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Large-Pore Ultrasmall Mesoporous Organosilica Nanoparticles: Micelle/Precursor Co-templating Assembly and Nuclear-Targeted Gene Delivery Meiying Wu, Qingshuo Meng, Yu Chen,* Yanyan Du, Lingxia Zhang, Yaping Li,* Linlin Zhang, and Jianlin Shi* Gene therapy is regarded as one of the most promising therapeutic strategies for combating many serious diseases, such as cancer and genetic disorders.[1] This therapeutic modality is generally based on the introduction of exogenous gene to living cells, which encodes a specific therapeutic protein to correct or modulate the diseases, thus eradicating the diseases at their sources.[2] It is well known that the success of gene therapy lies in the efficient transportation of large, fragile DNA molecules into the nucleus of the targeted cells without significant degradation by nucleases.[3] Extensive researches have been conducted to exploit efficient and safe nonviral vectors that can protect and release the genetic cargos at the site of action in the past decades.[4] Mesoporous silica nanoparticles (MSNs) have been recently developed as one of the most promising drug and gene delivery nanosystems due to a series of advantages in bioapplications.[5] However, in the most reported gene deliveries by MSNs, nucleic acids were generally conjugated or adsorbed onto the outer surface of MSNs due to the limitations by the relatively small pore sizes ( 100–300 nm), irregular morphologies, and severe aggregations among particles. Very recently, drug delivery directly into cell nucleus has been realized, which shows substantially enhanced therapeutic effect Dr. M. Wu, Dr. Y. Chen, Dr. Y. Du, Dr. L. Zhang, Dr. L. Zhang, Prof. J. Shi State Key Laboratory of High Performance Ceramics and Superfine Microstructure Shanghai Institute of Ceramics Chinese Academy of Sciences 1295 Ding-Xi Road, Shanghai 200050, PR China E-mail: [email protected]; [email protected] Dr. Q. Meng, Prof. Y. Li Shanghai Institute of Materia Medica Chinese Academy of Sciences 501 Haike Road, Shanghai 201203, PR China E-mail: [email protected]
DOI: 10.1002/adma.201404256
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in killing cancer cells and in overcoming multidrug resistance of cancer cells by a nuclear-targeted strategy using ultrasmall MSNs as drug carrier.[9] Unfortunately, these small MSNs usually have rather small pore size of ≈2–3 nm, therefore are not applicable for gene loading and delivery. Therefore, it is still a great challenge to develop a simple synthetic methodology to fabricate MSNs with large enough pore sizes and ultrasmall particle sizes for intranuclear gene delivery, as well as high biocompatibility and transfection efficiencies. Herein, we propose a facile and generalizable micelle/precursor co-templating assembly (M/P-CA) strategy to elaborately design and synthesize a unique kind of molecularly organic– inorganic hybrid nanosystem, i.e., mesoporous organosilica nanoparticles (MONs) with monodispersity, large nanopores, ultrasmall particle sizes, and enhanced biocompatibility. Furthermore, the cell-penetrating peptide transactivator of transcription (TAT) has been conjugated onto the outer surface of MONs for constructing a nuclear-targeted nanoplatform, which exhibits the high loading capacity, improved protection for loaded genes, and enhanced transfection efficiencies of plasmids. Traditional tetraethoxysilane (TEOS) and bissilylated organosilica precursors are chemically homologous, which have been successfully adopted for constructing various molecularly organic–inorganic hybrid mesoporous organosilicas, such as periodic mesoporous organosilicas.[10] It is well known that the organic disulfide bond is physiologically reducing-responsive, which has been extensively explored as the biocompatible groups for intelligent on-demand drug releasing.[11] In this respect, bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES), as a typical bissilylated organosilica precursor with thioether-bridged groups, was chosen to co-hydrolyze and co-condense with TEOS to fabricate unique molecularly organic–inorganic hybrid MONs. The thioether-bridged organic groups can be uniformly embedded within the framework of MONs at the atomic scale (Figure 1a). Cetyltrimethylammonium chloride (CTAC) and triethanolamine (TEA) were used as the structure-directing agent and the basic catalyst, respectively.[12] It has been reported that the hydrolysis and condensation rates of BTES are much lower than that of TEOS, due to the conformation and inductive effect of bulk organic spacer, as well as the steric effect of rigid ethoxy group.[13] Thus, the as-hydrolyzed BTES molecules with hydrophobic CH2CH2CH2 S S S S CH2CH2CH2 chains can penetrate into the hydrophobic domains of asformed surfactant CTAC micelles, resulting in the substantially enlarged micelle, and much enlarged pore sizes of MONs consequently (Figure 1b).
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Figure 1. a) Schematic representation for the molecularly organic–inorganic hybrid composition of thioether-bridged MONs. b) The proposed M/P-CA strategy to enlarge the micelle size of CTAC by incorporating the hydrophobic long organic chains of as-hydrolyzed BTES into the hydrophobic part of initially formed CTAC micelles.
