Article pubs.acs.org/Langmuir
Selective Gene Delivery to Cancer Cells Using an Integrated Cationic Amphiphilic Peptide Qiong Tang, Bin Cao, Haiyan Wu, and Gang Cheng* Department of Chemical and Biomolecular Engineering, University of Akron, Akron, Ohio 44325, United States ABSTRACT: Gene therapy provides a number of potential treatments that could be applied in clinic to prevent deaths from cancer. However, the transfer of gene therapy to the clinical application has proven difficult because many problems remain to be solved concerning the transfection efficiency, target specificity, and safety issues. To overcome these barriers, a peptide-based vector, K12H6V8SSQHWSYKLRP (KHVLHRH) that comprises four functional blocks, is studied in this work for the targeted delivery of a model gene drug to cancer cells. KHV-LHRH peptide, which contains a luteinizing hormone-releasing hormone (LHRH) sequence, can specifically target cancer cells expressing LHRH receptors. The gene expression, cytotoxicity, and cellular uptake mediated by this vector were evaluated against MCF-7 human breast cancer cells (LHRH-receptor-positive) and SKOV-3 human ovarian carcinoma cells (LHRH-receptor-negative) and compared to a peptide vector (K12H6V8) (KHV) without the LHRH ligand and poly(ethylenimine) (PEI). The results showed that KHV-LHRH enhanced the DNA internalization and induced significantly higher gene expression than KHV in LHRH-receptor-positive MCF7 cells. Also, the peptide-based vectors had low cytotoxicity compared to that of PEI. The high specificity and transfection efficiency of the integrated peptide-based vector make it a very promising material for targeted gene delivery in cancer therapy.
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desired functions. For example, lysine16 and arginine17 residue that are positively charged in a physiological environment are used for DNA binding. The histidine residue was introduced into peptides to aid DNA escape from the endosome and thus to improve the transfection efficiency.18,19 It is reported that adding hydrophobic amino acid residues to peptide vectors could induce the formation of micelle-like nanoparticles and thus increase the local cationic charge density in the solution, which allows for better complexation of DNA.20 Current amphiphilic cationic peptide vectors have shown a high transfection efficiency; however, they lack specificity for the cells.20 Localized therapeutic methods have been broadly used to enhance the drug delivery efficiency and to reduce the side effects of toxic drugs. Targeted gene delivery is highly desired for in vivo gene therapy because the expression of transgenes in nontarget cells may induce undesirable side effects.21 Localized gene delivery can be achieved via the enhanced permeability and retention (EPR) effect22 of tumor tissue or by using the targeting ligands. Targeting ligands have included sugars,23−25 lectins,26−28 antibodies29,30 and their fragments,31,32 peptides,33 and nonpeptide receptor ligands.34 Peptide receptors have attracted notable interest for targeted drug delivery and imaging because many peptide receptors are often preferentially expressed in disease tissues or specific organs.35 An everincreasing number of peptide receptors have been discovered by phage display or combinatorial library methods.36,37 In
INTRODUCTION Gene therapy has attracted significant attention in the past two decades because it holds great potential for the treatment of genetic disorders, such as severe combined immunodeficiency,1 chronic granulomatous disorder,2 and hemophilia,3 and acquired diseases including cancer4 and Parkinson’s disease.5 Because gene therapy has many potential applications, efficient gene delivery systems need to be developed for these clinical applications. These systems must have DNA protection, endosomal escape,6 and targeting of the specific cell/tissue to overcome the very challenging barriers in gene delivery.7,8 To achieve this aim, various delivery systems have been developed, and they can be grouped into two categories: viral and nonviral vectors. Although nonviral vectors are not as efficient as viral vectors, they provide safer delivery strategies that could avoid the problems associated with viruses, including complexity of production, immunogenicity, and mutagenesis.9 Various cationic lipids, synthetic polymers, and dendrimers as nonviral vectors have been investigated,10−12 and some of them could induce high gene transfection in vitro.13 However, the problems associated with these systems, such as cytotoxicity14 and low specificity, hold potential as gene delivery vectors in clinic. Recently, peptide-based vectors have been proposed as a promising candidate for gene delivery because of their advantages over other nonviral synthetic vectors.15 Peptide vectors are composed of natural amino acids, and they are compatible with the biological environment, less cytotoxic, and biodegradable. Also, peptide-based drug carriers are particularly amenable to rational design and development because amino acid building blocks with diverse properties provide us the freedom to develop multifunctional drug carriers with the © 2012 American Chemical Society
