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Intracellular Delivery of Peptide Nucleic Acid and Organic Molecules Using Zeolite-L Nanocrystals Alessandro Bertucci, Henning Lülf, Dedy Septiadi, Alex Manicardi, Roberto Corradini,* and Luisa De Cola* high affinity for DNA and RNA, their high sequence selectivity, and their stability towards degradation of both nucleases and peptidases. However, a persistent drawback for biomedical applications of PNAs is their poor cell membrane permeability.[8] For this reason numerous efforts have been done to improve their cellular uptake by their modification at the backbone level, or conjugating the PNA to cell penetrating peptides.[9–17] Recently, first examples of soft nanoparticles as PNA carriers have been reported in the literature.[18,19] To the best of our knowledge, however, there are no reports of non-polymeric and porous nanoparticles that were used as PNA carriers for in vitro or in vivo experiments. Porous nanoparticles are very popular for drug delivery applications and, to date, mesoporous silica nanoparticles (MSNPs) are the most prominent ones studied for this purpose.[20–22] Due to their high porosity and surface area, these materials show great potential in manufacturing tailor-made multifunctional nanocontainers. Besides the amorphous MSNPs, crystalline silica-based nanocontainers, such as zeolites, possess similar characteristics but they are much less studied for drug delivery or other biomedical applications.[23–26] Zeolite-L in particular is a porous, crystalline aluminosilicate that possesses a 1D channel system able to host a large number of guest molecules. The aspect ratio and size of zeolite-L crystals can be tuned over a wide range and single crystals with a size of just a few tenths of nanometers can be obtained,[27] which makes them very promising for in vitro and in vivo applications. Here, we report the first example of the use of porous zeolite-L nanoparticles as a multifunctional PNA and drug delivery system. We demonstrate that our nanomaterial is able to deliver high amounts of both a model drug and PNA to living cells.
The design and synthesis of smart nanomaterials can provide interesting potential applications for biomedical purposes from bioimaging to drug delivery. Manufacturing multifunctional systems in a way to carry bioactive molecules, like peptide nucleic acids able to recognize specific targets in living cells, represents an achievement towards the development of highly selective tools for both diagnosis and therapeutics. This work describes a very first example of the use of zeolite nanocrystals as multifunctional nanocarriers to deliver simultaneously PNA and organic molecules into living cells. Zeolite-L nanocrystals are functionalized by covalently attaching the PNA probes onto the surface, while the channel system is filled with fluorescent guest molecules. The cellular uptake of the PNA/Zeolite-L hybrid material is then significantly increased by coating the whole system with a thin layer of biodegradable poly-L-lysine. The delivery of DAPI as a model drug molecule, inserted into the zeolite pores, is also demonstrated to occur in the cells, proving the multifunctional ability of the system. Using this zeolite nanosystem carrying PNA probes designed to target specific RNA sequences of interest in living cells could open new possibilities for theranostic and gene therapy applications.
1. Introduction The development of novel tools for antisense-diagnostics and gene therapy represents a very active research field.[1–6] In this context, peptide nucleic acids (PNAs) have become a powerful tool and they are considered a promising platform for the development of novel gene therapy agents. PNAs are oligonucleotide mimics in which the natural negatively charged sugar-phosphate backbone is replaced by a neutral polyamide backbone composed of N-(2-aminoethyl)glycine units.[7] Key features that make PNAs highly suitable for biological applications are their A. Bertucci, H. Lülf, D. Septiadi, Prof. L. De Cola Institut de science et d’ingénierie supramoléculaire (ISIS), icFRC and CNRS Université de Strasbourg 8 Rue Gaspard Monge, BP 70028 67000 Strasbourg, France E-mail: [email protected] A. Bertucci, Dr. A. Manicardi, Prof. R. Corradini Dipartimento di Chimica Università di Parma Parco Area delle Scienze, 17/A 43124 Parma, Italy E-mail: [email protected]
DOI: 10.1002/adhm.201400116
Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201400116
2. Results and Discussion 2.1. Material Preparation and Characterization Zeolite-L crystals of 60 nm were synthesized following a literature protocol[27] and characterized by SEM, TEM, DLS, and zeta-potential measurements (see Figure 1 and Supporting
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Figure 1. A) Schematic representation of the synthetic pathway for the preparation of PNA/zeolite-L nanoparticles; B) TEM image of zeolite-L nanocrystals; C) DLS measurement showing the size distribution of the particles with different functionalizations; Table inset: Zeta-potential values obtained after each functionalization step.
