Efficient and safe gene transfection in fish spermatogonial stem cells using nanomaterials

Abstract

Multiwalled carbon nanotubes (MWCNTs), nanographene oxide (NGO), and gold nanorods (NRs) can be functionalized and complexed to DNA to promote efficient gene delivery to Nile tilapia spermatogonial stem cells inducing less cell death than electroporation and the commercial reagents tested. Therefore, nanomaterials can contribute to achieve fish transgenesis.

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A large number of experiments in the study of the developmental and cytophysiological processes of fish cells and other organisms involve the delivery of DNA fragments to cells, e.g. a DNA sequence to express a protein of interest or a RNA sequence to induce interference by siRNA. In order to perform these studies, a high efficiency of gene expression and avoidance of cell death are desirable in gene delivery methods.^1,2 However, some commercial reagents and strategies such as electroporation induce high cell death rates, and/or fail to efficiently transfect fish cells,³ especially in the case of primary cell culture, which are difficult-to-transfect cells (that can be obtained from different animal's tissues; e.g., cardiomyocytes⁴ and spermatogonial stem cells (SSCs)⁵).

With the advancement of nanobiotechnology, the challenge of developing new materials for the efficient transfection of fish and mammary primary culture cells has received considerable attention.^6,7 Functionalized nanomaterials could be useful alternatives for promoting gene delivery to fish SSCs in an efficient and nontoxic manner.

These cells are unique in their capacity to transmit genetic manipulations to future generations. SSCs can be obtained from immature and adult fish testes, modified in vitro, and transplanted to the testes of recipient animals,⁸ or maintained in culture⁹ to generate transgenic gametes. In particular, the offspring derived from transplanted modified SSCs comprise nonmosaic transgenic animals.¹⁰

However, the efficiency of transfection in fish SSCs is low when using common commercial transfection reagents, such as Lipofectamine 2000® (Lipo) or standard techniques like electroporation.¹¹ In this regard, we investigated if nanomaterials could be an interesting tool to promote the delivery of genes to fish germ cells inducing low rate of cell death and offering satisfactory copy numbers of mRNA synthesis from transgene.

Here we report the results obtained on the delivery of the cyan fluorescent protein gene to Nile tilapia SSCs using functionalized versions of nanographene oxide (NGO), multiwalled carbon nanotubes (fMWCNTs), gold nanorods (NRs), nanodiamonds (NDs), and phosphate based nanocomposites (NPC) as vehicles. Transfection was also assessed using the commercial reagents Lipofectamine 2000®, Gene Juice® (GJ), and X-tremeGENE™ (XT).

SSCs were isolated by using a previously published protocol¹² and in accordance with the guidelines approved by the local ethics committee on animal use-CEUA, UFMG (protocol# 89/2012). Briefly, the testes were removed from adult Nile tilapia (after anesthesia with quinaldine), fragmented in pieces of approximately 2 mm,³ and dissociated with 2% collagenase, (Sigma Aldrich, St. Louis, Missouri) in Dulbecco Modified Eagle Medium/Ham F-12 Medium (DMEM/F12 medium) (Gibco-BRL Life Technologies, Grand Island, NY) for four hours at 25 °C. Then, DNAseI and trypsin were added to the final concentrations of 0.03% and 0.25% and the mixture incubated at 25 °C for 30 minutes. The reaction was stopped with fetal bovine serum (FBS, Gibco) 1 : 1 v/v and submitted to centrifugation at 200g for 10 minutes at room temperature. An enriched type A spermatogonia cell suspension was obtained by Percoll® (Sigma Aldrich) gradient centrifugation (800g for 30 minutes at 18 °C) from which the upper cell band was collected. After enrichment, the cell suspension was pooled for differential plating on 1% gelatin-coated 6-well dishes (TPP, Switzerland) to remove eventual testicular somatic cells. A total of 1.5 × 10⁶ cells per well were cultured in DMEM/F12 supplemented with 10% FBS, 10 000 U L⁻¹ penicillin, 10 mg L⁻¹ streptomycin for 12 hours at 25 °C in an atmosphere of 5% CO₂.

fMWCNTs' synthesis is presented in a patent filing under the number: BR1020140139397 available at https://www.inpi.gov.br. They were synthesized from ethylene (Sigma Aldrich) (cobalt and iron were used as catalysts) through chemical vapor deposition. They were then functionalized using nitric/sulfuric acids (Sigma Aldrich – 1 : 3 v/v) as oxidizing agents. The resulting fMWCNTs were analyzed through Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and thermogravimetry (TG).

