Communication
EGFP-Based Protein Nanoparticles with Cell-Penetrating Peptide for Efficient siRNA Deliverya Xingang Guan, Xiuli Hu, Fengchao Cui, Yunqi Li,* Xiabing Jing, Zhigang Xie*
Development of an innovative nucleic acid nanocarriers still represents a challenge. In this study, we develop a protein nanoparticle (H6-TatEGFP) and examine its siRNA condensing activity. Gel retardation assay show that protein nanoparticle can condense siRNA into stable nanoparticle/siRNA complexes. UsingCy3labelled siRNA, we also evaluate siRNA transport characteristic of protein nanoparticles in tumor cells, the results indicate that H6-TatEGFP nanoparticle may be a potential nanocarrier for siRNA in tumor cells.
1. Introduction Delivery of siRNA into tumor cells on the basis of nanocarriers holds great potentials as anti-cancer agents in cancer therapy.[1,2] Nowadays, a variety of nanoparticles have been developed as vehicles for siRNA delivery, including polymer-based nanoparticles,[3–6] lipid-based nanoparticles,[7–10] gold nanoparticles,[11–14] magnetic nanoparticles,[15–17] mesoporous silica nanoparticles,[18–22] and carbon-based nanomaterials.[23–26] These nanocarriers could protect siRNA from degradation by serum nucleases
X. Guan, X. Hu, X. Jing, Z. Xie State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China E-mail: [email protected] X. Guan Life Science Research Center, Beihua University, Jilin 132013, P. R. China F. Cui, Y. Li Key Laboratory of Synthetic Rubber, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China E-mail: [email protected] Supporting Information is available online from the Wiley Online Library or from the author.
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and deliver siRNA to subcellular region of target cells. However, the application of these promising nanocarriers in cancer therapy has been hampered by their safety and biocompatibility concerns.[27] It is challenging and desirable to develop safe siRNA delivery systems without causing any untoward effects. Proteins, possessing perfect biocompatibility and biodegradability, low toxicity, and good tuning ability via genetic engineering, are highly promising biomaterials for drug delivery.[28] Due to possessing several advantages than synthetic polymers such as low cytotoxicity, easy production, and significant improvement in tumor targeting, protein-based nanocarriers represent promising nanocarriers for efficient drug and gene delivery. These nanocarriers can be hydrolyzed to peptides by digestive enzymes in vivo, while synthetic polymers may be degraded as harmful products. As related to the possible protein immunotoxicity, until now no antigenic reactions were reported in studies of albumin, gelatin, and some other protein-based
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DOI: 10.1002/mabi.201500163
EGFP-Based Protein Nanoparticles with Cell-Penetrating . . . www.mbs-journal.de
nanoparticles.[29] Several natural proteins can selfassemble into nanoparticles, including virus-like particles, nanocages, nanodisks, and others.[30–33] For example, Toita et al. developed a nanocage based on heat shock proteins (Hsp) for doxorubicin delivery, showing good cytotoxicity toward human hepatocellular carcinoma (HCC) cells and reduced cytotoxicity to normal hepatocytes.[34] Zhen et al. had developed a protein-only nanoparticle based on RGD4C-modified ferritin for delivering photosensitizers to human glioblastoma U87MG, showing a good capability of tumor inhibition.[35] Vazquez et al. demonstrated the DNA transduction capability of R9-GFP-H6 nanoparticles in HeLa cells.[36,37] However, using protein nanoparticles for siRNA delivery have been rarely reported. Tat peptide, which was discovered from HIV-1 TAT protein, is a well-known cell-penetrating peptide (CPP), widely used for efficient delivery of various cargoes into cells.[38,39] In this study, we designed a new protein nanoparticle based on H6-TatEGFP, which was prepared from self-assembly of enhanced green fluorescent protein (EGFP) protein derivatives. Six histidines (H6) were fused to the N-terminal regions of EGFP protein or Tat-EGFP fusion protein. The size and morphology, siRNA condensing and transport activities of the protein nanoparticles were examined.