information). The crystal channels were then filled with the desired guest molecules, DAPI (Supporting Information), and then the outer particle surface was functionalized with (3-aminopropyl)triethoxysilane (APTES). The functionalization was confirmed by a zeta-potential shift from negative to +15.70 mV and by a positive ninhydrin test. Subsequently, the amine groups were converted into carboxylic acid groups, as confirmed by a zeta-potential shift to −37.37 mV. Finally, these groups were converted into NHS esters and the PNA probes H-(AEEA)2-CTTTCCTTCACTGTT-NH2 (AEEA = 2-(2-aminoethoxy)ethoxyacetyl spacer) were covalently attached via acyl coupling reactions. The PNA chosen is full complementary to a DNA sequence bearing a single-point mutation (W1282X) implicated in human Cystic Fibrosis disease. It was already tested in a biosensing application[28] and represents a good example in view of possible further biomedical purposes. After PNA attachment, the remaining NHS esters were quenched with ethanolamine and the zeta potential of the PNA-functionalized particles was found to be −10.21 mV. The final overall negative zeta potential is crucial since a thin coating with polyL-lysine (PLL), which is a cationic polymer in physiological media, was finally performed to favor cell uptake. A schematic representation of the material preparation is shown in Figure 1. The size of the nanoparticles increased after each step and zetapotential changes were consistent with the addition of the corresponding layer for each step. After the PLL final coating, the positive zeta-potential observed was stable after 24 h; the average size of these NPs (123 nm) and the low dispersion of data (14% relative standard deviation) confirmed that no significant aggregation was present at this stage. To further prove the successful functionalization of the zeolite-L crystals with PNA, a hybridization test with a Cy3-labeled
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full-complementary DNA oligonucleotide was carried out, which led to a zeta-potential shift to −24.80 mV. Additionally, we used confocal microscopy to assess the co-localization of the Cy3 label (on the full-complementary DNA) and the fluorescent label (DAPI), entrapped in the zeolite-L pores (see Figure S2, Supporting Information). Finally, we estimated the number of PNA units bound to a single zeolite crystal to be about 150 (for details, see Supporting Information). For antisense purposes, it is crucial that the PNA probes maintain their biologically activity even when anchored to the zeolites. Thus, we determined whether the PNA retained its DNA/RNA-binding activity when attached to the zeolites. PNA/zeolite-L particles were therefore incubated with three different Cy3 labeled DNA strands: a full match, a single mismatch, and a 3-mismatch oligonucleotide. The particles were then washed and dissolved in HF and UV/vis spectroscopy was applied to determine the DNA concentration in the solution. A clear difference in DNA binding was observed depending on the sequence (Figure 2). The highest amount of DNA bound to the PNA/zeolite-L was obtained, when a fully complementary DNA strand was used. The introduction of one mismatch in the DNA led to a significant reduction of binding and finally no binding was observed with a 3-mismatch DNA strand. These results confirm that PNA retains its sequence selectivity, following the Watson-Crick base pairing scheme, also when covalently anchored to the surface of zeolite-L.
2.2. In Vitro Experiments with PNA/Zeolite-L To visualize the nanomaterials by fluorescence microscopy, the zeolites were filled with the fluorescent dye
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Figure 2. UV/vis spectra of dissolved zeolite-L/PNA particles after hybridization with three different DNA strands. Black line: full-match DNA strand; Red line: single mismatch DNA strand; Blue line: 3-mismatch DNA strand.
N,N ′-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarbodiimide (DXP),[29] chosen because of its insolubility in aqueous media, which prevents its release from the pores. The nanocontainers were then covalently functionalized with PNA and further covered with PLL to improve cell uptake, as shown in Figure 1. Indeed, it has been shown that the molecular corona surrounding nanoparticles is very important in the uptake process;[30,31] thus, biocompatible polymers can be used to increase the cellular uptake of nanoparticles[32–38] and PLL represents a good candidate.[39] Besides that, PLL is biodegradable[40,41] and therefore does not affect the overall activity of the system. PLL coating was confirmed by a zeta-potential shift, +2.82 mV, and a size increase to about 120 nm, as confirmed by DLS measurements (Figure 1). Cell experiments were carried out by using human cervical cancer cells (HeLa) (50 000 cells per well). Incubation was done for 1, 4, and 24 h using a particle concentration of 0.01 mg mL−1. After incubation, the cells were washed, fixed, and analyzed by confocal microscopy. Figure 3 shows confocal images after 1, 4, and 24 h incubation time. We obtained a very fast uptake of the PNA-functionalized zeolites, and already after an incubation time of 1 h, a high particle concentration can be seen inside the cells, as shown in Figure 3A–C. We proved the particle uptake by recording z-stacks after 1 h incubation (see Figure S3, Supporting Information). An increase in incubation time then leads to even higher particle concentrations inside the cells and after 24 h, a very high particle concentration is observed (Figure 3G–I). In all cases, the particles are equally distributed in the cytoplasm without entering the nucleus. Taking into account that PNA is very poor membrane permeable, this approach presents a very simple and straightforward way to efficiently deliver PNA into living cells. To prove that the PNA/zeolite-L particles are not toxic, a cell-viability test was carried out under the same conditions described above. Details can be found in the Supporting Information.
Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201400116
Figure 3. Confocal images of HeLa cells incubated with a 0.01 mg mL−1 dispersion of PLL-PNA-DXP-functionalized zeolites for A–C) 1 h; D–F) 4 h; and G–I) 24 h. A,D,G) Fluorescent image of cell nuclei stained with DAPI; B,E,H) Fluorescent image of DXP, showing the presence of the nanoparticles inside cells; C,F,I) Overlay of the previous pictures.