Received: August 14, 2012 Revised: October 5, 2012 Published: October 22, 2012 16126
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peptide and DNA. Here, the N/P ratio is a molar ratio of the nitrogen atom (N) in the side chains of the peptide to the phosphate group (P) in the DNA molecule. The phosphate content of the DNA molecules was estimated on the basis of the assumed 330 g/mol average molecular weight of the nucleotides, and the nitrogen content of the peptide molecule was estimated on the basis of the number of lysine residues in each peptide molecule. Size and Zeta Potential Measurements. Peptide/DNA complexes were prepared as described above at different N/P ratios. The size and zeta potential of peptide/DNA complexes were measured by a Zetasizer Nano-ZS from Malvern Instruments (U.K.). The size measurements were performed in Malvern disposable sizing cuvettes at a laser wavelength of 633 nm and a scattering angle of 173 °C, and the zeta potential measurements were performed in Malvern disposable zeta potential cells. Before the measurement, peptide/ DNA complexes were diluted 10-fold with PBS, and each measurement was repeated for 3 runs/sample. Gel Retardation Assay. Peptide/DNA complex solutions were prepared at N/P ratios ranging from 0 to 20. An aliquot (5 μL, 0.5 μg DNA) of each solution was mixed with loading dye and loaded into an agarose gel (0.8%). The loaded gel was exposed to 100 V for 40 min in 0.5× TBE buffer and was stained with ethidium bromide (0.5 μg/mL) for 30 min and destained with water for 15 min. Then the gel was visualized and documented using a BioDoc-It imaging system from UVP (Upland, CA, USA). Cell Culture. Both MCF-7 and SKOV-3 cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum, nonessential amino acids, sodium pyruvate, and penicillin-streptomycin and incubated at 37 °C in a humidified atmosphere of 5% CO2. SKOV-3 and MCF-7 cells were seeded at densities of 1.5 × 105 and 2 × 105 cells/mL, respectively, in 24-well plates (0.5 mL/well) or 96well plates (0.1 mL/well) and incubated for 24 h to reach 80−90% confluence before treatment. In Vitro Gene Transfection and Expression. A plasmid containing a firefly luciferase reporter gene from Photinus pyralis was used as the model gene drug to evaluate the efficiency of peptide carriers in vitro. SKOV-3 and MCF-7 cells were seeded on 24-well plates and incubated for 24 h. Then, old medium was replaced with 450 μL of fresh medium, and 50 μL of the complex solution (containing 2.5 μg DNA) was added to each well. After 4 h of incubation, cells were washed with PBS and incubated with fresh medium for 68 h. Then, cells were washed with PBS, and 0.2 mL of 1× Glo lysis buffer was added to each well to lyse cells. After being incubated at room temperature for 15 min and subjected to two cycles of freezing (80 °C for 30 min) and thawing (room temperature), cell lysates were centrifuged at 14 000 rpm at 4 °C for 15 min to remove cell debris. The supernatant (20 μL) was transferred to a luminometer tube and mixed with 100 μL of luciferase assay reagent, and the luciferase activity was immediately measured for a 10 s read by a Berthold Lumat LB9507 luminometer (Germany). The relative light unit (RLU) reading from the luminometer was normalized by the protein content in the supernatant, which was determined by BCA protein assay. All experiments were performed with six replicates. Cytotoxicity Test. The cytotoxicity of peptide/DNA complexes was evaluated against MCF-7 and SKOV-3 cell lines in a 96-well plate using Vybrant MTT cell proliferation assay. MCF-7 and SKOV-3 cells were seeded on 96-well plates and incubated for 24 h. After incubation for 24 h, the old medium in the 96-well plate was replaced with 90 μL of fresh growth medium, and 10 μL of the complex solution was added to each well. The cells were incubated at 37 °C for 4 h, washed with PBS, and incubated with fresh medium for 20 h. The medium was then replaced with 100 μL of the fresh medium and 10 μL of the MTT stock solution (5 mg/mL in PBS), and cells were incubated at 37 °C for 4 h. Finally, the medium was removed, and 150 μL of DMSO was added to each well to dissolve purple formazan crystals. The absorbance of the DMSO supernatant was measured at 570 nm using a Tecan Infinite 200 microplate reader (Switzerland). The cytotoxicity test was performed in eight replicates for each sample. The cells without any treatment were used as a control, and the cell viability was expressed as a percentage of the control.
