Deciphering the Role of Chondroitin Sulfate in Increasing the

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Deciphering the role of chondroitin sulphate in increasing the transfection efficiency of amphipathic peptide based nanocomplexes Daniel Nisakar, Manika Vij, Tanuja Pandey, Poornemaa Natarajan, Rajpal Sharma, Sarita Mishra, and Munia Ganguli ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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ACS Biomaterials Science & Engineering

Deciphering the role of chondroitin sulphate in increasing the transfection efficiency of amphipathic peptide based nanocomplexes 1#

1,2#

Daniel Nisakar , Manika Vij 1,2* Munia Ganguli .

1

1

1

1,2

, Tanuja Pandey , Poornemaa Natarajan , Rajpal Sharma , Sarita Mishra ,

1

CSIR- Institute of Genomics and Integrative Biology, South Campus, Mathura Road, Opp: Sukhdev Vihar Bus Depot, New Delhi-110020, India. 2 Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi-110001, India.

Correspondence should be addressed to: *Dr. Munia Ganguli ([email protected], [email protected]) New Delhi, India. # Both authors have equal contribution Present Address: Room No. 225/ Lab No. 219, Department of Structural Biology, CSIR Institute of Genomics and Integrative Biology, South Campus, Mathura Road, Opp. Sukhdev Vihar Bus Depot, New Delhi- 110 025, India. Tel: 91-11-29879 225 / +91 9871607437; Fax: 001 27667471; Email: [email protected], [email protected]

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ABSTRACT Glycosaminoglycans, both cell-surface and exogenous, can interfere with DNA delivery efficiency of non-viral carrier systems. In this work, we report an extensive comparative study to explore the effect of exogenously added chondroitin sulphate on biophysical characteristics, cellular uptake, transfection efficiency and intracellular trafficking of nanocomplexes formed using primary and secondary amphipathic peptides developed in our laboratory. Our results indicate that the presence of exogenous chondroitin sulphate exhibits differential enhancement in transfection efficiency of the amphipathic peptides depending upon their chemical nature. The enhancement was more pronounced in primary amphipathic peptide-based nanocomplexes as compared to the secondary counterpart. This difference can be attributed to possible alteration of the intracellular entry pathway in addition to increased extracellular stability, less cellular toxicity and assistance in nuclear accumulation. These results imply potential use of glycosaminoglycans like chondroitin sulphate to improve the transfection efficiency of primary amphipathic peptides for possible in vivo applications.

KEYWORDS Cell penetrating peptides, Amphipathic peptides, Glycosaminoglycans, Chondroitin sulphate, Transfection efficiency, Intracellular trafficking


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INTRODUCTION Gene therapy is considered to be a potential approach towards treatment of various genetically inherited as well as acquired disorders. For successful implementation of gene therapy, it is imperative to select an efficient delivery method1. Till now, a large number of viral as well as non-viral delivery systems have been developed and tested in vitro and in vivo2,3. Of these, non-viral peptide based delivery systems possess several advantages such as easy synthesis, low toxicity and amenability to modifications for suitable cellular targeting. However, efficiency of most of these peptide-based systems during DNA delivery is impeded by extracellular barriers (involving nucleases and the immune system), difficulty in traversing the plasma membrane, endosomal entrapment as well as difficulty in nuclear entry4-6. In addition to these well-established barriers towards mobility of peptide-DNA nanocomplexes within the cells, there are several other factors in the extracellular milieu such as serum proteins which can also impede their subsequent cellular entry7. Glycosaminoglycans (GAGs) constitute one such class of naturally occurring extracellular molecules which can alter the delivery efficiency of cationic peptidebased gene delivery systems8-11. GAGs are linear, differentially sulphated, negatively charged polysaccharides that are ubiquitously present either on cell surface as proteoglycans or in extracellular milieu where they exist as soluble molecules12. Till now, there is conflicting evidence on the contribution of interaction between gene delivery carriers and GAGs in determining downstream cellular effects like cellular uptake and intracellular trafficking mechanisms. In case of positively charged nanocomplexes containing cationic peptides or lipids, there is evidence to indicate that they interact with cell-surface GAGs through electrostatic interactions whereby they either get entrapped at the cell surface or are destabilized leading to reduced cellular entry9,13. On the other hand, few reports also mention this interaction as a pre-requisite for successful internalization of these nanocomplexes11. In addition to cell-surface GAGs, exogenous GAGs have also been used to impart better extracellular stability to cationic nanocomplexes (particularly in serum rich environment), increase their overall cellular uptake through receptor mediated interactions and to reduce the associated cytotoxicity of carriers without altering their delivery efficiency13-15.