In order to demonstrate that the PNA-zeolites are internalized without destruction of the oligonucleotide and that the PNA units are covalently bound to the nanocontainers after internalization, we have used fluorescence colocalization experiments. A PNA labeled with the yellow fluorescent TAMRA (H-TAMRA-(D-Lys)-GTAGATGA-NH2) was bound to the surface of zeolites filled with deep-red fluorescent oxazine 170. The necessity of using oxazine 170 as label for the nanocontainers is dictated by the need to have an emission color (deep red) that does not overlap with TAMRA (yellow) or the DAPI (blue), which was used to stain the nucleus. Again, the particles were coated with PLL and cell experiments were done as described above using an incubation of 24 h. The different fluorescent labels have then been localized by using different excitation wavelengths. As can be seen in Figure 4, the emitting zeolites, red, and the labeled PNA (shown in green) showed an identical pattern and a co-localization proves that the PNA is still bound to the zeolites after cell internalization. Also the images show that the nanomaterials are distributed in the cytoplasm with patterns similar (at least at the image resolution obtained) to that of PLL-PNA-DXP (Figure 3), with no particles observed inside the nucleus.
2.3. Delivery of DAPI as a Model Drug Having understood that the PNA nanocontainers can be internalized without releasing the PNA by degradation of the covalent bonds, we have exploited the possibility to combine the oligonucleotide activities with a drug that can be released
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Figure 4. Confocal image of HeLa cells treated with (TAMRA)-PNA-Oxazine-zeolites after 24 h of incubation using a 0.01 mg mL−1 concentration of the dispersion. A) Fluorescent image of cell nuclei stained with DAPI; B) Fluorescent image of TAMRA, showing the position of the labeled PNA probes; C) Fluorescent image of oxazine 170; D) Overlay of the previous pictures to prove co-localization of the PNA and the zeolites. Excitation was performed at 405 nm (DAPI), 543 nm (TAMRA), and 633 nm (Oxazine 170).
from the zeolites after degradation of the PLL. We have selected DAPI as a model drug, since its fluorescence can be easily detected and after the release, it will migrate into the nucleus where the particles cannot enter. DAPI was inserted into the channels by cationic exchange and the filled crystals were functionalized as described above. The DAPI loading was determined by thermogravimetric analysis (TGA) to be 16% ndye/nsite. Cell experiments have been performed under the same conditions of the other samples. After incubating the cells for 1 h (Figure 5A–C), we observed the internalization of the particles but no release of DAPI was observed, since the nucleus was unstained and the blue emission was observed only from the zeolites. This can be ascribed to the PLL coating, which prevents the diffusion of the DAPI from the zeolite pores and which has relatively slow kinetics of degradation inside the cells.[42] After 4 h of incubation, a notable amount of DAPI can be found in the nucleus (see Figure 5D–F). Indeed the release of the DAPI can occur at this time, since the PLL has been degraded and thus the channels are “opened,” allowing the leakage of the DAPI into the cell. The release is further increased when the incubation time is prolonged to 24 h (see Figure 5G–I). It is worth noticing that also the number of particles which are internalized is time dependent; thus, the final delivery of DAPI is the result of the increase in concentration of the internalized nanoparticles as well as the slow release of the fluorophore from them.
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Figure 5. Confocal images of HeLa cells incubated with a 0.01 mg mL−1 dispersion of PLL-PNA-DAPI-functionalized zeolites for A–C) 1 h; D–F) 4 h; G–I) 24 h. A,D,G) Fluorescent image recorded monitoring DAPI signal; B,E,H) Bright-field image; C,F,I) Overlay of the previous ones.
3. Conclusion We developed novel multifunctional hybrid nanoparticles to deliver simultaneous PNA and organic molecules into the cells. For the first time, we used inorganic nanoparticles as PNA carriers for this goal. A very efficient uptake, even after 1 h, can be achieved by coating the particles with a layer of PLL. The PNA remains intact even after the cell internalization. To prove the functions of the nanocontainers, a model drug, DAPI, was inserted into the pores, and the leakage was suppressed by coating the nanoparticles with PLL. After 4 h of incubation, the degradation of the PLL is complete and the DAPI can diffuse into the nucleus leading to its staining. The results are therefore opening interesting paths to the delivery of specific sequences of PNAs with a simultaneous combined drug effect.
