International Journal of Biological Macromolecules 72 (2015) 819–826
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Betaine conjugated cationic pullulan as effective gene carrier Lizebona August Ambattu, M.R. Rekha ∗ Biosurface Technology Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India
a r t i c l e
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Article history: Received 4 June 2014 Received in revised form 30 August 2014 Accepted 22 September 2014 Available online 7 October 2014 Keywords: Pullulan Betaine Hemocompatible Gene delivery p53
a b s t r a c t Polyethyleneimne (PEI) is a very efficient transfecting agent but is toxic due to high charge density. To generate a vector which is efficient and less cytotoxic, PEI was conjugated with pullulan (PPEI). Further conjugation was done on PPEI with zwitter ionic betaine which possess antifouling property. PEI of molecular weight 1.2, 2, and 10 kDa were used in the study. Buffering capacity of pullulan-PEI-betaine (PPB) conjugates was found to be sufficient enough for the polymers to make endosomal escape. The polymers proved to be less cytotoxic and highly hemocompatible than PEI. Nuclear localization of YOYO tagged DNA was observed with the nanoplexes developed using PPEI and PPBs of PEI 10 kDa. Transfection efficiency was evaluated using p53 expressing gene and the live dead assay demonstrated very high transfection efficiency with PPB conjugates of PEI 10 kDa. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy is considered as solution for every inherited or acquired disease known. Even after first clinical trial for severe combined immunodeficiency (SCID), progress in gene delivery has not been as growing as expected. The rate limiting step in successful gene therapy is preparation of efficient gene-delivery vector. Viral vectors are lately replaced by nonviral vectors owing to low immunogenicity and mutagenicity of nonviral vectors [1–3]. For a nonviral vector to be efficient in delivering its cargo, it should be able to escape extracellular nuclease activity, evade reticuloendothelial system, avoid non specific interaction with blood cells thereby avoiding immune response and inflammatory reaction and has to reach target cells. At the target cells, the nanoparticle has to be internalized by cell membrane, escape from endosome, and traverse through viscous cytoplasm and finally therapeutic gene has to be expressed [4]. Pullulan is a polymer of maltotriose unit, connected by an ␣ (1 → 4) glycosidic bond, where consecutive maltotriose units are connected to each other by an ␣ (1 → 6) glycosidic bond. Hydrophobized pullulan is reported as drug-delivery carriers. Biocompatible and nontoxic nature of pullulan has been exploited to produce gene-delivery vectors [5,6]. Liver targeting with pullulan is already exploited [7–9]. Cationized pullulan has been reported as genedelivery vector [10].
∗ Corresponding author.Tel.: O: +91 471 2520214. E-mail address: [email protected] (M.R. Rekha). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.043 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Polyethyleneimine (PEI) has been considered as potent transfection agent. The high positive charge due to protonable amine groups facilitates ionic interaction with nucleic acids. Primary amine being basic and most reactive has been modified to change the properties of PEI [11]. PEI also has another remarkable property among organic polymers. PEI forms amino ethylene unit when fully protonated which provides one of the highest possible charge density. So when partially protonated PEI on further protonation in endosomes causes membrane destabilization. This membrane destabilization may be the cause of high toxicity of PEI [12,13]. A new conjugate with the benefits of PEI but with reduced toxic effect were developed and reported from this lab earlier. The pullulan was cationized with PEI which was found to be hemocompatible and an excellent candidate for gene delivery to cells especially to liver cells [6]. Betaine, also known as trimethylglycine, is a zwitterionic highly polar naturally occurring molecule. One of the major role of betaine as an osmolyte is maintaining the cell volume [14]. Accumulation of betaine in cells leads to increase in volume of cytoplasmic water by 20–50% per unit of dry weight of cell [15]. It is proposed that the alteration in cell volume slows down rate of cell proliferation [16]. As mentioned earlier, highly viscous cytoplasm also poses a hurdle to successful gene delivery. Hence, the introduction of betaine thereby causes a decrease in cytoplasmic viscosity. Nonviral vectors of cationic polymers facilitated with zwitterionic compounds were developed by various groups. Zwitterionic polymers were found to be highly resistant to non specific protein adsorption owing to the electrostatically induced hydration. These polymers are known to reduce serum protein interactions [17–19]. Antifouling properties of zwitterionic polymers are also known
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[20,21]. Of the zwitterionic polymers studied those with betaine were in focus recently [22–25]. Several betaine-based polymers are reported which showed good nucleic acid condensation capability and better transfection efficiency in serum compared to PEI. Lower protein adsorption and reduced cytotoxicity are yet other properties favoring successful nonviral gene-delivery vector. Even though the transfection efficiency of PEI is high, its use as genetic delivery vector is limited by its high toxicity. So there is a need of a system which can easily be internalized by cell and escape endosomes and deliver its cargo with considerably low toxicity to the cells. The effect of pullulan conjugated to both low molecular weight and high molecular weight PEI were studied. It was also studied whether osmolyte like betaine when conjugated with PEI conjugated with pullulan can improve the transfection efficiency or compatibility.
2. Materials and Methods
2.4. Preparation of polyplexes and determination of size and zeta of polyplexes Polyplexes were prepared by vortexing varying concentration of polymer to a fixed concentration of calf thymus DNA (ctDNA). The w/w ratio of polymer to ctDNA was from 0.5:1 to 15:1. The size and zeta of the polyplexes were determined using dynamic light scattering measurement using Zetasizer Nano ZS (Malvern Instruments Ltd., UK) at a temperature of 25 ◦ C. 2.5. Determination of buffering capacity The buffering capacity of PEI of three different molecular weights, PPEIs and PPBs, saline and betaine were evaluated by acid base titration over a pH range of 10–4. The pH of the polymer solution (0.1 mg/mL in saline) was adjusted to 10 using 0.2 N NaOH then titrated against 0.01 N HCl, the pH being noted after each addition of a volume of 50 L of acid. A graph was plotted with pH against the volume of HCl.
2.1. Chemicals 2.6. Agarose gel electrophoresis Pullulan (Sigma–Aldrich), carbonyl diimidazole (CDI), polyethyleneimine (10 kDa, 1.2 kDa, 2 kDa, Polysciences), betaine (Polysciences), EDC,Agarose,Ethidium Bromide,), 3-(4,5-dimethylthialzol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium (DMEM), trypsin, ethylenediamine tetra acetic acid (EDTA), DNase I (Sigma–Aldrich Chemicals Co, USA.). Deoxyribonucleic acid sodium salt from calf thymus (ctDNA) (Worthington Biochemical Corp), YOYO iodide and Hoechst 33342 (Invitrogen). Fetal bovine serum (FBS) (GIBCO, USA). All other reagents were of analytical grade from Merck, India.
2.2. Preparation of polymers 2.2.1. Preparation of PPEI Pullulan in dimethylsulphoxide was activated using CDI at 37 ◦ C for 2 h. After the 2 h, PEI of desired molecular weight in 20 mM borax was added to the mixture and was constantly stirred at room temperature overnight. Later pullulan PEI conjugate (PPEI) was precipitated with acetone. Acetone washed PPEI precipitate is then dissolved in deionized water and dialyzed for 24 h with two changes to remove unreacted PEI.
2.2.2. Preparation Pullulan-PEI-Betaine (PPB) PPB was prepared by using EDC as cross linker between PPEI and betaine under constant stirring for overnight at room temperature. Unreacted materials are removed by overnight dialysis.
2.3. Characterization 2.3.1. 1 H NMR 1 H NMR spectra of pullulan and derivatives were measured in D2 O using a 500 MHz spectrometer (Bruker Avance NMR Spectrometer). ␦H (500 MHz; D2 O; Me4 Si)
2.3.2. Determination of Betaine The assay is based on the fact that betaine forms a periodite complex with iodide in acidic medium at low temperature [26]. Briefly, cold potassium iodide–iodine was allowed to react with PPB in presence of concentrated sulphuric acid. The pellet obtained was then dissolved in 1, 2- dichloromethane and absorbance were measured at 365 nm with dichloromethane as blank. Betaine of different concentration was used to get standard curve.
