Head on Comparison of Self- and Nano-assemblies of Gamma Peptide Nucleic Acid Amphiphiles

Abstract

Peptide nucleic acids (PNAs) are nucleic acid analogs with superior hybridization properties and enzymatic stability than deoxyribonucleic acid (DNA). In addition to gene targeting applications, PNAs have garnered significant attention as bio-polymer due to the Watson-Crick -based molecular recognition and flexibility of synthesis. Here, we engineered PNA amphiphiles using chemically modified gamma PNA (8 mer in length) containing hydrophilic diethylene glycol units at the gamma position and covalently conjugated lauric acid (C12) as a hydrophobic moiety. Gamma PNA (γPNA) amphiphiles self-assemble into spherical vesicles. Further, we formulate nano-assemblies using the amphiphilic γPNA as a polymer via ethanol injection-based protocols. We perform comprehensive head-on comparison of the physicochemical and cellular uptake properties of PNA derived self- and nano-assemblies. Small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) analysis reveal ellipsoidal morphology of γPNA nano-assemblies that results in superior cellular delivery compate to the spherical self-assembly. Next, we compare the functional activities of γPNA self-and nano-assemblies in lymphoma cells via multiple endpoints, including gene expression, cell viability, and apoptosis-based assays. Overall, we establish that γPNA amphiphile is a functionally active bio-polymer to formulate nano-assemblies for a wide range of biomedical applications.

Graphical Abstract

Herein, we engineered gamma Peptide Nucleic Acids (γPNA)-based amphiphiles by conjugating hydrophobic lauric acid domain with γPNA containing hydrophilic diethylene glycol scaffolds. γPNA self-assembles into large spherical vesicles with >200 nm diameter. In parallel comparison, we investigated that γPNA nano-assemblies, formulated via ethanol injection, formed small ellipsoidal vesicles (100 nm) with superior transfection features than γPNA self-assemblies.

1. Introduction

Peptide nucleic acids (PNAs), discovered three decades ago, are synthetic deoxyribonucleic acid (DNA) mimics with a pseudopeptide backbone.^[1] PNAs contain repeating units of uncharged N-(2-aminoethyl) glycine units linked by amide bonds as a backbone. Nucleobases are attached via a methylene carbonyl linker to the amino nitrogen of the PNA backbone.^[2] The structural features of PNA attributes to its unique enzymatic stability while maintaining resistance to both the proteases and nucleases.^[3] Further, PNAs can bind to the target DNA or RNA with high affinity and specificity via Watson-Crick base pairing.^[4] Due to the aforementioned properties, PNAs have been extensively used to target both duplex DNA^[5] and RNA^[6] for gene regulation and other diverse biomedical applications like DNA biosensing,^[7] polymerase chain reaction (PCR),^[8] diagnostics, and genomic barcoding.^[9] As compared to other nucleic acid analogs, the neutral backbone of PNA imparts it superior hybridization property towards the target DNA or RNA. However, classical PNAs are limited by numerous challenges, poor aqueous solubility, self-aggregation, non-specific interactions with macromolecules, low cellular transfection,^[10] and short in vivo elimination half-life (17 mins).^[11] Hence efforts have been made to overcome the aforementioned challenges of PNAs including conjugation with cell penetrating peptides,^[12] ligands,^[13] pH low insertion peptide (pHLIP),^[6b] or encapsulation in nanoparticles.^[14]

PNAs have also been explored as building blocks in the field of nucleic acid nanotechnology.^[15] Until recently, peptides^[16] and DNA^[17] have been utilized for supramolecular self-assemblies with wide range of biomedical applications. However, PNAs have gained significant attention as a biopolymer, driven by the dual advantage of Watson-Crick molecular recognition of nucleic acids along with the chemical versatility and flexibility of synthesis. Several PNA modifications; conjugation with lipophilic domains,^[18] peptide motifs,^[19] combining PNAs with the self-assembled DNA units,^[20] and self-organizing guanine containing dinucleotide PNAs, also known as minimalistic PNAs,^[21] have been investigated to induce its self-assembly into nano- or micron sized structures. In particular, PNA amphiphiles provide the flexible platform to assemble PNA into nanostructures for diverse biomedical applications. PNA amphiphiles contain a hydrophobic domain on one end and a hydrophilic unit on the other which can be assembled into fibers, hydrogels, or micelles with tunable dimensions.^[22] Schneider et al. conjugated 12-carbon (12C) alkane tails on to the short PNA sequences containing (Lys)₂ or (Glu)₄ peptides and investigated their self-assembly into the micelles.^[14b]. It was established that inclusion of 12C units do not affect the biophysical features of PNA amphiphiles (PNAA). Small angle X-ray scattering analysis (SAXS) of short PNA (7 nucleobase in length) conjugated with 12C units indicated formation of ellipsoidal micelles.^[23] Similarly, PNA-peptide amphiphile (PNA-PA) were formulated by conjugating a PNA sequence with small peptide sequence and alkyl chain which self-assembled into nanofibers with pH changes. PNA-PA nanofibers showed high binding affinity and specificity towards the complementary DNA and RNA targets with potential applications in gene silencing, purification, and biosensing.^[19] Another study reported self-assembly of bilingual PNAs into micelles.^[24] These bilingual PNAs were designed by introducing hydrophilic lysine and hydrophobic alanine side chains at the gamma positions of a 12-mer PNA sequence complementary to the oncomiR-21.

The pseudopeptide backbone of PNA is amiable to chemical modifications and has been explored extensively to improve the solubility and binding affinity of PNA. Gamma modified PNAs (γPNA) were developed by introducing a side chain at the gamma position of 2-(aminoethyl) glycine unit which induced chirality and improved binding properties contingent upon the stereochemistry.^[25] MiniPEG gamma PNA (^MPγPNA) contains a diethylene glycol unit at the gamma position which introduces a (R)-stereogenic center resulting in pre-organization of PNA into a right-handed helical conformation.^[26] In addition to improving the recognition properties, γPNAs also exhibit higher solubility, reduced self-aggregation, and superior biocompatibility in comparison to regular PNAs. The left and right handed (LH and RH) γPNAs, designed by introducing different stereogenic center at the γ position, exhibit orthogonality to each other for recognition while regular PNAs, lacking any helical conformation, can bind to either of the complementary γPNAs.^[27] This orthogonal property of LH and RH γPNAs has been exploited to organize small PNAs with 5 nucleobases into molecular self-assembly and was proposed as “all-in-one” platform for organization and molecular assembly.^[27] A recent study reported self-assembly of ^MPγPNAs into nano-fibers following the single stranded title approach in DNA nanotechnology using organic solvents.^[28] These studies have established the potential of γPNA to self-assemble into nanostructures.