4. Experimental Section PNA Oligomer Synthesis: The solid-phase synthesis of PNA sequence H-(AEEA)2-CTTTCCTTCACTGTT-NH2 was performed as reported in ref. [28] and that of labeled PNA sequence H-TAMRA-(D-Lys)GTAGATGA-NH2 was carried out as reported elsewhere.[43] Synthesis of Nanozeolite-L: For the synthesis of nanozeolite-L, a gel composition of 9.34 K2O—1.00 Al2O3—20.20 SiO2—412.84 H2O was used. First, a silica suspension was prepared by adding 12.02 g of silica source (Aerosil OX-50) to 28.04 g deionized water. The silica was dispersed by using an Ultra Turrax mixer (IKA T18 Basic) for 8 min at 18 000 rpm. A potassium silicate solution was prepared by adding 40.06 g of silica suspension to 7.23 g of potassium hydroxide dissolved in 21.68 g deionized water. A potassium aluminate solution was prepared by adding 20.00 g of deionized water to a mixture of 4.84 g potassium
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hydroxide and 1.56 g aluminum hydroxide. Both solutions were refluxed until clear solutions were obtained. After cooling to room temperature, the potassium aluminate solution was added to the potassium silicate solution. The mixture was stirred at room temperature for 10 min and then gel was transferred to a Teflon vessel. Crystallization was done at 170 °C for 6 h under dynamic conditions (16 rpm). The zeolites were cooled to room temperature and washed with deionized water until the pH of the supernatant was neutral. DAPI Insertion in Zeolite Channels: Zeolite-L nanocrystals (150 mg) were dispersed in 15 mL of a 0.3 × 10−3 M aqueous solution of DAPI and stirred overnight at 50 °C. The final fluorescent material was finally recovered by centrifugation washing with water until the supernatant was found no more emissive. After TGA, an amount of 0.97 of DAPI per 100 mg of zeolites was determined. DXP Insertion in Zeolite Channels: DXP was inserted from the gas phase at 290 °C. 100 mg of zeolite-L crystals were mixed with 0.5 mg DXP in a glass ampoule and dehydrated at 1.0 × 10−5 mbar for 6 h and sealed. The mixture was left at 290 °C for 24 h to obtain dye insertion. The ampoule was opened and the zeolites were washed with n-butanol until the supernatant did not show any fluorescence. This procedure affords a dye loading of around 7% (ndye/nsite). Insertion of Oxazine 170 in Zeolite Channels: Zeolite-L nanocrystals (200 mg) were dispersed in a 0.01 mmol solution of oxazine 170 perchlorate in water and stirred overnight at reflux. The fluorescent zeolites were then recovered by centrifugation, washing the solid residue with water until the supernatant resulted completely UV transparent. After TGA, a value of about 2 mg of oxazine per 100 mg of zeolites was found. Nanozeolites Surface Modification with PNA: Amino-modified zeolites were first prepared by sonication of the particles (80 mg) in toluene (10 mL) for 30 min to form a good dispersion. APTES (50 µL, 0.2 mmol) was added to the suspension in presence of a catalytic amount of TEA and the mixture was stirred at room temperature overnight. The particles were then recovered by centrifugation and washed three times with ethanol to remove impurities. The same protocol was carried out both for unfilled zeolite-L crystals and for Oxazine and DXP zeolites. To obtain carboxylic acid-functionalized nanoparticles, 70 mg of the former amino-modified zeolites was dispersed in a succinic anhydride solution in DMSO (0.1 M in 10 mL) and, after 30 min of sonication to get a good dispersion, the mixture was stirred overnight at room temperature. The material was then centrifuged and washed three times with DMSO. After that, succinimidyl-functionalized nanozeolites were obtained by reaction of the former material (50 mg) with a solution of DIC and NHS (both 0.25 M) in dry DMSO. The nanoparticles were dispersed in 10 mL of the reactant solution and stirred overnight at room temperature under nitrogen inert atmosphere. The final material was again recovered by centrifugation washing with DMSO. PNA attachment was subsequently carried out by dispersing 5 mg of the activated-esters-modified-zeolites in 500 µL of a PNA solution 30 × 10−6 M in a 100 × 10−3 M carbonate buffer, H2O:Acetonitrile 9:1, pH 9. The dispersion was stirred overnight at room temperature and PNA-zeolites were finally obtained by centrifuging and washing three times with water. The final quenching step was performed by stirring overnight at room temperature the PNAzeolites with a 100 × 10−3 M solution of ethanolamine in Tris buffer (100 × 10−3 M, pH 9). The final material was recovered by centrifugation and washing three times with water. Poly-L-lysine Coating: Poly-L-lysine coating was always carried out by dispersing 1 mg of PNA-zeolites in a 1% w/v solution of poly-L-lysine hydrobromide in water and stirring the mixture at room temperature for 1 h. The final material was recovered by centrifugation and washing with water. Cell Culture: HeLa cells were cultured inside media, which contain 88% Dulbecco’s modified Eagle medium (DMEM), 10% Fetal Bovine Serum, 1% Penicillin–Streptomycin, and 1% L-glutamine 200 × 10−3 M (all material was purchased from Gibco) under 37 °C and 5% of CO2 conditioning for 48 h until 70% to 80% cell confluency was reached. Subsequently, the cells were washed twice with phosphate buffer solution (PBS, Gibco) and treated with trypsin, then ≈50 000 cells were reseeded on the monolayer glass cover slip inside 6-well plate
