 5 November 2009 Received in revised form 11 December 2009 Accepted 15 December 2009 Available online 22 December 2009 Keywords: Lactoferrin Nanoparticles Brain-targeting Gene delivery
a b s t r a c t Lactoferrin (Lf)-modified nanoparticles (NPs) have been demonstrated to mediate efficient expression of exogenous genes in the brain via intravenous administration. The brain-targeting properties of Lfmodified NPs were investigated in this study. In vivo imaging results showed that the accumulation of Lfmodified NPs was higher in the brain but lower in the other organs than that of unmodified counterparts. The results of analytical transmission electron microscopy showed that some Lf-modified NPs crossed the blood–brain barrier (BBB) and reached the neural tissues, while some remained within the BBB. Similar results were observed in the distribution of exogenous gene products. All the results demonstrated the successful delivery of Lf-modified NPs into the brain. Lf-modified NPs could be exploited as potential brain-targeting delivery systems for exogenous genes, especially for those encoding secretive proteins. © 2009 Elsevier Inc. All rights reserved.
1. Introduction The blood–brain barrier (BBB), due to the presence of tight junctions and lack of fenestrae, is usually the rate-limiting factor for the penetration of proteins, peptides or genes into the central nervous system (CNS) [2]. Fortunately, the BBB possesses specific receptormediated transport mechanisms that potentially can be exploited as a means to target drugs to the brain [19,16]. Till date, receptors discovered on the BBB mainly include transferrin (Tf) receptors, insulin receptors, epidermal growth factor receptors, insulin-like growth factor receptors, and so on [10,11,23]. Ligand-mediated brain-targeting drug delivery is one of the focuses at present. The natural ligand, Tf, and the synthetic monoclonal antibody to Tf receptors, for example, have been extensively used as braintargeting ligands for constructing drug delivery systems to the brain [10,23,15,1]. Some other ligands such as Angiopep-2 to lowdensity lipoprotein receptor-related protein-1 (LRP1) and RVG29 to GABA (B) receptor have also been exploited for conjugating brain delivery systems [18,13,22,14]. Recently, lactoferrin (Lf) has been exploited as a novel braintargeting ligand in our lab. Lf is a single-chain iron-binding glycoprotein that belongs to the Tf family. One of the advantages of Lf to be used as a brain-targeting ligand is the low plasma concentration of endogenous Lf, approximately 5 nM [21]. The plasma concentration of Lf is much lower than the Kd of Lf receptors
∗ Corresponding author. Tel.: +86 21 5198 0079; fax: +86 21 5198 0079. E-mail address: [email protected] (C. Jiang). 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.12.008
in the BBB [9], efficiently avoiding the competitive inhibition of endogenous Lf to Lf-conjugated exogenous drug delivery systems. Furthermore, the transport of Lf across the BBB monolayer model was reported to be unidirectional, from the apical to the basolateral side [4]. This unidirectional transport might result in higher accumulation of Lf-conjugated drug delivery systems in the neuron, compared to Tf-conjugated counterparts. These advantages were partly reflected in a recent study, the results of which showed that exogenous gene expression of Lf-modified NPs in brains was about 2.3-fold of that of Tf-modified NPs [8]. Similar results were obtained in the biodistribution study of Lf-modified vectors and unmodified counterparts [8]. Hu et al. also reported that an approximate 3-fold of coumarin-6 was found in mice brains carried by Lf-modified NPs compared to that carried by unmodified counterparts [6]. In this study, the brain-targeting properties of Lf-modified NPs were characterized in vivo. The brain accumulation of different NPs was evaluated in macro-level using an in vivo imaging system and in microscopic level via an analytical transmission electron microscope. The expression of exogenous genes in the brain was also studied by staining Von Willebrand in the BBB. 2. Materials and methods 2.1. Materials Polyamidoamine (PAMAM) dendrimer [generation = 5, 21.43%, w/w solution in methanol, containing 128 surface primary amino groups (MW 28,826)], was purchased from Dentritech (Midland, MI, U.S.A.). ␣-Malemidyl--Nhydroxysuccinimidyl polyethyleneglycol (NHS-PEG-MAL, MW 3400) was obtained from Nektar Therapeutics (Huntsville, AL, U.S.A.). Ethidium monoazide bromide (EMA) was purchased from Molecular Probes (Eugene, OR, U.S.A.). Copper chloro-