In this study, we designed PNA amphiphiles using short length (8mer) ^MPγPNA containing the diethylene glycol units as the hydrophilic domain and lauric acid (C12 alkane) group as the hydrophobic tail. Covalent conjugation of ^MPγPNA with the lauric acid results in the self-assembly of amphiphilic PNA into nanostructures. The designed amphiphilic PNA was formulated into nano-assemblies (NA) using ethanol injection-based technique.

To investigate the functional activity, we designed a short 8mer PNA sequence complementary to the seed region (a functional region on the 5’ end from nucleotides 2 to 8) of oncomiR-155 using regular (PNA-155) and ^MPγPNA monomers (γPNA-155). OncomiRs are small 20–25 nucleotide length non-coding microRNAs or miRs that are highly upregulated in cancers.^[29] In prior work, we established that targeting the seed region of oncomiR-155 with 8mer PNAs can efficiently inhibit the tumor growth in a cell line-derived xenograft.^[6c] The inclusive physico-chemical characterization of PNA-155 and γPNA-155 using dynamic light scattering and electron microscopy indicated that only amphiphilic γPNA-155 self-assembles into micelles and large vesicular structures. A head-to-head comparison of γPNA-155 based self-assemblies (SA) and NA showed superior transfection efficiency of γPNA-155 NA in multiple cell lines. Further, small-angle X-ray scattering analysis (SAXS) and small-angle neutron scattering analysis (SANS) confirmed the oblate ellipsoidal morphology of γPNA-155 NA. Our results indicated the superior efficacy of γPNA-155 NA in lymphoma cells based on a series of gene expression, cellular viability, and apoptosis-based analysis.

Overall, we established that ^MPγPNA and lauric acid-based amphiphiles could be formulated into NA with superior transfection and functional activity in vitro. The proposed PNA amphiphile provides a novel platform to overcome the current challenges of limited cellular delivery and minimal efficacy of PNAs.

2. Results and Discussion

2.1. Design and Synthesis of PNA oligomers

We synthesized two 8 mer-PNA oligomers complementary to the seed region of oncomiR-155 as reported previously.^[30] To investigate the role of diethylene glycol unit for inducing self-assembly of PNAs, we synthesized γPNA-155 containing diethylene glycol moieties at the gamma position of each PNA monomer while PNA-155 was synthesized using regular PNA monomers (Figure 1A). Lauric acid (C12) was conjugated as the hydrophobic scaffold to the N-terminus of both γPNA-155 and PNA-155 using trioxo (OOO) as a flexible linker. To study cellular uptake and intra-cellular localization, carboxytetramethylrhodamine (TAMRA) dye was appended on to the C-terminus of both the PNA-155/γPNA-155 with trioxo as a linker (Figure 1B). PNAs were then purified using reversed-phase high performance liquid chromatography and molecular weight was confirmed using mass spectrometry analysis. Next, we examined the binding of the PNA-155 and γPNA-155 with the miR-155 target via thermal denaturation studies. PNA-155/γPNA-155 was incubated with the target miR-155 at 1:1 ratio in physiological conditions. Consistent with the prior studies,^[26a] γPNA-155 showed a higher thermal melting temperature (ΔT_m: 23°C) than the regular PNA-155 (Figure S1). Further, presence of lauric acid on the N-terminus did not interfere with the binding of PNA-155/γPNA-155 with the target miR-155.

Figure 1.

(A) Chemical structures of DNA, PNA, MiniPEGγPNA (^MPγPNA) monomers and lauric acid (LA). (B) The sequence of oncomiR-155 target and sequence of PNAs used in the study. The designed PNAs were complementary to the seed region of oncomiR-155 (underlined segment). γPNA-155 was synthesized using MiniPEG γPNA monomers and PNA-155 was synthesized using regular PNA monomers. Lauric acid was appended to N terminus or 5’ end and one lysine (K) was appended to the C terminus or 3’ end of both the PNAs. 5-Carboxytetramethylrhodamine (TAMRA) dye was appended to the C terminus or 3’ end of both the PNAs for visualization purposes. OOO is 11-amino-3,6,9-Trioxaundecanoic Acid linker.

Overall, we designed and synthesized lauric acid (C12) conjugated regular PNA-155 and γPNA-155 amphiphiles to target the seed region of oncomiR-155 which is upregulated in B-cell lymphomas.^[31] Prior studies have reported self-assembly of PNAs, however, here we designed γPNA-155 amphiphiles by conjugating γPNA with lauric acid (C12) and compared the self- and nano-assemblies of γPNA-155 for cellular delivery purposes.

2.2. Formulation and characterization of self- and nano-assemblies

We used ethanol injection method^[32] to formulate NA using PNA-155 and γPNA-155. The PNA-155 based nano-formulation showed significant precipitation of PNA-155 due to its hydrophobicity and poor solubility in water. Whereas, γPNA-155 nano-formulation showed minimal signs of self-aggregation. Formulations were centrifuged to remove the aggregates and supernatant was collected for further evaluation. The concentration of nano-formulations was determined using UV-Vis absorbance methods. Dynamic light scattering (DLS) was used to determine the hydrodynamic diameter and poly dispersity index (PDI). To confirm if only PNA-155 and γPNA-155 formed self assemblies (SA) due to the presence of lauric acid, we also characterized the PNA-155 and γPNA-155 dilutions in water at the same concentration as the NA. We noted that the hydrodynamic size of regular PNA-155 NA was higher (180 nm) than the only PNA-155 dilution (142 nm) in water. However, γPNA-155 showed a significant decrease in the hydrodynamic size (100 nm) after formulation into NA in comparison to only γPNA-155 dilution in water (500 nm) (Figure 2A). The PDI values were less than 0.2 for the γPNA-155 NA in comparison to the only γPNA-155 dilutions in water. These results indicated the rearrangement of γPNA-155 molecules into NA after formulation via ethanol injection technique (Figure 2B). We noted a change in the surface charge of PNA-155 and γPNA-155 formulations. γPNA-155 showed a negative zeta potential (−0.5 mV) while γPNA-155 NA showed positive (~+5 mV) surface charge density. Similarly, PNA-155 based NA showed an optimal increase in zeta potential (+20 mV) (Figure 2C).

