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Published as doi: 10.1096/fj.06-7380com.
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(The FASEB Journal. 2007;21:1117-1125.)
© 2007 FASEB

Efficient gene delivery targeted to the brain using a transferrin-conjugated polyethyleneglycol-modified polyamidoamine dendrimer

Rong-Qin Huang, Ying-Hua Qu, Wei-Lun Ke, Jian-Hua Zhu, Yuan-Ying Pei and Chen Jiang1

Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, China

1Correspondence: Department of Pharmaceutics, School of Pharmacy, Fudan University, P.O. Box 130, 200032, Shanghai, China. E-mail: jiangchen{at}shmu.edu.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The blood-brain barrier (BBB) poses great difficulties for gene delivery to the brain. To circumvent the BBB, we investigated a novel brain-targeting gene vector based on the nanoscopic high-branching dendrimer, polyamidoamine (PAMAM), in vitro and in vivo. Transferrin (Tf) was selected as a brain-targeting ligand conjugated to PAMAM via bifunctional polyethyleneglycol (PEG), yielding PAMAM-PEG-Tf. UV and nuclear magnetic resonance (NMR) spectroscopy were used to evaluate the synthesis of vectors. The characteristics and biodistribution of gene vectors were evaluated by fluorescent microscopy, flow cytometry, and a radiolabeling method. The transfection efficiency of vector/DNA complexes in brain capillary endothelial cells (BCECs) was evaluated by fluorescent microscopy and determination of luciferase activity. The potency of vector/DNA complexes was evaluated by using frozen sections and measuring tissue luciferase activity in Balb/c mice after i.v. administration. UV and NMR results demonstrated the successful synthesis of PAMAM-PEG-Tf. This vector showed a concentration-dependent manner in cellular uptake study and a 2.25-fold brain uptake compared with PAMAM and PAMAM-PEG in vivo. Transfection efficiency of PAMAM-PEG-Tf/DNA complex was much higher than PAMAM/DNA and PAMAM-PEG/DNA complexes in BCECs. Results of tissue expression experiments indicated the widespread expression of an exogenous gene in mouse brain after i.v. administration. With a PAMAM/DNA weight ratio of 10:1, the brain gene expression of the PAMAM-PEG-Tf/DNA complex was ~2-fold higher than that of the PAMAM/DNA and PAMAM-PEG/DNA complexes. These results suggested that PAMAM-PEG-Tf can be exploited as a potential nonviral gene vector targeting to brain via noninvasive administration.—Huang, R-Q., Qu, Y-H., Ke, W-L., Zhu, J-H., Pei, Y-Y., Jiang, C. Efficient gene delivery targeted to the brain using a transferrin-conjugated polyethyleneglycol-modified polyamidoamine dendrimer.


Key Words: brain targeting • PAMAM • transferrin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE BLOOD-BRAIN BARRIER (BBB) protects the central nervous system (CNS) from potentially harmful xenobiotics and endogenous molecules (1) . Despite this beneficial role, >98% of candidate brain-targeting drugs have been halted mid-development due to the poor permeability of the BBB, presenting a major problem to the pharmaceutical industry (2 , 3) . Another striking problem is to develop gene therapy drugs that offer the promise of an effective cure for both genetic and acquired brain diseases, since the majority of these diseases do not respond well to small molecule drugs and have no effective long-term therapy (4) . Most current gene vectors do not cross the BBB after an i.v. administration and must be given via craniotomy or intracerebral injection, which are considered to be highly invasive and unable to deliver exogenous genes to global areas of brain (4 , 5) . Brain gene targeting technology, including both viral and nonviral delivery, enables widespread expression of exogenous genes throughout the CNS after an i.v. injection (6) . But given the evident side effects of viral vectors, the goal of brain gene targeting technology is the efficient, noninvasive, and nonviral gene therapy of the brain.

The BBB does possess specific receptor-mediated transport mechanisms that potentially can be exploited as a means to target drugs to brain (7 , 8) . Receptors expressed on the BBB, including transferrin (Tf) receptor and insulin receptor, have been used for brain-targeting drug delivery research (9) . The Tf receptor is of particular interest because its expression in capillaries throughout the body is restricted to brain capillaries (10) . The most accomplished work in the field of brain targeting with colloidal gene carriers has been carried out with PEGylated immunoliposomes, which access the brain from blood via Tf receptor-mediated transcytosis and deliver exogenous genes into the brain parenchyma without damaging the BBB (11 , 12) . PEGylated immunonanoparticles formed by conjugation of an antitransferrin receptor monoclonal antibody (mAb) (OX26) to polyethyleneglycol (PEG)-poly(lactic acid) (PLA) have also been prepared and may enable the targeting delivery of gene medicines to brain (13) .