The well-defined and monodispersed flower-like MONs with radial ultralarge porous structure and ≈30 nm particle size can be clearly observed in the transmission electron microscopy (TEM) (Figure 2a–c) images and scanning transmission electron microscopy (STEM) (Figure 2e–g and Figure S1, Supporting Information) images. The N2 sorption isotherm implies that MONs possess the well-defined mesoporous structure with a large surface area of 613.9 m2 g–1 and an extraordinarily high pore volume of 2.19 cm3 g–1(Figure 2d). The corresponding pore size distribution curve further shows that the average pore size of MONs is about 6.2 nm (inset to Figure 2d). The diameter of pore openings is significantly larger than 6.2 nm observed from the TEM images, which reaches 8–13 nm (Figure 2a–c). The average hydrated particle size of MONs is 50.75 nm (polydispersity: 0.121), determined by dynamic light scattering (DLS) (Figure S2a, Supporting Information), due to the presence of hydrated layer on the particle surface. Raman spectroscopy, as a general sensitive tool for the detection of S S bonds, affirms the presence of stretching vibrations of S S and S C bonds located at 438, 488, and 636 cm−1, respectively (Figure 2h).[13] The absorptions of S S and S C (520–720 cm−1) bonds in the Fourier transform infrared (FTIR) spectrum of MONs also demonstrate the formation of silsesquioxane framework (Figure 2i),[14] as compared with MSNs of the same diameter (Figure 3c), where these characteristic absorptions were absent.
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The presence of sulfur signals in energy-dispersive X-ray spectroscopy spectrum further proves the incorporation of sulfur element within MONs (Figure S2b, Supporting Information). 29 Si magic-angle spinning (MAS) solid-state NMR spectrum of MONs exhibits the signals in the range of –100 to –110 ppm and –45 to –90 ppm, which derive from Q and T silicon sites, respectively (Figure 2j). The 13C cross-polarization MAS (CPMAS) spectrum of MONs (Figure 2k) shows the characteristic peaks at 10, 21, and 40 ppm, corresponding to the 1C, 2C, and 3C carbon species in Si 1CH22CH23CH2 S S S S 3CH22CH21CH2 Si , respectively, which further clarify the organic–inorganic hybrid compositions of MONs.[13,15] The effects of the alkaline catalyst and silicon precursor were investigated on the formation of mesoporous nanostructure during M/P-CA. Without the addition of BTES, the average size of MSNs with pure Si O Si framework decreases from 80 to 30 nm along with increasing the TEA amount (Figure 3a–c). When BTES is introduced, increasing the TEA amount but keeping the other parameters constant (e.g., Figure 3d–f, or g–i), the hydrolysis and condensation of TEOS and BTES can be accelerated, and more hydrolyzed BTES can be incorporated into the surfactant micelles, resulting in expanded CTAC micelles and correspondingly enlarged pore size of MONs. At the same TEA amount while elevating BTES amount (e.g., Figure 3d and g, or e and h, or f and i), more as-hydrolyzed
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COMMUNICATION Figure 2. a–c) TEM images of MONs at different magnifications (inset of c: the pore shape of MONs). d) N2 adsorption–desorption isotherm and (inset of d) the corresponding pore size distribution of MONs. e–g) Bright-field (e) and dark-field (f) STEM images and their corresponding SEM image (g) of MONs. h,i) Raman spectra (h) and FTIR spectra (i) of MSNs and MONs. j,k) Solid-state 29Si MAS NMR spectrum (j) and 13C CPMAS NMR spectrum (k) of MONs.
BTES penetrate in the micelles, resulting in the increased pore size of MONs (Figure S3, Supporting Information). Thus, the pore sizes of MONs can be simply tuned by varying the amount of either organosilica precursor or alkaline catalyst. Both synthesized MSNs and MONs of almost the same particle size (Figure 3c) were evaluated of their hematological biocompatibility. It can be found that traditional MSNs have caused significant hemolytic effect at elevated concentrations. Comparatively, MONs exhibit much lower hemolytic effect. The hemolysis percentage of MONs at an extremely high concentration of 1000 µg mL–1 is still low at 16.6%, much lower than that of MSNs (48%) at the same concentration (Figure S4, Supporting Information) due to the much decreased Si OH amounts on the surface of MONs, which is responsible for the hemolytic effect.[16] The in vivo long-term toxicity of MONs was further evaluated by intravenously administrating MONs saline solution into mice at dosages of 15 and 30 mg kg–1 for 30, 60, and 90 d. Hematoxylin and eosin staining (Figure S5, Supporting Information), blood chemistry, and complete blood panel analysis (Figure S6, Supporting Information) results show no significant differences from the control group, indicating that MONs are highly biocompatible at the dose up to 30 mg kg–1 for as long as 90 d. Nuclear-targeted genetic engineering facilitates the direct transport of the therapeutic genetic biomacromolecules into
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the nucleus, where nucleic acids can be exactly and efficiently expressed.[17] However, nuclear-targeted gene delivery using mesoporous materials has not been reported so far due to the substantial technical challenges in synthesizing large-pore and ultrasmall MSNs for gene encapsulation and intranuclear gene delivery. The highly biocompatible MONs are highly suitable for nuclear-targeted gene delivery attributing to their radial and highly open large mesopores and ultrasmall particle size. To concurrently realize the enhanced binding to nucleic acids, nuclear-targeted transport and high gene transfection efficiency, the surface of MONs was conjugated with polyethyleneimine (PEI) and TAT stepwise (Figure 4a) for enhanced gene loading and protection by PEI and the transport across the nuclear membrane via TAT mediation. Typically, MONs were firstly aminated through silanization (MONs–NH2), followed by the reaction with succinic acid to obtain MONs–COOH. Subsequently, MONs–COOH could react with amino groups of PEI (M.W. = 1.2 kDa) to enable the grafting of the PEI molecules (MONs–PEI). The low molecular weight of PEI endows MONs– PEI with low toxicity and enhanced uptake efficiency by cells. TAT peptide was then grafted onto the surface of MONs–PEI by an esterification reaction (designated as MONs–PTAT). Finally, the nanocomplexes (MONs–PTAT@pDNA) were obtained by loading pDNA into MONs–PTAT simply by a vortex process. MONs–PTAT@pDNA will accumulate in tumors via the typical
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Figure 3. TEM images of MSNs and thioether-bridged MONs synthesized using different TEA and BTES amounts: a–c) MSNs by the hydrolysis and condensation of TEOS without the addition of BTES but with different TEA amounts (a: 0.1 g, b: 0.2 g, and c: 0.8 g); d–f) MONs by using varied TEA amounts (d: 0.4 g, e: 0.6 g, and f: 0.8 g) while keeping the BTES amount constant (0.65 g); g–i) MONs by increasing the BTES amount to 1.3 g and changing the TEA amounts (g: 0.4 g, h: 0.6 g, and i: 0.8 g).