culture dish. New culture media (2 mL) were added gently and the cells were grown overnight. Incubation: Working solutions of culture medium containing PNAzeolites in 0.01 mg mL−1 concentration were added gently to the cells. After 1, 4, and 24 h incubation, the media were removed and cells were washed with PBS twice. Cells were fixed with 4% paraformaldehyde (PFA) and, subsequently, the cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). After staining, cells were washed once with PBS and then with water. Cells were removed from the well and mounted onto the rectangular glass cover for microscopy experiments.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements The author thank the ERC advanced grant award (no. 2009–247365), the International Center for Frontier Research in Chemistry (icFRC), and MIUR PRIN09 grant (20093N774P_001) for financial support, as well as the French Embassy in Italy and the French government for providing the MAEE grant for scientific cooperation between Italy and France (no. 778588G). Received: February 24, 2014 Revised: March 19, 2014 Published online:
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Intracellular Delivery of Peptide Nucleic Acid and Organic Molecules Using Zeolite-L Nanocrystals Alessandro Bertucci, Henning Lülf, Dedy Septiadi, Alex Manicardi, Roberto Corradini,* and Luisa De Cola* high affinity for DNA and RNA, their high sequence selectivity, and their stability towards degradation of both nucleases and peptidases. However, a persistent drawback for biomedical applications of PNAs is their poor cell membrane permeability.[8] For this reason numerous efforts have been done to improve their cellular uptake by their modification at the backbone level, or conjugating the PNA to cell penetrating peptides.[9–17] Recently, first examples of soft nanoparticles as PNA carriers have been reported in the literature.[18,19] To the best of our knowledge, however, there are no reports of non-polymeric and porous nanoparticles that were used as PNA carriers for in vitro or in vivo experiments. Porous nanoparticles are very popular for drug delivery applications and, to date, mesoporous silica nanoparticles (MSNPs) are the most prominent ones studied for this purpose.[20–22] Due to their high porosity and surface area, these materials show great potential in manufacturing tailor-made multifunctional nanocontainers. Besides the amorphous MSNPs, crystalline silica-based nanocontainers, such as zeolites, possess similar characteristics but they are much less studied for drug delivery or other biomedical applications.[23–26] Zeolite-L in particular is a porous, crystalline aluminosilicate that possesses a 1D channel system able to host a large number of guest molecules. The aspect ratio and size of zeolite-L crystals can be tuned over a wide range and single crystals with a size of just a few tenths of nanometers can be obtained,[27] which makes them very promising for in vitro and in vivo applications. Here, we report the first example of the use of porous zeolite-L nanoparticles as a multifunctional PNA and drug delivery system. We demonstrate that our nanomaterial is able to deliver high amounts of both a model drug and PNA to living cells.
The design and synthesis of smart nanomaterials can provide interesting potential applications for biomedical purposes from bioimaging to drug delivery. Manufacturing multifunctional systems in a way to carry bioactive molecules, like peptide nucleic acids able to recognize specific targets in living cells, represents an achievement towards the development of highly selective tools for both diagnosis and therapeutics. This work describes a very first example of the use of zeolite nanocrystals as multifunctional nanocarriers to deliver simultaneously PNA and organic molecules into living cells. Zeolite-L nanocrystals are functionalized by covalently attaching the PNA probes onto the surface, while the channel system is filled with fluorescent guest molecules. The cellular uptake of the PNA/Zeolite-L hybrid material is then significantly increased by coating the whole system with a thin layer of biodegradable poly-L-lysine. The delivery of DAPI as a model drug molecule, inserted into the zeolite pores, is also demonstrated to occur in the cells, proving the multifunctional ability of the system. Using this zeolite nanosystem carrying PNA probes designed to target specific RNA sequences of interest in living cells could open new possibilities for theranostic and gene therapy applications.
1. Introduction The development of novel tools for antisense-diagnostics and gene therapy represents a very active research field.[1–6] In this context, peptide nucleic acids (PNAs) have become a powerful tool and they are considered a promising platform for the development of novel gene therapy agents. PNAs are oligonucleotide mimics in which the natural negatively charged sugar-phosphate backbone is replaced by a neutral polyamide backbone composed of N-(2-aminoethyl)glycine units.[7] Key features that make PNAs highly suitable for biological applications are their A. Bertucci, H. Lülf, D. Septiadi, Prof. L. De Cola Institut de science et d’ingénierie supramoléculaire (ISIS), icFRC and CNRS Université de Strasbourg 8 Rue Gaspard Monge, BP 70028 67000 Strasbourg, France E-mail: [email protected] A. Bertucci, Dr. A. Manicardi, Prof. R. Corradini Dipartimento di Chimica Università di Parma Parco Area delle Scienze, 17/A 43124 Parma, Italy E-mail: [email protected]
DOI: 10.1002/adhm.201400116
Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201400116
2. Results and Discussion 2.1. Material Preparation and Characterization Zeolite-L crystals of 60 nm were synthesized following a literature protocol[27] and characterized by SEM, TEM, DLS, and zeta-potential measurements (see Figure 1 and Supporting
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Figure 1. A) Schematic representation of the synthetic pathway for the preparation of PNA/zeolite-L nanoparticles; B) TEM image of zeolite-L nanocrystals; C) DLS measurement showing the size distribution of the particles with different functionalizations; Table inset: Zeta-potential values obtained after each functionalization step.