polymer-based synthetic vectors, functional groups such as thiol, tetrazole,38 or carboxylate39 need to be incorporated into drug carriers to conjugate targeting ligands. Usually, excess residual unreacted functional groups can cause nonspecific protein adsorption and reduce the blood circulation time of nanosized drug carriers. To prepare very pure and potent drug carrier materials, other challenging factors include the effort expended to purify and the difficulty in precisely controlling the conjugation of multiple ligands to drug carriers. In peptidebased vectors, the targeting ligand can be seamlessly incorporated into the drug delivery carriers without affecting the other properties. In this study, an integrated peptide-based gene vector, NH2− K12H6V8SSQHWSYKLRP−OH (KHV-LHRH), was proposed for targeted gene delivery. The integrated peptide contains four blocks of amino acid residues. The lysine block is for DNA binding, the histidine block is for endolysosomal release, the valine block is a hydrophobic block to enhance DNA complexation, and the luteinizing hormone releasing hormone (LHRH) ligand block is used as a cancer-targeting moiety. The receptors for LHRH are overexpressed in cancer tissues33,40 but are not detectably expressed in most visceral organs.40 The utility of this multifunctional peptide-based vector for targeted gene delivery was investigated in LHRH-receptor-positive cell line MCF-7 and negative cell line SKOV-3 in comparison to that of peptide NH2−K12H6V8SSQHWSYKLRP−OH (KHV) without the LHRH ligand using a luciferase-encoding plasmid as a model gene drug.
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MATERIALS AND METHODS
Materials. Phosphate-buffered saline (PBS), paraformaldehyde, and poly(ethylenimine) (PEI, branched, 25 kDa) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris(hydroxymethyl)aminomethane (Tris) was obtained from J. T. Baker (Center Valley, PA, USA). Peptides NH2−KKKKKKKKKKKKHHHHHHVVVVVVVV−COOH (KHV) and NH2−KKKKKKKKKKKKHHHHHHVVVVVVVVSSQHWSYKLRP−COOH (KHV-LHRH) were designed by us and synthesized by NeoBioScience (Cambridge, MA, USA) at more than 95% purity. Agarose, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, nonessential amino acids, sodium pyruvate, L-glutamine, trypsinEDTA, Prolong gold antifade reagent, and Vybrant MTT cell proliferation assay kit were all purchased from Life Technologies (Carlsbad, CA, USA). Ethidium bromide and nuclease-free water were purchased from EMD Millipore (Billerica, MA, USA). DNA loading dye was purchased from New England Biolabs (Ipswich, MA, USA). Glolysis buffer and a luciferase assay system were purchased from Promega (Madison, WI, USA). A BCA protein assay kit was purchased from Thermo Scientific (Waltham, MA, USA). Plasmid DNA encoding the 5.2 kb firefly luciferase (pCMV-luc-1038) was purchased from Elim Biopharmaceuticals (St. Hayward, CA, USA), amplified in Escherichia coli strain NovaBlue from EMD Millipore, and purified with a plasmid maxi kit supplied by Omega Bio-Tek (Norcross, GA, USA). For confocal microscopy analysis, label IT tracker intracellular nucleic acid localization kit-Cy3 was purchased from Mirus Bio (Madison, WI, USA), and Hoechst 433342 nucleic acid stain was purchased from Life Technologies. MCF-7 and SKOV-3 cell lines were purchased from ATCC (St. Cloud, MN, USA). Preparation of Peptide/DNA Complexes. The peptide solution was prepared at 10 mg/mL in deionized (DI) water, and plasmid DNA was dissolved in nuclease-free water at a concentration of 1 mg/mL. The complexes were formed by directly mixing equal volumes of peptide and plasmid DNA stock solution in 10 mM Tris buffer (pH 7.4) at the intended N/P ratios and incubating for 20 min at room temperature to allow for complete electrostatic interaction between 16127