Polyamidoamine (PAMAM) dendrimers have emerged as a new class of nanoscopic, spherical polymers that have captured the interest of researchers in various scientific disciplines over the past few years. It is becoming increasingly evident that PAMAM is a multifunctional polymer for which several applications have been discovered. PAMAM dendrimers have been reported recently as carriers for anticancer drugs (e.g., 5-fluorouracil) (14) , as solubility enhancers for ketoprofen (15) , and as delivery vehicles for antisense and siRNA oligonucleotides (16) . In addition, PAMAM has proved to be an efficient gene carrier itself (17) . PAMAM with primary amino surface groups has the inherent ability to associate with and condense DNA, and has been used for biocompatible high-efficiency DNA delivery (18 , 19) . Enhanced transfection efficiency has been reported by surface modification of PAMAM with L-arginine (20) . Moreover, the primary amines located on the surface of PAMAM make it possible to conjugate suitable ligands, such as Tf, for efficient brain-targeting gene delivery.

The objective of this work was to develop an efficient vector for gene transfer into the brain. Tf-conjugated, PEG-modified PAMAM, designated PAMAM-PEG-Tf, was synthesized. Cellular uptake, body distribution, and exogenous gene expression of conjugated or unconjugated PAMAM complexes with DNA were evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The plasmid pEGFP-N2 (Clontech, Palo Alto, CA, USA) and pGL2-control vector (Promega, Madison, WI, USA) were purified using QIAGEN Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). PAMAM G5 dendrimer [5% w/w solution in methyl alcohol, containing 128 surface primary amino groups (MW 28,826)], human holo-transferrin, Bolton and Hunter reagent, and 2-iminothiolane hydrochloride (Traut’s reagent) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5,5-Dithiobis(2-nitrobenzoic acid) (Ellmann’s reagent) was purchased from Acros (Geel, Belgium). {alpha}-Malemidyl-{omega}-N-hydroxysuccinimidyl polyethyleneglycol (NHS-PEG-MAL, MW 3400) was obtained from Nektar Therapeutics (Huntsville, AL, USA). The BODIPY fluorophore (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, sulfosuccinimidyl ester, sodium salt), and 4',6-diamidino-2-phenylindole (4',6'-diam idino-2-phenylidole (DAPI)) were purchased from Molecular Probes (Eugene, OR, USA). DNase I was purchased from Takara (Dalian, China).

Animals
Male Balb/c mice (4–5 wk old) of 20–25 g body weight were purchased from the Department of Experimental Animals, Fudan University, and maintained under standard housing conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University.

Cell line
Brain capillary endothelial cells (BCECs) were kindly provided by Prof. J. N. Lou (the Clinical Medicine Research Institute of the Chinese-Japanese Friendship Hospital). Primary BCECs were cultured as described previously (21) . Briefly, BCECs were expanded and maintained in special Dulbecco’s modified Eagle medium (Sigma-Aldrich) supplemented with 20% heat-inactivated fetal calf serum (FCS), 100 µg/ml epidermal cell growth factor, 2 mmol/L L-glutamine, 20 µg/ml heparin, 40 µU/ml insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin and cultured at 37°C under a humidified atmosphere containing 5% CO2. All cells used in this study were between passage 10 and passage 20.

125I-radiolabeling of PAMAM
Bolton and Hunter reagent (3 µg) was radiolabeled with 125I (3.96x107 Bq) as described previously with an 81.4% yield (22) . 125I-Labeled Bolton and Hunter reagent in benzene was carefully dried under a stream of nitrogen. PAMAM (10 mg) was dissolved in borate buffer (pH 8.5, 0.1 M), added to the 125I-labeled Bolton and Hunter reagent (2.74x107 Bq), and allowed to react for 15 min on ice, with period mixing. The 125I-labeled PAMAM solution was carefully purified by dialysis against NaCl (1%) and stored at 4°C until use.