The condensation property of nanoparticles on ctDNA (calf thymus DNA) was assessed by agarose gel electrophoresis. Nanoplexes of desired ratios was prepared (0.5:1, 3:1, 5:1) with ctDNA. The stability of nanoplexes in the presence of plasma was also assessed by incubating nanoplexes with 20 L of plasma for 30 min. The ability of polymers to protect pDNA from endonuclease degradation was evaluated by treating the nanoplexes with DNAse enzyme. The polyplexes were incubated with DNase of concentration 853 U/mL at 37 ◦ C for 30 min. The reaction was stopped with termination buffer. Naked ctDNA with and without DNase 1 treatment were positive and negative control, respectively. Electrophoresis was carried out in 1× TAE buffer, staining with 2 L of 10 mg/mL ethidium bromide in a Bio Rad electrophoresis system (Bio-Rad laboratories, CA, USA) at 75 V for 45 min. The DNA bands were visualized and the gel was photographed using Image Analyzer (LAS 4000, Fuji). 2.7. Polymer–plasma protein interactions-PAGE studies Native PAGE analysis was performed to assess the interaction of polymer with plasma proteins. The polymer (1 mg/mgL) was incubated with 20 Lof plasma for 30 min. PEI interaction with plasma protein was marked as negative control and plasma as positive. After 30 min, the complexes were subjected to centrifugation at 8000 rpm for 10 min. Supernatant was collected and mixed with sample buffer at 2:1 ratio. Electrophoresis was carried out in a MiniPROTEAN II electrophoresis cell (Bio-RAD, CA, USA). Gel was later stained with 0.2% coomassie brilliant blue R 250 for 2 h and then destained using destaining buffer containing acetic acid, methanol, and distilled water in the ratio. The gel was then photographed using an image analyzer (LAS 4000, Fuji). 2.8. Blood compatibility studies Blood from the unmedicated healthy volunteer was collected in a tube containing 3.8% sodium citrate. Ratio of blood to anticoagulant was 9:1. 2.8.1. Hemolysis RBC devoid of plasma was diluted a ratio 1:9. A total of 100 L of RBC was then added to polymers taken at four different concentrations 25, 50, 75, and 100 g and incubated for 2 h at room temperature. The mixture was then centrifuged at 700 rpm for 10 min. Absorbance of 100 L from the supernatant made up to
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1 mL with normal saline was taken at 541 nm. RBC incubated with Triton X-100 and saline were taken as positive and negative controls. 2.8.2. RBC Aggregation Polymers at two different concentrations 50 and 100 g was incubated with 100 L of RBC (diluted at ratio 1:8) at room temperature for 30 min. Cells treated with PEI and normal saline were taken as positive and negative controls, respectively. Images were captured using LeicaDMI3000 B (Lexica, Germany). Blood with anticoagulant overlaid on Histopaque was centrifuged at 800 rpm for 20 min to separate WBC and platelet. 2.8.3. WBC Aggregation A total of 50 and 100 g of the polymers incubated with 100 L of WBC for 20 min. PEI and plasma incubated with WBC were taken as positive and negative control, respectively. Presence of aggregation if any was detected through LeicaDMI3000 B (Leica, Germany). 2.8.4. Platelet Aggregation A total of 100 L platelet was incubated with 50 and 100 g of polymer for 30 min. Platelet incubated with PEI and saline were the positive and negative controls, respectively. Presence of aggregation if any was detected through LeicaDMI3000 B (Lexica, Germany). 2.9. Cytotoxicity Toxicity of the PPEIs and PPBs were assessed using MTT assay on C6 glioma cells. The cells were trypsinized using 0.25% trypsinEDTA. Cells were seeded at a density of 1 × 103 cells/well on a 96 well plate and incubated for 24 h in CO2 incubated at 37 ◦ C under 5% CO2 atmosphere. After 24 h, the medium was removed and polymers were added at a concentration of 25, 50, 100 g/mL per well along with positive and negative controls and incubated again for 24 h at 37 ◦ C in 5% CO2 atmosphere. After incubation, the samples were removed and MTT reagent 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide was added at a concentration of 0.2 mg/mL to each well and placed for incubation for 3 h. The reagent was removed and dimethyl suphoxide (DMSO) was added to dissolve the MTT formazan crystals. The absorbance was measured using automated microplate reader at 570 nm. Toxicity of PEI 10 kDa at a concentration of 25, 50, and 100 g/mL per well was also done in triplicate. Cell viability was expressed as the mean percentage of sample absorbance relative to untreated cells: Cell viability =
absorbance of sample × 100. absorbance of control
2.10. Cellular uptake studies Nanoplexes with PPEI 10k was formed at ratio 1:5 with ctDNA while a ratio of 1:4 was taken in case of PPBs. Polymers with PEI1.2k and 2k was taken at a ratio of 1:14. C6 glioma cells were incubated with nanoplexes with YOYO tagged ctDNA for 3 h at 37 ◦ C in DMEM/Ham’s F12:MEM (1:1) medium with 10% FBS. Nuclear staining was performed using Hoechst 33342 by incubating for 30 min. Later cells were washed with phosphate buffered saline and fixed with 1% formaldehyde. Cellular uptake was visualized and the image was captured using fluorescence microscope (Leica DM IRB, Germany). Flow cytometry analysis was done to quantify the cellular internalization using BD FACS ARIA.
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2.11. Polymer localization studies PPB 10k (30) was tagged using amine reactive labeling reagent, TRITC (tetramethylrhodamine isothiocyanate). Cellular uptake at a ratio of 1:4 was done with the polymer tagged with TRITC as mentioned earlier. Polymer trafficking was visualized and photographed using confocal microscope (Nikon AIR).
2.12. In vitro transfection assay (live dead assay) C6 cells were seeded into 4 well plates and were incubated overnight under 5% CO2 at 37 ◦ C. The cells were then incubated with nanoplexes formed with p53 plasmid (nanoplexes were formed at 1:5 ratio was used for PPEI 10k and 1:4 for PPBs) for 4 h after which the medium was replaced with fresh medium and the cells were then incubated for an additional 24 h. The cells were washed thrice with phosphate buffered saline. The Live and Dead assay was performed using the kit protocol. The cells were then visualized and photographed using fluorescence microscope (Leica DM IRB, Germany). Cell death was quantified by flow cytometry analysis by BD FACS ARIA.
3. Results and Discussion Many hurdles have to be overcome by a nonviral gene delivery system to impart its role. Carriers have to evade immune system, internalized by cells, escape from lysosomal degradation and be released from endosome, traverse viscous cytoplasm, avoid nucleases and should facilitate gene expression [27].
3.1. Preparation and characterization of polymer Pullulan was cationized with three different molecular weight PEI. The obtained polymers were named as PPEI 1.2k, PPEI 2k, PPEI 10k with the numeric representing the molecular weight of PEI used. Each PPEI was then conjugated with betaine at varying concentration to obtain PPB (Supplementary—Scheme 1). The weight by weight ratio of PPEI to betaine was 100:30, 100:60, and 100:100. Based on the amount of betaine used and molecular weight of PEI used, second conjugates were named as PPB 1.2k (30), PPB 2k (30), PPB 2k (100), PPB 10K (30), PPB 10k (60), PPB 10k (100) (Fig. 1). Fig. 1 Representation of 1 H NMR of pullulan (A), PPEI (B) and PPB (C). The conjugations were established by 1 H NMR spectroscopy. 1 H NMR spectrum representatives of pullulan, PPEI, and betaine conjugated to PPEI was shown (Fig. 1). The characteristic peaks of hydroxyl group attributed to pullulan lies between ␦ 4.46 and ␦ 5.56 was found in 1 H NMR spectrum of pullulan. The peaks from ␦ 2.5 to ␦ 2.7 are characteristics of PEI attributed to amines. The obtained results were similar to that reported earlier [6]. The reduced peak intensity at hydroxyl groups in comparison between A and B indicates conjugation of PEI to pullulan while peak intensity change in B and C indicated conjugation between PPEI and betaine. Since polymers with PEI of low molecular weight were incompetent most of the data shown are that of polymers with PEI 10 kDa unless otherwise mentioned.
3.2. Determination of betaine Amount of betaine present in the PPB conjugates were determined quantitatively and is given in Supplementary Table 1. PPBs of PEI 10 kDa were found to have high amount of betaine compared to low molecular weight PPBs.
