Chapter 16 Cellular Delivery of Peptide Nucleic Acids (PNAs) Takehiko Shiraishi and Peter E. Nielsen Abstract Cellular delivery methods are a prerequisite for cellular studies with PNA. This chapter describes PNA cellular delivery using cell-penetrating peptide (CPP)–PNA conjugates and transfection of PNA–ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures employing chloroquine treatment or photochemical internalization (PCI) for significantly improving PNA delivery efficacy are described. Key words Cell-penetrating peptides (CPPs), Ligand, Peptide nucleic acid (PNA), Photochemical internalization (PCI), Transfection
1
Introduction Unaided cellular uptake of synthetic antisense oligomers (AOs), including charge neutral peptide nucleic acids (PNAs) [1], is generally negligible, and therefore efficient and robust cellular delivery methods are a prerequisite for cellular studies with AOs [2]. Routinely, cationic lipid formulations (lipoplexes) are used as effective delivery vehicles for anionic oligonucleotides, whereas lipoplexes are only efficient with non-charged AOs if these are conjugated to a lipophilic or an anionic domain or are hybridized to a carrier oligonucleotide [3]. However, cellular uptake of AOs can also be dramatically improved by chemical conjugation to cell-penetrating peptides (CPPs) [4] without interfering with their (antisense) activity and sequence specificity [5–7]. This chapter describes two PNA cellular delivery methods by using either CPP– PNA conjugates or PNA–ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures (chloroquine treatment and photochemical internalization (PCI) treatment) for improvement of AO delivery efficacy are described.
1.1 Cellular Delivery of PNA by CellPenetrating Peptide
CPPs are short (typically less than 30 amino acid residue) cationic or amphipathic peptides derived from many different origins (e.g., viruses, shuttling proteins, chimeric or totally synthetic, etc.)
Peter E. Nielsen and Daniel H. Appella (eds.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 1050, DOI 10.1007/978-1-62703-553-8_16, © Springer Science+Business Media New York 2014
193
194
Takehiko Shiraishi and Peter E. Nielsen
[8–10]. Upon covalent chemical conjugation or by simple complexation they can transport various bioactive molecules into mammalian cells by primarily endocytotic pathway(s) both in vitro and in vivo. Because of this remarkable cell-penetrating feature as well as their relatively nontoxic nature (compared to liposome-based methods), CPPs are promising vectors for a wide range of ligands and drugs, although the cellular uptake efficiency (and uptake mechanism/pathway) strongly depends on the specific CPP used as well as the cargo molecule [11–14]. To date, many different CPPs have been tested for various AOs including PNA, and significant improvement of cellular uptake without interfering with biological activity and sequence specificity has been demonstrated. Thus CPP-mediated delivery, in particular using chemical conjugation, is a promising strategy also for in vivo applications and drug discovery [12–14]. However, the efficacy of CPP-mediated delivery is generally limited due to the accumulation of CPP conjugates in endosomal compartments without direct access to the cytosol/ nucleus as these conjugates use endocytotic pathways as main uptake routes. Because of this entrapment (accumulation) of CPP conjugates in the endosomes, relatively high concentrations (micromolar) of the conjugates are typically needed to obtain significant biological responses. However, many studies have shown that the delivery efficacy of CPP conjugates can be dramatically improved by facilitating the release of CPP conjugates from the endosomes thereby allowing them to reach their intracellular targets in the cytoplasm or the nucleus. Improved endosomal release can be achieved by further chemical modification of CPP conjugates using fusogenic peptides [15, 16] or lipidic ligands such as cholesterol or fatty acids [17, 18], or by co-treatment with endosomolytic agents [19–21]. 1.2 Cellular Delivery of PNA Conjugates by Cationic Lipids