2. Results and Discussion Histidine-tagged EGFP and Tat-EGFP fusion protein prepared by a convenient genetic engineering approach (Figure 1A). We found that H6-EGFP proteins could selfassemble into nanosized structures with a diameter of 11.7 1.07 nm, which were not detected by previous studies.[31,37] Moreover, when Tat peptide was inserted between H6 and EGFP to form H6-TatEGFP proteins, this protein could also self-assemble into nanoparticles with a diameter of 13.5 1.35 nm. We investigated the biocompatibility and cell uptake of H6-EGFP and H6-TatEGFP nanoparticles in vitro. Taking into account, the key role of Tat peptides in gene and drug delivery,[40–43] we speculated that our protein nanoparticle with Tat may be used as siRNA delivery system. The DNA-condensing ability and siRNA transfection mediated by H6-TatEGFP nanoparticles were also examined. Taken together, our study implicates H6-TatEGFP as a novel siRNA nanocarrier for tumor cells. Both, H6-EGFP and H6-TatEGFP were prepared according to Escherichia coli protein expression system (pET28a). To confer the stronger ability of absorption with anionic siRNA, Tat peptide (YGRRARRRRRR) was added to the amino-terminal region of EGFP. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) analysis (Figure S1, ESI†) and the fluorescence
Figure 1. Illustration of genetically engineered H6-EGFP and H6-Tat-EGFP proteins and characterization of two protein-only nanoparticles. (A) Description of H6-EGFP and H6-TatEGFP proteins. H6, six histidines; L, linker peptide; Tat, YGRRARRRRRR; EGFP, enhanced green fluorescent protein. (B) Size distribution of H6-EGFP and H6-TatEGFPin HBS and elution buffer determined by DLS. (C) Zeta potential analysis of two proteins. (D) TEM images of two protein nanoparticles (The high resolution TEM of H6-TatEGFP nanoparticle was shown. Scale Bar ¼ 5 nm.).
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emission spectra (Figure S2, ESI†) showed that both proteins were successfully prepared with enough purity. To determine when two protein nanoparticles were assembled, the size distribution of H6-EGFP and H6-TatEGFP nanoparticles in HBS (low salt) and elution buffer (high salt) were analyzed by dynamic light scattering (DLS). The results indicated that the nanosized structure of H6-EGFP disaggregated in solution with high salt and transformed into monomer; however, the size of H6-TatEGFP nanoparticles increased to 28.2 nm, suggesting the stability of H6-TatEGFP protein nanoparticles. Based on these results, we speculated that two protein nanoparticles may emerge in process of protein expression (Figure 1B). The zeta potential of H6-EGFP and H6-TatEGFP Figure 2. Molecular modeling of H6-EGFP (A) and H6-Tat-EGFP (B) nanoparticles through nanoparticles were –11.11 and –0.18 mV, protein–protein docking algorithm HADDOCK. The diameter and thickness of two respectively (Figure 1C). The decrease in protein nanoparticles were shown above and below, respectively. negative charge of H6-TatEGFP nanoparticles was ascribed to the addition of Tat sequence including eight arginines. Transmission bromide (MTT) assay on HeLa cells. As shown in electron microscope (TEM) analysis showed that both Figure S3, both proteins showed at least 85% cell viability nanoparticles displayed nanosized structure as shown in even at the concentration of 400 mg ml1. Then we determined if these nanoparticles could penetrate mamFigure 1D. These results confirmed well-defined nanostructures formed by two proteins. malian cell membranes while preserving the particulate Then the fined self-assemble structures of H6-EGFP organization and EGFP fluorescence. The cellular uptake and H6-Tat-EGFP were modelled through protein–protein experiments were performed on HeLa cells and EGFP docking algorithm HADDOCK.