Synthesis of PAMAM derivatives
PAMAM was reacted with NHS-PEG3400-MAL 1:2 (mol/mol) in PBS (pH 8.0) for 15 min at room temperature. The primary amino groups on the surface of PAMAM were specifically reacted with the NHS groups of the bifunctional PEG derivative. The resulting conjugate, PAMAM-PEG, was purified by ultrafiltration through a membrane (cutoff=5 kDa) and the buffer was changed to PBS (pH 7.0). At the same time, Tf was thiolated using Traut’s reagent as described (23) , and the extent of thiolation was determined by Ellmann’s reagent (24) (~3 thiol groups per Tf). Then PAMAM-PEG was reacted with thiolated Tf, 1:1 (mol/mol) in PBS (pH 7.0) for 24 h at room temperature. The MAL groups of PAMAM-PEG were specifically reacted with the thiol groups of thiolated Tf. For synthesis of BODIPY-labeled conjugates, PAMAM was first reacted with BODIPY in 100 mM NaHCO3 for 12 h at 4°C, then purified and identified as described (16) . The corresponding conjugates—BODIPY-labeled PAMAM, BODIPY-labeled PAMAM-PEG, and BODIPY-labeled PAMAM-PEG-Tf—were synthesized as described above. For the synthesis of 125I-labeled conjugates, 125I-labeled PAMAM was used.

Characterization of PAMAM-PEG-Tf
The characteristics of PAMAM-PEG-Tf were analyzed by UV-Vis and nuclear magnetic resonance (NMR) spectroscopy. For the UV-Vis study, three samples (BODIPY-labeled PAMAM-PEG, BODIPY-labeled PAMAM-PEG-Tf, and BODIPY-labeled PAMAM-PEG+free Tf) were scanned in the range of 200–600 nm in a UV 2401PC spectrophotometer (Shimadzu, Japan). For NMR analysis, PAMAM-PEG-Tf was solubilized in D2O and analyzed in a 400 MHz spectrometer (Varian, Palo Alto, CA, USA).

Cellular uptake of dendrimers
BCECs were seeded at a density of 8 x 104 cells/well in 6-well plates (Corning-Coaster, Tokyo, Japan), incubated for 72 h, and checked under the microscope for confluency and morphology. After this, BCECs were incubated with BODIPY-labeled PAMAM derivatives (BODIPY-labeled PAMAM, BODIPY-labeled PAMAM-PEG, or BODIPY-labeled PAMAM-PEG-Tf) in a PAMAM concentration range of 0.035 to 1.750 µM for 60 min. The cells were washed three times with PBS (pH 7.4) and visualized under an IX2-RFACA fluorescent microscope (Olympus, Osaka, Japan). For quantitative analysis, BCECs treated as described above were trypsinized and centrifuged at 1600 rpm for 8 min to obtain a cell pellet, which was subsequently resuspended in PBS (pH 7.4) and analyzed using a flow cytometer (FACSCalibur, BD Biosciences, Bedford, MA, USA) equipped with an argon ion laser (488 nm) as the excitation source. The fluorescence of BODIPY was collected at 520 nm (FL1). For each sample, 10,000 events were collected and data were analyzed with CELLQuest software. BCECs cultured under normal conditions served as the control. Living cells were defined by gating the major population of the cells, and only cells within this gate were analyzed. The mean fluorescence intensity of the cells was calculated using the histogram plot.

Body distribution of 125I-labeled dendrimers
Balb/c mice were injected via the tail vein with 125I-labeled dendrimers (125I-PAMAM, 125I-PAMAM-PEG, or 125I-PAMAM-PEG-Tf) at a dose of 1.48 x 106 Bq/mouse. At 2 h after injection, a blood sample (0.2 ml) was collected. Then mice were anesthetized with diethyl ether, killed by decapitation, and the principal organs (including brain, heart, liver, spleen, lung, and kidney) were removed and weighed. The radioactivity of 125I in the blood and organ samples was assessed using a gamma counter and the results expressed as percentage of dose administered accumulating in each organ (% ID/g).

Preparation of dendrimer/DNA complexes
Dendrimers (PAMAM, PAMAM-PEG, and PAMAM-PEG-Tf) were freshly prepared and diluted to appropriate concentrations in distilled water. DNA solution (100 µg DNA/ml 50 mM sodium sulfate solution) was added to obtain specified weight ratios (6:1 or 10:1, PAMAM to DNA, w/w) and immediately vortexed for 30 s at room temperature. Agarose gel electrophoresis was carried out to verify the complete complexation of dendrimers with DNA. Freshly prepared complexes were used in the experiments that follow.

Stability of complexes against DNase I digestion
Each complex solution with PAMAM to DNA at weight ratio of 6:1 was divided into equal triplicates. One served as a control and the other two were incubated with DNase I at 37°C for 2 h at a final DNase I concentration of 50 U/g plasmid DNA. Three microliters of EDTA (0.5 M) was added to one of these two solutions to immediately stop DNA degradation, then 3 µl SDS (10%, w/v) was added to displace DNA. Naked DNA, with and without the DNase I treatment, served as a control. Finally, 0.7% agarose gel electrophoresis was performed to evaluate the integrity of DNA in the complexes.