Figure 2.

(A) The hydrodynamic diameter, (B) polydispersity index (PDI), and (C) surface charge of γPNA-155, γPNA-155 nano-assemblies (NA), PNA-155 and PNA-155 NA. The results are presented as mean ± SD (n=3). The 10x dilution of γPNA-155 NA and PNA-155 NA was used for characterization. γPNA-155 and PNA-155 dilutions were prepared at the same concentration as the 10x dilutions of their respective NAs. (D) Transmission electron microscopy (TEM) images of γPNA-155, γPNA-155 NA, PNA-155, and PNA-155 NA.

Next, we used transmission electron microscopy (TEM) to compare the morphology of PNA-155 and γPNA-155 based NA with the respective PNA dilutions in water. The TEM results explained that γPNA-155 self-assembles into vesicular structures ranging from 100–600 nm (Figure 2D). These results are consistent with the DLS characterization of γPNA-155 which also showed high hydrodynamic size and PDI indicating heterogeneous distribution. The PNA-155 dilution in water showed only self-aggregation of PNA. Hence, presence of lauric acid does not induce self-assembly of regular PNA oligomer, however conjugation of lauric acid on hydrophilic diethylene glycol modified γPNA induces their self-assembly (SA) into spherical vesicles. The TEM analysis of PNA-155 NA showed heterogenous distribution of different structures ranging from micelles <100 nm to liposomal shaped structures >200 nm (Figure 2D). The major caveat of PNA-155 NA was the low concentration of supernatant after centrifugation as most of the PNA-155 is precipitated out. Hence, we further investigated only γPNA-155 SA and NA.

2.3. Small-angle neutron and X-ray scattering analysis (SANS and SAXS)

Figure 3A shows the SANS data of γPNA-155. The oscillation at the low q possibly represents a core-shell structure. The lacking of low-q plateau suggests large radius (>1000 Å) beyond the probing q range. The attempt of using a single core-shell spherical (CSS) model (Figure S2) to fit the SANS data was not successful as the best fit completely misses the high q data. It is expected since the TEM image (Figure 2B) showed two populations. The one with a smaller size should yield scattering signal at high-q range. Moreover, the SANS data of γPNA-155 NA (Figure 3B) indicate a similar feature (for q > 0.06 Å⁻¹), suggesting both assemblies may contain similar types of smaller-sized particles. To characterize the smaller-sized vesicles, SAXS was applied to the sample because the highest probing q value extends to q = 1.0 Å⁻¹. Figure 3C shows the SAXS data of γPNA-155 NA. A clear intensity shoulder is observed at q ~ 0.3 Å⁻¹, which cannot be clearly resolved in SANS, resembling the scattering feature of a core-shell spheres. Therefore, we applied another CSS model to best fit the SAXS data and were able to describe the data of q > 0.05 Å⁻¹ (as shown in dark dashed line in Figure 3C). The low-q mismatch is due to larger-size particles from nano-assemblies. The smaller particles are presumably micelles with a best fitting radius of 13 (±1) Å and shell thickness of 10 (±1) Å (Figure 3E). The structural dimensions of the micelles were then directly applied to describe the SANS data over the high-q range. As for the larger particles of the γPNA-155 NA, (Figure 3B) the low–q intensity approaches plateau, suggesting that the dimension is smaller than of the SA. A core-shell ellipsoidal (CSE) model shown in Figure 3D and S2 provides a reasonable fit for the SANS data. The half lengths along the long- and short- axes of the inner water core are a and b, and the shell thicknesses along the corresponding axes are represented as d and c, respectively. Note that the rotating axis is the short one (b axis), forming an oblate-shell morphology. The best fitting structure yields an oblate shell and the dimension of its equator radius [~ 530 (±23) Å] agrees with the TEM images (Figure 2B) with a shorter polar radius of 43 (±2) Å. The same dimension of the oblate structures was directly applied to the analysis of SAXS data (Figure 3C) to match the low-q initial intensity decay (the orange dashed line); however, the dimension of large particles cannot be obtained due to the inaccessible lower q range. The SANS data of γPNA-155 can be best fit by two CSS models of different sizes. The similar structural parameters of smaller-sized particle from the γPNA-155 NA were applied to the SANS data of γPNA-155 because of the similar scattering pattern over the high-q range. The sum of the smaller-sized CSS model (accounting for micelles) and another CSS model with larger size (accounting for vesicles) was applied to fit the whole range of SANS data (Figure 3A), yielding a shell thickness of 45 (±16) Å and a vesicular radius > 1000 Å (Figure 3E). Compared to the TEM image of the γPNA-155, which shows a wide size distribution of layered structure (100–600 nm), we presume that the planar layers are the shells of the large vesicles. As a result, the combination of SANS and SAXS data allows us to access the structures of both particles which are consistent with the TEM images because of their different probing q ranges.

Figure 3.

The SANS patterns of (A) γPNA-155 and (B) γPNA-155 NA. (C) The SAXS pattern of γPNA-155 NA. (D) The cross section of the oblate ellipsoid shape of γPNA-155 NA. (E) The fitting results of γPNA-155 (SANS) and γPNA-155 NA (SANS and SAXS). The lighter and darker dashed lines correspond to the contributions of the larger and smaller morphologies, respectively.

2.4. Binding and stability study of γPNA-155 NA

First, we examined the binding of only γPNA-155 and γPNA-155 NA with the miR-155 target containing predicted binding sites by gel-shift assay. We tested different ratios of γPNA-155 with miR-155 from 1:1 to 4:1. We noted significant bound fraction (>20%) of γPNA: miR-155 heteroduplex with γPNA-155 and γPNA-155 NA at 1:1 ratio. The fraction of γPNA: miR-155 heteroduplex increased with higher γPNA-155 content (Figure S4). We observed >90% binding at 4:1 ratio of γPNA-155 and miR-155 for both the γPNA-155 and γPNA-155 NA. Hence, the formulation of γPNA-155 into the nano-assemblies did not impact its binding affinity with miR-155 target site.