enhanced permeability and retention (EPR) effect of tumors from blood stream. The endocytosed MONs–PTAT@pDNA can escape from the endosomes and actively target the nuclear pore complexes (NPCs) due to the combined proton sponge effect of PEI and the nuclear localization signal of TAT peptide on the surface of MONs, respectively (Figure 4b). The successful conjugations of PEI and TAT are confirmed by the moderate increase in the particle size of MONs determined by the DLS technique, zeta potential changes (Figure S7 and Table S1, Supporting Information), Raman spectroscopy (Figure S8a, Supporting Information), FTIR spectroscopy (Figure S8b, Supporting Information), and thermogravimetric analysis (TGA) (Figure S8c, Supporting Information). Moreover, the porous structures of MONs are well maintained after the step-by-step modifications (Figure S9 and S10, and Table S2, Supporting Information). Furthermore, in vivo histocompatibility evaluations of MONs–PTAT show that there are no apparent pathological changes of mice after receiving MONs– PTAT compared with the control group (Figure S11, Supporting Information), indicating that the MONs–PTAT hold a high biocompatibility as a nonviral gene vector for gene therapy.
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The plasmid DNA (pUC57 DNA, M.W. = 2710 bp) was chosen as a model gene. Gel retardation assay shows that pDNA could be condensed completely at the nanomaterials to pDNA mass ratio (MR) over 20 for MONs–PEI while 15 for MONs–PTAT (Figure S12a,b, Supporting Information), which indicates that the PEI–TAT conjugates can significantly enhance the pDNA loading capacity of MONs.[18] The pDNA loading amount of MONs–PTAT is 66.67 µg mg–1 (Figure S12b, Supporting Information), much higher than that by MSNs– PTAT (33.3 µg mg–1, Figure S12e,f, Supporting Information) and those of large pore MSNs-based nanovectors,[19] most probably due to its unique radial large mesopores and the specific PEI–TAT comodifications. The cell viabilities of MONs–PEI, MONs–PTAT, MONs–PEI@pDNA, and MONs–PTAT@pDNA were further assessed by a conventional MTT assay. All materials employed do not cause significant cytotoxicities to HeLa cells at the concentrations up to 400 µg mL–1 (Figure S12c,d, Supporting Information). In addition, DNase I was introduced to detect the degradation behavior of pDNA under varied conditions (Figure S13, Supporting Information). Thus, MONs– PTAT can efficiently protect pDNA from enzymatic degradation
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COMMUNICATION Figure 4. a) Schematic illustration showing the TAT peptide tethering approach onto the surface of MONs, including covalent bonding of succinic acid with amino group of MONs–NH2 to produce MONs–COOH, the conjugation of branched PEI realized by esterification reaction (MONs–PEI) and final anchoring of the TAT peptide onto the surface of MONs–PEI to form MONs–PTAT; b) Schematic representation of nuclear-targeted gene delivery procedure mediated by MONs–PTAT@pDNA.