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Intracellular Uptake of Peptide/DNA Complexes. To evaluate the ability of peptide-based vectors to transport plasmid DNA to MCF-7 and SKOV-3 cells, plasmid DNA was labeled with Cy3 fluorescent dye using Label IT tracker intracellular nucleic acid localization kit, and cell nucleus DNA was stained with Hoechst 33342. The cellular uptake of carrier/DNA complexes was visualized with an Olympus Fluoview FV1000 confocal laser scanning microscopy (CLSM) (Japan). MCF-7 and SKOV-3 cells were seeded on a Nalgene Lab-Tek II eight-well chamber slide (Waltham, MA, USA) at densities of 8 × 104 and 2 × 104 cells/well, respectively, and incubated for 24 h. Then the medium was replaced with 180 μL of fresh medium, and 20 μL of the peptide/DNA complex solution containing 1 μg Cy3-labeled plasmid was subsequently added to each well. Naked DNA and PEI/DNA at an N/P ratio of 10 were used as the control. Peptide/DNA complexes were formed at an N/P ratio of 30. After transfection for 0.5 and 2 h, the medium was removed, and the cells were washed with cold PBS three times. The cells were fixed in 3.7% paraformaldehyde for 30 min. Then the cells were washed with PBS three times and incubated for 30 min in 100 μL of Hoechst 33342 (2 μg/mL) for each well. For microscopy analysis, the chamber slide was mounted with Prolong gold antifade solution and covered with a glass coverslip. The slide was then examined by differential interference contrast (DIC) microscopy and CLSM with excitation at 405 and 559 nm for Hoechst 33342 and Cy3, respectively. The images were recorded and processed with Olympus FV10-ASW software. Statistical Analysis. Data were analyzed using single-factor analysis of variance (ANOVA) and expressed as the mean ± standard deviation for four to eight replicates. The comparison among groups was performed using the student’s t test. A p value of less than 0.05 was considered to be statistically significant.
negatively charged DNA solution, peptide/DNA complexes were formed via electrostatic interaction. The size of peptide/ DNA complexes is controlled by two factors, the binding ability of vector to DNA and the surface charge of peptide/DNA complexes, but the two factors cannot be completely separated. At low N/P ratios (1 and 5), the interaction was not strong enough to form compact peptide/DNA complexes because of the low peptide concentration. At higher concentrations of both peptides (above the N/P ratio of 5), more peptides can bind DNA molecules to condense DNA and form much smaller complexes. Also for the particles/proteins with high surface charge, the repulsion force between particles/proteins prevents their aggregation. During the transition between negative and positive or around their isoelectric point, particles/proteins tend to aggregate because of the weaker repulsion force. In our system, the surface charge of peptide/DNA complexes changes from negative to positive when the N/P ratio increases from 1 to 5. Among all tested N/P ratios, the smallest particle was formed at an N/P ratio of 40 for both peptides, and the particles sizes are 190 nm for the KHV/DNA complexes and 188 nm for the KHV-LHRH/DNA complexes. The particle size is even more important to in vivo gene delivery. It is reported that nanoparticles with a size of less than 200 nm in diameter are desired for drug blood retention and tumor extravasation.44,45 The zeta potential of carrier/DNA complexes is also a significant factor affecting the efficiency of gene transfection. The net positive charge of carrier/DNA complexes is preferred to enhance cellular uptake because of the interaction of the negatively charged cell membrane and the positively charged carrier/DNA complexes. However, if the net positive charge density of the carrier/DNA is too high (∼>30 mV), then it