Next, we evaluated the stability of γPNA-155 NA at refrigerated conditions (4°C) to avoid the impact of freeze-thaw cycles on the γPNA-155 NA stability. We performed dynamic light scattering (DLS) to measure the changes in hydrodynamic size and polydispersity index (PDI) of the samples at regular interval of two days for a period of two weeks which was then followed by weekly measurements. We did not notice any change in the hydrodynamic size of γPNA-155 NA over the period of the stability study. However, we noted an increase in PDI (>0.2) after 28 days (Figure S5). Hence, the γPNA-155 NA were stable for a month when stored under refrigerated conditions. We also tested the impact of ultrafiltration and lyophilization, processes routinely employed during the processing of nano-formulations, on the stability of γPNA-155 NA. The ultrafiltration technique is used for separation, concentration, as well as quantification of encapsulation efficiency of liposomes.^[33] Similarly, lyophilization is critical to ensure the long-term stability of nano-formulations.^[34] We measured the DLS size, PDI, and the surface charge of the γPNA-155 NA both pre- and post-ultrafiltration and -lyophilization. We observed aggregation of γPNA-155 NA when reconstituted after lyophilization as indicated by the increase in DLS size, PDI, and zeta potential (Figure S6). These results are consistent with aggregation of nanoparticles observed after lyophilization in the absence of cryoprotectants.^[35] However, ultrafiltration showed the minimal impact on the physicochemical characteristics of γPNA-155 NA (Figure S5). Hence, ultra-filtration can be used to concentrate the γPNA-155 NA while maintaining their characteristics.

2.5. γPNA-155 NA exhibit superior cellular uptake

The cellular uptake of γPNA-155 and γPNA-155 NA was studied in two cell lines including HeLa, a cervical cancer cell line, and U2932, a B-cell lymphoma cell line to establish the potential for targeting diverse cancer cells. The uptake of both the γPNA-155 and γPNA-155 NA was visualized via confocal microscopy and quantified using flow cytometry analysis. HeLa and U2932 cells were incubated with γPNA-155 and γPNA-155 NA at the same PNA equivalent dose of 0.5, 1, and 2 μM for 24 hours. We noted dose dependent increase in the cellular transfection of γPNA-155 and γPNA-155 NA in HeLa cells (Figure 4A). Since γPNA-155 self-assembles into large heterogeneous structures (100–600 nm), we observed reduced cellular uptake in comparison to the γPNA-155 NA. Confocal microscopy images of live HeLa cells showed higher TAMRA intensity in γPNA-155 NA treated cells in comparison to only γPNA-155 particularly at 0.5 and 1 μM (Figure 4A). The flow cytometry analysis indicated superior transfection of γPNA-155 NA in HeLa cells at 0.5 uM dose (Figure 4B, 4C and S7).

Figure 4.

(A) Confocal microscopy images of live HeLa cells indicating uptake of γPNA-155 and γPNA-155 nano-assemblies (NA) at different PNA equivalent dose after 24 hours of treatment. The scale bar represents 80 pixels. Blue indicates nucleus and red indicates TAMRA. (B) The flow cytometry histograms showing uptake of γPNA-155 and γPNA-155 NA. (C) The quantification γPNA-155 and γPNA-155 NA containing HeLa cells at indicated doses via flow cytometry. The results are presented as mean ±SD (n=3) and t-test was used for statistical analysis. (D) Confocal microscopy images of live U2932 cells after 24 hours of treatment with γPNA-155 and γPNA-155 NA at different PNA equivalent doses. The scale bar represents 80 pixels. Blue represents nucleus and red is TAMRA. (E) Flow cytometry histograms depicting the uptake of γPNA-155 and γPNA-155 NA in U2932 cells after 24 hours of treatment. (F) The quantitative representation of γPNA-155 and γPNA-155 NA containing U2932 cells. The results are represented as mean±SD (n=3) and t-test was used for statistical analysis. **p<0.01, ***p<0.001.

Interestingly, we noted higher uptake of γPNA-155 NA in U2932 lymphoma cells in comparison to the HeLa cells in particular at lower concentration. The differential uptake pattern of PNAs has been reported in different cell lines especially adherent and suspended cells.^[36] We observed higher cellular accumulation of γPNA-155 NA than γPNA-155 at 0.5 μM PNA equivalent dose via both confocal and flow cytometry (Figure 4B and 4C). Flow cytometry results showed more than 2-fold higher cellular uptake of γPNA-155 NA than γPNA-155 in U2932 cells. However, the fold change in uptake of γPNA-155 NA in comparison to γPNA-155 reduced with increase in the dose (Figure 4D and S8). Receptor mediated endocytosis has been reported to undergo saturation leading to reduced uptake at higher concentrations.^[37] Hence, the low dose treatment of γPNA-155 NA can attain superior therapeutic efficacy in the cancer cells in comparison to the only γPNA-155. In addition, saturation of uptake at high dose indicate endocytosis as possible mechanism for cellular transport of γPNA-155 and γPNA-155 NA. Further, we correlated the structure of γPNA-155 NA and SA with their cellular uptake. Note that both γPNA-155 NA and SA have similar small micelles; therefore the distinct cellular uptake is likely attributed to the larger-size particles. We hypothesize that small oblate morphology of γPNA-155 NA enhances the cellular uptake compared to the large spherical vesicles noted in the SA of γPNA-155. This hypothesis is consistent with previous reports on the enhanced cellular uptake of lipid nanodisc (or bicelle) in contrast to spherical lipid vesicles made of the same compositions.^{[6d, 38]} The positive charge density of γPNA-155 NA also plays a role in the enhancement of cellular uptake.