information). The crystal channels were then filled with the desired guest molecules, DAPI (Supporting Information), and then the outer particle surface was functionalized with (3-aminopropyl)triethoxysilane (APTES). The functionalization was confirmed by a zeta-potential shift from negative to +15.70 mV and by a positive ninhydrin test. Subsequently, the amine groups were converted into carboxylic acid groups, as confirmed by a zeta-potential shift to −37.37 mV. Finally, these groups were converted into NHS esters and the PNA probes H-(AEEA)2-CTTTCCTTCACTGTT-NH2 (AEEA = 2-(2-aminoethoxy)ethoxyacetyl spacer) were covalently attached via acyl coupling reactions. The PNA chosen is full complementary to a DNA sequence bearing a single-point mutation (W1282X) implicated in human Cystic Fibrosis disease. It was already tested in a biosensing application[28] and represents a good example in view of possible further biomedical purposes. After PNA attachment, the remaining NHS esters were quenched with ethanolamine and the zeta potential of the PNA-functionalized particles was found to be −10.21 mV. The final overall negative zeta potential is crucial since a thin coating with polyL-lysine (PLL), which is a cationic polymer in physiological media, was finally performed to favor cell uptake. A schematic representation of the material preparation is shown in Figure 1. The size of the nanoparticles increased after each step and zetapotential changes were consistent with the addition of the corresponding layer for each step. After the PLL final coating, the positive zeta-potential observed was stable after 24 h; the average size of these NPs (123 nm) and the low dispersion of data (14% relative standard deviation) confirmed that no significant aggregation was present at this stage. To further prove the successful functionalization of the zeolite-L crystals with PNA, a hybridization test with a Cy3-labeled
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full-complementary DNA oligonucleotide was carried out, which led to a zeta-potential shift to −24.80 mV. Additionally, we used confocal microscopy to assess the co-localization of the Cy3 label (on the full-complementary DNA) and the fluorescent label (DAPI), entrapped in the zeolite-L pores (see Figure S2, Supporting Information). Finally, we estimated the number of PNA units bound to a single zeolite crystal to be about 150 (for details, see Supporting Information). For antisense purposes, it is crucial that the PNA probes maintain their biologically activity even when anchored to the zeolites. Thus, we determined whether the PNA retained its DNA/RNA-binding activity when attached to the zeolites. PNA/zeolite-L particles were therefore incubated with three different Cy3 labeled DNA strands: a full match, a single mismatch, and a 3-mismatch oligonucleotide. The particles were then washed and dissolved in HF and UV/vis spectroscopy was applied to determine the DNA concentration in the solution. A clear difference in DNA binding was observed depending on the sequence (Figure 2). The highest amount of DNA bound to the PNA/zeolite-L was obtained, when a fully complementary DNA strand was used. The introduction of one mismatch in the DNA led to a significant reduction of binding and finally no binding was observed with a 3-mismatch DNA strand. These results confirm that PNA retains its sequence selectivity, following the Watson-Crick base pairing scheme, also when covalently anchored to the surface of zeolite-L.
2.2. In Vitro Experiments with PNA/Zeolite-L To visualize the nanomaterials by fluorescence microscopy, the zeolites were filled with the fluorescent dye
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Figure 2. UV/vis spectra of dissolved zeolite-L/PNA particles after hybridization with three different DNA strands. Black line: full-match DNA strand; Red line: single mismatch DNA strand; Blue line: 3-mismatch DNA strand.
N,N ′-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarbodiimide (DXP),[29] chosen because of its insolubility in aqueous media, which prevents its release from the pores. The nanocontainers were then covalently functionalized with PNA and further covered with PLL to improve cell uptake, as shown in Figure 1. Indeed, it has been shown that the molecular corona surrounding nanoparticles is very important in the uptake process;[30,31] thus, biocompatible polymers can be used to increase the cellular uptake of nanoparticles[32–38] and PLL represents a good candidate.[39] Besides that, PLL is biodegradable[40,41] and therefore does not affect the overall activity of the system. PLL coating was confirmed by a zeta-potential shift, +2.82 mV, and a size increase to about 120 nm, as confirmed by DLS measurements (Figure 1). Cell experiments were carried out by using human cervical cancer cells (HeLa) (50 000 cells per well). Incubation was done for 1, 4, and 24 h using a particle concentration of 0.01 mg mL−1. After incubation, the cells were washed, fixed, and analyzed by confocal microscopy. Figure 3 shows confocal images after 1, 4, and 24 h incubation time. We obtained a very fast uptake of the PNA-functionalized zeolites, and already after an incubation time of 1 h, a high particle concentration can be seen inside the cells, as shown in Figure 3A–C. We proved the particle uptake by recording z-stacks after 1 h incubation (see Figure S3, Supporting Information). An increase in incubation time then leads to even higher particle concentrations inside the cells and after 24 h, a very high particle concentration is observed (Figure 3G–I). In all cases, the particles are equally distributed in the cytoplasm without entering the nucleus. Taking into account that PNA is very poor membrane permeable, this approach presents a very simple and straightforward way to efficiently deliver PNA into living cells. To prove that the PNA/zeolite-L particles are not toxic, a cell-viability test was carried out under the same conditions described above. Details can be found in the Supporting Information.
Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201400116
Figure 3. Confocal images of HeLa cells incubated with a 0.01 mg mL−1 dispersion of PLL-PNA-DXP-functionalized zeolites for A–C) 1 h; D–F) 4 h; and G–I) 24 h. A,D,G) Fluorescent image of cell nuclei stained with DAPI; B,E,H) Fluorescent image of DXP, showing the presence of the nanoparticles inside cells; C,F,I) Overlay of the previous pictures.