Amphipathic cell penetrating peptides have largely been used for delivery of biomolecules to multiple cell types; however, little work has been reported in literature to understand the contribution of GAGs in internalization of amphipathic peptide-DNA nanocomplexes. Recently, few reports demonstrated the ability of amphipathic peptides to interact with cell surface GAGs where overall GAG content on the cell surface determines the efficiency of cellular uptake16. Moreover, it has been shown that interaction of amphipathic peptides, bare or as a nanocomplex, with cell surface GAGs may lead to conformational changes in the peptide i.e. from random coil to alpha-helix, thereby providing better membrane traversing ability to the peptide. There could also be clustering of GAGs for rapid cellular entry of nanocomplexes1719

. However, effect of exogenous GAGs on biophysical characteristics, cellular internalization,

intracellular trafficking and cytotoxicity of amphipathic peptide-based nanocomplexes still remains elusive. In this manuscript, we have studied the effect of exogenous GAGs on size, surface charge, stability, cellular uptake, transfection efficiency, intracellular movement as well as cytotoxicity of nanocomplexes formed using two different amphipathic peptides Mgpe9, a secondary amphipathic peptide and Mgpe10, a primary amphipathic peptide. Our overall aim was to decipher whether exogenously added GAGs can enhance the transfection efficiency of amphipathic peptides and if the amphipathicity of the peptide is important. We rationalized that amphipathic peptides with different physico-chemical properties may differ in terms of their interaction with exogenous GAGs, hence we selected two peptides which have same chemical composition and differ only in the overall amphipathicity and compared the mechanisms involved in cellular uptake and intracellular trafficking upon interaction with GAGs. It has been shown in a previous study from our laboratory that Mgpe9 has superior DNA delivery efficiency as compared to Mgpe10 which exhibits low transfection and poor cell viability of the DNA nanocomplexes-particularly at high charge ratio 20,21. In addition to the general effect of GAGs on the gene delivery efficiency of these peptides, we also specifically wanted to check if the presence of exogenous GAGs can improve DNA transfer ability of Mgpe10.


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Materials and Methods Both

the

amphipathic

peptides,

Mgpe9

(CRRLRHLRHHYRRRWHRFRC)

and

Mgpe10

(CLLYWFRRRHRHHRRRHRRC), were custom synthesized by Beijing SBS Genetech Co., Ltd. (Beijing, China) with greater than 95% purity. The plasmids pEGFP-C1, 4.7 kb (Clontech) and pMIRREPORT™ Luciferase, 6.47 kb (Ambion) were amplified in E.coli DH5-α and purified using GenElute HP Endotoxin-Free Plasmid MaxiPrep Kit (Sigma). Cell viability assay kit CellTiter-Glo and Luciferase assay kit were obtained from Promega. Unlabeled Chondroitin sulphate A (CS) sodium salt (from bovine trachea) was purchased from Sigma. Labeled Chondroitin sulphate A (FITC-CS) was purchased from Cosmo Bio Co. Ltd., Japan. All other chemicals were obtained from Sigma unless stated otherwise.

Cell line used CHO-K1 (Chinese Hamster Ovary) cells were obtained from ATCC to perform all the cellular studies. It is a derivative cell line of CHO cells which is often used as transfection host and is less prone to transformation. Cells were maintained using Ham's F-12K medium with 10% (v/v) fetal bovine serum (Life Technologies, USA) supplement and kept at 37°C with 5% CO2 in a humidified incubator.

Preparation of Peptide-DNA nanocomplexes Peptide-DNA nanocomplexes were prepared in MilliQ water using Mgpe9 and Mgpe10 peptide at charge ratio 5, where charge ratio is the peptide nitrogen per nucleic acid phosphate. For selected experiments, nanocomplexes were also prepared at charge ratio 10. The plasmid DNA stock was diluted in MilliQ to a concentration of 20ng/µl (for Dynamic Light Scattering, Atomic Force Microscopy, gel studies, Ethidium Bromide release study) or 40ng/µl (for cellular uptake, transfection efficiency, cell viability studies) respectively and added drop wise to an equal volume of the appropriate peptide dilution while vortexing. These nanocomplexes were incubated for 30 minutes at room temperature followed by addition of CS at different weight/weight (wt/wt) ratio of GAG/peptide as per requirement. Nanocomplexes were then further incubated at room temperature for 30 minutes before performing any experiment.