2.6. Uptake mechanism of γPNA-155 and γPNA-155 NA

Next, we investigated the uptake mechanism of γPNA-155 and γPNA-155 NA in the adherent HeLa cells which are widely used for the mechanistic endocytic studies. To ascertain the endocytosis role in cellular uptake of γPNA-155 based SA and NA, we compared their transfection efficiency at low (4°C) and physiological temperature (37°C). It is well known that the production of ATP is reduced at 4°C resulting in the inhibition of endocytosis-based transport across the cell membranes.^[39] Hence, we treated HeLa cells with 1 μM equivalent dose of γPNA-155 and γPNA-155 NA at 37°C and 4°C for 2 hour and used flow cytometry to quantify the uptake. γPNA-155 and γPNA-155 NA (Figure 5A) showed a decline in uptake at 4°C in HeLa cells. γPNA-155 showed 80% decrease in the cellular uptake while the transfection efficiency of γPNA-155 NA decreased more than 90%. Aforementioned results confirmed that the endocytosis-based pathways contribute towards the uptake of γPNA-155 and γPNA-155 NA. Further, we also investigated the contribution of different endocytic transport pathways for cellular delivery of γPNA-155 and γPNA-155 NA. We performed uptake studies in the presence of endocytic pathway inhibitors for mechanistic understanding of the routes for cellular transport of γPNA-155 and γPNA-155 NA in HeLa cells. Prior studies established that chlorpromazine (CPZ) inhibits clathrin mediated endocytic pathway,^[40] genistein prevents the caveolae based pathway,^[41] and amiloride interferes with the macropinocytosis based transport.^[42] We pre-incubated HeLa cells in the presence of different endocytic inhibitors for 1 hour followed by treatment with γPNA-155 and γPNA-155 NA.

Figure 5.

(A) The quantitative representation of γPNA-155 and γPNA-155 nano-assemblies (NA) uptake in HeLa cells at 37°C and 4°C after 2 hour of incubation. The results are represented as mean±SD (n=3) and t-test was used for statistical analysis. ****p<0.0001. (B) Graph representing the change in cellular transport of γPNA-155 and γPNA-155 NA in HeLa cells via flow cytometry analysis after inhibition of endocytic pathways using genistein, chlorpromazine, and amiloride which inhibits caveolae-mediated, clathrin-mediated, and macropinocytosis cellular transport respectively. The results are represented as mean±SD (n=3) and t-test was used for statistical analysis. **p<0.01, ***p<0.001, ****p<0.0001. (C) The fluorescent images of HeLa cells showing uptake of γPNA-155 and γPNA-155 NA after small molecule (chlorpromazine, genistein, amiloride) mediated inhibition of endocytic pathways and 2 hour treatment duration. Scale bar represents 80 pixels. Blue indicates nucleus and red is TAMRA.

Further, we examined the cellular uptake via flow cytometry and confocal microscopy-based techniques. Flow cytometry results indicated the substantial role of macropinocytosis in transport of γPNA-155 and γPNA-155 NA (Figure 5B). Whereas, γPNA-155 NA also undergoes uptake via clathrin and caveolae-mediated pathways. CPZ and genistein-mediated inhibition of clathrin and caveolae endocytic pathways, respectively in HeLa cells resulted in 30% lower cellular internalization of γPNA-155 NA. Overall, our results indicated that γPNA-155 NA undergoes uptake via three major endocytic pathways including macropinocytosis, being the dominant route, followed by caveolae and clathrin mediated transport. We confirmed our results by confocal imaging where we noted maximum reduction in cellular uptake of γPNA-155 NA after amiloride mediated inhibition of macropinocytosis (Figure 5C). However, HeLa cells treated with only γPNA-155 showed 50% reduced uptake after inhibition of macropinocytosis and less than 25% decrease in uptake after inhibition of caveolae or clathrin mediated pathways. These results are consistent with previous report, where peptide conjugated PNAs utilized macropinocytosis, clathrin, and caveolae mediated pathways for cellular transport.^[43] For the future studies, it will be interesting to combine the different endocytic pathway inhibitors and study their cumulative effect on internalization of γPNA-155 and γPNA-155 NA.

2.7. Safety and efficacy evaluation of γPNA-155 and γPNA-155 NA

The nucleic acids (DNA or RNA)^[44] as well as lipid nanoparticles containing positively charged lipids^[45] have been reported to activate the toll-like receptor (TLR) mediated immune response. Peripheral blood mononuclear cells (PBMC) consists of heterogenous cell population including lymphocytes, monocytes, and macrophages which are critical components of innate and adaptive immune response.^[46] Hence, we tested the safety of γPNA-155 and γPNA-155 NA in PBMC in vitro. We treated the PBMC cells with increasing doses of γPNA-155 and γPNA-155 NA at the PNA equivalent doses of 0.5, 1, and 2 μM. We measured the viability of PBMC cells after 48 hours of treatment via luminescence-based assay. The results showed no impact of γPNA-155 NA on the viability of PBMC cells in comparison to the control and γPNA-155 treated cells at all indicated doses (Figure 6A).

Figure 6.

(A) The graph representing cellular viability of peripheral blood mononuclear cells (PBMC) after 48-hour treatment with γPNA-155 and γPNA-155 nano-assemblies (NA). Untreated cells were used as control. Results are presented as mean±SD (n=5). (B) The fold change in U2932 miR-155 levels after 48-hour treatment with γPNA-155 and γPNA-155 NA at indicated doses. The results are presented as mean±SD (n=3) and t-test was used for statistical analysis. *p<0.05, **p<0.01, ****p<0.0001. (C) The fold change in levels of downstream targets of miR-155 in U2932 cells after 48-hour treatment with γPNA-155 and γPNA-155 NA at 0.5 uM dose. The results are presented as mean±SD (n=3) and t-test was used for statistical analysis. *p<0.05, **p<0.01. (D) The change in percentage of live U2932 cells after treatment with γPNA-155 and γPNA-155 NA at 0.5 uM PNA equivalent dose. The results are presented as mean±SD (n=3) and t-test was used for statistical analysis. *p<0.05, ****p<0.0001.