R. Huang et al. / Brain Research Bulletin 81 (2010) 600–604 phyll was supplied by Qingdao Green Source Bioengineering Co., Ltd., China. Rabbit polyclonal to Von Willebrand Factor was purchased from Abcam (Cambridge, U.K.). Rhodamine labeled goat anti-rabbit IgG was obtained from Kirkegaard & Perry Laboratories (Maryland, U.S.A.). The plasmid DNA, pEGFP-N2 (Clontech, U.S.A.) coding green fluorescent protein (GFP) and pGL2-Control Vector (Promega, U.S.A.) coding luciferase, were purified by using QIAGEN Plasmid Mega Kit (Qiagen GmbH, Germany). Lactoferrin (Lf) from bovine colostrum, and other reagents, if not specified, were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Male balb/c mice (4–5 weeks old) of 20–25 g body weight and male nude mice (4–6 weeks old) of 18–20 g body weight were supplied by the Department of Experimental Animals, Fudan University, and maintained under standard housing conditions. All animal experiments were carried out in accordance with the guidelines evaluated and approved by the ethics committee of Fudan University. 2.2. Synthesis of Lf-modified vectors and preparation of Lf-modified NPs A 1:2:1 (mol/mol/mol) conjugate of PAMAM, PEG and Lf (designated as Lfmodified vectors) was synthesized successfully and the structure was characterized, as described previously [8]. Lf-modified vectors were freshly synthesized and diluted to appropriate concentrations in distilled water. In 50 mM sodium sulfate solution, plasmid DNA solution was added to obtain specified weight ratios (10:1, PAMAM to DNA, w/w) and immediately vortexed for 30 s at room temperature. Report genes encoding GFP were used to complex with Lf-modified vectors, formulating Lfmodified NPs. PAMAM/DNA complexes were designated as unmodified NPs. Agarose gel electrophoresis was carried out to verify the complete complexation of vectors with DNA. The immuno-gold staining technique was adopted to confirm the covalent conjugation of Lf in Lf-modified NPs [7]. Freshly prepared NPs were used in the following experiments. Plasmid DNA was covalently labeled with fluorescent dye, EMA, when necessary. Fresh plasmid DNA solution (1 mg/ml 0.05 M Tris–HCl buffer, pH 8.0) was diluted with aqueous solution of EMA (0.1 mg/ml) and incubated for 30 min in dark. The complex was then exposed to UV light for 1 h, and the resulting solution was precipitated by the addition of ethanol to a final concentration of 30% (v/v). The precipitate was collected by centrifugation and further dissolved in 50 mM sodium sulfate solution to obtain EMA-labeled plasmid DNA. 2.3. In vivo imaging analysis Lf-modified and unmodified NPs loading EMA-labeled pGL2-Control Vector (10:1, PAMAM to DNA, w/w) were injected into the tail vein of nude mice at a dose of 50 g DNA/mouse. About 4 h later, the mice were anesthetized and visualized using Cri in vivo imaging system (Cri, MA, U.S.A.). Then the mice were humanely killed, following which the principle organs (including brain, heart, liver, spleen, lung and kidney) were removed and also visualized using Cri in vivo imaging system. 2.4. Tracing Lf-modified NPs in the brain Lf-modified vectors were complexed with copper chlorophyll, 1:15 (mol/mol), for 4 h at room temperature in darkness. The resulting complexes were purified by ultrafiltration through a membrane (cutoff MW = 5 kDa) to remove free copper chlorophyll. The copper chlorophyll-labeled Lf-modified vectors were used to prepare copper chlorophyll-labeled Lf-modified NPs for in vivo tracking. The copper chlorophyll-labeled Lf-modified NPs loading pGL2-Control Vector (10:1, PAMAM to DNA, w/w) were injected into the tail vein of balb/c mice at a dose of 50 g DNA/mouse. About 1 h later, the animals were humanely sacrificed. The