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Fig. 1.
1
H NMR spectra of pullulan (A), PPEI (B) and PPB (C).
3.3. Size and zeta potential measurements To investigate the physical properties of polymer, the size and zeta potential of complex of polymer and calf thymus DNA were studied. The size of the nanoplexes was found to be between 97 nm to 222 nm (Supplementary Table 2). Nanoplexes made from PPEI and PPBs of PEI 10 kDa gave smaller size with better polydispersity index (PDI). The nanoplex size for PPB from PEI 2k had lower size when compared with that of PPB from PEI 1.2k. The zeta potential of the nanoplexes varied from −13.6 to +14. 5 mV (Supplementary Table 2). Better size and zeta potential was found in nanoplexes of polymer developed with PEI 10 kDa. A slight increase in size was found with PPBs of PEI 10 kDA on comparison with that PPEI 10k. Conjugation of betaine to the PPEI 10k reduced its zeta potential. However, size and zeta potential of PPB 10k (30), PPB 10k (60), and PPB 10k (100) were found to be significantly similar to each other. The crucial step in development of nonviral gene-delivery vector is the ability of the polymer to package DNA strand. The polyplex formed by polymer and DNA is by electrostatic interaction between negatively charged phosphate backbone of the DNA and positively charged amine groups of polymer [28]. The overall charge of the polyplex plays a key role in internalization of the polyplex into cell. The negatively charged heparin sulfate proteoglycan (HsPGs) interacts with the positively charged polyplexes enabling the internalization [4]. It is already established that in a polyplex with PEI, the amount of DNA involved in condensation is lower than that of the polymer. The interaction between PEI and DNA may not cause neutralization of charge. The localized bending and distortion of DNA facilitates the DNA condensation [29]. The positive charge of the complex may be explained by the presence of polymer molecules on the surface [30].
3.4. Buffering capacity The transfection efficiency of polymers has been found to be related to the buffering capacity of polymer. The internalized polymers have to escape from endosome before being degraded by lysosomes. Therefore a polymer should possess enough buffering capacity to have timely endosomal escape and prevent lysosomal
degradation. The buffering capacity of polymers prepared in the study was investigated (Supplementary Figure 1). The high density of protonable primary, secondary, and tertiary amines gives the buffering capacity [31]. PPEI 10k, PPB 10k (30), PPB 10k (60), and PPB 10k (100) exhibited better buffering capacities than others. Amongst the PPEI 10k and its PPBs slight increase in buffering capacity was found with the amount of betaine.
3.5. Agarose gel electrophoresis Negatively charged plasma proteins in blood may interact with polyplexes which may lead to reduced polymer/DNA association. So the effect of plasma on nanoplexes was checked. Both intracellular and extracellular nucleases pose another threat to the stability of nanoplexes. Hence the stability of polyplexes was checked in presence of DNase 1. The ability of polymers to condense DNA was assessed by gel retardation assay. The conjugates of low molecular weight PEI displayed very low DNA condensation ability. The ability to retard DNA increased with increasing amounts of polymer. In case of polymers with low molecular weight the DNA leached out from the nanoplexes at lower ratio (Data not shown). PPEI and PPBs of PEI 10 kDa has shown excellent DNA condensation ability (Supplementary Figure 2). In case of polymers involving PEI 10 kDa, uncomplexed DNA leeched out at ratio 0.5:1, slight leaching was present at ratio 3:1 also. But at ratio 5:1 the band was absent probably because of the absence of uncomplexed DNA for EtBr to intercalate. On comparison with intensity observed with DNA alone the fluorescence exhibited in lanes representing ratios 0.5, 3, and 5 was significantly low indicating reduced availability of bases available for EtBr intercalation. The results were found to be similar to absence of fluorescent band reported by Singh et al. [32]. Hence it may be understood that the whole DNA was complexed and the condensation ability of PPEI and PPBs of PEI 10 kDa was excellent. Interaction with plasma proteins is imperative during in vivo transfection. Hence stability of DNA/polymer nanoplexes was checked by incubating them with plasma protein. Stability of polymer/DNA nanoplexes in presence of plasma was studied at ratio 0.5:1, 3:1, and 5:1. It could be noticed that the plasma proteins have
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3.7. Blood compatibility studies
Fig. 2. Cytotoxicity of PEI 10 kDa, PPEI 10k, PPB 10k (30), PPB 10k (60), and PPB 10k (100) were studied on C6 cell lines by MTT assay. The quantity of polymers used for study included 25, 50, and 100 g/mL.
affected the DNA condensation ability of polymer at ratio 0.5:1 and 3:1. Band observed at ratio 5:1 was similar to that observed in the absence of serum. This indicates that the polymer/DNA nanocomplexes at higher ratio were stable even in the presence of serum. One of the main barriers to efficient gene delivery is the intracellular nuclease activity of DNase (lysosomal endonuclease) and extracellular serum nucleases. In polyplex formed at ratio 5:1, DNA was found to be protected against enzymatic digestion while at lower ratios of 0.5:1 and 3:1, the DNA was found to be degraded, resembling the lane with naked DNA and DNase alone (Supplementary Figure 2). Thus it was established that the polymers prepared were able to protect the DNA from DNase.
3.6. Polyacrylamide gel electrophoresis Plasma proteins play a key role in clearance of polymers from blood during in vivo transfection. Binding of plasma proteins is the mechanism by which reticulo endothelial system recognizes nanoparticles [33]. Once recognized, these nanoparticles are rapidly cleared from the blood stream and thus unable to reach target cells. Hence association with plasma proteins has to be reduced to improve the chance of nanoparticles to reach the target cells. Plasma protein interaction with polymers was studied by PAGE (Supplementary Figure 3). On comparison it was noted that PPBs showed less interactions with protein than PPEI. This was detected by presence of bands that were absent or less pronounced in PPEI (lane PPEI). The ability of betaine to resist non specific protein interaction might be the reason for such observation. Betaine has antifouling property and resists the non specific protein binding which subsequently reduces the inflammatory responses and immunological responses [34]. Hydration of non fouling moieties is one of the proposed mechanisms of this property. The bound water molecules form a physical and energy barrier for the protein to penetrate and adsorb. Trimethylglycine or betaine was found to have contiguous hydration shell across the molecule [35,36]. Hydration layers formed by hydroxyl groups of pullulan and trimethyl ammonion group of betaine might be the reason for less non specific protein interactions. Substantial reduction in non specific binding of protiens with pullulan PEI conjugate is reported earlier by Rekha. et al. [6]. Hence, it may be elucidated that this cationized pullulan has low affinity to blood proteins unlike PEI.