Currently, employment of transfection agents is the most common (and probably the most efficient) nonviral transfection method for the delivery of negatively charged AOs (and their derivatives) into mammalian cells [22, 23]. Most transfection reagents are cationic lipids or cationic polymer-based compounds capable of complexation with negatively charged AOs via electrostatic interactions (forming lipoplexes or polyplexes, respectively). These delivery approaches by cationic carriers are quite simple to perform and extremely useful for many cells in vitro, although their application in vivo is still limited owing to their low in vivo efficacy, acute immune responses, and toxicity to cells [24]. In contrast to the success with negatively charged AOs, unmodified aminoethylglycyl PNA (aegPNA) are charge neutral and do not interact with cationic lipids (or polymers) and consequently are not efficiently delivered to the cells by these agents. In order to use lipoplex carriers for charge neutral PNAs, several approaches have been introduced to increase the affinity of the PNA oligomers for the
Cellular Delivery of Peptide Nucleic Acids (PNAs)
195
cationic lipids and thus allow formation of the PNA/lipid complexes required for transfection. The first approach exploited a PNA complementary oligodeoxynucleotide to specifically form a PNA/ DNA heteroduplex [3] thereby utilizing the anionic oligonucleotide as a carrier for a complexation with cationic lipids. This method is active even at low nanomolar concentrations and does not require further chemical modification of the PNA. However, very careful optimization of complementary DNA oligomers (both oligomer length and length of complementary region) is necessary for each individual PNA. More recently other approaches have been devised for complexing the PNA with cationic lipids using covalent PNA conjugates. These include use of lipidic ligands such as cholesterol, cholic acid, or polyheteroaromates [5–7, 25], or negatively charged ligands such as bis-phosphonate lysine peptides, allowing complex formation between PNA and cationic lipids via hydrophobic or electrostatic interactions, respectively. Among these PNA conjugates, bis-phophonate-PNAs showed the highest potency exhibiting sub-nanomolar (EC50) activity in mammalian cells. Nonetheless, other lipidic PNA conjugates are also active at low nanomolar concentrations, and importantly these PNA conjugates are more readily synthetically available. The present chapter describes the use of PNA conjugates for cellular delivery and antisense targeting in mammalian cells in culture including cellular transfection of PNA conjugates by the cationic lipid LipofectAMINE2000 using a simple co-incubation protocol. 1.3 Improvement of PNA Cellular Delivery by Endosomolytic Treatment
The poor inherent cellular uptake of PNA can be significantly improved by either simple conjugation to CPPs and can be further enhanced by co-conjugation to lipidic ligands such as fatty acids or cholesterol [17, 18]. However, the efficacy of these PNA conjugates is still limited by significant endosomal entrapment [15, 26, 27]. It has been shown that the bioavailability of CPP–PNA conjugates can be (significantly) improved by the use of auxiliary agents such as chloroquine, Ca2+ treatment, sucrose, or photosensitizers (photochemical internalization (PCI)) [19–21] that promote release of endosomal contents to the cytosol via different mechanisms dependent on the particular endosomolytic treatment. In the present protocol, we describe two endosomolytic procedures namely chloroquine (CQ, Fig. 1) treatment and PCI using photosensitizers (AlPcS2a or TPPS2a, Fig. 1) [28]. CQ [29] can be used by simple supplementation to the transfection medium as it shows significant accumulation (entrapment) in acidic cellular compartments (such as endosome/lysosome) and induce disruption of the endosome (presumably through a proton sponge effect resulting in an increase of osmotic pressure in the endosome). PCI treatment consists of treatment with a lipophilic photosensitizer combined with irradiation (visible light absorbed by the photosensitizer) of the cells resulting in transient pore formation in the
196
Takehiko Shiraishi and Peter E. Nielsen
OH N HN
O
N
Cl
HO
NH
OH
Cholate
Chloroquine
SO3SO3-
-O3S
N N N
NH
N
SO3-
N
Al N
N
N
N
HN
N
AlPcS2a
TPPS2a
Fig. 1 Structures of chloroquine, cholate (conjugated to amino end of a PNA), and photosensitizers (disulfonated aluminum phthalocyanines (AlPcS2a), disulfonated tetraphenylporphyrin (TPPS2a))
endosomal membrane through perturbation (oxidation) of lipids by short lived, reactive oxygen species (ROS, mainly singlet oxygen in this case) thereby facilitating release of endosomal content into the cytosol. As mentioned above, release of the delivered cargo from the endosomal compartment, appears one of the main challenges for efficient and functional delivery of antisense oligomers, and in particular PNA, to mammalian cells. The methods describe here can easily be applied to other AOs such as morpholino oligomers and also other cargoes (e.g., siRNA) to improve intracellular bioavailability by liberating cargoes from endosome/lysosome compartments.