Efficiency of gene expression in BCECs
BCECs were seeded in 24-well plates at a density of 2.5 x 104 cells/well. Cultured for 48 h, cells reached 70% confluence. Complexes containing pEGFP, with PAMAM to DNA at weight ratios of 6:1 and 10:1, were added to the cells in FCS-free medium and the mixture was incubated at 37°C for 1 h. After 48 h, fluorescence images were acquired and photographed under a fluorescence microscope. The efficiency of gene transfer for quantitative analysis was determined by luciferase activity using pGL2-control vector. Two days post-transfection, the activity of luciferase, the gene product, was quantified by the Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, cells were lysed in a sufficient volume of cell culture lysis buffer (Promega). The lysate was vortexed for 15 s, then centrifuged at 12,000 g for 2 min at 4°C. The supernatant was assayed for luciferase activity and the emitted light was measured for 10 s using a chemiluminescence analyzer (BPCL, Peking, China) yielding light units. The light units were normalized to protein amounts in the cell extracts, determined by the Bio-Rad Protein Assay Kit (25) , to light units/mg protein.

Distribution of gene expression qualitatively in mouse brain
Complexes containing EGFP (10:1, PAMAM to DNA, w/w) were injected into the tail vein of mice at a dose of 50 µg DNA/mouse. About 48 h later, animals were anesthetized with diethyl ether and killed by decapitation. The brains were removed, fixed in 4% paraformaldehyde for 48 h, placed in 15% sucrose PBS solution until subsidence (~6 h), then in 30% sucrose until subsidence (~24 h). After this, brains were frozen in OCT embedding medium (Sakura, Torrance, CA, USA) at –80°C. Frozen sections of 20 µm thickness were prepared with a cryotome Cryostat (Leica, CM 1900, Wetzlar, Germany) and stained with 300 nM DAPI for 10 min at room temperature. After washing twice with PBS (pH 7.4), the sections were immediately examined under the fluorescence microscope.

In vivo gene quantitative expression
Complexes containing the pGL2 control vector (10:1, PAMAM to DNA, w/w) were injected into the tail vein of mice at a dose of 50 µg DNA/mouse. At 48 h after injection, the mice were humanely decapitated and the principal organs (including brain, heart, liver, spleen, lung, and kidney) were extirpated. The organs were carefully washed with distilled water and homogenized in 1 ml lysis reagent (Promega, Madison, WI, USA) using a JY92-II tissue homogenizer. The homogenate was centrifuged at 14,000 g for 20 min at 4°C. Luciferase activity and cellular proteins in the supernatant were quantified by a Luciferase Assay System and Bio-Rad Protein Assay Kit, respectively. The results were expressed as light units/mg protein.

Statistical analysis
All of the quantitative measurements were collected in quadruplicate and the experiments were repeated four times. The data are expressed as mean ± SE. Statistical analysis was performed by one-way ANOVA, followed by Bonferroni’s post-test using Stata software (version 7.0).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of PAMAM-PEG-Tf
The UV peaks characteristic of BODIPY and Tf were present at 504 nm and 280 nm (data not shown), respectively. As shown in Fig. 1 A, BODIPY-labeled PAMAM-PEG, BODIPY-labeled PAMAM-PEG-Tf, and BODIPY-labeled PAMAM-PEG plus free Tf all demonstrated the BODIPY peak at 504 nm. BODIPY-labeled PAMAM-PEG demonstrated no characteristic peak of Tf. The mixture of BODIPY-labeled PAMAM-PEG and free Tf demonstrated the characteristic peak of Tf at 280 nm. The spectrum of BODIPY-labeled PAMAM-PEG-Tf was different from that of BODIPY-labeled PAMAM-PEG and the mixture, which preliminarily demonstrated the synthesis of conjugates. In NMR spectra, the solvent peak of D2O was found at 4.65 ppm. The methylene protons of branching units of PAMAM and a series of protons of Tf have multiple peaks between 2.2 and 3.4 ppm. The NMR spectrum of PAMAM-PEG had a characteristic peak of the MAL group in PEG at 6.7 ppm (data not shown). However, the MAL peak disappeared in the NMR spectrum of PAMAM-PEG-Tf, whereas the repeat units of PEG still presented a sharp peak at 3.6 ppm (Fig. 1B ), showing that the MAL group had reacted with the thiol group of Tf. The UV and NMR spectra together provide a good picture of the conjugate structure.