In order to demonstrate that the PNA-zeolites are internalized without destruction of the oligonucleotide and that the PNA units are covalently bound to the nanocontainers after internalization, we have used fluorescence colocalization experiments. A PNA labeled with the yellow fluorescent TAMRA (H-TAMRA-(D-Lys)-GTAGATGA-NH2) was bound to the surface of zeolites filled with deep-red fluorescent oxazine 170. The necessity of using oxazine 170 as label for the nanocontainers is dictated by the need to have an emission color (deep red) that does not overlap with TAMRA (yellow) or the DAPI (blue), which was used to stain the nucleus. Again, the particles were coated with PLL and cell experiments were done as described above using an incubation of 24 h. The different fluorescent labels have then been localized by using different excitation wavelengths. As can be seen in Figure 4, the emitting zeolites, red, and the labeled PNA (shown in green) showed an identical pattern and a co-localization proves that the PNA is still bound to the zeolites after cell internalization. Also the images show that the nanomaterials are distributed in the cytoplasm with patterns similar (at least at the image resolution obtained) to that of PLL-PNA-DXP (Figure 3), with no particles observed inside the nucleus.
2.3. Delivery of DAPI as a Model Drug Having understood that the PNA nanocontainers can be internalized without releasing the PNA by degradation of the covalent bonds, we have exploited the possibility to combine the oligonucleotide activities with a drug that can be released
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Figure 4. Confocal image of HeLa cells treated with (TAMRA)-PNA-Oxazine-zeolites after 24 h of incubation using a 0.01 mg mL−1 concentration of the dispersion. A) Fluorescent image of cell nuclei stained with DAPI; B) Fluorescent image of TAMRA, showing the position of the labeled PNA probes; C) Fluorescent image of oxazine 170; D) Overlay of the previous pictures to prove co-localization of the PNA and the zeolites. Excitation was performed at 405 nm (DAPI), 543 nm (TAMRA), and 633 nm (Oxazine 170).
from the zeolites after degradation of the PLL. We have selected DAPI as a model drug, since its fluorescence can be easily detected and after the release, it will migrate into the nucleus where the particles cannot enter. DAPI was inserted into the channels by cationic exchange and the filled crystals were functionalized as described above. The DAPI loading was determined by thermogravimetric analysis (TGA) to be 16% ndye/nsite. Cell experiments have been performed under the same conditions of the other samples. After incubating the cells for 1 h (Figure 5A–C), we observed the internalization of the particles but no release of DAPI was observed, since the nucleus was unstained and the blue emission was observed only from the zeolites. This can be ascribed to the PLL coating, which prevents the diffusion of the DAPI from the zeolite pores and which has relatively slow kinetics of degradation inside the cells.[42] After 4 h of incubation, a notable amount of DAPI can be found in the nucleus (see Figure 5D–F). Indeed the release of the DAPI can occur at this time, since the PLL has been degraded and thus the channels are “opened,” allowing the leakage of the DAPI into the cell. The release is further increased when the incubation time is prolonged to 24 h (see Figure 5G–I). It is worth noticing that also the number of particles which are internalized is time dependent; thus, the final delivery of DAPI is the result of the increase in concentration of the internalized nanoparticles as well as the slow release of the fluorophore from them.
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Figure 5. Confocal images of HeLa cells incubated with a 0.01 mg mL−1 dispersion of PLL-PNA-DAPI-functionalized zeolites for A–C) 1 h; D–F) 4 h; G–I) 24 h. A,D,G) Fluorescent image recorded monitoring DAPI signal; B,E,H) Bright-field image; C,F,I) Overlay of the previous ones.
3. Conclusion We developed novel multifunctional hybrid nanoparticles to deliver simultaneous PNA and organic molecules into the cells. For the first time, we used inorganic nanoparticles as PNA carriers for this goal. A very efficient uptake, even after 1 h, can be achieved by coating the particles with a layer of PLL. The PNA remains intact even after the cell internalization. To prove the functions of the nanocontainers, a model drug, DAPI, was inserted into the pores, and the leakage was suppressed by coating the nanoparticles with PLL. After 4 h of incubation, the degradation of the PLL is complete and the DAPI can diffuse into the nucleus leading to its staining. The results are therefore opening interesting paths to the delivery of specific sequences of PNAs with a simultaneous combined drug effect.