[44] It is a top ranked without H6 was used as a control. As shown in Figure 3, both two nanoparticles could penetrate the cell membrane and approach for protein–protein complex structure prediction distribute in the region near nucleus of HeLa cells. As a that utilizing the information from identified or predicted contrast, the EGFP protein, which was lack of his-tag at protein interfaces in ambiguous interaction restraints to the N-terminus, remained fully dispersed in the extradrive a flexible docking. The assemblies of both H6-EGFP cellular region. These results showed that only welland H6-Tat-EGFP have pentamers and arrange in a starassembled H6-EGFP and H6-TatEGFP nanoparticles but shape. The assembly of H6-EGFP is constructed through not EGFP proteins could be internalized by tumor cells. the interaction between N-terminal and C-terminal tails, Although Tat peptides has been widely used in develand through the interaction between a-helix in the oping efficient nanocarriers for delivery of siRNA or other N-terminal and the coils in both terminals in the complex therapeutic molecules,[46,47] to our knowledge protein of H6-Tat-EGFP. The predicted values of radius of gyration (Rg) were 5.23 and 5.48 nm for the assembly structures of nanoparticles based exclusively on a single protein construct for siRNA delivery had never been explored. MoreH6-EGFP and H6-Tat-EGFP. Their diameters were 11.90 over, the Tat peptide in this study (YGRRARRRRR) designed and 15.50 nm by converting the radius of gyration to a by Kim et al. and named cytoplasmic transduction disk with equal volume. The thickness of the disks were peptide (CTP), could translocate its payloads to the 3.90 and 4.10 nm, which are close to the cross-sections of cytoplasm rather than to the nucleus after transduction.[48] EGFP monomer (Figure 2). The size and the geometry of the predicted structures agree with our recent measureThen, we examined if our protein-only nanoparticles could ments using small angle X-ray scattering. be used as a novel nanocarrier for siRNA delivery to tumor The biocompatibility of nanomaterial is very important cells. An efficient siRNA vehicle should condense siRNA into stable nanosized complexes. A gel retardation assay for their biomedical applications. We investigated the cytotoxicity of H6-EGFP and H6-TatEGFP nanoparticles using was performed to investigate complexes formation of the 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium two protein nanoparticles with Cy3-labeled siRNA at
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Figure 3. Cell uptake of EGFP, H6-EGFP, and H6-Tat-EGFP proteins by HeLa cells analyzed. (A) CLSM results. The cell nuclear was stained with DAPI (blue signal) and three types of EGFP were analyzed by their inherent green fluorescence; (B) The cell uptake rate of H6-EGFPand H6Tat-EGFP nanoparticles were determined by flow cytometry results indicated.
different protein/siRNA ratio (wt/wt) from 1 to 100. As shown in Figure 4A, the intensity of migrating free siRNA nearly unchanged for H6-EGFP protein even at the ratio of 100:1; while the free siRNA bands of H6-TatEGFP decreased gradually with the increasing protein/siRNA ratio from 1 to 5, and finally disappeared at the ratio of 10, 25, 50, and 100.
These results suggested that despite the cationic nature conferred by six histidines, H6-EGFP nanoparticle was unable to condense siRNA into stable complex as revealed by unchanging migrating free siRNA; while the H6-TatEGFP nanoparticle with Tat peptide at the amino-terminus could condense siRNA into protein/siRNA complex as indicated by altering siRNA mobility in gel electrophoresis. DLS analysis indicated that the size of H6-TatEGFP protein/siRNA complex was nearly same to that of H6TatEGFP nanoparticles (Figure 4B), which also showed the siRNA-condensing property of H6-TatEGFP proteins. The transfection of Cy3-labelled siRNA mediated by protein nanoparticles in vitro was performed on HeLa cells and commercial liposome Lipofectamine 2000 (Invitrogen) was as a positive control. As shown in Figure 4C, H6EGFP/siRNA complexes were fully distributed in the extracellular region adherent to the cell membrane, suggesting a failure in siRNA delivery into HeLa cells; but H6-TatEGFP/siRNA and Lipo/siRNA complexes displayed red punctate structures in cytosol adjacent to the nucleus. Different from Lipo/siRNA group, some red fluorescence in H6-TatEGFP group was distributed in entire nucleus, suggesting the unique property of Tat peptides in nucleus targeting which has been proved by previous reports.[49–51]
Figure 4. Evaluation of siRNA-condensing properties of H6-EGFP and H6-TatEGFP nanoparticles. (A) Retardation of siRNA migration in 1% agarose gelelectrophoresis mediated by increasing ratio (wt/wt) of protein/siRNA. (B) Size distributions of the H6TatEGFP protein/siRNA complex at different ratio. (C) Transfection of Cy3-labelled siRNA by H6-EGFP and H6-Tat-EGFP nanoparticles in HeLa cells by CLSM. The nucleus was stained by DAPI (blue signal), and the siRNA was detected by Cy3 fluorescence (red signal).