might induce cellular toxicity.46 Also, the high charge density of carrier/DNA complexes will reduce their specificity to target cells because of the elevated nonspecific binding to serum protein and cells. As listed in Table 1, the zeta potentials of both KHV and KHV-LHRH peptide/DNA complexes are negative at an N/P ratio of 1, but they became positive, 12 mV for KHV/DNA and 14.7 mV for KHV-LHRH/DNA, at an N/P ratio of 5. Above an N/P ratio of 5, the zeta potentials of peptide/DNA complexes did not change very much and stayed around 13 mV. The results show that both KHV and KHVLHRH peptides can efficiently condense DNA to the desired size and surface potential. DNA Binding Ability of Peptides. The peptide/DNA complexes were formed at different N/P ratios and the DNA binding ability of peptides was evaluated by the agarose gel retardation assay. As shown in Figure 1, carrier/DNA complexes formed at the N/P of 1 traveled a slightly shorter distance than did naked DNA across the gel, which indicates incomplete retardation of DNA at an N/P ratio of 1. The complete retardation of DNA was achieved at a relative low N/ P ratio of 2 for both KHV and KHV-LHRH peptides. Although the sizes of complexes at an N/P ratio of 5 for both peptides were large, which indicated incomplete condensation of peptides and DNA, the electrostatic interaction between peptides and DNA was still strong enough to prevent DNA from traveling through the gel. Gene Transfection. To study whether the LHRH ligand can enhance transgene expression, in vitro transfection efficiencies of KHV-LHRH and KHV peptides were studied in the MCF-7 human breast cancer cell line and the SKOV-3 human ovarian carcinoma cell line. MCF-7 cells have been
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RESULTS AND DISCUSSION Particle Size and Zeta Potential. Particle size is one of the most important factors that influence the efficiency of gene transfection in vitro and in vivo. In general, nanoparticles have a higher chance of being taken up by cells than do microparticles.42,43 As shown in Table 1, the size of peptide/DNA Table 1. Size and Zeta Potential of KHV/DNA and KHVLHRH/DNA Complexes vector KHV
KHV-LHRH
N/P ratio 0 1 5 10 20 30 40 blank peptide 0 1 5 10 20 30 40 blank peptide
size (d, nm) 142 149 1928 375 244 286 190 31 142 138 1757 340 429 217 188 30
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
10 7 150 22 16 20 15 2 10 11 105 27 34 13 13 2
zeta potential (mV) −18.4 −14.2 12.0 12.1 12.5 11.7 12.8 8.9 −18.4 −43.8 14.7 13.0 13.7 13.6 13.8 12.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.3 0.7 1.0 0.7 0.9 0.9 0.9 0.6 1.3 2.6 1.3 0.9 1.1 1.0 1.0 1.0
complexes increases to its maximum values of 1928 nm for the KHV/DNA complex and 1757 nm for the KHV-LHRH/DNA complex, respectively, at an N/P ratio of 5. A general decreasing trend in particle size appeared as the N/P ratio was increased from 5 to 40. Initially, the size of naked DNA is 142 nm. As the positively charged peptides were added to the 16128
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achieved at an N/P ratio of 30. For SKOV-3 cells, there was no significant difference in luciferase expression between cells treated with KHV/DNA complexes and cells treated with KHV-LHRH/DNA complexes because SKOV-3 cells have no LHRH receptors to facilitate the specific uptake of KHVLHRH/DNA complexes (Figure 2B). In SKOV-3 cells, the luciferase expression increased slightly for both KHV and KHVLHRH as the N/P ratio increased from 10 to 30, and the optimal expression for both peptides was achieved at an N/P ratio of 30 compared to the expression of naked DNA. This might be explained by the decreased size of the complexes as the N/P ratio increased. Although KHV/DNA complexes at the N/P ratio of 40 were formed with a smaller size (∼190 nm), the