NRs were synthesized through an adaptation of the Nikoobakht and El-Sayed method¹³ by reducing cetyltrimethylammonium bromide (CTAB – Sigma Aldrich, as same as the other reagents in this synthesis) and chloroauric acid (HAuCl₄) with sodium borohydride (NaBH₄) (seed solution), and then adding silver nitrate (AgNO₃), 0.1 M ascorbic acid, and 1 M hydrochloric acid (HCl) to this solution. NRs were obtained by centrifugation at 5600g and subjected to TEM. The functionalization with phospholipid phosphatidylcholine (PC) was performed using chloroform to extract the CTAB,¹⁴ and confirmed by spectrophotometry and FTIR.

NDs were synthesized by hydrothermal treatment (180 °C) of a mixture of 2% acetic acid and chitosan (Sigma Aldrich), followed by centrifugation at 8000g for 15 minutes. The nanomaterial obtained was analyzed with TEM, before being functionalized in an ultrasonic bath with PEI (Sigma Aldrich), and analyzed by FTIR.

NGO was synthesized from graphite by acid treatment (3.2 M nitric acid and 0.9 M sulfuric acid – Sigma Aldrich) under heated reflux in a microwave oven. After ultrasonication and pH adjustment in an ice bath, the resulting solution was filtered through a 0.22 µm membrane (Merck-Millipore, Billerica, Massachusetts, USA) and analyzed by TEM. Functionalization was performed with polyethylenimine (PEI – poly(ethylenimine) solution ∼50% in H₂O – Sigma Aldrich) in an ultrasonic bath for 30 minutes, followed by analysis by FTIR.

NPCs' synthesis is presented in two patent filing under the numbers: BR 102012032493-8 and BR 102013032731-0 available at https://www.inpi.gov.br. They were synthesized in liquid media using 7 mmol of Na₄P₂O₇·10H₂O (Sigma Aldrich, as same as the other reagents in this synthesis), 5 mmol of CaCl₂·2H₂O, 5 mmol of MgCl₂·6H₂O, and 3 mmol of CrCl₃·6H₂O in a controlled environment. The suspension was centrifuged at 3560g for 10 minutes; the precipitate was washed with absolute ethanol and dried at 60 °C for 48 hours. The sample was analyzed by TEM and FTIR.

The plasmid DNA pAmCyan1-N1 (Clontech Laboratories Inc, Mountain View, CA), containing the cyan fluorescent protein AmCyan1 gene under the control of cytomegalovirus (CMV) promoter, was complexed to either functionalized nanomaterials or to one of the commercial reagents, Lipo (Life Technologies, Carlsbad, CA), GJ (Novagen, San Diego, CA) and XT (Roche, Basel, Switzerland).

For nanomaterials, the complex was formed in an ultrasonic bath (25 kHz, 100 W for 30 minutes) on ice. The capacity of the nanomaterials to bind plasmid DNA (ESI Fig. S1†) was previously determined.¹⁵ The death rates for all the delivery vehicles were determined by the Alamar Blue® assay, in different serial concentrations.¹⁵ For commercial reagents, the nanocomplex was formed according to the manufacturer's recommendation. The final DNA (4.7 kb) concentration was 20 nM for all reagents and nanomaterials.

SSCs were exposed to nanomaterials, Lipo, GJ or XT in optimal concentration, either complexed or not complexed to plasmid DNA. The nanocomplexes were added to the 6-well plate in drops, thereby ensuring a homogeneous medium. Electroporation (225 V and 50 µF) was performed using the MicroPulser™ Electroporation Apparatus (BioRad, Hercules, CA). The plates were incubated for 24 hours at 28 °C and 5% CO₂ (Esco's incubator). The experiments were performed in triplicate.

After 24 hours of cell culture in the presence of the delivery vehicles, cell viability was determined by the Alamar Blue® assay. The percentages of dead and living cells were determined according to manufacturer's recommendation. After that, the percentage of cell death for each nanomaterial without DNA or electroporation in the absence of DNA was compared to the one induced by each commercial reagent, also in the absence of DNA. The cell dead rate induced by the nanocomplexes (nanomaterial–DNA) or electroporation in the presence of plasmid, were compared to the rates observed for each commercial reagent–DNA complex. Statistically significant data were determined through χ² test (p < 0.05).