(Figure S13a, Supporting Information) while pDNA is completely degraded in the case of MSNs–PTAT (Figure S13b, Supporting Information). The significant difference in DNase I protection assays is attributed to the fact that pDNA has been well trapped into the pore channels of MONs–PTAT but only absorbed onto the outer surface of MSNs–PTAT. To demonstrate the intranuclear gene transport, HeLa cells were preincubated with MONs–PEI@pDNA and MONs– PTAT@pDNA for 12 and 24 h, and observed using confocal laser scanning microscopy (CLSM) (Figure 5a–p). MONs–PTAT with green fluorescence and pDNA with red fluorescence, as labeled by FITC and TOTO-3, respectively, could be observed in the nucleoplasm after MONs–PTAT-mediated plasmid delivery (Figure 5i–p), while the green and red fluorescences are only present in the cytoplasm and perinuclear region in the case of MONs–PEI (Figure 5a–h). BioTEM images further indicate that MONs–PTAT uptaken by HeLa cells could enter the nucleus while the MONs–PEI are located mostly in the cytoplasm or perinuclear area (Figure 5q and r). More convincingly, the intranuclear silicon amounts of MONs–PTAT in HeLa cells were found to be 21.6- and 18.1-folds higher than those incubated
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with MONs–PEI in 12 and 24 h, respectively. While the total intracellular silicon accumulations in HeLa cells induced by MONs–PTAT are also significantly higher than those by MONs–PEI, though the difference of the intracellular silicon accumulations between the two types of nanoparticles is not as remarkable as those in the nucleus, which show only 2.4and 1.7-fold increments in 12 and 24 h, respectively (Figure 5s). The above results undoubtedly prove the role of TAT peptide in elevating the cellular uptake of the carriers, especially into the nucleus. The transfection efficiencies of pDNA were evaluated by transfecting an EGFP plasmid (pEGFP), encoding enhanced green fluorescent protein, into HeLa cells mediated by MONs– PEI, MONs–PTAT, and MSNs–PTAT at varied MRs after coincubation for 72 h. The cells transfected by MONs–PTAT@ pEGFP exhibit significantly brighter green fluorescence than those by MONs–PEI@pEGFP and MSNs–PTAT@pEGFP (Figure 6a and Figure S14a, Supporting Information), indicating the enhanced transfection efficiency due to the intranuclear gene delivery. The quantitative results, as evidenced by the mean fluorescent intensity (Figure 6b and Figure S14b,
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Figure 5. CLSM images of HeLa cells after the incubation with (a–h) MONs–PEI@pDNA and (i–p) MONs–PTAT@pDNA for 12 and 24 h. (a), (e), (i), and (m) represent nuclei stained with DAPI (blue fluorescence); (b), (f), (j), and (n) represent MONs–PEI and MONs–PTAT labeled with green fluorescein FITC, respectively; (c), (g), (k), and (o) represent pDNA labeled by TOTO-3 emitting red fluorescene; d, h, l, and p are the merged images. a2-p2 are 3D confocal fluorescence reconstruction images. BioTEM images of HeLa cells after incubation with (q) MONs–PEI and (r) MONs-PTAT for 24 h. s) Cellular and nuclear uptake amounts of MONs–PEI and MONs–PTAT by HeLa cells in 12 and 24 h incubations.
Figure 6. Enhanced pEGFP transfection efficiency mediated by MONs–PTAT. a) Fluorescent images of EGFP expression of HeLa cells after coincubation with MONs–PEI@pEGFP and MONs–PTAT@ pEGFP at varied carrier/pDNA MRs of 15, 20, and 25 for 72 h (the scale bars of all the images are 100 µm); Transfection efficiency as illustrated by (b) fluorescent intensity and (c) proportion of cells in the population showing EGFP expressions. Data are given as mean ± standard deviation of three independent experiments (n = 3). *p < 0.05 and **p < 0.01 are compared with control group.
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements M.W. and Q.M. contributed equally to this work. This work was supported by the National Natural Science Foundation of China (Grant No. 51132009, 51302293), the Natural Science Foundation of Shanghai (13ZR1463500), the Shanghai Rising-Star Program (14QA1404100),
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Supporting Information) and the percentage of transfected cells (Figure 6c and S14c, Supporting Information), show that the transfection efficiency mediated by MONs–PTAT is 36.5% at MR = 25, substantially higher than that by MONs–PEI (13.5%) and MSNs–PTAT (4.97%). Such a significant increase in transfection efficiency further demonstrate the significant contributions by the coconjugated PEI and TAT peptide and the unique radial large pores. The nuclear-targeting gene-delivery nanoplatform MONs– PTAT is superior to traditional MSNs of small pore sizes and most organic nanosystems featured with low stability, inefficient cellular internalization, poor nuclear translocation, and potential cytotoxicity, thanks to the following three specific features for gene transport by MONs–PTAT. Firstly, the significantly improved biocompatibility of MONs with molecularly organic–inorganic hybridization can guarantee the biosafety in their biomedical applications and further clinical translations. Secondly, the unique radial large pores and about 30 nm particulate sizes can effectively protect the adsorbed pDNA from enzymatic degradation and deliver pDNA through NPCs on the nuclear membrane. Thirdly, elaborately designed PEI–TAT complex grafted on the surface of MONs–PTAT can promote the endosome escape due to the PEI proton sponge effect and mediate the transport to cross the nuclear membrane by TAT peptide. These unique characteristics of MONs–PTAT endow them with enhanced gene transfection efficiency and bright potential for clinical translations. In summary, a novel, highly efficient and versatile micelle/ precursor co-templating assembly (M/P-CA) strategy has been successfully developed to construct unique molecularly organic–inorganic hybrid MONs with ultrasmall particulate size and large radial mesopore. Such a specific thioether-hybrid composition endows MONs with significantly improved biocompatibility. The unique large mesopores and ultrasmall particle sizes of the MONs have been successfully employed for highly efficient intranuclear gene delivery after the stepwise surface conjugations with PEI and TAT, which demonstrate a much enhanced loading capacity of plasmids, satisfactory protection of plasmids from nuclease-mediated degradation, and corresponding intranuclear high plasmid transfection efficiency. The successful chemical construction of molecularly organic–inorganic hybrid MONs with controllable key structural/compositional features is a substantial advance over traditional MSNs, and is expected to be highly promising in biomedical applications, such as drug/gene delivery for cancer therapy.