expression level of luciferase in SKVO-3 is still lower than that at an N/P ratio of 30, which might be caused by the higher zeta potential of KHV/DNA complexes formed at an N/ P of 40. At the N/P ratio of 30, the size and zeta potential of peptide/DNA complexes reached an optimal balance. The luciferase expression levels in SKOV-3 cells induced by both KHV/DNA and KHV-LHRH/DNA complexes are comparable to or slightly higher than the expression levels induced by KHV/DNA complexes in MCF-7 cells, whereas the expression levels induced by KHV-LHRH/DNA complexes in MCF-7 cells were much higher because of the receptor−ligand interaction. In LHRH-receptor-positive MCF-7 cells, the absolute transfection efficiency of the KHV-LHRH peptide at the N/P ratio of 10 is lower than that at higher N/P ratios, but it shows the second highest specificity, which is also highly desired for targeted drug delivery. At this N/P ratio, the luciferase expression level of KHV-LHRH/DNA complexes in nontarget SKOV-3 cells is close to that of naked DNA, and the luciferase expression level of KHV-LHRH/DNA complexes in target MCF-7 cells is 280 times higher than that of naked DNA. These results indicated that the LHRH ligand block in the peptides played an important role in gene transfection in LHRH-receptor-positive cells. Another study needs to be conducted to optimize the peptides to improve their transfection efficiency further and maintain their high specificity. Cytotoxicity of Peptide/DNA Complexes. The cytotoxicity of peptide/DNA complexes was determined against SKOV-3 and MCF-7 cell lines. Naked DNA was used as a negative control, and PEI/DNA complexes at an N/P ratio of 10, at which concentration PEI achieved an optimal gene expression, were used as a positive control. In MCF-7 cells, the cell viability of KHV-LHRH/DNA exhibited a slightly decreasing trend as the N/P ratio was increased. The cytotoxicity of KHV-LHRH/DNA complexes reached its lowest value at an N/P ratio of 10 and was close to that of naked DNA and significantly lower than that of PEI. Even at an N/P ratio of 40, it is still lower than that of the positive control (PEI/DNA complexes) at an N/P ratio of 10. For the KHV peptide, the MCF-7 cell viability also exhibited a slightly decreasing trend as the N/P ratio was increased, and it was slightly lower than that of PEI/DNA complexes at an N/P of 40 (Figure 3A). However, the peptide/DNA complexes had a relatively low cytotoxicity to SKOV-3 cells at all tested N/P ratios (Figure 3B). The cell viability of the peptide/DNA complexes is above 90%, which is significantly higher than that of PEI/DNA complexes (around 75%). In general, the cell viability of MCF-7 cells was lower than that of SKOV-3 cells for naked DNA, PEI/DNA, and peptide/DNA complexes at the same N/P ratios. The difference might be explained by the fact
Figure 1. Electrophoresis mobility of DNA in peptide/DNA complexes formed at various N/P ratios as specified. (Top) KHV/ DNA complexes. (Bottom) KHV-LHRH/DNA complexes.
found to overexpress LHRH receptors.40 The LHRH-receptornegative ovarian SKOV-3 cell line47 was used as a negative control in this study. In MCF-7 cells, 50- to 183-fold higher luciferase expression was observed at tested N/P ratios in cells treated with KHV-LHRH/DNA complexes, compared to that for cells treated with KHV/DNA complexes (Figure 2A). The highest ratio of the luciferase expression level in cells induced by KHV-LHRH to that induced by KHV was 183, which was
Figure 2. Luciferase expression level induced by KHV/DNA and KHV-LHRH/DNA complexes in (A) MCF-7 and (B) SKOV-3 cell lines at N/P ratios of 10, 20, 30 and 40 compared to naked DNA and PEI/DNA at an N/P ratio of 10 (* and **, P < 0.05, n = 6). 16129