midbrains were rapidly removed, fixed in 2.5% glutaraldehyde for 3 h, and divided to nubs of 1 mm3 . The nubs were fixed in 2.5% glutaraldehyde for another 3 h, rinsed with PBS, and further postfixed in 1% osmium tetroxide solution. Such pretreated brain nubs were dehydrated gradiently and then embedded in the epoxy resin. The dried embedded nubs were cut into ultra-thin sections (60 nm) with an ultramicrotome (LKB, Sweden). After being stained with uranyl acetate and lead citrate, the specimens were observed in an analytical transmission electron microscope (Philips, Germany). Normal mice without the administration of NPs were treated as described above and served as controls. 2.5. Gene expression of Lf-modified NPs in the brain Lf-modified NPs loading pEGFP-N2 (10:1, PAMAM to DNA, w/w) were injected into the tail vein of balb/c mice at a dose of 50 g DNA/mouse. Two days later, the animals were humanely sacrificed. The brains were rapidly removed, fixed in cold 4% paraformaldehyde for 48 h, and then transferred to PBS containing 30% sucrose at 4 ◦ C until subsidence. Frozen coronal sections of the midbrain of 20 m thickness were cut and rinsed in PBS with a cryostat. Then the sections were incubated in 0.25% Triton X-100 for 30 min and then blocked with 2% normal goat serum (in 5% BSA) for 2 h. After that, the sections were incubated with anti-Von Willebrand Factor polyclonal antibody (1:200) overnight at 4 ◦ C, PBS rinsed, then incubated with secondary rhodamine labeled antibody for 1 h at 37 ◦ C. Finally, the sections were rinsed with PBS and mounted on slides. Digital photomicrographs were captured
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with an Olympus IX51 microscope. Sections not treated with anti-Von Willebrand Factor polyclonal antibody were used as controls.
3. Results 3.1. In vivo imaging of mice administrated with different NPs Nude mice were administered with EMA-labeled Lf-modified NPs, and unmodified NPs as control. In vivo fluorescent images were taken at 4 h after injection. As shown in Fig. 1, EMA-labeled DNA was obviously accumulated in the brain of the mouse treated with Lf-modified NPs (Fig. 1B and F), the fluorescence of which was much stronger than that of unmodified NPs (Fig. 1A and E). Furthermore, the fluorescence relatively concentrated in the central region of the brain, which might be mainly dependent on the distribution of Lf receptors. On the contrast, the accumulation of Lf-modified NPs in the liver (Fig. 1D and F) was much lower than that of unmodified NPs (Fig. 1C and E), indicating the potential function of PEGylation and Lf modification. Strong fluorescence observed in the right abdomen of mice (Fig. 1C and D) might be the autofluorescence of food. 3.2. In vivo tracking of Lf-modified NPs The specimens of brain tissues of those intravenously injected mice and normal mice were observed using an analytical transmission electron microscope. The result showed that some particles with the information of copper located at the wall of brain capillary (Fig. 2B) and some distributed within the neural tissues (Fig. 2C). The size of these compact and spherical particles was around 200 nm, concluded to be copper chlorophyll-labeled Lf-modified NPs. No similar particles were found in brain tissues of control mice (Fig. 2A). 3.3. Gene expression of Lf-modified NPs in the brain The distribution of GFP in midbrain sections of balb/c mice was observed under a fluorescent microscope, using rhodamine-labeled anti-Von Willebrand Factor polyclonal antibody to stain the brain capillaries. As shown in Fig. 3, part of green fluorescence of gene product, GFP, did not co-localized with the red fluorescence of rhodamine-labeled brain capillaries (Fig. 3A–C and G), indicating that part of Lf-modified NPs crossed the BBB and GFP could be found in neural tissues. Otherwise, part of NPs remained within the BBB and directly mediated the gene expression of Lf-modified NPs in the BBB (Fig. 3C and H). No red fluorescence was found in sections without the treatment of anti-Von Willebrand Factor polyclonal antibody (Fig. 3D), while the gene expression could still be observed (Fig. 3E and F). 