the Natural Science Foundation of Shanghai (13ZR1463500) and the National Natural Science Foundation of China (81373359). Received: September 14, 2014 Published online:
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Large-Pore Ultrasmall Mesoporous Organosilica Nanoparticles: Micelle/Precursor Co-templating Assembly and Nuclear-Targeted Gene Delivery Meiying Wu, Qingshuo Meng, Yu Chen,* Yanyan Du, Lingxia Zhang, Yaping Li,* Linlin Zhang, and Jianlin Shi* Gene therapy is regarded as one of the most promising therapeutic strategies for combating many serious diseases, such as cancer and genetic disorders.[1] This therapeutic modality is generally based on the introduction of exogenous gene to living cells, which encodes a specific therapeutic protein to correct or modulate the diseases, thus eradicating the diseases at their sources.[2] It is well known that the success of gene therapy lies in the efficient transportation of large, fragile DNA molecules into the nucleus of the targeted cells without significant degradation by nucleases.[3] Extensive researches have been conducted to exploit efficient and safe nonviral vectors that can protect and release the genetic cargos at the site of action in the past decades.[4] Mesoporous silica nanoparticles (MSNs) have been recently developed as one of the most promising drug and gene delivery nanosystems due to a series of advantages in bioapplications.[5] However, in the most reported gene deliveries by MSNs, nucleic acids were generally conjugated or adsorbed onto the outer surface of MSNs due to the limitations by the relatively small pore sizes ( 100–300 nm), irregular morphologies, and severe aggregations among particles. Very recently, drug delivery directly into cell nucleus has been realized, which shows substantially enhanced therapeutic effect Dr. M. Wu, Dr. Y. Chen, Dr. Y. Du, Dr. L. Zhang, Dr. L. Zhang, Prof. J. Shi State Key Laboratory of High Performance Ceramics and Superfine Microstructure Shanghai Institute of Ceramics Chinese Academy of Sciences 1295 Ding-Xi Road, Shanghai 200050, PR China E-mail: [email protected]; [email protected] Dr. Q. Meng, Prof. Y. Li Shanghai Institute of Materia Medica Chinese Academy of Sciences 501 Haike Road, Shanghai 201203, PR China E-mail: [email protected]
DOI: 10.1002/adma.201404256
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in killing cancer cells and in overcoming multidrug resistance of cancer cells by a nuclear-targeted strategy using ultrasmall MSNs as drug carrier.[9] Unfortunately, these small MSNs usually have rather small pore size of ≈2–3 nm, therefore are not applicable for gene loading and delivery. Therefore, it is still a great challenge to develop a simple synthetic methodology to fabricate MSNs with large enough pore sizes and ultrasmall particle sizes for intranuclear gene delivery, as well as high biocompatibility and transfection efficiencies. Herein, we propose a facile and generalizable micelle/precursor co-templating assembly (M/P-CA) strategy to elaborately design and synthesize a unique kind of molecularly organic– inorganic hybrid nanosystem, i.e., mesoporous organosilica nanoparticles (MONs) with monodispersity, large nanopores, ultrasmall particle sizes, and enhanced biocompatibility. Furthermore, the cell-penetrating peptide transactivator of transcription (TAT) has been conjugated onto the outer surface of MONs for constructing a nuclear-targeted nanoplatform, which exhibits the high loading capacity, improved protection for loaded genes, and enhanced transfection efficiencies of plasmids. Traditional tetraethoxysilane (TEOS) and bissilylated organosilica precursors are chemically homologous, which have been successfully adopted for constructing various molecularly organic–inorganic hybrid mesoporous organosilicas, such as periodic mesoporous organosilicas.[10] It is well known that the organic disulfide bond is physiologically reducing-responsive, which has been extensively explored as the biocompatible groups for intelligent on-demand drug releasing.[11] In this respect, bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES), as a typical bissilylated organosilica precursor with thioether-bridged groups, was chosen to co-hydrolyze and co-condense with TEOS to fabricate unique molecularly organic–inorganic hybrid MONs. The thioether-bridged organic groups can be uniformly embedded within the framework of MONs at the atomic scale (Figure 1a). Cetyltrimethylammonium chloride (CTAC) and triethanolamine (TEA) were used as the structure-directing agent and the basic catalyst, respectively.[12] It has been reported that the hydrolysis and condensation rates of BTES are much lower than that of TEOS, due to the conformation and inductive effect of bulk organic spacer, as well as the steric effect of rigid ethoxy group.[13] Thus, the as-hydrolyzed BTES molecules with hydrophobic CH2CH2CH2 S S S S CH2CH2CH2 chains can penetrate into the hydrophobic domains of asformed surfactant CTAC micelles, resulting in the substantially enlarged micelle, and much enlarged pore sizes of MONs consequently (Figure 1b).
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Figure 1. a) Schematic representation for the molecularly organic–inorganic hybrid composition of thioether-bridged MONs. b) The proposed M/P-CA strategy to enlarge the micelle size of CTAC by incorporating the hydrophobic long organic chains of as-hydrolyzed BTES into the hydrophobic part of initially formed CTAC micelles.