4. Experimental Section PNA Oligomer Synthesis: The solid-phase synthesis of PNA sequence H-(AEEA)2-CTTTCCTTCACTGTT-NH2 was performed as reported in ref. [28] and that of labeled PNA sequence H-TAMRA-(D-Lys)GTAGATGA-NH2 was carried out as reported elsewhere.[43] Synthesis of Nanozeolite-L: For the synthesis of nanozeolite-L, a gel composition of 9.34 K2O—1.00 Al2O3—20.20 SiO2—412.84 H2O was used. First, a silica suspension was prepared by adding 12.02 g of silica source (Aerosil OX-50) to 28.04 g deionized water. The silica was dispersed by using an Ultra Turrax mixer (IKA T18 Basic) for 8 min at 18 000 rpm. A potassium silicate solution was prepared by adding 40.06 g of silica suspension to 7.23 g of potassium hydroxide dissolved in 21.68 g deionized water. A potassium aluminate solution was prepared by adding 20.00 g of deionized water to a mixture of 4.84 g potassium
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hydroxide and 1.56 g aluminum hydroxide. Both solutions were refluxed until clear solutions were obtained. After cooling to room temperature, the potassium aluminate solution was added to the potassium silicate solution. The mixture was stirred at room temperature for 10 min and then gel was transferred to a Teflon vessel. Crystallization was done at 170 °C for 6 h under dynamic conditions (16 rpm). The zeolites were cooled to room temperature and washed with deionized water until the pH of the supernatant was neutral. DAPI Insertion in Zeolite Channels: Zeolite-L nanocrystals (150 mg) were dispersed in 15 mL of a 0.3 × 10−3 M aqueous solution of DAPI and stirred overnight at 50 °C. The final fluorescent material was finally recovered by centrifugation washing with water until the supernatant was found no more emissive. After TGA, an amount of 0.97 of DAPI per 100 mg of zeolites was determined. DXP Insertion in Zeolite Channels: DXP was inserted from the gas phase at 290 °C. 100 mg of zeolite-L crystals were mixed with 0.5 mg DXP in a glass ampoule and dehydrated at 1.0 × 10−5 mbar for 6 h and sealed. The mixture was left at 290 °C for 24 h to obtain dye insertion. The ampoule was opened and the zeolites were washed with n-butanol until the supernatant did not show any fluorescence. This procedure affords a dye loading of around 7% (ndye/nsite). Insertion of Oxazine 170 in Zeolite Channels: Zeolite-L nanocrystals (200 mg) were dispersed in a 0.01 mmol solution of oxazine 170 perchlorate in water and stirred overnight at reflux. The fluorescent zeolites were then recovered by centrifugation, washing the solid residue with water until the supernatant resulted completely UV transparent. After TGA, a value of about 2 mg of oxazine per 100 mg of zeolites was found. Nanozeolites Surface Modification with PNA: Amino-modified zeolites were first prepared by sonication of the particles (80 mg) in toluene (10 mL) for 30 min to form a good dispersion. APTES (50 µL, 0.2 mmol) was added to the suspension in presence of a catalytic amount of TEA and the mixture was stirred at room temperature overnight. The particles were then recovered by centrifugation and washed three times with ethanol to remove impurities. The same protocol was carried out both for unfilled zeolite-L crystals and for Oxazine and DXP zeolites. To obtain carboxylic acid-functionalized nanoparticles, 70 mg of the former amino-modified zeolites was dispersed in a succinic anhydride solution in DMSO (0.1 M in 10 mL) and, after 30 min of sonication to get a good dispersion, the mixture was stirred overnight at room temperature. The material was then centrifuged and washed three times with DMSO. After that, succinimidyl-functionalized nanozeolites were obtained by reaction of the former material (50 mg) with a solution of DIC and NHS (both 0.25 M) in dry DMSO. The nanoparticles were dispersed in 10 mL of the reactant solution and stirred overnight at room temperature under nitrogen inert atmosphere. The final material was again recovered by centrifugation washing with DMSO. PNA attachment was subsequently carried out by dispersing 5 mg of the activated-esters-modified-zeolites in 500 µL of a PNA solution 30 × 10−6 M in a 100 × 10−3 M carbonate buffer, H2O:Acetonitrile 9:1, pH 9. The dispersion was stirred overnight at room temperature and PNA-zeolites were finally obtained by centrifuging and washing three times with water. The final quenching step was performed by stirring overnight at room temperature the PNAzeolites with a 100 × 10−3 M solution of ethanolamine in Tris buffer (100 × 10−3 M, pH 9). The final material was recovered by centrifugation and washing three times with water. Poly-L-lysine Coating: Poly-L-lysine coating was always carried out by dispersing 1 mg of PNA-zeolites in a 1% w/v solution of poly-L-lysine hydrobromide in water and stirring the mixture at room temperature for 1 h. The final material was recovered by centrifugation and washing with water. Cell Culture: HeLa cells were cultured inside media, which contain 88% Dulbecco’s modified Eagle medium (DMEM), 10% Fetal Bovine Serum, 1% Penicillin–Streptomycin, and 1% L-glutamine 200 × 10−3 M (all material was purchased from Gibco) under 37 °C and 5% of CO2 conditioning for 48 h until 70% to 80% cell confluency was reached. Subsequently, the cells were washed twice with phosphate buffer solution (PBS, Gibco) and treated with trypsin, then ≈50 000 cells were reseeded on the monolayer glass cover slip inside 6-well plate
culture dish. New culture media (2 mL) were added gently and the cells were grown overnight. Incubation: Working solutions of culture medium containing PNAzeolites in 0.01 mg mL−1 concentration were added gently to the cells. After 1, 4, and 24 h incubation, the media were removed and cells were washed with PBS twice. Cells were fixed with 4% paraformaldehyde (PFA) and, subsequently, the cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). After staining, cells were washed once with PBS and then with water. Cells were removed from the well and mounted onto the rectangular glass cover for microscopy experiments.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements The author thank the ERC advanced grant award (no. 2009–247365), the International Center for Frontier Research in Chemistry (icFRC), and MIUR PRIN09 grant (20093N774P_001) for financial support, as well as the French Embassy in Italy and the French government for providing the MAEE grant for scientific cooperation between Italy and France (no. 778588G). Received: February 24, 2014 Revised: March 19, 2014 Published online:
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