γPNA-155 was designed to target the seed region of oncomiR-155, which is highly overexpressed in diffuse large B cell lymphoma (DLBCL)^[47] and burkitt lymphoma.^[48] Hence, we tested the efficacy of γPNA-155 NA in U2932 cells, a DLBCL cell line which overexpresses the oncomiR-155 in comparison to the non-tumor reactive lymph nodes.^[47] Since γPNA-155 NA showed superior cellular transfection at low doses (0.5 and 1 μM), we treated U2932 cells with both the γPNA-155 and γPNA-155 NA at two PNA equivalent doses including 0.5 and 1 μM. qRT-PCR results indicated 70% knockdown of miR-155 after treatment with γPNA-155 NA at 0.5 μM dose in comparison to the control group (Figure 6B). However, we noted only 35% and 60% knockdown of miR-155 in γPNA-155 treated U2932 cells at 0.5 and 1 μM dose, respectively (Figure 6B). γPNA-155 NA reached maximum knockdown (70%) of the target miR-155 at 0.5 μM as we did not observe an increase in knockdown efficiency of miR-155 at 1 μM. These observations are consistent with the reports where receptor mediated endocytosis plays a major role for cellular transport at lower doses and is saturated at higher doses.^[49] However, only γPNA-155 showed a dose dependent increase in the cellular delivery as well as the knockdown efficiency. These results further establish that only γPNA-155 utilizes macropinocytosis as the major cellular uptake pathway which is not saturated at the tested concentration (0.5 and 1 μM).

Further, we evaluated the levels of tumor suppressor genes using γPNA-155 and γPNA-155 NA at 0.5 μM equivalent dose in U2932 cells. The level of genes including forkhead box O3 (FOXO3A), phosphatase and tensin homolog (PTEN), colony stimulator factor-1 receptor (CSF1R), and jumonji- and AT-rich interactive domain 2 (JARID2) have been reported to be upregulated after knockdown of miR-155 in multiple diffuse large B-cell lymphoma cell lines.^[50] We observed higher upregulation of FOXO3A, PTEN, CSF1R, and JARID2 in γPNA-155 NA treated U2932 cells in comaprison to only γPNA-155 (Figure 6C). These results are consistent with the uptake study of γPNA-155 NA in U2932 where we noted more than 2 fold higher uptake of γPNA-155 NA (82%) at 0.5 uM when compared against γPNA-155 (32%) via flow cytometry analysis. Hence, the qRT-PCR results corroborate our observations in cellular uptake studies, establishing that superior transfection properties of γPNA-155 NA results in higher knockdown efficiency of miR-155 in comparison to γPNA-155 at 0.5 μM.

The high levels of oncomiR-155 are critical for the proliferation of lymphoma cells.^[51] Hence, we investigated the impact of miR-155 knockdown on U2932 cell viability. We used luminescence-based assay to determine the viability of U2932 cells after 48 hours of treatment with γPNA-155 and γPNA-155 NA at the PNA equivalent doses of 0.5, 1, 2, and 4 μM. We observed significant decline in the viability of γPNA-155 NA treated U2932 cells in comparison to the only γPNA-155 when tested at the same dose (0.5, 1, 2, and 4 μM) (Figure S9). These results further support that γPNA-155 NA mediated superior knockdown of oncomiR-155 results in the reduced viability of U2932 cells in comparison to γPNA-155 treated cells. Next, we performed annexin V assay to study the role of γPNA-155 NA mediated apoptosis in the U2932 cells. Consistent with the cellular viability studies, we observed 25% decrease in the live U2932 cells after 48 hours of treatment with γPNA-155 NA, while γPNA-155 treated U2932 cells showed only 15% decrease in live cells when compared against the untreated cells (Figure 6D). Further, annexin V assay results showed that U2932 cells undergo early or late apoptosis after treatment with γPNA-155 and γPNA-155 NA. We observed higher percentage increase of apoptotic cells (22%) in PNA-155 NA treated U2932 cells in comparison to only γPNA-155 (15%) treated cells at the PNA equivalent dose of 0.5 μM (Figure S10). The results from apoptosis and cell viability study together with the miR-155 knockdown in U2932 cells confirmed the higher efficacy of γPNA-155 NA in comparison to γPNA-155 in vitro.

3. Conclusion

The cellular delivery of PNAs has been a major challenge towards their clinical success. Until now the focus has been on utilizing nanoparticles,^[52] peptide based approaches^[53] or liposomes for improving the cellular delivery of PNAs. Here we developed a simple approach based upon nucleic acid nanotechnology to improve the cellular transport of PNAs. The superior efficacy of chemically modified γPNAs containing diethylene glycol units have been established in multiple studies in vivo for both gene targeting^[54] and gene editing^{[5a, 5c]} applications. Most recently, few studies have also reported self-assembly of γPNAs containing diethylene glycol side-chains into nanofibers^[55] or complex nanostructures.^{[28, 56]} However, in this study we designed amphiphilic γPNAs which can be formulated into NA with superior cellular transfection and functional properties. Hence, this strategy opens a new avenue for γPNA mediated gene targeting applications where γPNAs can be delivered without the need of a carrier system. It would be interesting to delivery multiple PNA sequences formulated into NA to target different RNA (coding or non-coding) for treating cancer and metabolic disorders. It would also be interesting to study the stability, safety, and efficacy of these amphiphilic γPNA based NA in vivo.

Overall, we established that amphiphilic γPNAs can be successfully formulated into NA which exhibit superior cellular transfection and functional activity in vitro. We exploited the potential of γPNA as a biopolymer to formulate biologically active NA using a simple and easily scalable approach. Unlike DNA, PNAs are enzymatically stable and non-immunogenic, hence PNA based NA can be utilized for diverse biomedical applications. This study provides the evidence that PNAs can be engineered as the compatible biomaterial to design the next generation of functionally active nanostructures.

4. Experimental Section

Materials

The boc protected regular and mini-PEG γPNA monomers (A, T, C, and G) were purchased from ASM Chemicals (Germany). Lauric acid and Boc-Mini-PEG-3 linker was obtained from Sigma Aldrich (USA). TAMRA or carboxytetramethylrhodamine dye was obtained from VWR International. Fmoc-Lysine (Boc)-OH was purchased from Sigma Aldrich (USA) and 4-methylbenzhydrylamine (MBHA) resin (100–200 mesh) was obtained from Peptide International (USA). The cells used in the study including HeLa (ATCC CCL-2) and peripheral blood mononuclear cells (PBMC) were obtained from American type culture collection (ATCC, USA). U2932 cells were purchased from DSMZ (ACC-633, Germany). Eagle’s minimum essential medium (EMEM, 30-2003), Roswell park memorial institute medium (RPMI-1640, 30-2001), and fetal bovine serum (FBS, 30-2020) were also bought from ATCC (USA).