Figure 1
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  		Figure 1. A) UV-Vis spectra of BODIPY-labeled PAMAM-PEG (a), BODIPY-labeled PAMAM-PEG-Tf (b), and BODIPY-labeled PAMAM-PEG plus free Tf (c) in the range of 200–600 nm. Peak 1 is from BODIPY and peak 2 is from Tf. B) NMR spectra of BODIPY-labeled PAMAM-PEG-Tf in D2O at 400 MHz.

Uptake characteristics of dendrimers by BCECs in vitro
BODIPY-labeled dendrimers were used to investigate cellular uptake characteristics, the results of which are shown qualitatively using fluorescent images and quantitatively as a percentage of BODIPY-positive cells (% BODIPY-positive cells). BCECs treated with BODIPY-labeled PAMAM-PEG-Tf exhibited fluorescence intensity corresponding to concentrations of BODIPY-labeled PAMAM-PEG-Tf (Fig. 2 ). When the concentration of BODIPY-labeled PAMAM-PEG-Tf ranged from 0.035 to 1.750 µM after 60 min exposure, the % BODIPY-positive cells increased from 32.97% to 79.70% and mean fluorescence intensity increased from 30.61 to 93.91 (Fig. 2) . The cellular uptake of BODIPY-labeled PAMAM, BODIPY-labeled PAMAM-PEG, and BODIPY-labeled PAMAM-PEG-Tf all exhibited concentration-dependent modes. When the concentration of dendrimers changed from 0.350 to 1.050 µM, both the % BODIPY-positive cells and mean fluorescence intensity increased correspondingly (Fig. 3 ). At a fixed concentration, BODIPY-labeled PAMAM-PEG was taken up by BCECs more efficiently than BODIPY-labeled PAMAM, and the uptake was significantly reduced when modified with Tf (Fig. 3) .


Figure 2
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  		Figure 2. Cellular uptake of BODIPY-labeled PAMAM-PEG-Tf in the concentration of 0.035 µM (B, E), 0.105 µM (C, F), 0.350 µM (G, J), 1.050 µM (H, K), and 1.750 µM (I, L) was examined by fluorescent microscopy and flow cytometry after a 60 min incubation. Normal BCECs without any treatment served as the control (A, D). The number of BODIPY-positive cells was analyzed by setting a gate according to the control. Numbers shown in inset refer to % BODIPY-positive cells and mean fluorescence intensity, respectively. Green: BODIPY. Original magnification: x200.


Figure 3
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  		Figure 3. Cellular uptake of BODIPY-labeled PAMAM (A, D, G, J), BODIPY-labeled PAMAM-PEG (B, E, H, K) and BODIPY-labeled PAMAM-PEG-Tf (C, F, I, L) in the concentrations of 0.350 µM (A–F) and 1.050 µM (G–L) was examined by fluorescent microscopy and flow cytometry after a 60 min incubation. The number of BODIPY-positive cells was analyzed by setting a gate according to the control. Numbers shown in inset refer to % BODIPY-positive cells and mean fluorescence intensity, respectively. Green: BODIPY. Original magnification: x200.

Body distribution of 125I-labeled dendrimers
The biodistribution of 125I-PAMAM 2 h after i.v. administration was almost unchanged when modified with PEG. The concentrations of 125I-PAMAM-PEG-Tf in the brain, heart, liver, spleen, lung, and kidney were significantly increased (P<0.01) compared with that of 125I-PAMAM and 125I-PAMAM-PEG, whereas the blood clearance was not markedly changed (P>0.05) (Fig. 4 ). Using 125I-PAMAM as a comparison, 125I-PAMAM-PEG-Tf increased the brain level by 2.25-fold, heart level by 1.98-fold, the liver level by 4.35-fold, the spleen level by 5.96-fold, lung level by 4.58-fold, and the kidney level by 3.69-fold at 2 h after injection.


Figure 4
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  		Figure 4. Plots of the biodistribution of 125I-labeled dendrimers in Balb/c mice 2 h after i.v. administration. Percentages of the injected dose per gram of tissue (% ID/g) in brain (A), blood, and other principal organs (B) are shown. ***P < 0.001. Data are expressed as mean ± SE (n=4).