As mentioned earlier, positive charge facilitates the cellular internalization. Yet, the same positive charge may attract non specific and undesirable interactions between polyplex and blood cells. Blood is a complex tissue comprising plasma and cells. Erythrocytes (RBC) are the most rigid ones but are prone to rupture and undergo hemolysis. Impact of polymer with RBC can be extrapolated to that of with other cells since RBC have typical mammalian cell membrane structure. Blood platelets are less abundant than RBC and maintain the vascular hemostasis. Platelets provide vascular integrity sealing off any blood leaks. These platelets are the primary factor leading to thrombosis which forms leading obstacle in use of any biomaterials. Leukocytes (WBC) may also be activated attracting other components of blood. Hemocompatibility assessment of any biomaterials thereby includes hemolysis assay, aggregation studies, and C3 activation assay. Hemolytic activity of any biomaterials relates to its toxicity and relates with inhibition of cell growth. Hemolysis induced by all the polymers was assessed and was found in the acceptable range of less than 1%. Aggregation studies were also carried out. The polymers were found to be compatible with all three blood cell fractions (Supplementary Figure 4, 5, 6). The blood compatibility might be because of pullulan along with betaine might have reduced the toxicity of amine groups of PEI. The pullulan in the polymers might have reduced the overall positive charge thereby rendering the hemocompatibility. 3.8. Cytotoxicity The internalization of polymers is often hindered by the cell membrane. Hence internalization of materials occurs by slight modification of cell membrane which protects the integrity of the cell which in turn leads to cytotoxicity. Balancing the disruption of integrity of cell membrane and cytotoxicity is one of crucial steps in gene delivery. The cytotoxicity of nanoparticles prepared in the study was investigated (Fig. 2). The results revealed that in comparison to PEI 10 kDa all polymers were nontoxic. Cytotoxicity of PPEI 10k and PPBs 10k was found to be lower than that of PEI 10 kDa. In polyplexes with PEI, some PEI remains in free form, which may be the reason for cytotoxicity. PEI binding as huge clusters on the outer surface of the cell membrane may be the reason for toxicity [37–39]. These clusters may alter the cell membrane leading to cell death by necrosis. So a decrease in the free amine groups might reduce the harmful effect. The betaine in PPBs reduced the cytotoxicity when compared with PEI 10 kDa. This might be because the incorporation of pullulan and betaine might have reduced the cation density of PEI thereby reducing the damage to cell membrane. But cytotoxicity was found to be more with PPB 10k (60) and PPB 10k (100). The results were supported by the same effect observed with group in which cytotoxicity increased with increase in degree of betaine substitution [37]. 3.9. Cellular uptake studies The ability of nanoparticles to penetrate the plasma membrane is studied by cell uptake assay. It was found out that cellular uptake of polymer with low molecular weight was very poor (Supplementary figure 7). However, YOYO tagged DNA carried by PPEI 1.2k were able to reach nucleus even though cell uptake was very poor. At the same time betaine conjugate of the same were unable to reach nucleus. Uptake efficiency of PPB 1.2k (30) was better compared to that of PPEI 1.2k. PPEI 2k and PPB 2k (30) were similar to PPB 1.2 kDa as both were able to reach cytoplasm rather than nucleus. PPB 2k (100) showed no cellular internalization capability. Though zeta potentials of PPEI 1.2k, PPB1.2k (30), PPEI
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Fig. 3. PPEI 10k, PPB 10k (30), PPB 10k (60), and PPB 10k (100) internalization by C6 glioma cells are shown in panel A, B, C, and D. The YOYO tagged DNA was internalized (green fluorescence) with all the polymers.
2k and PPB 2k (30) were negative, they managed to be internalized despite the unfavorable interaction between negatively charged cell surface. This might be facilitated by cationic sites, though less in number, in the plasma membrane [40]. It is reported
that negatively charged particles are internalized by non specific binding to the limited cationic sites on the plasma membrane [40]. As low molecular weight conjugate showed lesser cellular internalization ability, focus was given to high molecular weight
Fig. 4. Confocal and confocal/DIC combined micrographs of TRITC labeled PPB30 nanoplexes with YOYO tagged ctDNA. The nanoplexes were formed in the ratio of 1:4. Panels A, B, and C depicts Hoechst 33342 nuclear staining, YOYO tagged ctDNA, TRITC labeled polymer in the cells, respectively. The lower D shows merged image of all three stained cells. Panel F shows overlaid image of differential interference contrast (DIC) micrograph E and D. The magnification is 60×.
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Fig. 5. Transfection capability of PPEI 10k, PPB 10k (30), PPB 10k (60), and PPB 10k (100) are represented in panel A to D, respectively.
conjugates (Fig. 3). PPEI 10k showed excellent internalization and all the PPB 10k polymers also showed good internalization. Polymers with PEI 10 kDa had better charge density compared to that of polymers with low molecular weight PEI. This might have resulted in improved stability of polymers and thus favored interaction with cell membrane facilitating internalization. Cellular internalization of the nanoplexes were quantified by flow cytometry. More than 90% internalization was found with all polymers (Supplementary Figure 8). The percentage of internalization was in order of PPEI 10k > PPB 10k (30) > PPB 10k (60) >PPB 10k (100).
buffering capacity exhibited by PPEIs and PPBs of low molecular weights might have failed them from escaping from endosome. As per proton sponge theory, PEI was able to counteract endosome acidification thereby delaying the lysosomal degradation [41]. It is reported that endosomal membrane rupture with decrease in pH may happen only when a fraction of free polymer is present which means that a range of uncomplexed polymer is essential for successful endosomal escape [42]. Based on above results it was concluded that PPB 10k polymers as efficient and hemcocompatible gene-delivery vector.
3.10. Polymer localization studies
4. Conclusion
The study was also extended to learn how far polymer is transported within the cell. Only PPB 10k (30) was used for this phase of study (Fig. 4). It can be clearly seen that TRITC tagged polymer remained in the cytoplasm and near cell membrane. The DNA was found inside the nucleus. The interaction of polymer with endosomal membrane and cytoplasmic components might have reduced the electrostatic interaction between the polymer and pDNA causing the genetic material to be dissociated from polymer.
The high transfection efficiency of any cationic polymers is impaired by its equally high cytotoxicity. The present study was able to develop a vector with pullulan which is hemocompatible and nontoxic while ensuring remarkable transfection efficiency. Conjugation of pullulan and betaine to PEI has considerably reduced the buffering capacity yet it was sufficient enough for endosomal escape and translocation of pDNA to nucleus. The high molecular weight polymers were found to be superior to low molecular weight polymers. Amongst high molecular weight polymers low betaine conjugated polymer was found to be superior. Conjugation of betaine to PPEI 10k found to increase the buffering capacity but produced low toxicity. Hence it may be concluded that betaine in right amount when used provides additional benefits to pullulan cationized with PEI.
3.11. In vitro transfection studies Transfection efficiency of polymers were studied by delivering the gene encoding the wild type p53 (pCMV-p53 Vector) as nanoplexes with the polymers. It was clear that polymers of lower molecular weight PEI exhibited very poor transfection efficiency. No cell death was observed (Data not shown). On the other hand, nanoplexes developed from polymeric conjugates of PEI 10 kDa exhibited excellent transfection efficiency (Fig. 5). The transfection efficiency was quantified by flow cytometry and found that more than 90% of the cells were dead (Supplementary Figure 9). The buffering capacity enables PEI or PEI conjugated polymers to act as proton sponge and hence escape from endosomes. Low
Acknowledgement We express our sincere gratitude to The Director, SCTIMST and The Head, BMT Wing for the facilities provided. Authors are thankful to the financial support from Bio-CARe, DBT, New Delhi. Dr. Lissy K Krishnan is acknowledged for providing the facility for flow cytometry analysis. The authors acknowledge the support by
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NIIST, Thiruvananthapuram for NMR analysis and Zeta determination. The support by RGCB, Thiruvananthapuram for Confocal Microscopy is also acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2014. 09.043. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