4. Discussion The brain-targeting delivery of Lf-modified NPs was verified in this study. The results fully demonstrated the brain-targeting potential of Lf-modified NPs via several separate means. Several lines of evidence demonstrated the presence of specific Lf receptors in the brain [21,3,20]. Talukder et al. carried out a binding assay for Lf receptors with 125 I-Lf and revealed a Kd of 0.11 M of epithelial membranes of the choroid plexus in young calves [21]. Our previous studies also provided direct evidence of receptor-mediated endocytosis for Lf in brain capillary endothelial cells (BCECs) and membrane preparations isolated from the brain of mice [9]. The results showed that Lf receptors exhibited at least two classes of binding sites, with high or low affinity to Lf, in the BBB and brain tissues [9]. Based on these studies, Lf was
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Fig. 1. In vivo imaging of nude mice administrated with different NPs. A was the dorsal image of mice injected with unmodified NPs while C was the corresponding ventral image. B was the dorsal image of mice injected with Lf-modified NPs while D was the corresponding ventral image. E and F were main organs obtained from unmodified and Lf-modified NPs, respectively. Images were taken 4 h after treatments.
Fig. 2. Observation of Lf-modified NPs using analytical transmission electron micrographs. The midbrain tissues of balb/c mice without any treatment (A) and intravenously injected with copper chlorophyll-labeled Lf-modified NPs (B and C) were shown. The NPs were indicated by black arrows in B and C. Bar = 200 nm.
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Fig. 3. Localization of gene expression of Lf-modified NPs in the midbrain using a fluorescent microscope. Brain sections were immunostained with (A) or without (D) rhodamine-labeled anti-Von Willebrand Factor polyclonal antibody. B and E were images of GFP expression. C was the merged picture of A and B, while F was that of D and E. The circles indicated GFP expressed in neural cells, and the quadrangles in white indicated the brain capillaries without gene expression (C). Green: GFP. Red: rhodamine. Bar = 100 m. G and H were magnified insets (bar = 20 m) of quadrangles in orange in C. The arrow in H indicated the GFP expression in the BBB (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
exploited as a novel ligand for designing brain-targeting drug delivery systems and exhibited great brain delivery effects (Fig. 1) [8,6]. Detailed brain delivery and localization of Lf-modified NPs was also demonstrated in this study. In fact, the distribution of Lf receptors in the brain differs in some pathological conditions. In parkinsonian patients, Lf receptors have been reported to be increased on neurons and microvessels and more pronounced in those regions of the midbrain where the loss of dopaminergic neurons is severe (PD) [3,5]. On the other hand, almost all the current drug delivery systems would remain in part within the BBB via intravenous administrations. Although the transport of Lf across the BCECs monolayer was reported as unidirectional, from the apical to the basolateral side [4], this phenomenon still exists. Lf-modified NPs were found to locate at both the BBB and neural tissues (Fig. 2), and similar result was observed in the distribution of gene products (Fig. 3). Enhanced brain delivery of therapeutic genes might at the same time increase their accumulation within the BBB, which was not the final target. Therefore, therapeutic genes encoding secreted proteins such as the glial cell line-derived neurotrophic factor (GDNF) are preferred. GDNF has been reported to functionalize in nerve cells in a paracrine and autocrine manner [24,12]. It will meet the therapeutic demand in whatever kind of cells that express these neurotrophic factors, because they will finally secrete out for their function. Moreover, GDNF have been in the forefront of PD treatments [17]. In conclusion, Lf-modified NPs might be one of the most potential delivery systems for gene therapy of PD.
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