The well-defined and monodispersed flower-like MONs with radial ultralarge porous structure and ≈30 nm particle size can be clearly observed in the transmission electron microscopy (TEM) (Figure 2a–c) images and scanning transmission electron microscopy (STEM) (Figure 2e–g and Figure S1, Supporting Information) images. The N2 sorption isotherm implies that MONs possess the well-defined mesoporous structure with a large surface area of 613.9 m2 g–1 and an extraordinarily high pore volume of 2.19 cm3 g–1(Figure 2d). The corresponding pore size distribution curve further shows that the average pore size of MONs is about 6.2 nm (inset to Figure 2d). The diameter of pore openings is significantly larger than 6.2 nm observed from the TEM images, which reaches 8–13 nm (Figure 2a–c). The average hydrated particle size of MONs is 50.75 nm (polydispersity: 0.121), determined by dynamic light scattering (DLS) (Figure S2a, Supporting Information), due to the presence of hydrated layer on the particle surface. Raman spectroscopy, as a general sensitive tool for the detection of S S bonds, affirms the presence of stretching vibrations of S S and S C bonds located at 438, 488, and 636 cm−1, respectively (Figure 2h).[13] The absorptions of S S and S C (520–720 cm−1) bonds in the Fourier transform infrared (FTIR) spectrum of MONs also demonstrate the formation of silsesquioxane framework (Figure 2i),[14] as compared with MSNs of the same diameter (Figure 3c), where these characteristic absorptions were absent.
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The presence of sulfur signals in energy-dispersive X-ray spectroscopy spectrum further proves the incorporation of sulfur element within MONs (Figure S2b, Supporting Information). 29 Si magic-angle spinning (MAS) solid-state NMR spectrum of MONs exhibits the signals in the range of –100 to –110 ppm and –45 to –90 ppm, which derive from Q and T silicon sites, respectively (Figure 2j). The 13C cross-polarization MAS (CPMAS) spectrum of MONs (Figure 2k) shows the characteristic peaks at 10, 21, and 40 ppm, corresponding to the 1C, 2C, and 3C carbon species in Si 1CH22CH23CH2 S S S S 3CH22CH21CH2 Si , respectively, which further clarify the organic–inorganic hybrid compositions of MONs.[13,15] The effects of the alkaline catalyst and silicon precursor were investigated on the formation of mesoporous nanostructure during M/P-CA. Without the addition of BTES, the average size of MSNs with pure Si O Si framework decreases from 80 to 30 nm along with increasing the TEA amount (Figure 3a–c). When BTES is introduced, increasing the TEA amount but keeping the other parameters constant (e.g., Figure 3d–f, or g–i), the hydrolysis and condensation of TEOS and BTES can be accelerated, and more hydrolyzed BTES can be incorporated into the surfactant micelles, resulting in expanded CTAC micelles and correspondingly enlarged pore size of MONs. At the same TEA amount while elevating BTES amount (e.g., Figure 3d and g, or e and h, or f and i), more as-hydrolyzed
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COMMUNICATION Figure 2. a–c) TEM images of MONs at different magnifications (inset of c: the pore shape of MONs). d) N2 adsorption–desorption isotherm and (inset of d) the corresponding pore size distribution of MONs. e–g) Bright-field (e) and dark-field (f) STEM images and their corresponding SEM image (g) of MONs. h,i) Raman spectra (h) and FTIR spectra (i) of MSNs and MONs. j,k) Solid-state 29Si MAS NMR spectrum (j) and 13C CPMAS NMR spectrum (k) of MONs.
BTES penetrate in the micelles, resulting in the increased pore size of MONs (Figure S3, Supporting Information). Thus, the pore sizes of MONs can be simply tuned by varying the amount of either organosilica precursor or alkaline catalyst. Both synthesized MSNs and MONs of almost the same particle size (Figure 3c) were evaluated of their hematological biocompatibility. It can be found that traditional MSNs have caused significant hemolytic effect at elevated concentrations. Comparatively, MONs exhibit much lower hemolytic effect. The hemolysis percentage of MONs at an extremely high concentration of 1000 µg mL–1 is still low at 16.6%, much lower than that of MSNs (48%) at the same concentration (Figure S4, Supporting Information) due to the much decreased Si OH amounts on the surface of MONs, which is responsible for the hemolytic effect.[16] The in vivo long-term toxicity of MONs was further evaluated by intravenously administrating MONs saline solution into mice at dosages of 15 and 30 mg kg–1 for 30, 60, and 90 d. Hematoxylin and eosin staining (Figure S5, Supporting Information), blood chemistry, and complete blood panel analysis (Figure S6, Supporting Information) results show no significant differences from the control group, indicating that MONs are highly biocompatible at the dose up to 30 mg kg–1 for as long as 90 d. Nuclear-targeted genetic engineering facilitates the direct transport of the therapeutic genetic biomacromolecules into
Adv. Mater. 2014, DOI: 10.1002/adma.201404256
the nucleus, where nucleic acids can be exactly and efficiently expressed.[17] However, nuclear-targeted gene delivery using mesoporous materials has not been reported so far due to the substantial technical challenges in synthesizing large-pore and ultrasmall MSNs for gene encapsulation and intranuclear gene delivery. The highly biocompatible MONs are highly suitable for nuclear-targeted gene delivery attributing to their radial and highly open large mesopores and ultrasmall particle size. To concurrently realize the enhanced binding to nucleic acids, nuclear-targeted transport and high gene transfection efficiency, the surface of MONs was conjugated with polyethyleneimine (PEI) and TAT stepwise (Figure 4a) for enhanced gene loading and protection by PEI and the transport across the nuclear membrane via TAT mediation. Typically, MONs were firstly aminated through silanization (MONs–NH2), followed by the reaction with succinic acid to obtain MONs–COOH. Subsequently, MONs–COOH could react with amino groups of PEI (M.W. = 1.2 kDa) to enable the grafting of the PEI molecules (MONs–PEI). The low molecular weight of PEI endows MONs– PEI with low toxicity and enhanced uptake efficiency by cells. TAT peptide was then grafted onto the surface of MONs–PEI by an esterification reaction (designated as MONs–PTAT). Finally, the nanocomplexes (MONs–PTAT@pDNA) were obtained by loading pDNA into MONs–PTAT simply by a vortex process. MONs–PTAT@pDNA will accumulate in tumors via the typical
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Figure 3. TEM images of MSNs and thioether-bridged MONs synthesized using different TEA and BTES amounts: a–c) MSNs by the hydrolysis and condensation of TEOS without the addition of BTES but with different TEA amounts (a: 0.1 g, b: 0.2 g, and c: 0.8 g); d–f) MONs by using varied TEA amounts (d: 0.4 g, e: 0.6 g, and f: 0.8 g) while keeping the BTES amount constant (0.65 g); g–i) MONs by increasing the BTES amount to 1.3 g and changing the TEA amounts (g: 0.4 g, h: 0.6 g, and i: 0.8 g).