Stability of complexes against DNase I digestion
Naked DNA was digested when treated with DNase I (Fig. 5 ). All three complexes could encapsulate DNA completely and protect it from digestion by DNase I. When treated further with SDS (an intensely electronegative substance that can displace DNA in the complexes) after DNase I digestion, plasmid DNA recovered from the complexes remained nearly intact.


Figure 5
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  		Figure 5. The stability of DNA-loaded complexes against DNase I digestion. Lane 1: DNA Marker, HindIII digested; lane 2: naked plasmid DNA; lane 3: naked plasmid DNA treated with DNase I. Agarose gel electrophoresis was performed independently to evaluate the amount of DNA in PAMAM/DNA, PAMAM-PEG/DNA, and PAMAM-PEG-Tf/DNA complexes without any treatment (lanes 4–6), with treatment with DNase I (lanes 7–9) and with further treatment with SDS (lanes 10–12).

Efficiency of gene expression in BCECs
Two days post-transfection, the green fluorescence of the green fluorescent protein (GFP) was observed qualitatively using an optical microscope and luciferase activity was quantitatively analyzed by flow cytometry of BCECs. For the different complexes (PAMAM/DNA, PAMAM-PEG/DNA, and PAMAM-PEG-Tf/DNA), an increase in the weight ratio of PAMAM to DNA resulted in an increase in gene expression in BCECs. Using BCECs treated with PAMAM/DNA complex as a comparison, the number of GFP-positive cells did not change markedly after treatment with the PAMAM-PEG/DNA complex, but increased when treated with the PAMAM-PEG-Tf/DNA complex at a fixed PAMAM-to-DNA weight ratio (Fig. 6 A–F). This was verified by a luciferase activity assay. With a PAMAM to DNA weight ratio of 6:1, the luciferase activity in BCECs treated with PAMAM-PEG-Tf/DNA complex was 4.19 ± 0.73 x 105 units/mg protein, which was ~1.8-fold of that with PAMAM/DNA complex (2.29±0.13x105 units/mg protein) and PAMAM-PEG/DNA complex (2.41±0.33x105 units/mg protein). With a PAMAM/DNA weight ratio of 10:1, luciferase activity in BCECs treated with PAMAM-PEG-Tf/DNA complex was 6.57 ± 0.89 x 105 units/mg protein, a ~2-fold higher gene expression than that with the PAMAM/DNA complex (3.31±0.63x105 units/mg protein) and the PAMAM-PEG/DNA complex (3.75±0.25x105 units/mg protein) (Fig. 6G ).


Figure 6
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  		Figure 6. The qualitative and quantitative evaluation of gene expression in vitro. The fluorescence images of GFP expression in BCECs were taken 48 h post-transfection with PAMAM/DNA (A, D), PAMAM-PEG/DNA (B, E), and PAMAM-PEG-Tf/DNA (C, F) complexes, with a PAMAM to DNA weight ratio of 6:1 (A–C) and 10:1 (D–F), respectively. BCECs were incubated with different complexes for 1 h. Green: GFP. Original magnification: x200. G) Luciferase activity was measured 48 h post-transfection, as described in Materials and Methods, and was expressed as light units per mg protein. **P < 0.01. Data represent the mean ± SE (n=4).

Qualitative analysis of distribution of gene expression in the mouse brain
GFP expression in sections of the cortical layer, hippocampus, caudate putamen, substantia nigra, and 4th ventricle at 48 h after i.v. injection of 50 µg/mouse of DNA complexed with PAMAM, PAMAM-PEG, or PAMAM-PEG-Tf are shown in Fig. 7 A–O. For PAMAM/DNA and PAMAM-PEG/DNA complexes, the pEGFP-N2 gene was expressed in the hippocampus, substantia nigra, and 4th ventricle, but was almost nonexistent in the cortical layer and caudate putamen (Fig. 7A-J ). The gene expression of the PAMAM-PEG/DNA complex in the substantia nigra was higher than that of the PAMAM/DNA complex (Fig. 7D, I ). For the PAMAM-PEG-Tf/DNA complex, gene expression was observed in all the five regions and was much higher than that of the PAMAM/DNA and PAMAM-PEG/DNA complexes (Fig. 7K-O ).