The synthesis of cyan fluorescent protein was observed through fluorescence microscopy 24 hours after the transfection. For quantification of the transcript mRNA, total RNA was isolated from each sample using the TRIzol® reagent (Invitrogen, Carlsbad, CA), and the cyan fluorescent protein mRNA transcription rate was assessed by RT-PCR and q-PCR. The PCR amplifications were performed using the StepOnePlus Real-Time PCR System (Life Technologies) as previously described,¹⁶ and the following primers for the AmCyan1 sequence: FWD-TTCGAGAAGATGACCGTGTG, and REV-AGGTGTGGAACTGGCATCTGTA (synthesized by Integrated DNA Technologies (Coralville, IA)). β-Actin served as a control and was detected by the following primers: FWD-CGGTATGGAGTCTTGTGGTATC, and REV-AGCACAGTGTTGGCGTATAA. To exclude contamination of nonspecific PCR products such as primer dimers, the dissociation curve analysis was applied to all products at the end of the cycle. Relative quantification of the expression of the target genes was performed using the comparative CT method, as previously described.¹⁷ Fold-changes in gene expression of the target genes are equivalent to 2^−ΔΔCT. The data were statistically validated through Student's t test comparing the gene expression for each nanomaterial–DNA complex or the one from electroporation in the presence of DNA to the gene expression induced by commercial reagent complexed to DNA.

All the nanomaterials were successfully synthesized. fMWCNTs (ESI Fig. S2a†) had an average outer diameter of 40 nm, and the NRs (ESI Fig. S2d†), an average length of 45 nm. The NDs were obtained with an average diameter of 5 nm (ESI Fig. S2b†), and the NGOs (ESI Fig. S2c†) were generated as thin, large-area films.

In FTIR spectra (ESI Fig. S3†), the typical band related to the stretching of the carbon nanotube skeleton at approximately 1600 cm⁻¹ was seen in the analysis of fMWCNTs. We were also able to observe bands due to the stretching of the carbonyl functional group (∼1770 cm⁻¹) and carboxylate anion (∼1500 cm⁻¹), as well as the deformation of the hydroxyl group (∼1000 cm⁻¹).¹⁸ In the case of NDs (ESI Fig. S4†), bands related to PEI and ND-PEI spectra were also observed. As examples, we found bands at ∼1700 cm⁻¹ corresponding to carbonyl from carboxylic acid stretch, at ∼1600 cm⁻¹ from amine N–H bending, and at ∼1400 cm⁻¹ due to amide bending from CH₂.¹⁹ The FTIR spectra of NGO-PEI (ESI Fig. S5†) also showed bands from NGO and PEI. We were able to identify the bands at ∼1600 cm⁻¹ and at ∼2900 cm⁻¹ as those from the stretching of amide and methylene groups, respectively, on PEI.²⁰ In NRs (ESI Fig. S6†), bands related to PC (at ∼3400 cm⁻¹ due to hydroxyl stretching, at ∼1200 cm⁻¹ due to PO₂ stretching, and at ∼2800 cm⁻¹ due to CH₃ stretching) were observed in the NR-PC spectra, demonstrating the success of functionalization.²¹ NPC FTIR spectra (ESI Fig. S7†) revealed bands from P–O binding at ∼1100 cm⁻¹ due to P=O stretching, and at ∼900 cm⁻¹ due to P–OR stretching.

In the present study, we observed that all nanomaterials, commercial reagents and electroporation were able to perform the delivery of plasmid DNA to Nile tilapia SSCs. Twenty-four hours after exposure to nanocomplexes, cyan fluorescence was observed for all vehicles and techniques of gene delivery, including the commercial reagents (Fig. 1d, f, h, j, l, n, p, r and t). There was no passive transfection of Nile tilapia SSCs in the absence of vehicles (Fig. 1b), and, as expected, no fluorescence was observed in the absence of plasmid DNA (Fig. 1c, e, g, i, k, m, o, q and s).

Fig. 1 Fluorescence microscopy images of SSCs culture; (a) control; (b) DNA 20 nM and no vehicle; (c) fMWCNTs; (d) fMWCNTs + DNA 20 nM; (e) electroporation; (f) electroporation + DNA 20 nM; (g) NPC; (h) NPC + DNA 20 nM; (i) NGO; (j) NGO + DNA 20 nM; (k) ND; (l) ND + DNA 20 nM; (m) NR; (n) NR + DNA 20 nM; (o) Lipofectamine 2000®; (p) Lipofectamine 2000® + DNA 20 nM. (q) GJ; (r) GJ + DNA20 nM; (s) XT; (t) XT + DNA bar = 50 µm.