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3. Conclusion In summary, we prepared two types of protein-only nanoparticles based on H6EGFP and H6-Tat-EGFP. The studies of
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Vazquez E could not detect the nanostructure of GFPH6,[10,14] considering the nearly same amino acid sequence between GFP and EGFP, we speculated that the structure differences of GFP-H6 and H6-EGFP were ascribed to the location of H6 in fusion proteins. For genetic engineer, H6 is just used as tag fused with interesting proteins which was easy to purify the proteins, and there seems no difference in fusion sites of H6 (at the amino or carboxyl terminal). However, our study showed that the site of H6 may play a role in formation of nanostructure. More importantly, we developed a novel siRNA delivery system based on H6-Tat-EGFP protein nanoparticles. H6-Tat-EGFP nanoparticles were internalized by HeLa cells with good biocompatibility. H6-TatEGFP nanoparticlescould condense siRNA into stable protein/siRNA complexes and deliver siRNA to cytosol. The H6-Tat-EGFP nanoparticle may be a novel siRNA nanocarrier for not only HeLa cells but also other tumors difficult to transfect.
Abbreviations EGFP enhanced green fluorescent protein CPP cell-penetrating peptide Hsp heat shock proteins DLS dynamic light scattering TEM transmission electron microscope
Acknowledgment: The project was supported by the National Natural Science Foundation of China (Project No. 91227118 and 51373167, 21374117).
Received: April 29, 2015; Revised: June 8, 2015; Published online: June 24, 2015; DOI: 10.1002/mabi.201500163 Keywords: protein nanoparticle; siRNA delivery; tat
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EGFP-Based Protein Nanoparticles with Cell-Penetrating Peptide for Efficient siRNA Deliverya Xingang Guan, Xiuli Hu, Fengchao Cui, Yunqi Li,* Xiabing Jing, Zhigang Xie*
Development of an innovative nucleic acid nanocarriers still represents a challenge. In this study, we develop a protein nanoparticle (H6-TatEGFP) and examine its siRNA condensing activity. Gel retardation assay show that protein nanoparticle can condense siRNA into stable nanoparticle/siRNA complexes. UsingCy3labelled siRNA, we also evaluate siRNA transport characteristic of protein nanoparticles in tumor cells, the results indicate that H6-TatEGFP nanoparticle may be a potential nanocarrier for siRNA in tumor cells.
1. Introduction Delivery of siRNA into tumor cells on the basis of nanocarriers holds great potentials as anti-cancer agents in cancer therapy.[1,2] Nowadays, a variety of nanoparticles have been developed as vehicles for siRNA delivery, including polymer-based nanoparticles,[3–6] lipid-based nanoparticles,[7–10] gold nanoparticles,[11–14] magnetic nanoparticles,[15–17] mesoporous silica nanoparticles,[18–22] and carbon-based nanomaterials.[23–26] These nanocarriers could protect siRNA from degradation by serum nucleases
X. Guan, X. Hu, X. Jing, Z. Xie State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China E-mail: [email protected] X. Guan Life Science Research Center, Beihua University, Jilin 132013, P. R. China F. Cui, Y. Li Key Laboratory of Synthetic Rubber, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China E-mail: [email protected] Supporting Information is available online from the Wiley Online Library or from the author.