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[17] V. Buttun, A.B. Lowe, N.C. Billingham, S.P. Armes, J. Am. Chem. Soc. 121 (1999) 4288–4289. [18] Z. Cao, L. Zhang, S. Jiang, Langmuir 28 (2012) 11625–11632. [19] J. Ladd, Z. Zhang, S. Chen, J.C. Hower, S. Jiang, Biomacromolecules 9 (2008) 1357–1361. [20] D.A. Barrett, M.S. Hartshorne, M.A. Hussain, P.N. Shaw, M.C. Davies, Anal. Chem. 73 (2001) 5232–5239. [21] W.K. Cho, B. Kong, I.S. Choi, Langmuir 23 (2007) 5678. [22] K.M. Xiu, N.N. Zhao, W.T. Yang, F.J. Xu, Acta Biomater. 9 (2013) 7439–7448. [23] J. Sun, F. Zeng, H. Jiana, S. Wu, Polym Chem. 4 (2013) 5810. [24] V. Floch, N. Legros, S. Loisel, C. Guillaume, J. Guilbot, T. Benvegnu, V. Ferrieres, D. Plusquellec, C. Ferec, Biochem. Biophys. Res. Commun. 251 (1998) 360–365. [25] F. Dai, W. Liu, Biomaterials 32 (2011) 628. [26] C.M. Greive, S.R. Grattan, Plant Soil. 70 (1983) 303–307. [27] D.J. Glover, L. Glouchkova, H.J. Lipps, D.A. Jans, Mol. Cell Therapy 1 (2007) 126. [28] C. Volcke, S. Pirotton, Ch. Grandfils, C. Humbert, P.A. Thiry, I. Ydens, P. Dubois, M. Raes, J. Biotechnol. 125 (2006) 11–21. [29] V.A. Bloomfield, Biopolymers 31 (1991) 1471–1481. [30] W.T. Godbey, K.K. Wu, G.J. Hirasaki, A.G. Mikos, Gene Ther. 6 (1999) 1380–1388. [31] W.T. Godbey, K.K. Wu, A.G. Mikos, J Control. Release 60 (1999) 149. [32] R. Singh, D. Pantarotto, D. Mccarthy, O. Chaloin, J. Hoebeke, C.D. Partidos, J.P. Briand, M. Prato, A. Bianco, K. Kostarelos, J. Am. Chem. Soc. 127 (2005) 4388. [33] S.-D. Li, L. Huang, J Control Release 145 (2010) 178–181. [34] A. White, S. Jiang, J. Phys. Chem. B. 115 (2011) 660–667. [35] S. Chen, L. Li, C. Zhao, J. Zheng, Polymers 51 (2010) 5283–5293. [36] D. Fischer, T. Bieber, Y. Li, H.P. Elsasser, T. Kissel, Pharm. Res. 16 (1999) 1273–1279. [37] Y. Gao, Z. Zhang, L. Chen, W. Gu, Y. Li, Int. J. Pharm. 371 (2009) 156–162. [38] W.T. Godbey, A.G. Mikos, J Control Release 72 (2001) 115–125. [39] W.T. Godbey, K.K. Wu, A.G. Mikos, Biomaterials 22 (2001) 471–480. [40] A. Verma, F. Stellacci, Small 6 (2010) 12. [41] S. Yang, S. May, J. Chem. Phys. 129 (2008) 185105. [42] N.D. Sonawane, F.C. Szoka Jr., A.S. Verkman, J. Biol.Chem. 278 (2003) 44826–44831.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Betaine conjugated cationic pullulan as effective gene carrier Lizebona August Ambattu, M.R. Rekha ∗ Biosurface Technology Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India
a r t i c l e
i n f o
Article history: Received 4 June 2014 Received in revised form 30 August 2014 Accepted 22 September 2014 Available online 7 October 2014 Keywords: Pullulan Betaine Hemocompatible Gene delivery p53
a b s t r a c t Polyethyleneimne (PEI) is a very efficient transfecting agent but is toxic due to high charge density. To generate a vector which is efficient and less cytotoxic, PEI was conjugated with pullulan (PPEI). Further conjugation was done on PPEI with zwitter ionic betaine which possess antifouling property. PEI of molecular weight 1.2, 2, and 10 kDa were used in the study. Buffering capacity of pullulan-PEI-betaine (PPB) conjugates was found to be sufficient enough for the polymers to make endosomal escape. The polymers proved to be less cytotoxic and highly hemocompatible than PEI. Nuclear localization of YOYO tagged DNA was observed with the nanoplexes developed using PPEI and PPBs of PEI 10 kDa. Transfection efficiency was evaluated using p53 expressing gene and the live dead assay demonstrated very high transfection efficiency with PPB conjugates of PEI 10 kDa. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy is considered as solution for every inherited or acquired disease known. Even after first clinical trial for severe combined immunodeficiency (SCID), progress in gene delivery has not been as growing as expected. The rate limiting step in successful gene therapy is preparation of efficient gene-delivery vector. Viral vectors are lately replaced by nonviral vectors owing to low immunogenicity and mutagenicity of nonviral vectors [1–3]. For a nonviral vector to be efficient in delivering its cargo, it should be able to escape extracellular nuclease activity, evade reticuloendothelial system, avoid non specific interaction with blood cells thereby avoiding immune response and inflammatory reaction and has to reach target cells. At the target cells, the nanoparticle has to be internalized by cell membrane, escape from endosome, and traverse through viscous cytoplasm and finally therapeutic gene has to be expressed [4]. Pullulan is a polymer of maltotriose unit, connected by an ␣ (1 → 4) glycosidic bond, where consecutive maltotriose units are connected to each other by an ␣ (1 → 6) glycosidic bond. Hydrophobized pullulan is reported as drug-delivery carriers. Biocompatible and nontoxic nature of pullulan has been exploited to produce gene-delivery vectors [5,6]. Liver targeting with pullulan is already exploited [7–9]. Cationized pullulan has been reported as genedelivery vector [10].
∗ Corresponding author.Tel.: O: +91 471 2520214. E-mail address: [email protected] (M.R. Rekha). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.043 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Polyethyleneimine (PEI) has been considered as potent transfection agent. The high positive charge due to protonable amine groups facilitates ionic interaction with nucleic acids. Primary amine being basic and most reactive has been modified to change the properties of PEI [11]. PEI also has another remarkable property among organic polymers. PEI forms amino ethylene unit when fully protonated which provides one of the highest possible charge density. So when partially protonated PEI on further protonation in endosomes causes membrane destabilization. This membrane destabilization may be the cause of high toxicity of PEI [12,13]. A new conjugate with the benefits of PEI but with reduced toxic effect were developed and reported from this lab earlier. The pullulan was cationized with PEI which was found to be hemocompatible and an excellent candidate for gene delivery to cells especially to liver cells [6]. Betaine, also known as trimethylglycine, is a zwitterionic highly polar naturally occurring molecule. One of the major role of betaine as an osmolyte is maintaining the cell volume [14]. Accumulation of betaine in cells leads to increase in volume of cytoplasmic water by 20–50% per unit of dry weight of cell [15]. It is proposed that the alteration in cell volume slows down rate of cell proliferation [16]. As mentioned earlier, highly viscous cytoplasm also poses a hurdle to successful gene delivery. Hence, the introduction of betaine thereby causes a decrease in cytoplasmic viscosity. Nonviral vectors of cationic polymers facilitated with zwitterionic compounds were developed by various groups. Zwitterionic polymers were found to be highly resistant to non specific protein adsorption owing to the electrostatically induced hydration. These polymers are known to reduce serum protein interactions [17–19]. Antifouling properties of zwitterionic polymers are also known
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[20,21]. Of the zwitterionic polymers studied those with betaine were in focus recently [22–25]. Several betaine-based polymers are reported which showed good nucleic acid condensation capability and better transfection efficiency in serum compared to PEI. Lower protein adsorption and reduced cytotoxicity are yet other properties favoring successful nonviral gene-delivery vector. Even though the transfection efficiency of PEI is high, its use as genetic delivery vector is limited by its high toxicity. So there is a need of a system which can easily be internalized by cell and escape endosomes and deliver its cargo with considerably low toxicity to the cells. The effect of pullulan conjugated to both low molecular weight and high molecular weight PEI were studied. It was also studied whether osmolyte like betaine when conjugated with PEI conjugated with pullulan can improve the transfection efficiency or compatibility.
2. Materials and Methods
2.4. Preparation of polyplexes and determination of size and zeta of polyplexes Polyplexes were prepared by vortexing varying concentration of polymer to a fixed concentration of calf thymus DNA (ctDNA). The w/w ratio of polymer to ctDNA was from 0.5:1 to 15:1. The size and zeta of the polyplexes were determined using dynamic light scattering measurement using Zetasizer Nano ZS (Malvern Instruments Ltd., UK) at a temperature of 25 ◦ C. 2.5. Determination of buffering capacity The buffering capacity of PEI of three different molecular weights, PPEIs and PPBs, saline and betaine were evaluated by acid base titration over a pH range of 10–4. The pH of the polymer solution (0.1 mg/mL in saline) was adjusted to 10 using 0.2 N NaOH then titrated against 0.01 N HCl, the pH being noted after each addition of a volume of 50 L of acid. A graph was plotted with pH against the volume of HCl.
2.1. Chemicals 2.6. Agarose gel electrophoresis Pullulan (Sigma–Aldrich), carbonyl diimidazole (CDI), polyethyleneimine (10 kDa, 1.2 kDa, 2 kDa, Polysciences), betaine (Polysciences), EDC,Agarose,Ethidium Bromide,), 3-(4,5-dimethylthialzol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium (DMEM), trypsin, ethylenediamine tetra acetic acid (EDTA), DNase I (Sigma–Aldrich Chemicals Co, USA.). Deoxyribonucleic acid sodium salt from calf thymus (ctDNA) (Worthington Biochemical Corp), YOYO iodide and Hoechst 33342 (Invitrogen). Fetal bovine serum (FBS) (GIBCO, USA). All other reagents were of analytical grade from Merck, India.