enhanced permeability and retention (EPR) effect of tumors from blood stream. The endocytosed MONs–PTAT@pDNA can escape from the endosomes and actively target the nuclear pore complexes (NPCs) due to the combined proton sponge effect of PEI and the nuclear localization signal of TAT peptide on the surface of MONs, respectively (Figure 4b). The successful conjugations of PEI and TAT are confirmed by the moderate increase in the particle size of MONs determined by the DLS technique, zeta potential changes (Figure S7 and Table S1, Supporting Information), Raman spectroscopy (Figure S8a, Supporting Information), FTIR spectroscopy (Figure S8b, Supporting Information), and thermogravimetric analysis (TGA) (Figure S8c, Supporting Information). Moreover, the porous structures of MONs are well maintained after the step-by-step modifications (Figure S9 and S10, and Table S2, Supporting Information). Furthermore, in vivo histocompatibility evaluations of MONs–PTAT show that there are no apparent pathological changes of mice after receiving MONs– PTAT compared with the control group (Figure S11, Supporting Information), indicating that the MONs–PTAT hold a high biocompatibility as a nonviral gene vector for gene therapy.
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The plasmid DNA (pUC57 DNA, M.W. = 2710 bp) was chosen as a model gene. Gel retardation assay shows that pDNA could be condensed completely at the nanomaterials to pDNA mass ratio (MR) over 20 for MONs–PEI while 15 for MONs–PTAT (Figure S12a,b, Supporting Information), which indicates that the PEI–TAT conjugates can significantly enhance the pDNA loading capacity of MONs.[18] The pDNA loading amount of MONs–PTAT is 66.67 µg mg–1 (Figure S12b, Supporting Information), much higher than that by MSNs– PTAT (33.3 µg mg–1, Figure S12e,f, Supporting Information) and those of large pore MSNs-based nanovectors,[19] most probably due to its unique radial large mesopores and the specific PEI–TAT comodifications. The cell viabilities of MONs–PEI, MONs–PTAT, MONs–PEI@pDNA, and MONs–PTAT@pDNA were further assessed by a conventional MTT assay. All materials employed do not cause significant cytotoxicities to HeLa cells at the concentrations up to 400 µg mL–1 (Figure S12c,d, Supporting Information). In addition, DNase I was introduced to detect the degradation behavior of pDNA under varied conditions (Figure S13, Supporting Information). Thus, MONs– PTAT can efficiently protect pDNA from enzymatic degradation
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COMMUNICATION Figure 4. a) Schematic illustration showing the TAT peptide tethering approach onto the surface of MONs, including covalent bonding of succinic acid with amino group of MONs–NH2 to produce MONs–COOH, the conjugation of branched PEI realized by esterification reaction (MONs–PEI) and final anchoring of the TAT peptide onto the surface of MONs–PEI to form MONs–PTAT; b) Schematic representation of nuclear-targeted gene delivery procedure mediated by MONs–PTAT@pDNA.