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of complexes (naked DNA, PEI/DNA, KHV/DNA, and KHVLHRH/DNA) against LHRH-receptor-positive MCF-7 cells and LHRH-receptor-negative SKOV-3 cells were investigated by CLSM using Cy3-labeled DNA and Hoechst 44432 nucleic acid stain. The internalization of carrier/DNA complexes could be observed 0.5 h after transfection (Figure 4). Cy3-labled naked DNA cannot be observed in either cell line (Figure 4A), but Cy3-labeled DNA condensed with PEI or peptides could penetrate both cells lines (Figure 4B−D). The degrees of cellular uptake of KHV/DNA and KHV-LHRH/DNA complexes are similar in LHRH-receptor-negative SKOV-3 cells. In contrast, the internalization of DNA delivered by KHVLHRH was substantially higher than that delivered by KHV in LHRH-receptor-positive MCF-7 cells. These results agreed with the results from the gene expression experiment and also confirmed that a gene vector with the targeting ligand (KHVLHRH) could enhance the internalization of DNA in LHRHreceptor-positive cells. Internalized DNA was found in the cytoplasma and in the perinuclear region 0.5 h after transfection. After 2 h, the red regions obviously increased in MCF-7 cells treated with both KHV/DNA and KHV-LHRH/ DNA complexes (Figure 5), which indicates that both of them could be efficiently internalized by cells. The maximal accumulation of DNA for both peptides was found in the perinuclear region. The DNA delivered by KHV-LHRH was widely dispersed around the nucleus and some even entered the nucleus. In contrast, the DNA in cells treated with KHV/DNA complexes aggregated in the perinuclear region and barely penetrated the nucleus, indicating the endosomal/lysosomal capture of DNA. The result is consistent with the gene expression result that a significantly higher luciferase expression was detected in LHRH-positive MCF-7 cells treated with KHVLHRH/DNA complexes when compared to nontargeting KHV/DNA complexes. In this cellular uptake experiment, cells transfected with PEI/DNA had less accumulation of DNA in the cytoplasma than cells treated with peptide/DNA complexes but achieved much higher gene expression. This
Figure 3. Viability of (A) MCF-7 and (B) SKOV-3 cells after being treated with KHV/DNA and KHV-LHRH/DNA complexes at specified N/P ratios compared to naked DNA and the PEI/DNA complex at an N/P ratio of 10 (*, P < 0.05, n = 8).
that MCF-7 cells were more sensitive to foreign materials than were SKOV-3 cells. For both MCF-7 and SKOV-3 cells, there is no significant difference in cytotoxicity between KHV/DNA and KHV-LHRH/DNA complexes. Overall, the cell viability of peptide/DNA complexes was comparable to or higher than that of PEI/DNA at an N/P ratio of 10. In Vitro Ligand-Mediated Uptake and Intracellular Distribution. The internalization and intracellular distribution
Figure 4. Confocal microscopy images of (A1−D1) LHRH-receptor-positive MCF-7 cells and (A2−D2) LHRH-receptor-negative SKOV-3 cells incubated for 0.5 h with (A1, A2) naked DNA, (B1, B2) PEI/DNA, (C1, C2) KHV/DNA, and (D1, D2) KHV-LHRH/DNA. The CLSM observation was performed using a 40× objective. Cy3-labeled DNA is shown in red, and the nucleus is shown in blue. 16130
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Figure 5. Confocal microscopy images of LHRH-receptor-positive MCF-7 cells incubated for 2 h with (A) KHV/DNA and (B) KHV-LHRH/DNA. The CLSM observation was performed using the 40× objective. Cy3-labeled DNA is shown in red, and the nucleus is shown in blue. Locations where DNA penetrates the nucleus are marked with a yellow arrow. (2) Grez, M.; Reichenbach, J.; Schwable, J.; Seger, R.; Dinauer, M. C.; Thrasher, A. J. Gene Therapy of Chronic Granulomatous Disease: The Engraftment Dilemma. Mol. Ther. 2011, 19, 28−35. (3) Mannucci, P. M.; Tuddenham, E. G. D. Medical Progress - The Hemophilias - From Royal Genes to Gene Therapy. N. Engl. J. Med. 