Dynamic light scattering (DLS) The hydrodynamic diameter, zeta potential and polydispersity index of nanocomplexes prepared using Mgpe9 and Mgpe10 at charge ratio 5, with and without exogenous addition of CS at different CS:peptide wt/wt ratios of 0.25 and 1, were measured using Zetasizer Nano-ZS (Malvern Instruments) at a fixed angle of 90° at 25°C. At least 3 readings were recorded for each sample in duplicates (each reading had 15 sub measurements).

Agarose gel electrophoresis Agarose gel electrophoresis was used to check the stability of nanocomplexes in presence of increasing concentration of GAGs. 20µl nanocomplexes were prepared using Mgpe9 and Mgpe10 peptide containing 200ng of plasmid DNA at charge ratio 5. After 30 minutes of incubation at room temperature, increasing amounts of CS was added to these nanocomplexes followed by further incubation for 30 minutes before loading them on 1% agarose gel. Electrophoresis was carried out at 100V in Tris acetate-EDTA buffer for 30 minutes. The amount of the plasmid DNA released from the nanocomplexes was compared with same amount of bare plasmid DNA.

Ethidium Bromide (EtBr) Intercalation Assay Stability of nanocomplexes was determined using EtBr intercalation assay. CS in increasing amounts was added to 96-well black plate (Nunc) followed by addition of 40µl of nanocomplexes (prepared as described above) and 4.22µl of EtBr (10ng/µl) which was then incubated for 5 minutes at room temperature in the dark. Fluorescence intensity was measured in a DTX 880 Multimode detector (Beckman Coulter) using 535 SL EXP 1 excitation and 595 SL EMP 1 emission filters. The fluorescence of plasmid DNA with EtBr was taken as the maximum, i.e. 100%, and the relative percentage increase in fluorescence signal was calculated at increasing concentration of CS.

Nuclease Degradation Assay


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DNaseI was reconstituted in buffer containing 50mM Tris-HCl (pH 7.5), 1mM CaCl2, 10mM MgCl2 and 50% glycerol. Nanocomplexes were prepared as mentioned before with and without CS at CS:peptide wt/wt ratio of 0.25. The nanocomplexes were then treated with DNaseI (1U) for 30 minutes at 37ºC in reaction buffer consisting of 10mM TrisHCl, pH 7.5, 0.1mM CaCl2 and 2.5mM MgCl2. DNaseI was inactivated by heating at 75ºC for 10 minutes. To release the protected DNA from the nanocomplexes, heparin (5µg) was added to the above reaction mixture and was further incubated for 30 minutes at room temperature. Heparin was used to provide the anionic challenge to the nanocomplexes so that electrostatic interaction which holds the peptide and DNA together gets disturbed and DNA is released. This was followed by addition of 1% trypsin and another incubation for 30 minutes to degrade the left over peptide and avoid possibility of hydrophobic interactions thereby ensuring complete release of DNA. The mixture was then analyzed on 1% agarose gel. The integrity and amount of plasmid DNA protected was compared with plasmid DNA released from control nanocomplexes (after addition of heparin and trypsin) that were not subjected to DNaseI treatment.

Atomic Force Microscopy (AFM) Nanocomplexes were prepared using both the peptides independently at charge ratio 5 in presence and absence of exogenously added CS at CS:peptide wt/wt ratio of 0.25. 6µl of nanocomplexes was then deposited on mica surface and air-dried following which imaging was carried out with 5500 Scanning Probe Microscope (Agilent Technologies, Inc., AZ) using Picoview software 1.4.4. Images were taken in AAC mode in air with silicon cantilevers having resonance frequency of 75 kHz and force constant of 2.8N/m. Scan speed was 1 line/s. Minimum image processing was employed and image analysis was done using Picoview software 1.6.

Labeling of plasmid DNA using fluorophores Labeling of plasmid DNA with Rhodamine or FITC fluorescent dyes was performed using Label IT® Tracker Rhodamine or Fluorescein kit (Mirus Bio Corp.) respectively, at a ratio of 0.75:1 (v:w) i.e. 0.75



µl of labeling reagent/µg of DNA according to the manufacturer's protocol.