(Figure S13a, Supporting Information) while pDNA is completely degraded in the case of MSNs–PTAT (Figure S13b, Supporting Information). The significant difference in DNase I protection assays is attributed to the fact that pDNA has been well trapped into the pore channels of MONs–PTAT but only absorbed onto the outer surface of MSNs–PTAT. To demonstrate the intranuclear gene transport, HeLa cells were preincubated with MONs–PEI@pDNA and MONs– PTAT@pDNA for 12 and 24 h, and observed using confocal laser scanning microscopy (CLSM) (Figure 5a–p). MONs–PTAT with green fluorescence and pDNA with red fluorescence, as labeled by FITC and TOTO-3, respectively, could be observed in the nucleoplasm after MONs–PTAT-mediated plasmid delivery (Figure 5i–p), while the green and red fluorescences are only present in the cytoplasm and perinuclear region in the case of MONs–PEI (Figure 5a–h). BioTEM images further indicate that MONs–PTAT uptaken by HeLa cells could enter the nucleus while the MONs–PEI are located mostly in the cytoplasm or perinuclear area (Figure 5q and r). More convincingly, the intranuclear silicon amounts of MONs–PTAT in HeLa cells were found to be 21.6- and 18.1-folds higher than those incubated
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with MONs–PEI in 12 and 24 h, respectively. While the total intracellular silicon accumulations in HeLa cells induced by MONs–PTAT are also significantly higher than those by MONs–PEI, though the difference of the intracellular silicon accumulations between the two types of nanoparticles is not as remarkable as those in the nucleus, which show only 2.4and 1.7-fold increments in 12 and 24 h, respectively (Figure 5s). The above results undoubtedly prove the role of TAT peptide in elevating the cellular uptake of the carriers, especially into the nucleus. The transfection efficiencies of pDNA were evaluated by transfecting an EGFP plasmid (pEGFP), encoding enhanced green fluorescent protein, into HeLa cells mediated by MONs– PEI, MONs–PTAT, and MSNs–PTAT at varied MRs after coincubation for 72 h. The cells transfected by MONs–PTAT@ pEGFP exhibit significantly brighter green fluorescence than those by MONs–PEI@pEGFP and MSNs–PTAT@pEGFP (Figure 6a and Figure S14a, Supporting Information), indicating the enhanced transfection efficiency due to the intranuclear gene delivery. The quantitative results, as evidenced by the mean fluorescent intensity (Figure 6b and Figure S14b,
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Figure 5. CLSM images of HeLa cells after the incubation with (a–h) MONs–PEI@pDNA and (i–p) MONs–PTAT@pDNA for 12 and 24 h. (a), (e), (i), and (m) represent nuclei stained with DAPI (blue fluorescence); (b), (f), (j), and (n) represent MONs–PEI and MONs–PTAT labeled with green fluorescein FITC, respectively; (c), (g), (k), and (o) represent pDNA labeled by TOTO-3 emitting red fluorescene; d, h, l, and p are the merged images. a2-p2 are 3D confocal fluorescence reconstruction images. BioTEM images of HeLa cells after incubation with (q) MONs–PEI and (r) MONs-PTAT for 24 h. s) Cellular and nuclear uptake amounts of MONs–PEI and MONs–PTAT by HeLa cells in 12 and 24 h incubations.
Figure 6. Enhanced pEGFP transfection efficiency mediated by MONs–PTAT. a) Fluorescent images of EGFP expression of HeLa cells after coincubation with MONs–PEI@pEGFP and MONs–PTAT@ pEGFP at varied carrier/pDNA MRs of 15, 20, and 25 for 72 h (the scale bars of all the images are 100 µm); Transfection efficiency as illustrated by (b) fluorescent intensity and (c) proportion of cells in the population showing EGFP expressions. Data are given as mean ± standard deviation of three independent experiments (n = 3). *p < 0.05 and **p < 0.01 are compared with control group.
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements M.W. and Q.M. contributed equally to this work. This work was supported by the National Natural Science Foundation of China (Grant No. 51132009, 51302293), the Natural Science Foundation of Shanghai (13ZR1463500), the Shanghai Rising-Star Program (14QA1404100),
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Supporting Information) and the percentage of transfected cells (Figure 6c and S14c, Supporting Information), show that the transfection efficiency mediated by MONs–PTAT is 36.5% at MR = 25, substantially higher than that by MONs–PEI (13.5%) and MSNs–PTAT (4.97%). Such a significant increase in transfection efficiency further demonstrate the significant contributions by the coconjugated PEI and TAT peptide and the unique radial large pores. The nuclear-targeting gene-delivery nanoplatform MONs– PTAT is superior to traditional MSNs of small pore sizes and most organic nanosystems featured with low stability, inefficient cellular internalization, poor nuclear translocation, and potential cytotoxicity, thanks to the following three specific features for gene transport by MONs–PTAT. Firstly, the significantly improved biocompatibility of MONs with molecularly organic–inorganic hybridization can guarantee the biosafety in their biomedical applications and further clinical translations. Secondly, the unique radial large pores and about 30 nm particulate sizes can effectively protect the adsorbed pDNA from enzymatic degradation and deliver pDNA through NPCs on the nuclear membrane. Thirdly, elaborately designed PEI–TAT complex grafted on the surface of MONs–PTAT can promote the endosome escape due to the PEI proton sponge effect and mediate the transport to cross the nuclear membrane by TAT peptide. These unique characteristics of MONs–PTAT endow them with enhanced gene transfection efficiency and bright potential for clinical translations. In summary, a novel, highly efficient and versatile micelle/ precursor co-templating assembly (M/P-CA) strategy has been successfully developed to construct unique molecularly organic–inorganic hybrid MONs with ultrasmall particulate size and large radial mesopore. Such a specific thioether-hybrid composition endows MONs with significantly improved biocompatibility. The unique large mesopores and ultrasmall particle sizes of the MONs have been successfully employed for highly efficient intranuclear gene delivery after the stepwise surface conjugations with PEI and TAT, which demonstrate a much enhanced loading capacity of plasmids, satisfactory protection of plasmids from nuclease-mediated degradation, and corresponding intranuclear high plasmid transfection efficiency. The successful chemical construction of molecularly organic–inorganic hybrid MONs with controllable key structural/compositional features is a substantial advance over traditional MSNs, and is expected to be highly promising in biomedical applications, such as drug/gene delivery for cancer therapy.
the Natural Science Foundation of Shanghai (13ZR1463500) and the National Natural Science Foundation of China (81373359). Received: September 14, 2014 Published online:
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