Figure 7
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  		Figure 7. The qualitative and quantitative evaluation of gene expression in vivo. Distribution of gene expression in brains of Balb/c mice treated with PAMAM/DNA (A–E), PAMAM-PEG/DNA (F–J), and PAMAM-PEG-Tf/DNA (K–O) complexes 2 days after i.v. administration. Frozen sections (20 µm thick) of cortical layer (A, F, K), hippocampus (B, G, L), caudate putamen (C, H, M), substantia nigra (D, I, N), and 4th ventricle (E, J, O) were examined by fluorescent microscopy. The sections were stained with 300 nM DAPI for 10 min at room temperature. Green: GFP. Blue: cell nuclei. Original magnification: x100. P, Q) Luciferase expression 48 h after i.v. administration of PAMAM/DNA, PAMAM-PEG/DNA, and PAMAM-PEG-Tf/DNA complexes into Balb/c mice at a dose of 50 µg DNA/mouse. Luciferase expression is plotted as light units per mg protein. ***P < 0.001 compared with the PAMAM/DNA complex. Data are expressed as mean ± SE (n=4).

Quantitative in vivo gene expression
To examine the transfection efficiencies of PAMAM/DNA, PAMAM-PEG/DNA, and PAMAM-PEG-Tf/DNA complexes in animals, complexes were injected via the tail veins of Balb/c mice, and the expression of pGL2-control vector in principal organs was measured after 48 h (Fig. 7P, Q ). The brain gene expression of the PAMAM-PEG-Tf/DNA complex was 3.08 ± 0.38 x 103 units/mg protein, ~2-fold higher than that of the PAMAM/DNA complex (1.46±0.41x103 units/mg protein) and PAMAM-PEG/DNA complex (1.35±0.20x103 units/mg protein) (Fig. 7P ). Gene expression of the PAMAM-PEG-Tf/DNA complex in the heart, lung, and kidney was significantly increased (P<0.01) compared with that of PAMAM/DNA and PAMAM-PEG/DNA complexes, whereas liver and spleen levels were not changed markedly (P>0.05) (Fig. 7Q ). Using the luciferase level of the PAMAM/DNA complex as a comparison, the PAMAM-PEG-Tf/DNA complex increased the heart level by 2.32-fold, liver level by 1.87-fold, lung level by 2.45-fold, and kidney level by 2.01-fold 48 h after injection. The PAMAM-PEG/DNA complex increased the spleen level by 1.64-fold. For all three kinds of complexes, gene expression in the heart was the highest.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The slow development of gene delivery vector is a major limiting factor of brain gene therapy. The goals of this work were to construct a Tf-mediated, brain-targeting, highly efficient gene delivery vector, PAMAM-PEG-Tf conjugate, and to investigate its characteristics in vitro and in vivo.

Tf is considered to possess great potential as a brain-targeting ligand (26) . The transferrin receptor is expressed both at the BBB and neuronal cell membrane (4) . The trans-vascular delivery of genes is possible by accessing transferrin-conjugated transport systems, which might cross the BBB and deliver genes to the doorstep of every neuron in the brain (4 , 11 , 12) . However, little information is presently available about the application of Tf as a ligand for gene delivery to the brain in vivo. In this study, PAMAM was chosen as the basic polymer vector and further modified with PEG and Tf to form a brain-targeting drug carrier nanoscale in size. PAMAM has recently received increasing attention in gene therapy as a new class of nanoscopic spherical polymer. PAMAM G5 closely mimics the size of biomolecules and has been used in several medical research areas, especially gene delivery in vitro (27 , 28) . The biocompatibility of the gene vector is further enhanced by modification with PEG, a widely used hydrophilic polymer in drug delivery systems (29) . In this study, PEG also served as a spacer in transferrin-modified conjugate. The conjugation of Tf at the tip of the PEG tail profoundly increased the targeting capability of PAMAM-PEG-Tf to the brain.

The results of UV and NMR spectroscopy demonstrated the successful synthesis of PAMAM-PEG-Tf. The characteristics and behavior of different vectors were investigated using BCECs and visualized with BODIPY, respectively. The rank order of cellular uptake was PAMAM-PEG > PAMAM > PAMAM-PEG-Tf (Fig. 3) . It has been reported that the modification of low-generation PAMAM with biocompatible PEG molecules would create a conjugate of PAMAM core with flexible PEG chains, which mimics the fractured high-generation dendrimer and produces high transfection efficiency (18) . Thus, PAMAM-PEG was taken up by BCECs more efficiently than PAMAM. However, modification with Tf caused a significant decrease in cellular uptake (Fig. 3) . The association of PAMAM-PEG-Tf with BCECs increased depending on both concentration and time (data not shown), whereas association of PAMAM and PAMAM-PEG was less dependent on concentration. This implies that PAMAM-PEG-Tf was internalized into BCECs via a different mechanism from PAMAM and PAMAM-PEG. The modification with PEG and Tf, which increased steric hindrance and decreased the positive charges, might make the cellular uptake mechanisms of PAMAM-PEG-Tf more complicated. In other words, it may be based mainly on receptor-mediated endocytosis and less on adsorptive-mediated mechanisms.