Methods

Synthesis of PNA oligomer

Fmoc-Lysine (Boc) was conjugated to the MBHA resin to use as a solid support for PNA synthesis. Boc-protected regular and Mini-PEG γPNA monomers (A, T, C, and G) were then added to the Fmoc-Lysine (Boc) conjugated resin using solid phase synthesis as reported previously.^[30b] Lauric acid was conjugated at the N terminus of PNAs using Mini-PEG-3 as a linker. Further TAMRA was conjugated to the C terminus of PNAs also using Mini-PEG-3 as a linker. The cocktail containing trifluoroacetic acid: trifluoromethanesulfonic acid: thioanisole: m-cresol was used at 6:2:1:1 ratio to cleave the PNA oligomers from the resin. Next, diethylether was used to precipitate PNA from the cleavage cocktail and was washed twice with diethyl ether. PNAs were then vacuum dried overnight and reconstituted in acetonitrile and water. The purification of PNAs was performed using reverse phase high performance liquid chromatography (RP-HPLC, Shimadzu, Japan) and an XBridge C18 OBD prep column (Waters, USA). 0.1% (v/v) TFA in acetonitrile was used as the organic phase and 0.1% (v/v) TFA in water as the aqueous mobile phase. The pure fraction of PNAs were then freeze-dried (Labconco, USA) and reconstituted in water. The PNA concentration was determined via UV-vis spectroscopy on a Nanodrop (Thermo Scientific, USA) and were stored at −80°C. Further the molecular weight of PNAs was confirmed using matrix assisted laser desorption-ionization time of flight (MALDI) spectrometry.

Formulation of PNA nano-assemblies (NA)

The NA were formulated using ethanol injection method as reported previously for liposome formulation.^[32] Both the PNA-155 and γPNA-155 containing lauric acid (50 nmoles) were completely dissolved in 0.5 ml ethanol and added dropwise to the 5 ml aqueous phase (0.2 micron filtered purified water) using a 28-gauze syringe. The aqueous phase was continuously stirred at 700 RPM (×0.016667 Hz) while maintaining a temperature of 75°C. After the addition of PNA to the aqueous phase, it was covered with aluminum foil and allowed to stir overnight at room temperature (RT) inside the chemical hood. The formulation was then centrifuged at 15000 RPM for 10 mins at 4°C to remove the large aggregates. The supernatant was collected in a separate sterile tube and stored in 4°C protected from light for further evaluation.

In vitro binding studies

Gel shift:

γPNA-155 and γPNA-155 NA were incubated with the miR-155 target at indicated ratios of PNA: miR-155 ranging from 1:1 to 4:1. Samples were prepared in physiological conditions (10 mM sodium phosphate, 150 mM KCl, and 2 mM MgCL₂) and incubation was done at 37°C for 16 hours in thermal cycler (Bio-Rad, USA). The samples were then separated on non-denaturing 8% polyacrylamide gel at 120V for 35 mins. The bound and unbound fraction of the miR-155 were stained with SYBR gold (Invitrogen, USA) and visualized using the Gel-Doc EZ imager (Bio-Rad, USA).

Thermal melting analysis:

Both the regular PNA-155 and γPNA-155 were incubated with the target miR-155 in physiological conditions at 1:1 ratio. The samples were subjected to a temperature ramp from 95°C to 25°C and 25°C to 95°C at 1°C per min. The absorbance was recorded every 0.5°C increase in temperature at 260 nm using UV-Vis spectrophotometer.

Characterization of PNA SA and NA

Dynamic light scattering technique was used to determine the size and polydispersity index (PDI) of the PNA based SA and NA. PNA NAs were diluted in 0.2 micron filtered purified water to a 10x dilution and hydrodynamic size was measured using the Nano-ZS from Malvern Panalytical (USA). The size and PDI of PNA-155 or γPNA-155 SA was also determined at the same concentration as 10x dilution of PNA NA using purified water as a diluent. The measurements were performed at 25°C using 1.33 as refractive index. The surface charge of PNA SA and NA was determined by measuring the zeta potential at 25°C utilizing laser doppler micro-electrophoresis technique.

Transmission electron microscopy (TEM)

The γPNA-155 and PNA-155 based SA and NA were stained with 1% (w/v) uranyl acetate at RT for 5 mins on carbon grid (400 mesh copper) followed by washing with water. The samples were allowed to dry for 10 mins followed by imaging using FEI Technai at 80 kV voltage.

Cell culture

HeLa cells were cultured in 10 cm petridishes using EMEM media supplemented with 10% (v/v) FBS and 1% (v/v) Pen-Strep. The HeLa cells were passaged as they reached 90% confluency. U2932 and PBMC cells were cultured in T-75 flasks using RPMI media supplemented with 10% (v/v) FBS and 1% (v/v) Pen-Strep. PBMC cells were used within 48 hours of thawing the cells. The cells were kept in the incubator at 37°C and 5% CO₂.

Cellular uptake studies

The uptake of γPNA-155 and γPNA-155 NA were studied in HeLa as well as U2932 cells. The cellular uptake was visualized using confocal microscopy and quantification was confirmed using flow cytometry analysis.

Confocal microscopy:

8-well chamber slides were used to grow the HeLa cells (50,000 cells per well). The HeLa cells were then treated with the γPNA-155 and γPNA-155 NA at the PNA equivalent doses of 0.5, 1, and 2 μM. After 24 hours of treatment, PBS was used to wash the extracellular γPNA-155 and γPNA-155 NA followed by staining of the nucleus with hoechst dye 33342 (R37605, Thermo Fisher, USA). The cells were washed again with PBS and suspended in the CO₂ independent media (Gibco, USA). The live cells were immediately imaged at 60x on the Nikon A1R microscope.

24 well plates were used to grow 100,000 U2932 cells per well. PNA equivalent doses of γPNA-155 and γPNA-155 NA at 0.5, 1, and 2 μM were added to the wells. 24 hours post-treatment, cells were washed with PBS and nucleus was stained with the hoechst dye for 25–30 mins. Cells were washed again with PBS and final pellet was resuspended in 15 μl of growth media. The resuspended cell pellet was then transferred to the glass slide and covered with the 1 mm coverslip. The samples were imaged right away to visualize the cellular uptake in U2932 cells.