Cellular entry of CS coated and uncoated nanocomplexes CHO-K1 cells were seeded at a density of 1.2x105 in 35mm µ-dishes (ibidi, Germany) and incubated for 24 hours. To visualize cellular entry of nanocomplexes in presence and absence of CS, nanocomplexes formed using rhodamine labeled plasmid DNA and unlabeled peptide, coated with FITC labeled CS at CS:peptide wt/wt ratio of 0.25,were added to the cells in serum-free media and incubated at 37°C for 4 hours. For assessing nuclear accumulation and lysosomal co-localization, labeled nanocomplexes (formed using FITC labeled plasmid DNA and unlabeled peptide) coated with unlabeled CS were added to the cells in serum-free media and incubated at 37°C for 4 hours in similar manner. Following treatments, cells were washed thrice with ice cold phosphate buffered saline (1X PBS) containing heparin (1mg/ml). For lysosomal co-localization studies, recommended concentration of 50nM of Lysotracker Deep Red dye was added to the cells during last one hour incubation. For nuclear staining, cells were treated with 2µg/ml of DAPI solution and incubated for 2 minutes at 37°C. The entry of the nanocomplexes was visualized in live cells using confocal microscopy (TCS SP8 from Leica) and z-stack images were collected at intervals of 3-5µm to ensure nuclear accumulation of nanocomplexes. Images were combined to form a 3D image using projection function.

Cellular uptake of CS coated and uncoated nanocomplexes The cellular uptake studies were carried out using nanocomplexes made of FITC labeled plasmid DNA and unlabeled peptide at charge ratio 5, either uncoated or coated with unlabeled CS at CS:peptide wt/wt ratio of 0.25, using flow cytometry. Plasmid DNA was labeled with FITC using Label IT® Tracker Fluorescein Kit at a ratio of 0.75:1 (v/w) according to the manufacture’s protocol. CHO-K1 cells were seeded in a 24 well plate 24 hours before treatment and incubated with the nanocomplexes at 37°C for 4 hours in serum free media (Opti-MEM, Invitrogen). For 4°C treatment, cells were incubated with nanocomplexes for 1 hour only to avoid excessive cell death. After this, the media containing


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nanocomplexes was removed and cells were washed twice with 1X PBS supplemented with heparin (1mg/ml). The cells were then treated with 0.4% trypan blue in 1X PBS to quench the extracellular fluorescence. The cells were collected by trypsinization using 0.25% trypsin and resuspended in 500µl of 1X PBS. Cells were analyzed on BD Accuri C6 flow cytometer using BD Accuri C6 software. 10,000 events were acquired for each sample.

Transfection and Luciferase gene expression assay CHO-K1 cells were seeded in a 24-well plate at density of 48,000 cells per well, 24 hours before the experimental treatment. 200µl nanocomplexes were prepared at charge ratio 5 as mentioned before and incubated for 1 hour at room temperature. Indicated amounts of CS expressed as GAG/peptide (wt/wt ratio), were added to the nanocomplexes after 30 minutes of incubation and kept for further 30 minutes. 100µl of nanocomplex (2µg of DNA/well) was added to cells (70% confluency) in serum free media (Opti-MEM, Invitrogen). For comparison, nanocomplexes containing commercial transfection agentsLipofectamineTM2000 (Invitrogen)/ Cellfectin® (Invitrogen)/SuperFect (Qiagen) and plasmid DNA were prepared at 2:1 ratio (wt/wt of transfection agent:DNA) as per manufacturer's instruction. After 20 minutes post incubation at room temperature, complexes were added to the cells followed by transfection efficiency analysis using standard protocol (as given below). After 4 hours of incubation at 37°C, the medium was aspirated and supplemented with 500µl of complete growth medium. After 24 and 48 hours of transfection, cells were washed with 1X PBS and lysed with 100µl of cell culture lysis buffer (1X CCLR, Promega). Luciferase expression was measured in 50µl of cell lysate supernatant using the luciferase assay substrate (Promega). Light emission was measured by integration over 10 seconds in Orion microplate luminometer (Berthold Detection System, Germany). Luciferase activity was normalized with total protein content of the cells estimated using Bicinchoninic acid assay (BCA) protein assay (Pierce) kit.

Analysis of cellular uptake pathway at different temperatures and in presence of various