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and deliver siRNA to subcellular region of target cells. However, the application of these promising nanocarriers in cancer therapy has been hampered by their safety and biocompatibility concerns.[27] It is challenging and desirable to develop safe siRNA delivery systems without causing any untoward effects. Proteins, possessing perfect biocompatibility and biodegradability, low toxicity, and good tuning ability via genetic engineering, are highly promising biomaterials for drug delivery.[28] Due to possessing several advantages than synthetic polymers such as low cytotoxicity, easy production, and significant improvement in tumor targeting, protein-based nanocarriers represent promising nanocarriers for efficient drug and gene delivery. These nanocarriers can be hydrolyzed to peptides by digestive enzymes in vivo, while synthetic polymers may be degraded as harmful products. As related to the possible protein immunotoxicity, until now no antigenic reactions were reported in studies of albumin, gelatin, and some other protein-based
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nanoparticles.[29] Several natural proteins can selfassemble into nanoparticles, including virus-like particles, nanocages, nanodisks, and others.[30–33] For example, Toita et al. developed a nanocage based on heat shock proteins (Hsp) for doxorubicin delivery, showing good cytotoxicity toward human hepatocellular carcinoma (HCC) cells and reduced cytotoxicity to normal hepatocytes.[34] Zhen et al. had developed a protein-only nanoparticle based on RGD4C-modified ferritin for delivering photosensitizers to human glioblastoma U87MG, showing a good capability of tumor inhibition.[35] Vazquez et al. demonstrated the DNA transduction capability of R9-GFP-H6 nanoparticles in HeLa cells.[36,37] However, using protein nanoparticles for siRNA delivery have been rarely reported. Tat peptide, which was discovered from HIV-1 TAT protein, is a well-known cell-penetrating peptide (CPP), widely used for efficient delivery of various cargoes into cells.[38,39] In this study, we designed a new protein nanoparticle based on H6-TatEGFP, which was prepared from self-assembly of enhanced green fluorescent protein (EGFP) protein derivatives. Six histidines (H6) were fused to the N-terminal regions of EGFP protein or Tat-EGFP fusion protein. The size and morphology, siRNA condensing and transport activities of the protein nanoparticles were examined.
2. Results and Discussion Histidine-tagged EGFP and Tat-EGFP fusion protein prepared by a convenient genetic engineering approach (Figure 1A). We found that H6-EGFP proteins could selfassemble into nanosized structures with a diameter of 11.7 1.07 nm, which were not detected by previous studies.[31,37] Moreover, when Tat peptide was inserted between H6 and EGFP to form H6-TatEGFP proteins, this protein could also self-assemble into nanoparticles with a diameter of 13.5 1.35 nm. We investigated the biocompatibility and cell uptake of H6-EGFP and H6-TatEGFP nanoparticles in vitro. Taking into account, the key role of Tat peptides in gene and drug delivery,[40–43] we speculated that our protein nanoparticle with Tat may be used as siRNA delivery system. The DNA-condensing ability and siRNA transfection mediated by H6-TatEGFP nanoparticles were also examined. Taken together, our study implicates H6-TatEGFP as a novel siRNA nanocarrier for tumor cells. Both, H6-EGFP and H6-TatEGFP were prepared according to Escherichia coli protein expression system (pET28a). To confer the stronger ability of absorption with anionic siRNA, Tat peptide (YGRRARRRRRR) was added to the amino-terminal region of EGFP. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) analysis (Figure S1, ESI†) and the fluorescence
Figure 1. Illustration of genetically engineered H6-EGFP and H6-Tat-EGFP proteins and characterization of two protein-only nanoparticles. (A) Description of H6-EGFP and H6-TatEGFP proteins. H6, six histidines; L, linker peptide; Tat, YGRRARRRRRR; EGFP, enhanced green fluorescent protein. (B) Size distribution of H6-EGFP and H6-TatEGFPin HBS and elution buffer determined by DLS. (C) Zeta potential analysis of two proteins. (D) TEM images of two protein nanoparticles (The high resolution TEM of H6-TatEGFP nanoparticle was shown. Scale Bar ¼ 5 nm.).