2.2. Preparation of polymers 2.2.1. Preparation of PPEI Pullulan in dimethylsulphoxide was activated using CDI at 37 ◦ C for 2 h. After the 2 h, PEI of desired molecular weight in 20 mM borax was added to the mixture and was constantly stirred at room temperature overnight. Later pullulan PEI conjugate (PPEI) was precipitated with acetone. Acetone washed PPEI precipitate is then dissolved in deionized water and dialyzed for 24 h with two changes to remove unreacted PEI.
2.2.2. Preparation Pullulan-PEI-Betaine (PPB) PPB was prepared by using EDC as cross linker between PPEI and betaine under constant stirring for overnight at room temperature. Unreacted materials are removed by overnight dialysis.
2.3. Characterization 2.3.1. 1 H NMR 1 H NMR spectra of pullulan and derivatives were measured in D2 O using a 500 MHz spectrometer (Bruker Avance NMR Spectrometer). ␦H (500 MHz; D2 O; Me4 Si)
2.3.2. Determination of Betaine The assay is based on the fact that betaine forms a periodite complex with iodide in acidic medium at low temperature [26]. Briefly, cold potassium iodide–iodine was allowed to react with PPB in presence of concentrated sulphuric acid. The pellet obtained was then dissolved in 1, 2- dichloromethane and absorbance were measured at 365 nm with dichloromethane as blank. Betaine of different concentration was used to get standard curve.
The condensation property of nanoparticles on ctDNA (calf thymus DNA) was assessed by agarose gel electrophoresis. Nanoplexes of desired ratios was prepared (0.5:1, 3:1, 5:1) with ctDNA. The stability of nanoplexes in the presence of plasma was also assessed by incubating nanoplexes with 20 L of plasma for 30 min. The ability of polymers to protect pDNA from endonuclease degradation was evaluated by treating the nanoplexes with DNAse enzyme. The polyplexes were incubated with DNase of concentration 853 U/mL at 37 ◦ C for 30 min. The reaction was stopped with termination buffer. Naked ctDNA with and without DNase 1 treatment were positive and negative control, respectively. Electrophoresis was carried out in 1× TAE buffer, staining with 2 L of 10 mg/mL ethidium bromide in a Bio Rad electrophoresis system (Bio-Rad laboratories, CA, USA) at 75 V for 45 min. The DNA bands were visualized and the gel was photographed using Image Analyzer (LAS 4000, Fuji). 2.7. Polymer–plasma protein interactions-PAGE studies Native PAGE analysis was performed to assess the interaction of polymer with plasma proteins. The polymer (1 mg/mgL) was incubated with 20 Lof plasma for 30 min. PEI interaction with plasma protein was marked as negative control and plasma as positive. After 30 min, the complexes were subjected to centrifugation at 8000 rpm for 10 min. Supernatant was collected and mixed with sample buffer at 2:1 ratio. Electrophoresis was carried out in a MiniPROTEAN II electrophoresis cell (Bio-RAD, CA, USA). Gel was later stained with 0.2% coomassie brilliant blue R 250 for 2 h and then destained using destaining buffer containing acetic acid, methanol, and distilled water in the ratio. The gel was then photographed using an image analyzer (LAS 4000, Fuji). 2.8. Blood compatibility studies Blood from the unmedicated healthy volunteer was collected in a tube containing 3.8% sodium citrate. Ratio of blood to anticoagulant was 9:1. 2.8.1. Hemolysis RBC devoid of plasma was diluted a ratio 1:9. A total of 100 L of RBC was then added to polymers taken at four different concentrations 25, 50, 75, and 100 g and incubated for 2 h at room temperature. The mixture was then centrifuged at 700 rpm for 10 min. Absorbance of 100 L from the supernatant made up to
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1 mL with normal saline was taken at 541 nm. RBC incubated with Triton X-100 and saline were taken as positive and negative controls. 2.8.2. RBC Aggregation Polymers at two different concentrations 50 and 100 g was incubated with 100 L of RBC (diluted at ratio 1:8) at room temperature for 30 min. Cells treated with PEI and normal saline were taken as positive and negative controls, respectively. Images were captured using LeicaDMI3000 B (Lexica, Germany). Blood with anticoagulant overlaid on Histopaque was centrifuged at 800 rpm for 20 min to separate WBC and platelet. 2.8.3. WBC Aggregation A total of 50 and 100 g of the polymers incubated with 100 L of WBC for 20 min. PEI and plasma incubated with WBC were taken as positive and negative control, respectively. Presence of aggregation if any was detected through LeicaDMI3000 B (Leica, Germany). 2.8.4. Platelet Aggregation A total of 100 L platelet was incubated with 50 and 100 g of polymer for 30 min. Platelet incubated with PEI and saline were the positive and negative controls, respectively. Presence of aggregation if any was detected through LeicaDMI3000 B (Lexica, Germany). 2.9. Cytotoxicity Toxicity of the PPEIs and PPBs were assessed using MTT assay on C6 glioma cells. The cells were trypsinized using 0.25% trypsinEDTA. Cells were seeded at a density of 1 × 103 cells/well on a 96 well plate and incubated for 24 h in CO2 incubated at 37 ◦ C under 5% CO2 atmosphere. After 24 h, the medium was removed and polymers were added at a concentration of 25, 50, 100 g/mL per well along with positive and negative controls and incubated again for 24 h at 37 ◦ C in 5% CO2 atmosphere. After incubation, the samples were removed and MTT reagent 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide was added at a concentration of 0.2 mg/mL to each well and placed for incubation for 3 h. The reagent was removed and dimethyl suphoxide (DMSO) was added to dissolve the MTT formazan crystals. The absorbance was measured using automated microplate reader at 570 nm. Toxicity of PEI 10 kDa at a concentration of 25, 50, and 100 g/mL per well was also done in triplicate. Cell viability was expressed as the mean percentage of sample absorbance relative to untreated cells: Cell viability =
absorbance of sample × 100. absorbance of control
2.10. Cellular uptake studies Nanoplexes with PPEI 10k was formed at ratio 1:5 with ctDNA while a ratio of 1:4 was taken in case of PPBs. Polymers with PEI1.2k and 2k was taken at a ratio of 1:14. C6 glioma cells were incubated with nanoplexes with YOYO tagged ctDNA for 3 h at 37 ◦ C in DMEM/Ham’s F12:MEM (1:1) medium with 10% FBS. Nuclear staining was performed using Hoechst 33342 by incubating for 30 min. Later cells were washed with phosphate buffered saline and fixed with 1% formaldehyde. Cellular uptake was visualized and the image was captured using fluorescence microscope (Leica DM IRB, Germany). Flow cytometry analysis was done to quantify the cellular internalization using BD FACS ARIA.
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2.11. Polymer localization studies PPB 10k (30) was tagged using amine reactive labeling reagent, TRITC (tetramethylrhodamine isothiocyanate). Cellular uptake at a ratio of 1:4 was done with the polymer tagged with TRITC as mentioned earlier. Polymer trafficking was visualized and photographed using confocal microscope (Nikon AIR).
2.12. In vitro transfection assay (live dead assay) C6 cells were seeded into 4 well plates and were incubated overnight under 5% CO2 at 37 ◦ C. The cells were then incubated with nanoplexes formed with p53 plasmid (nanoplexes were formed at 1:5 ratio was used for PPEI 10k and 1:4 for PPBs) for 4 h after which the medium was replaced with fresh medium and the cells were then incubated for an additional 24 h. The cells were washed thrice with phosphate buffered saline. The Live and Dead assay was performed using the kit protocol. The cells were then visualized and photographed using fluorescence microscope (Leica DM IRB, Germany). Cell death was quantified by flow cytometry analysis by BD FACS ARIA.
3. Results and Discussion Many hurdles have to be overcome by a nonviral gene delivery system to impart its role. Carriers have to evade immune system, internalized by cells, escape from lysosomal degradation and be released from endosome, traverse viscous cytoplasm, avoid nucleases and should facilitate gene expression [27].