To further track the vectors’ fate after systemic administration in Balb/c mice, PAMAM labeled with 125I was used to synthesize different dendrimer derivatives. Modification by Tf greatly increased the biodistribution of PAMAM-PEG-Tf in the mouse brain (Fig. 4A ), probably due to the large amount of Tf receptors expressed on the BBB. All the conjugates were most densely accumulated in the kidney. This tropism is in good agreement with an earlier work where 111In- or 88Y-labeled PAMAM showed mainly renal accumulation, which was attributed to the strongly positive property of PAMAM by the researchers (30) . At 2 h after injection, the systemic distribution patterns of PAMAM and PAMAM-PEG were indistinguishable, suggesting that PEGylation alone did not affect the relative distribution of the dendrimers. The systemic distribution of dendrimers indicates a good potential of PAMAM-PEG-Tf to be exploited as an efficient carrier for drugs, including genes targeted to the brain.

In this study, different cationic dendrimers were used as gene vectors to complex DNA. All the dendrimers could completely encapsulate and protect DNA from degradation by DNase I (Fig. 5) . The PAMAM-PEG-Tf/DNA complex showed higher GFP expression and up to a 2-fold increase in luciferase activity compared with PAMAM/DNA and PAMAM-PEG/DNA complexes (Fig. 6) , suggesting that Tf facilitates the expression of DNA complexed with PAMAM-PEG-Tf in BCECs, presumably by receptor-mediated endocytosis. The ligand/receptor interaction is considered a type of physiological process, and the Tf-conjugated DNA-loaded complexes are more easily released into the cytoplasm (31) . Moreover, due to the low-density charges of PEG molecules, DNA is more likely to escape from the PAMAM-PEG-Tf/DNA complex once inside cells (18) .

Results of the in vivo studies in brain gene expression are also consistent with the in vitro transfection studies. When tested in vivo, the highest gene expression in the brain was again produced by the PAMAM-PEG-Tf/DNA complex vs. that with the PAMAM/DNA and PAMAM-PEG/DNA complexes (Fig. 7A-P ). In vivo gene targeting to organs such as brain that have continuous capillaries of restricted permeability has two main barriers: the microvascular endothelial barrier and the plasma membrane barrier of target cells (11) . The presence of the two barriers profoundly limits the expression of exogenous genes after transvascular administration. It was reported that exogenous gene expression could be observed in limited regions around cerebral ventricles due to their relatively weak BBB (32) . Ding et al. demonstrated that StarburstTM PAMAM dendrimer/DNA complexes could penetrate the BBB quickly and showed much more evident GFP expression in brain than that of a naked DNA group (33) . This might be attributed to the transepithelial and endothelial capability of PAMAM dendrimers (34) . In this study, the conjugation of Tf with PAMAM-PEG greatly increased gene expression in the brain. The high expression of the Tf receptors in both barriers (the BBB and the neuronal plasma membrane) enables the widespread expression of an exogenous gene encapsulated in PAMAM-PEG-Tf after i.v. administration. This is in harmony with the fact that OX26 PEGylated immunoliposomes exhibited extended gene expression in brain via i.v. administration (11) . In addition, we noted an interesting discrepancy in the biodistribution of 125I-labeled dendrimers and dendrimer/DNA complexes. Whereas 125I-labeled dendrimers mainly accumulated in the kidney, gene expression of the dendrimer/DNA complexes was primarily in the heart. The in vivo fate of carriers and gene expression of the complexes are affected by complicated factors. The detailed mechanisms of this discrepancy are undergoing investigation.

In conclusion, brain-specific expression of an exogenous gene is possible with the combined use of a brain-targeting ligand and a cationic gene delivery system using PAMAM, PAMAM-PEG-Tf. There are three important features of this novel gene vector: 1) it is produced from a low generation of PAMAM (generation=5); 2) it uses a biocompatible hydrophilic molecule (PEG); and 3) it exploits a brain-targeting ligand (Tf). Although the mechanisms of this efficient gene delivery vector are not fully understood and are under investigation, we believe that PAMAM-PEG-Tf holds great promise for efficient, noninvasive, and brain-targeting gene delivery.


   ACKNOWLEDGMENTS
 
This work was supported by the grant from National Natural Science Foundation of China (30400570).

Received for publication September 19, 2006. Accepted for publication November 9, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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