2001, 344, 1773−1779. (4) El-Aneed, A. An Overview of Current Delivery Systems in Cancer Gene Therapy. J. Controlled Release 2004, 94, 1−14. (5) Kaplitt, M. G.; Feigin, A.; Tang, C.; Fitzsimons, H. L.; Mattis, P.; Lawlor, P. A.; Bland, R. J.; Young, D.; Strybing, K.; Eidelberg, D.; During, M. J. Safety and Tolerability of Gene Therapy with an AdenoAssociated Virus (AAV) Borne GAD Gene for Parkinson’s Disease: An Open Label, Phase I Trial. Lancet 369, 2097−2105. (6) Wiethoff, C. M.; Middaugh, C. R. Barriers to Nonviral Gene Delivery. J. Pharm. Sci. 2003, 92, 203−217. (7) Gupta, B.; Levchenko, T. S.; Torchilin, V. P. Intracellular Delivery of Large Molecules and Small Particles by Cell-Penetrating Proteins and Peptides. Adv. Drug Delivery Rev. 2005, 57, 637−651. (8) Vyas, S. P.; Singh, A.; Sihorkar, V. Ligand-Receptor-Mediated Drug Delivery: An Emerging Paradigm in Cellular Drug Targeting. Crit. Rev. Ther. Drug Carrier Syst. 2001, 18, 1−76. (9) Verma, I. M.; Somia, N. Gene Therapy - Promises, Problems and Prospects. Nature 1997, 389, 239−242. (10) Mintzer, M. A.; Simanek, E. E. Nonviral Vectors for Gene Delivery. Chem. Rev. 2008, 109, 259−302. (11) Pathak, A.; Patnaik, S.; Gupta, K. C. Recent Trends in Non-Viral Vector-Mediated Gene Delivery. Biotechnol. J. 2009, 4, 1559−1572. (12) Nguyen, D. N.; Green, J. J.; Chan, J. M.; Langer, R.; Anderson, D. G. Polymeric Materials for Gene Delivery and DNA Vaccination. Adv. Mater. 2009, 21, 847−867. (13) Akinc, A.; Anderson, D. G.; Lynn, D. M.; Langer, R. Synthesis of Poly(β-amino ester)s Optimized for Highly Effective Gene Delivery. Bioconjugate Chem. 2003, 14, 979−988. (14) Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. Toxicity of Cationic Lipids and Cationic Polymers in Gene Delivery. J. Controlled Release 2006, 114, 100−109. (15) Martin, M.; Rice, K. Peptide-Guided Gene Delivery. AAPS J. 2007, 9, E18−E29. (16) Zauner, W.; Ogris, M.; Wagner, E. Polylysine-Based Transfection Systems Utilizing Receptor-Mediated Delivery. Adv. Drug Delivery Rev. 1998, 30, 97−113. (17) Rothbard, J. B.; Kreider, E.; Vandeusen, C. L.; Wright, L.; Wylie, B. L.; Wender, P. A. Arginine-Rich Molecular Transporters for Drug Delivery: Role of Backbone Spacing in Cellular Uptake. J. Med. Chem. 2002, 45, 3612−3618.
may be due to the efficient endosomal escape ability of PEI, which helps DNA molecules avoid being degraded in the late endosomes/lysosomes, thus having a greater chance to enter the nucleus to be expressed.
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CONCLUSIONS In this study, an integrated cationic peptide (KHV-LHRH) has been investigated as a carrier for targeted gene delivery. The integrated cationic peptide had a strong DNA-binding ability at a relatively low N/P ratio. At a higher N/P ratio of 40, more compact peptide/DNA complexes were formed with a diameter of 188 nm and a zeta potential of 13.8 mV, which is preferable for cellular uptake. Although peptide/DNA complexes were positively charged, the cell viability was comparable to or even higher than that of PEI/DNA. However, the peptide with the LHRH ligand (KHV-LHRH) induced a much higher and specific gene expression in LHRH-receptor positive MCF-7 cells than did the peptide (KHV) without ligand. In additional, the cellular uptake results confirmed that the internalization of Cy3-DNA by MCF-7 cells was enhanced by KHV-LHRH compared to that induced by KHV. These results indicate that the integrated peptide with the LHRH ligand may provide a promising solution for safe and efficient cancer therapy.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the University of Akron Research Foundation and the National Science Foundation (NSF DMR1206923).
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REFERENCES
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