Flow cytometry:

To quantify the uptake of γPNA-155 and γPNA-155 NA, 24 well plates were used to grow HeLa and U2932 cells. γPNA-155 and γPNA-155 NA were added to the cells at the PNA equivalent doses of 0.5, 1, and 2 μM. After 24 hours, HeLa cells were washed twice with PBS and trypsinized by adding 100 μl of 0.25% trypsin (Gibco, USA) followed by incubation at 37°C and 5% CO₂ for 5–10 mins. The HeLa cells were then collected in the 1.5 ml tubes by centrifuging (2000 RPM) at 4°C. The treated and untreated U2932 cells (suspended) were directly transferred to the 1.5 ml tube and centrifuged at 4°C. The U2932 cell pellet was washed with PBS and centrifuged again. The washed U2932 cells were resuspended in 300 μl of PBS. The cell suspension of both HeLa and U2932 were transferred to the FACS tubes and maintained on ice. Without any delay, samples were evaluated using Fortessa cell analyzer (BD Biosciences, USA) to quantify the uptake of γPNA-155 and γPNA-155 NA.

Endocytosis inhibition study

The uptake of γPNA-155 and γPNA-155 NA was also studied at 4°C (low temperature) in HeLa cells. HeLa cell lines were pre-incubated at 4°C for 1 hour followed by treatment with γPNA-155 and γPNA-155 NA. After 2 hours, treated and untreated HeLa cells were washed, trypsinized, and transferred to the FACS tubes. The uptake was quantified using Fortessa X-20 cell analyzer (BD Biosciences, USA). Further, different endocytic pathways were inhibited using chlorpromazine at 10 μg/ml, genistein at 200 μM, and amiloride at 1 mM in HeLa cells for 1 hour (pre-treatment) at 37°C. γPNA-155 and γPNA-155 NA were then added to the pre-treated HeLa cells. After 2 hours, HeLa cells were trypsinized and transferred to the FACS tube for flow cytometry analysis. For confocal microscopy, the nucleus of treated HeLa cells was stained with hoechst dye and images were taken using a Nikon A1R confocal microscope.

Quantitative real time polymerase chain reaction (qRT-PCR)

12 well plates were used to seed 200,000 U2932 cells per well. γPNA-155 and γPNA-155 NA were used at the equivalent dose of 0.5 and 1 μM. After 48 hours of treatment, cells were transferred to the 1.5 ml tube and washed with PBS once. The total RNA was extracted from the treated and untreated U2932 cell pellets using Qiagen RNAeasy kit (Germany). cDNA was synthesized using the target specific primers (miR-155 assay ID: 467534_mat and U6 assay ID: 001973) and following the conditions recommended in high capacity cDNA reverse transcription kit from applied biosystems (USA). The cDNA was then amplified using target specific primers and universal master mix II (Applied Biosystems, USA) under the recommended conditions using CFX connect RT-PCR detection system (Bio-Rad, USA). U6 snRNA was used as the reference gene for miR-155 quantification. The taqman gene expression assays for PTEN (Hs02621230), FOXO3A (Hs00818121_m1), CSF1R (Hs00911250_m1), and JARID2 (Hs01004467_m1) were used for PCR. GAPDH (Hs02786624_g1) was used as the reference gene for downstream mRNA target evaluation.

Cell viability assay

The viability of PBMC and U2932 cells after treatment with γPNA-155 and γPNA-155 NA was determined using CellTiter-Glo® luminescence-based assay (G7570, Promega, USA). 96 well plate was used to seed 20,000 PBMC and U2932 cells per well. γPNA-155 and γPNA-155 NA were added at indicated PNA equivalent doses. After 48 hours, cells were incubated at RT for 30 mins and 100 μl of CellTiter-Glo reagent was added. The plate was kept on a shaker for 30 secs and kept at RT for 10 mins. The luminescence signal was then captured using the Tecan plate reader.

Apoptosis assay

Annexin V apoptosis detection kit (BD Biosciences, USA) was used to determine the apoptosis in U2932 cells. γPNA-155 and γPNA-155 NA were added at 0.5 μM PNA equivalent dose. After 48 hours, cells were collected in the 1.5 ml tubes and centrifuged at 2000 RPM for 3 mins at 4°C. The treated and untreated U2932 cells were counted using the cell counter (Bio-Rad, USA). U2932 cells (100,000) were then transferred to the FACS tubes and stained with 10 μl of each 7-AAD and Annexin V PE. The cells were incubated in dark for 15 mins followed by addition of 400 ul Annexin V binding buffer to the FACS tubes. The samples were analyzed using the Fortessa Cell analyzer (BS Bioscience, USA) using unstained cells, only 7-AAD stained and Annexin V stained cells as controls. The results were analyzed using flowjo analysis software.

Small-angle neutron scattering (SANS)

γPNA-155 NA was formulated using D₂O and ethanol injection method to obtain 5 mg/ml concentration. The SANS setup at National Institute of Science and Technology (NIST) Center for Neutron Research (NCNR, Gaithersburg, MD). The average wavelength, λ of 6 Å was used with a spread of 12.5%. This yielded a q range $[q\equiv \frac{4\pi }{\lambda }\text{sin}\left(\frac{\theta }{2}\right)$, where θ is the scattering angle] from 0.001 Å⁻¹ to 0.4 Å⁻¹. The front and middle detectors were used for low and high q data collection, respectively. The 2D raw data were corrected by the detector sensitivity, background, scattering and transmission of empty cells and sample transmission. The corrected data were then circularly averaged, yielding 1D profiles and the intensity was put on the absolutely scale using the measured incident beam flux. The data reduction package was provided by NIST using the IGOR macros with IGOR Pro software. The SANS data is analyzed by using SASView 4.2.2.

Small-angle X-Ray scattering (SAXS)

SAXS experiments were performed with the 16ID-LiX Beamline at National Synchrotron Light Source II at the Brookhaven National Laboratory (Upton, NY). Samples were loaded and measured with the high throughput flow cell solution scattering setup with an X-ray energy of 13.5 keV. The scattering images were acquired with 1s of exposure time for twice and averaged to yield the 2D raw data, which were then circularly averaged and reduced to 1-D profiles as a function of scattering vector, SAXS intensity is presented as a function of scattering vector, q varying from 0.005 to 2.5 Å⁻¹. To reduce the intensity of hydrogen bond, transmission correction and background subtraction was done at q ~2.0 Å⁻¹. The SAXS data is also analyzed by using SASView 4.2.2.