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emission spectra (Figure S2, ESI†) showed that both proteins were successfully prepared with enough purity. To determine when two protein nanoparticles were assembled, the size distribution of H6-EGFP and H6-TatEGFP nanoparticles in HBS (low salt) and elution buffer (high salt) were analyzed by dynamic light scattering (DLS). The results indicated that the nanosized structure of H6-EGFP disaggregated in solution with high salt and transformed into monomer; however, the size of H6-TatEGFP nanoparticles increased to 28.2 nm, suggesting the stability of H6-TatEGFP protein nanoparticles. Based on these results, we speculated that two protein nanoparticles may emerge in process of protein expression (Figure 1B). The zeta potential of H6-EGFP and H6-TatEGFP Figure 2. Molecular modeling of H6-EGFP (A) and H6-Tat-EGFP (B) nanoparticles through nanoparticles were –11.11 and –0.18 mV, protein–protein docking algorithm HADDOCK. The diameter and thickness of two respectively (Figure 1C). The decrease in protein nanoparticles were shown above and below, respectively. negative charge of H6-TatEGFP nanoparticles was ascribed to the addition of Tat sequence including eight arginines. Transmission bromide (MTT) assay on HeLa cells. As shown in electron microscope (TEM) analysis showed that both Figure S3, both proteins showed at least 85% cell viability nanoparticles displayed nanosized structure as shown in even at the concentration of 400 mg ml1. Then we determined if these nanoparticles could penetrate mamFigure 1D. These results confirmed well-defined nanostructures formed by two proteins. malian cell membranes while preserving the particulate Then the fined self-assemble structures of H6-EGFP organization and EGFP fluorescence. The cellular uptake and H6-Tat-EGFP were modelled through protein–protein experiments were performed on HeLa cells and EGFP docking algorithm HADDOCK.[44] It is a top ranked without H6 was used as a control. As shown in Figure 3, both two nanoparticles could penetrate the cell membrane and approach for protein–protein complex structure prediction distribute in the region near nucleus of HeLa cells. As a that utilizing the information from identified or predicted contrast, the EGFP protein, which was lack of his-tag at protein interfaces in ambiguous interaction restraints to the N-terminus, remained fully dispersed in the extradrive a flexible docking. The assemblies of both H6-EGFP cellular region. These results showed that only welland H6-Tat-EGFP have pentamers and arrange in a starassembled H6-EGFP and H6-TatEGFP nanoparticles but shape. The assembly of H6-EGFP is constructed through not EGFP proteins could be internalized by tumor cells. the interaction between N-terminal and C-terminal tails, Although Tat peptides has been widely used in develand through the interaction between a-helix in the oping efficient nanocarriers for delivery of siRNA or other N-terminal and the coils in both terminals in the complex therapeutic molecules,[46,47] to our knowledge protein of H6-Tat-EGFP. The predicted values of radius of gyration (Rg) were 5.23 and 5.48 nm for the assembly structures of nanoparticles based exclusively on a single protein construct for siRNA delivery had never been explored. MoreH6-EGFP and H6-Tat-EGFP. Their diameters were 11.90 over, the Tat peptide in this study (YGRRARRRRR) designed and 15.50 nm by converting the radius of gyration to a by Kim et al. and named cytoplasmic transduction disk with equal volume. The thickness of the disks were peptide (CTP), could translocate its payloads to the 3.90 and 4.10 nm, which are close to the cross-sections of cytoplasm rather than to the nucleus after transduction.[48] EGFP monomer (Figure 2). The size and the geometry of the predicted structures agree with our recent measureThen, we examined if our protein-only nanoparticles could ments using small angle X-ray scattering. be used as a novel nanocarrier for siRNA delivery to tumor The biocompatibility of nanomaterial is very important cells. An efficient siRNA vehicle should condense siRNA into stable nanosized complexes. A gel retardation assay for their biomedical applications. We investigated the cytotoxicity of H6-EGFP and H6-TatEGFP nanoparticles using was performed to investigate complexes formation of the 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium two protein nanoparticles with Cy3-labeled siRNA at
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Figure 3. Cell uptake of EGFP, H6-EGFP, and H6-Tat-EGFP proteins by HeLa cells analyzed. (A) CLSM results. The cell nuclear was stained with DAPI (blue signal) and three types of EGFP were analyzed by their inherent green fluorescence; (B) The cell uptake rate of H6-EGFPand H6Tat-EGFP nanoparticles were determined by flow cytometry results indicated.
different protein/siRNA ratio (wt/wt) from 1 to 100. As shown in Figure 4A, the intensity of migrating free siRNA nearly unchanged for H6-EGFP protein even at the ratio of 100:1; while the free siRNA bands of H6-TatEGFP decreased gradually with the increasing protein/siRNA ratio from 1 to 5, and finally disappeared at the ratio of 10, 25, 50, and 100.