3.1. Preparation and characterization of polymer Pullulan was cationized with three different molecular weight PEI. The obtained polymers were named as PPEI 1.2k, PPEI 2k, PPEI 10k with the numeric representing the molecular weight of PEI used. Each PPEI was then conjugated with betaine at varying concentration to obtain PPB (Supplementary—Scheme 1). The weight by weight ratio of PPEI to betaine was 100:30, 100:60, and 100:100. Based on the amount of betaine used and molecular weight of PEI used, second conjugates were named as PPB 1.2k (30), PPB 2k (30), PPB 2k (100), PPB 10K (30), PPB 10k (60), PPB 10k (100) (Fig. 1). Fig. 1 Representation of 1 H NMR of pullulan (A), PPEI (B) and PPB (C). The conjugations were established by 1 H NMR spectroscopy. 1 H NMR spectrum representatives of pullulan, PPEI, and betaine conjugated to PPEI was shown (Fig. 1). The characteristic peaks of hydroxyl group attributed to pullulan lies between ␦ 4.46 and ␦ 5.56 was found in 1 H NMR spectrum of pullulan. The peaks from ␦ 2.5 to ␦ 2.7 are characteristics of PEI attributed to amines. The obtained results were similar to that reported earlier [6]. The reduced peak intensity at hydroxyl groups in comparison between A and B indicates conjugation of PEI to pullulan while peak intensity change in B and C indicated conjugation between PPEI and betaine. Since polymers with PEI of low molecular weight were incompetent most of the data shown are that of polymers with PEI 10 kDa unless otherwise mentioned.
3.2. Determination of betaine Amount of betaine present in the PPB conjugates were determined quantitatively and is given in Supplementary Table 1. PPBs of PEI 10 kDa were found to have high amount of betaine compared to low molecular weight PPBs.
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Fig. 1.
1
H NMR spectra of pullulan (A), PPEI (B) and PPB (C).
3.3. Size and zeta potential measurements To investigate the physical properties of polymer, the size and zeta potential of complex of polymer and calf thymus DNA were studied. The size of the nanoplexes was found to be between 97 nm to 222 nm (Supplementary Table 2). Nanoplexes made from PPEI and PPBs of PEI 10 kDa gave smaller size with better polydispersity index (PDI). The nanoplex size for PPB from PEI 2k had lower size when compared with that of PPB from PEI 1.2k. The zeta potential of the nanoplexes varied from −13.6 to +14. 5 mV (Supplementary Table 2). Better size and zeta potential was found in nanoplexes of polymer developed with PEI 10 kDa. A slight increase in size was found with PPBs of PEI 10 kDA on comparison with that PPEI 10k. Conjugation of betaine to the PPEI 10k reduced its zeta potential. However, size and zeta potential of PPB 10k (30), PPB 10k (60), and PPB 10k (100) were found to be significantly similar to each other. The crucial step in development of nonviral gene-delivery vector is the ability of the polymer to package DNA strand. The polyplex formed by polymer and DNA is by electrostatic interaction between negatively charged phosphate backbone of the DNA and positively charged amine groups of polymer [28]. The overall charge of the polyplex plays a key role in internalization of the polyplex into cell. The negatively charged heparin sulfate proteoglycan (HsPGs) interacts with the positively charged polyplexes enabling the internalization [4]. It is already established that in a polyplex with PEI, the amount of DNA involved in condensation is lower than that of the polymer. The interaction between PEI and DNA may not cause neutralization of charge. The localized bending and distortion of DNA facilitates the DNA condensation [29]. The positive charge of the complex may be explained by the presence of polymer molecules on the surface [30].
3.4. Buffering capacity The transfection efficiency of polymers has been found to be related to the buffering capacity of polymer. The internalized polymers have to escape from endosome before being degraded by lysosomes. Therefore a polymer should possess enough buffering capacity to have timely endosomal escape and prevent lysosomal
degradation. The buffering capacity of polymers prepared in the study was investigated (Supplementary Figure 1). The high density of protonable primary, secondary, and tertiary amines gives the buffering capacity [31]. PPEI 10k, PPB 10k (30), PPB 10k (60), and PPB 10k (100) exhibited better buffering capacities than others. Amongst the PPEI 10k and its PPBs slight increase in buffering capacity was found with the amount of betaine.
3.5. Agarose gel electrophoresis Negatively charged plasma proteins in blood may interact with polyplexes which may lead to reduced polymer/DNA association. So the effect of plasma on nanoplexes was checked. Both intracellular and extracellular nucleases pose another threat to the stability of nanoplexes. Hence the stability of polyplexes was checked in presence of DNase 1. The ability of polymers to condense DNA was assessed by gel retardation assay. The conjugates of low molecular weight PEI displayed very low DNA condensation ability. The ability to retard DNA increased with increasing amounts of polymer. In case of polymers with low molecular weight the DNA leached out from the nanoplexes at lower ratio (Data not shown). PPEI and PPBs of PEI 10 kDa has shown excellent DNA condensation ability (Supplementary Figure 2). In case of polymers involving PEI 10 kDa, uncomplexed DNA leeched out at ratio 0.5:1, slight leaching was present at ratio 3:1 also. But at ratio 5:1 the band was absent probably because of the absence of uncomplexed DNA for EtBr to intercalate. On comparison with intensity observed with DNA alone the fluorescence exhibited in lanes representing ratios 0.5, 3, and 5 was significantly low indicating reduced availability of bases available for EtBr intercalation. The results were found to be similar to absence of fluorescent band reported by Singh et al. [32]. Hence it may be understood that the whole DNA was complexed and the condensation ability of PPEI and PPBs of PEI 10 kDa was excellent. Interaction with plasma proteins is imperative during in vivo transfection. Hence stability of DNA/polymer nanoplexes was checked by incubating them with plasma protein. Stability of polymer/DNA nanoplexes in presence of plasma was studied at ratio 0.5:1, 3:1, and 5:1. It could be noticed that the plasma proteins have
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3.7. Blood compatibility studies
Fig. 2. Cytotoxicity of PEI 10 kDa, PPEI 10k, PPB 10k (30), PPB 10k (60), and PPB 10k (100) were studied on C6 cell lines by MTT assay. The quantity of polymers used for study included 25, 50, and 100 g/mL.
affected the DNA condensation ability of polymer at ratio 0.5:1 and 3:1. Band observed at ratio 5:1 was similar to that observed in the absence of serum. This indicates that the polymer/DNA nanocomplexes at higher ratio were stable even in the presence of serum. One of the main barriers to efficient gene delivery is the intracellular nuclease activity of DNase (lysosomal endonuclease) and extracellular serum nucleases. In polyplex formed at ratio 5:1, DNA was found to be protected against enzymatic digestion while at lower ratios of 0.5:1 and 3:1, the DNA was found to be degraded, resembling the lane with naked DNA and DNase alone (Supplementary Figure 2). Thus it was established that the polymers prepared were able to protect the DNA from DNase.
3.6. Polyacrylamide gel electrophoresis Plasma proteins play a key role in clearance of polymers from blood during in vivo transfection. Binding of plasma proteins is the mechanism by which reticulo endothelial system recognizes nanoparticles [33]. Once recognized, these nanoparticles are rapidly cleared from the blood stream and thus unable to reach target cells. Hence association with plasma proteins has to be reduced to improve the chance of nanoparticles to reach the target cells. Plasma protein interaction with polymers was studied by PAGE (Supplementary Figure 3). On comparison it was noted that PPBs showed less interactions with protein than PPEI. This was detected by presence of bands that were absent or less pronounced in PPEI (lane PPEI). The ability of betaine to resist non specific protein interaction might be the reason for such observation. Betaine has antifouling property and resists the non specific protein binding which subsequently reduces the inflammatory responses and immunological responses [34]. Hydration of non fouling moieties is one of the proposed mechanisms of this property. The bound water molecules form a physical and energy barrier for the protein to penetrate and adsorb. Trimethylglycine or betaine was found to have contiguous hydration shell across the molecule [35,36]. Hydration layers formed by hydroxyl groups of pullulan and trimethyl ammonion group of betaine might be the reason for less non specific protein interactions. Substantial reduction in non specific binding of protiens with pullulan PEI conjugate is reported earlier by Rekha. et al. [6]. Hence, it may be elucidated that this cationized pullulan has low affinity to blood proteins unlike PEI.