2 2.1
Materials Cell Culture
1. Growth medium: RPMI1640 supplemented with 10 % fetal bovine serum (FBS) and 1 % Glutamax (Gibco). 2. Adherent cells in culture (i.e., HeLa pLuc705 cells). 3. Cell culture flask (T-25).
Cellular Delivery of Peptide Nucleic Acids (PNAs)
197
4. Multiple well plate (e.g., 24-well plate or 96-well plate). 5. Silicon cell scraper. 2.2 PNA Transfection
1. PNA conjugate solution at 200 μM (this solution is preferably in water but also could be in organic solvents such as DMSO, DMF in case of PNA conjugate with low water solubility). PNA conjugates were synthesized, HPLC-purified, characterized (MALDI-TOF Mass), and stored at 4 °C until use. Synthesis of PNA (conjugates) is described in Subheading 4 and also in Chapters 1 and 5. Synthesis of other PNA conjugates (e.g., cholic acid–PNA, cholesterol–PNA, and bis-phosphonate-PNA) are described in the literature [5–7, 25]. 2. OPTI-MEM medium (Invitrogen). 3. Cationic lipid (e.g., LipofectAMINE2000 (Invitrogen)). 4. Growth medium: RPMI1640 supplemented with 1 % Glutamax (Gibco) and FBS (20 % for supplementation and 10 % for the replacement).
2.3 Improvement of PNA Cellular Delivery Efficacy by Endosomolytic Treatment
1. OPTI-MEM medium with chloroquine (CQ): 100 μM chloroquine (CQ) in OPTI-MEM. 2. Growth medium with a photosensitizer: RPMI1640 (10 % FBS, 1 % Glutamax) containing aluminum phthalocyanine (AlPcS2a, Frontier Scientific) (5 μg/ml) or tetraphenyl porphyrin (TPPS2a, Frontier Scientific) (2 μg/ml). 3. Equipment (specially required): Light tubes for photosensitizer irradiation: Red light irradiation for AlPcS2a (fluorescence tube FO18 (Arcadia)) or blue light irradiation for TPPS2a (fluorescent light tube 40 W/03 (Phillips) with maximum emission at 420 nm).
2.4
PNA Synthesis
1. 4-Mehylbenzhydryl amine resin (MBHA resin,
1
Introduction Unaided cellular uptake of synthetic antisense oligomers (AOs), including charge neutral peptide nucleic acids (PNAs) [1], is generally negligible, and therefore efficient and robust cellular delivery methods are a prerequisite for cellular studies with AOs [2]. Routinely, cationic lipid formulations (lipoplexes) are used as effective delivery vehicles for anionic oligonucleotides, whereas lipoplexes are only efficient with non-charged AOs if these are conjugated to a lipophilic or an anionic domain or are hybridized to a carrier oligonucleotide [3]. However, cellular uptake of AOs can also be dramatically improved by chemical conjugation to cell-penetrating peptides (CPPs) [4] without interfering with their (antisense) activity and sequence specificity [5–7]. This chapter describes two PNA cellular delivery methods by using either CPP– PNA conjugates or PNA–ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures (chloroquine treatment and photochemical internalization (PCI) treatment) for improvement of AO delivery efficacy are described.
1.1 Cellular Delivery of PNA by CellPenetrating Peptide
CPPs are short (typically less than 30 amino acid residue) cationic or amphipathic peptides derived from many different origins (e.g., viruses, shuttling proteins, chimeric or totally synthetic, etc.)