These results suggested that despite the cationic nature conferred by six histidines, H6-EGFP nanoparticle was unable to condense siRNA into stable complex as revealed by unchanging migrating free siRNA; while the H6-TatEGFP nanoparticle with Tat peptide at the amino-terminus could condense siRNA into protein/siRNA complex as indicated by altering siRNA mobility in gel electrophoresis. DLS analysis indicated that the size of H6-TatEGFP protein/siRNA complex was nearly same to that of H6TatEGFP nanoparticles (Figure 4B), which also showed the siRNA-condensing property of H6-TatEGFP proteins. The transfection of Cy3-labelled siRNA mediated by protein nanoparticles in vitro was performed on HeLa cells and commercial liposome Lipofectamine 2000 (Invitrogen) was as a positive control. As shown in Figure 4C, H6EGFP/siRNA complexes were fully distributed in the extracellular region adherent to the cell membrane, suggesting a failure in siRNA delivery into HeLa cells; but H6-TatEGFP/siRNA and Lipo/siRNA complexes displayed red punctate structures in cytosol adjacent to the nucleus. Different from Lipo/siRNA group, some red fluorescence in H6-TatEGFP group was distributed in entire nucleus, suggesting the unique property of Tat peptides in nucleus targeting which has been proved by previous reports.[49–51]
Figure 4. Evaluation of siRNA-condensing properties of H6-EGFP and H6-TatEGFP nanoparticles. (A) Retardation of siRNA migration in 1% agarose gelelectrophoresis mediated by increasing ratio (wt/wt) of protein/siRNA. (B) Size distributions of the H6TatEGFP protein/siRNA complex at different ratio. (C) Transfection of Cy3-labelled siRNA by H6-EGFP and H6-Tat-EGFP nanoparticles in HeLa cells by CLSM. The nucleus was stained by DAPI (blue signal), and the siRNA was detected by Cy3 fluorescence (red signal).
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3. Conclusion In summary, we prepared two types of protein-only nanoparticles based on H6EGFP and H6-Tat-EGFP. The studies of
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Vazquez E could not detect the nanostructure of GFPH6,[10,14] considering the nearly same amino acid sequence between GFP and EGFP, we speculated that the structure differences of GFP-H6 and H6-EGFP were ascribed to the location of H6 in fusion proteins. For genetic engineer, H6 is just used as tag fused with interesting proteins which was easy to purify the proteins, and there seems no difference in fusion sites of H6 (at the amino or carboxyl terminal). However, our study showed that the site of H6 may play a role in formation of nanostructure. More importantly, we developed a novel siRNA delivery system based on H6-Tat-EGFP protein nanoparticles. H6-Tat-EGFP nanoparticles were internalized by HeLa cells with good biocompatibility. H6-TatEGFP nanoparticlescould condense siRNA into stable protein/siRNA complexes and deliver siRNA to cytosol. The H6-Tat-EGFP nanoparticle may be a novel siRNA nanocarrier for not only HeLa cells but also other tumors difficult to transfect.
Abbreviations EGFP enhanced green fluorescent protein CPP cell-penetrating peptide Hsp heat shock proteins DLS dynamic light scattering TEM transmission electron microscope
Acknowledgment: The project was supported by the National Natural Science Foundation of China (Project No. 91227118 and 51373167, 21374117).
Received: April 29, 2015; Revised: June 8, 2015; Published online: June 24, 2015; DOI: 10.1002/mabi.201500163 Keywords: protein nanoparticle; siRNA delivery; tat
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