Regarding toxicity, there are some nanomaterials that were already been described as prone to cause chromosomal damage or to interfere in gene expression pattern in fish and other aquatic species (for example titanium dioxide nanoparticles^22–24). Our findings shows that, the rate of cell death achieved in transfections using NRs and fMWCNTs was significantly lower (p < 0.05) than that induced by all the commercial reagents tested (Fig. 2–4). Electroporation was the technique that induced the highest cell death rate, being more aggressive to cells than all commercial reagents. MWCNTs functionalized with nitric acid were also considered non toxic to juvenile Nile tilapia at concentrations ranging from 0.1 to 3.0 mg L⁻¹.²⁵ Thus, the possibility to use these nanotubes to achieve the gene delivery to SSCs in vitro opens a new scenario to the future use of this nanomaterial to achieve transgenesis, avoiding risks related to the used of them in vivo. It could be risky to use functionalized MWCNTs in fishes into water containing toxic substances even in low concentration. This nanomaterial can enhance pesticide ecotoxicity²⁵ and potentiate lead toxic effects.²⁶

Fig. 2 Dead and living SSCs percentage obtained through Alamar Blue® assay for the different methods of gene delivery tested in comparison to Lipofectamine (asterisk, p < 0.05, χ² test).

Fig. 3 Dead and living SSCs percentage obtained through Alamar Blue® assay for the different methods of gene delivery tested in comparison to GJ (GeneJuice) (asterisk, p < 0.05, χ² test).

Fig. 4 Dead and living SSCs percentage obtained through Alamar Blue® assay for the different methods of gene delivery tested in comparison to XT (X-treme) (asterisk, p < 0.05, χ² test).

Regarding the delivery efficiency, as they were able to offer higher transcript production rate, fMWCNTs, NGO, and NR were better options than Lipo (a lipofection reagent) (Fig. 5). Only NPC among the nanomaterials had a lower efficiency than this commercial reagent. ND and electroporation showed transfection efficiencies comparable to those of Lipo. GJ, a transfection reagent based on proteins and polyamine had higher efficiencies than electroporation and most nanomaterials. Nevertheless, fMWCNTs showed a capacity to deliver genes to Nile tilapia SSCs that was comparable to that of GJ (Fig. 6). This finding is of interest because it is already known that the gene delivery based on carbon nanotubes is influenced by charge: in the nanomaterial surface²⁷ and in cell surface.²⁸ Unlike the commercial reagent, that is ready-to-use, the fMWCNTs synthesis/functionalization protocol can be adjusted in order to offer the desirable surface charge to enhance the delivery efficiency. Besides that it is also possible to optimize the delivery using fMWCNTs by combining it to other charge dependent gene delivery strategy: electroporation for example (ESI Fig. S8†). Another fact important to highlight is that fMWCNTs are lesser expensive than the commercial GJ. XT, the other lipofection commercial reagent tested, delivered genes to Nile tilapia SSCs more efficiently than all other delivery strategies (ESI Fig. S9†), but this reagent was more cytotoxic than the nanomaterials (Fig. 4).

Fig. 5 Dosage of transgene expression by qPCR. AmCyan1 mRNA synthesis in relation to β-actin using different strategies to promote transfection of SSCs. The results were compared to those using the commercial reagent Lipofectamine 2000 (asterisk, p < 0.05, t test).

Fig. 6 Dosage of transgene expression by qPCR. AmCyan1 mRNA synthesis in relation to β-actin using different strategies to promote transfection of SSCs. The results were compared to those from the commercial reagent GeneJuice (asterisk, p < 0.05, t test).

Conclusions

The transfection of Nile tilapia SSCs using functionalized nanomaterials is possible. In addition to this feasibility, the gene delivery can be performed in an efficient manner inducing also low cytotoxicity. Some nanomaterials can even surpass the gene delivery capacity of commercial reagents used for this purpose. This characteristic allows fMWCNTs, NGOs, and NRs to be promising alternatives to be used in studies that require the transfection of fish cells. Due to the ample range of possibilities offered by nanobiotechnology, functionalization of these nanomaterials can be an useful tool as transfection agents. It is also possible to modify their synthesis and or functionalization to further increase in efficiency, and also reduction in cost. Therefore, using nanotechnology developed to deliver genes to fish SSCs can offer innovative and efficient strategies toward the development of transgenic fish.

Acknowledgements

We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Instituto Nacional de Ciência e Tecnologia em Nanomateriais de Carbono and Instituto Nanocell for grants support.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra07010h

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