Peter E. Nielsen and Daniel H. Appella (eds.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 1050, DOI 10.1007/978-1-62703-553-8_16, © Springer Science+Business Media New York 2014
193
194
Takehiko Shiraishi and Peter E. Nielsen
[8–10]. Upon covalent chemical conjugation or by simple complexation they can transport various bioactive molecules into mammalian cells by primarily endocytotic pathway(s) both in vitro and in vivo. Because of this remarkable cell-penetrating feature as well as their relatively nontoxic nature (compared to liposome-based methods), CPPs are promising vectors for a wide range of ligands and drugs, although the cellular uptake efficiency (and uptake mechanism/pathway) strongly depends on the specific CPP used as well as the cargo molecule [11–14]. To date, many different CPPs have been tested for various AOs including PNA, and significant improvement of cellular uptake without interfering with biological activity and sequence specificity has been demonstrated. Thus CPP-mediated delivery, in particular using chemical conjugation, is a promising strategy also for in vivo applications and drug discovery [12–14]. However, the efficacy of CPP-mediated delivery is generally limited due to the accumulation of CPP conjugates in endosomal compartments without direct access to the cytosol/ nucleus as these conjugates use endocytotic pathways as main uptake routes. Because of this entrapment (accumulation) of CPP conjugates in the endosomes, relatively high concentrations (micromolar) of the conjugates are typically needed to obtain significant biological responses. However, many studies have shown that the delivery efficacy of CPP conjugates can be dramatically improved by facilitating the release of CPP conjugates from the endosomes thereby allowing them to reach their intracellular targets in the cytoplasm or the nucleus. Improved endosomal release can be achieved by further chemical modification of CPP conjugates using fusogenic peptides [15, 16] or lipidic ligands such as cholesterol or fatty acids [17, 18], or by co-treatment with endosomolytic agents [19–21]. 1.2 Cellular Delivery of PNA Conjugates by Cationic Lipids
Currently, employment of transfection agents is the most common (and probably the most efficient) nonviral transfection method for the delivery of negatively charged AOs (and their derivatives) into mammalian cells [22, 23]. Most transfection reagents are cationic lipids or cationic polymer-based compounds capable of complexation with negatively charged AOs via electrostatic interactions (forming lipoplexes or polyplexes, respectively). These delivery approaches by cationic carriers are quite simple to perform and extremely useful for many cells in vitro, although their application in vivo is still limited owing to their low in vivo efficacy, acute immune responses, and toxicity to cells [24]. In contrast to the success with negatively charged AOs, unmodified aminoethylglycyl PNA (aegPNA) are charge neutral and do not interact with cationic lipids (or polymers) and consequently are not efficiently delivered to the cells by these agents. In order to use lipoplex carriers for charge neutral PNAs, several approaches have been introduced to increase the affinity of the PNA oligomers for the
Cellular Delivery of Peptide Nucleic Acids (PNAs)
195
cationic lipids and thus allow formation of the PNA/lipid complexes required for transfection. The first approach exploited a PNA complementary oligodeoxynucleotide to specifically form a PNA/ DNA heteroduplex [3] thereby utilizing the anionic oligonucleotide as a carrier for a complexation with cationic lipids. This method is active even at low nanomolar concentrations and does not require further chemical modification of the PNA. However, very careful optimization of complementary DNA oligomers (both oligomer length and length of complementary region) is necessary for each individual PNA. More recently other approaches have been devised for complexing the PNA with cationic lipids using covalent PNA conjugates. These include use of lipidic ligands such as cholesterol, cholic acid, or polyheteroaromates [5–7, 25], or negatively charged ligands such as bis-phosphonate lysine peptides, allowing complex formation between PNA and cationic lipids via hydrophobic or electrostatic interactions, respectively. Among these PNA conjugates, bis-phophonate-PNAs showed the highest potency exhibiting sub-nanomolar (EC50) activity in mammalian cells. Nonetheless, other lipidic PNA conjugates are also active at low nanomolar concentrations, and importantly these PNA conjugates are more readily synthetically available. The present chapter describes the use of PNA conjugates for cellular delivery and antisense targeting in mammalian cells in culture including cellular transfection of PNA conjugates by the cationic lipid LipofectAMINE2000 using a simple co-incubation protocol. 1.3 Improvement of PNA Cellular Delivery by Endosomolytic Treatment