As mentioned earlier, positive charge facilitates the cellular internalization. Yet, the same positive charge may attract non specific and undesirable interactions between polyplex and blood cells. Blood is a complex tissue comprising plasma and cells. Erythrocytes (RBC) are the most rigid ones but are prone to rupture and undergo hemolysis. Impact of polymer with RBC can be extrapolated to that of with other cells since RBC have typical mammalian cell membrane structure. Blood platelets are less abundant than RBC and maintain the vascular hemostasis. Platelets provide vascular integrity sealing off any blood leaks. These platelets are the primary factor leading to thrombosis which forms leading obstacle in use of any biomaterials. Leukocytes (WBC) may also be activated attracting other components of blood. Hemocompatibility assessment of any biomaterials thereby includes hemolysis assay, aggregation studies, and C3 activation assay. Hemolytic activity of any biomaterials relates to its toxicity and relates with inhibition of cell growth. Hemolysis induced by all the polymers was assessed and was found in the acceptable range of less than 1%. Aggregation studies were also carried out. The polymers were found to be compatible with all three blood cell fractions (Supplementary Figure 4, 5, 6). The blood compatibility might be because of pullulan along with betaine might have reduced the toxicity of amine groups of PEI. The pullulan in the polymers might have reduced the overall positive charge thereby rendering the hemocompatibility. 3.8. Cytotoxicity The internalization of polymers is often hindered by the cell membrane. Hence internalization of materials occurs by slight modification of cell membrane which protects the integrity of the cell which in turn leads to cytotoxicity. Balancing the disruption of integrity of cell membrane and cytotoxicity is one of crucial steps in gene delivery. The cytotoxicity of nanoparticles prepared in the study was investigated (Fig. 2). The results revealed that in comparison to PEI 10 kDa all polymers were nontoxic. Cytotoxicity of PPEI 10k and PPBs 10k was found to be lower than that of PEI 10 kDa. In polyplexes with PEI, some PEI remains in free form, which may be the reason for cytotoxicity. PEI binding as huge clusters on the outer surface of the cell membrane may be the reason for toxicity [37–39]. These clusters may alter the cell membrane leading to cell death by necrosis. So a decrease in the free amine groups might reduce the harmful effect. The betaine in PPBs reduced the cytotoxicity when compared with PEI 10 kDa. This might be because the incorporation of pullulan and betaine might have reduced the cation density of PEI thereby reducing the damage to cell membrane. But cytotoxicity was found to be more with PPB 10k (60) and PPB 10k (100). The results were supported by the same effect observed with group in which cytotoxicity increased with increase in degree of betaine substitution [37]. 3.9. Cellular uptake studies The ability of nanoparticles to penetrate the plasma membrane is studied by cell uptake assay. It was found out that cellular uptake of polymer with low molecular weight was very poor (Supplementary figure 7). However, YOYO tagged DNA carried by PPEI 1.2k were able to reach nucleus even though cell uptake was very poor. At the same time betaine conjugate of the same were unable to reach nucleus. Uptake efficiency of PPB 1.2k (30) was better compared to that of PPEI 1.2k. PPEI 2k and PPB 2k (30) were similar to PPB 1.2 kDa as both were able to reach cytoplasm rather than nucleus. PPB 2k (100) showed no cellular internalization capability. Though zeta potentials of PPEI 1.2k, PPB1.2k (30), PPEI
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Fig. 3. PPEI 10k, PPB 10k (30), PPB 10k (60), and PPB 10k (100) internalization by C6 glioma cells are shown in panel A, B, C, and D. The YOYO tagged DNA was internalized (green fluorescence) with all the polymers.
2k and PPB 2k (30) were negative, they managed to be internalized despite the unfavorable interaction between negatively charged cell surface. This might be facilitated by cationic sites, though less in number, in the plasma membrane [40]. It is reported
that negatively charged particles are internalized by non specific binding to the limited cationic sites on the plasma membrane [40]. As low molecular weight conjugate showed lesser cellular internalization ability, focus was given to high molecular weight
Fig. 4. Confocal and confocal/DIC combined micrographs of TRITC labeled PPB30 nanoplexes with YOYO tagged ctDNA. The nanoplexes were formed in the ratio of 1:4. Panels A, B, and C depicts Hoechst 33342 nuclear staining, YOYO tagged ctDNA, TRITC labeled polymer in the cells, respectively. The lower D shows merged image of all three stained cells. Panel F shows overlaid image of differential interference contrast (DIC) micrograph E and D. The magnification is 60×.
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Fig. 5. Transfection capability of PPEI 10k, PPB 10k (30), PPB 10k (60), and PPB 10k (100) are represented in panel A to D, respectively.
conjugates (Fig. 3). PPEI 10k showed excellent internalization and all the PPB 10k polymers also showed good internalization. Polymers with PEI 10 kDa had better charge density compared to that of polymers with low molecular weight PEI. This might have resulted in improved stability of polymers and thus favored interaction with cell membrane facilitating internalization. Cellular internalization of the nanoplexes were quantified by flow cytometry. More than 90% internalization was found with all polymers (Supplementary Figure 8). The percentage of internalization was in order of PPEI 10k > PPB 10k (30) > PPB 10k (60) >PPB 10k (100).
buffering capacity exhibited by PPEIs and PPBs of low molecular weights might have failed them from escaping from endosome. As per proton sponge theory, PEI was able to counteract endosome acidification thereby delaying the lysosomal degradation [41]. It is reported that endosomal membrane rupture with decrease in pH may happen only when a fraction of free polymer is present which means that a range of uncomplexed polymer is essential for successful endosomal escape [42]. Based on above results it was concluded that PPB 10k polymers as efficient and hemcocompatible gene-delivery vector.
3.10. Polymer localization studies
4. Conclusion
The study was also extended to learn how far polymer is transported within the cell. Only PPB 10k (30) was used for this phase of study (Fig. 4). It can be clearly seen that TRITC tagged polymer remained in the cytoplasm and near cell membrane. The DNA was found inside the nucleus. The interaction of polymer with endosomal membrane and cytoplasmic components might have reduced the electrostatic interaction between the polymer and pDNA causing the genetic material to be dissociated from polymer.
The high transfection efficiency of any cationic polymers is impaired by its equally high cytotoxicity. The present study was able to develop a vector with pullulan which is hemocompatible and nontoxic while ensuring remarkable transfection efficiency. Conjugation of pullulan and betaine to PEI has considerably reduced the buffering capacity yet it was sufficient enough for endosomal escape and translocation of pDNA to nucleus. The high molecular weight polymers were found to be superior to low molecular weight polymers. Amongst high molecular weight polymers low betaine conjugated polymer was found to be superior. Conjugation of betaine to PPEI 10k found to increase the buffering capacity but produced low toxicity. Hence it may be concluded that betaine in right amount when used provides additional benefits to pullulan cationized with PEI.
3.11. In vitro transfection studies Transfection efficiency of polymers were studied by delivering the gene encoding the wild type p53 (pCMV-p53 Vector) as nanoplexes with the polymers. It was clear that polymers of lower molecular weight PEI exhibited very poor transfection efficiency. No cell death was observed (Data not shown). On the other hand, nanoplexes developed from polymeric conjugates of PEI 10 kDa exhibited excellent transfection efficiency (Fig. 5). The transfection efficiency was quantified by flow cytometry and found that more than 90% of the cells were dead (Supplementary Figure 9). The buffering capacity enables PEI or PEI conjugated polymers to act as proton sponge and hence escape from endosomes. Low
Acknowledgement We express our sincere gratitude to The Director, SCTIMST and The Head, BMT Wing for the facilities provided. Authors are thankful to the financial support from Bio-CARe, DBT, New Delhi. Dr. Lissy K Krishnan is acknowledged for providing the facility for flow cytometry analysis. The authors acknowledge the support by
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NIIST, Thiruvananthapuram for NMR analysis and Zeta determination. The support by RGCB, Thiruvananthapuram for Confocal Microscopy is also acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2014. 09.043. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
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