The poor inherent cellular uptake of PNA can be significantly improved by either simple conjugation to CPPs and can be further enhanced by co-conjugation to lipidic ligands such as fatty acids or cholesterol [17, 18]. However, the efficacy of these PNA conjugates is still limited by significant endosomal entrapment [15, 26, 27]. It has been shown that the bioavailability of CPP–PNA conjugates can be (significantly) improved by the use of auxiliary agents such as chloroquine, Ca2+ treatment, sucrose, or photosensitizers (photochemical internalization (PCI)) [19–21] that promote release of endosomal contents to the cytosol via different mechanisms dependent on the particular endosomolytic treatment. In the present protocol, we describe two endosomolytic procedures namely chloroquine (CQ, Fig. 1) treatment and PCI using photosensitizers (AlPcS2a or TPPS2a, Fig. 1) [28]. CQ [29] can be used by simple supplementation to the transfection medium as it shows significant accumulation (entrapment) in acidic cellular compartments (such as endosome/lysosome) and induce disruption of the endosome (presumably through a proton sponge effect resulting in an increase of osmotic pressure in the endosome). PCI treatment consists of treatment with a lipophilic photosensitizer combined with irradiation (visible light absorbed by the photosensitizer) of the cells resulting in transient pore formation in the
196
Takehiko Shiraishi and Peter E. Nielsen
OH N HN
O
N
Cl
HO
NH
OH
Cholate
Chloroquine
SO3SO3-
-O3S
N N N
NH
N
SO3-
N
Al N
N
N
N
HN
N
AlPcS2a
TPPS2a
Fig. 1 Structures of chloroquine, cholate (conjugated to amino end of a PNA), and photosensitizers (disulfonated aluminum phthalocyanines (AlPcS2a), disulfonated tetraphenylporphyrin (TPPS2a))
endosomal membrane through perturbation (oxidation) of lipids by short lived, reactive oxygen species (ROS, mainly singlet oxygen in this case) thereby facilitating release of endosomal content into the cytosol. As mentioned above, release of the delivered cargo from the endosomal compartment, appears one of the main challenges for efficient and functional delivery of antisense oligomers, and in particular PNA, to mammalian cells. The methods describe here can easily be applied to other AOs such as morpholino oligomers and also other cargoes (e.g., siRNA) to improve intracellular bioavailability by liberating cargoes from endosome/lysosome compartments.
2 2.1
Materials Cell Culture
1. Growth medium: RPMI1640 supplemented with 10 % fetal bovine serum (FBS) and 1 % Glutamax (Gibco). 2. Adherent cells in culture (i.e., HeLa pLuc705 cells). 3. Cell culture flask (T-25).
Cellular Delivery of Peptide Nucleic Acids (PNAs)
197
4. Multiple well plate (e.g., 24-well plate or 96-well plate). 5. Silicon cell scraper. 2.2 PNA Transfection
1. PNA conjugate solution at 200 μM (this solution is preferably in water but also could be in organic solvents such as DMSO, DMF in case of PNA conjugate with low water solubility). PNA conjugates were synthesized, HPLC-purified, characterized (MALDI-TOF Mass), and stored at 4 °C until use. Synthesis of PNA (conjugates) is described in Subheading 4 and also in Chapters 1 and 5. Synthesis of other PNA conjugates (e.g., cholic acid–PNA, cholesterol–PNA, and bis-phosphonate-PNA) are described in the literature [5–7, 25]. 2. OPTI-MEM medium (Invitrogen). 3. Cationic lipid (e.g., LipofectAMINE2000 (Invitrogen)). 4. Growth medium: RPMI1640 supplemented with 1 % Glutamax (Gibco) and FBS (20 % for supplementation and 10 % for the replacement).
2.3 Improvement of PNA Cellular Delivery Efficacy by Endosomolytic Treatment
1. OPTI-MEM medium with chloroquine (CQ): 100 μM chloroquine (CQ) in OPTI-MEM. 2. Growth medium with a photosensitizer: RPMI1640 (10 % FBS, 1 % Glutamax) containing aluminum phthalocyanine (AlPcS2a, Frontier Scientific) (5 μg/ml) or tetraphenyl porphyrin (TPPS2a, Frontier Scientific) (2 μg/ml). 3. Equipment (specially required): Light tubes for photosensitizer irradiation: Red light irradiation for AlPcS2a (fluorescence tube FO18 (Arcadia)) or blue light irradiation for TPPS2a (fluorescent light tube 40 W/03 (Phillips) with maximum emission at 420 nm).
2.4
PNA Synthesis
1. 4-Mehylbenzhydryl amine resin (MBHA resin,