endocytosis pathway inhibitors CHO-K1 cells were seeded in a 24 well plate at a density of 48,000 cells per well. Before addition of nanocomplexes, cells were pre-incubated for 1 hour at 37°C with different endocytosis pathway inhibitors at the respective concentrations as established in our earlier studies22 and also standardized using fluorescently labeled positive markers (Alexa Fluor 633-Transferrin (5µg/mL) for clathrin pathway; BODIPY FL C5-lactosylceramide (1µM) for caveolae pathway; Neutral Dextran-tetramethyl rhodamine (1mg/mL) for macropinocytosis). The final concentration of inhibitors used was Chlorpromazine (10µg/mL), Genistein (200µM) and Dimethyl amiloride (200µM) suspended in 300µl of serum free medium, i.e., Opti-MEM (Invitrogen) in all cases. Following this, 100µl of FITC labeled nanocomplexes, formed using FITC labeled plasmid DNA and unlabeled peptide, with or without CS coating, was added to the cells (nanocomplexes containing 2µg of plasmid DNA/well). Positive controls for different pathways were given as independent treatments 15 minutes prior to completion of 4 hours incubation at 37°C. After 4 hours of incubation at 37°C, cells were processed as mentioned in cellular uptake protocol followed by flow cytometry measurements using FACS Caliber (Becton Dickinson).

Cell Viability assay Cell viability was evaluated using CellTiter- Glo Luminescent Cell Viability Assay (Promega). CHO-K1 cells were seeded in a 96-well plate 24 hours before treatment. 20µl of nanocomplexes prepared at charge ratio 5 (for Mgpe9 and Mgpe10) and 10 (for Mgpe10 only) with/without CS was added to the cells in serum-free medium for 4 hours and cell viability was evaluated. For an assay at 24 hours, the cells were incubated with nanocomplexes for 4 hours, after which the medium was aspirated and cells were supplemented with 100µl of complete growth medium. Thereafter, cell viability was evaluated according to the manufacturers’ protocol. Untreated cells were taken to be 100% viable. For comparison, nanocomplexes containing LipofectamineTM2000 (Invitrogen) and plasmid DNA were prepared at 2:1 ratio (wt/wt of transfection agent:DNA) as per manufacturer's instruction. After 20 minutes post incubation at room temperature, complexes were added to the cells followed by cell viability analysis


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using standard protocol.

Transfection efficiency of CS coated Mgpe10 nanocomplexes in presence of serum CHO-K1 cells were seeded in a 24-well plate and nanocomplexes containing Mgpe10 peptide were prepared at charge ratio 5 as mentioned before. 100µl of nanocomplex (2µg of DNA/well) was added to cells (70% confluency) in complete media with 10% serum (Opti-MEM, Invitrogen). For concentration dependent study, different percentage of serum i.e. 10%, 20%, 30% ,40%, 50% was used. After 5 hours of incubation at 37°C, the medium was aspirated and supplemented with 500µl of complete growth medium. After 24 hours of transfection, cells were washed with 1X PBS and lysed with 100µl of cell culture lysis buffer (1X CCLR, Promega). Luciferase expression was measured as explained previously and Luciferase activity was normalized with total protein content of the cells estimated using Bicinchoninic acid assay (BCA) protein assay (Pierce) kit.

Statistical Analysis Statistical analysis was performed using unpaired student's t-test (unpaired) in GraphPad Prism and pvalue was calculated.

RESULTS Peptide-DNA nanocomplexes remain compact and stable at low concentrations of exogenously added CS. Biophysical characteristics of nanocomplexes play a crucial role in determining their interaction with extracellular components as well as effective internalization into the cells. Therefore we first analyzed the effect of addition of exogenous CS on size and surface charge density of peptide-DNA nanocomplexes formed using two different amphipathic peptides Mgpe9 (secondary amphipathic) and Mgpe10 (primary amphipathic). CS was exogenously added to the nanocomplexes at two different CS:peptide wt/wt ratio of 0.25 and 1 as described in materials and methods section. Size, zeta potential and polydispersity index of



the nanocomplexes was then measured using Zetasizer Nano ZS (Fig.1A, B, S1A). When low quantities of CS was added to the nanocomplexes (at a CS:peptide wt/wt ratio of 0.25), we observed an increase in size of nanocomplexes along with decrease in the overall surface charge presumably because of the binding of CS on surface of the nanocomplex. The changes seemed to be more evident for Mgpe9 nanocomplexes as compared to that observed for Mgpe10. At higher CS:peptide wt/wt ratio of 1, the size of the Mgpe9 nanocomplexes is similar to that of the native one, while for Mgpe10, they are still higher in size with respect to the native nanocomplexes. Polydispersity index values for both the nanocomplexes were low even in the presence of CS at both the ratios, thereby indicating homogenous nature of the samples. However both the nanocomplexes containing high CS exhibited high negative surface charge which could indicate tendency towards destabilization of these nanocomplexes leading to release of plasmid DNA.

We next wanted to assess the stability of these nanocomplexes in presence of different amounts of CS. Agarose gel electrophoresis was carried out to monitor the release of plasmid DNA from the nanocomplexes (Fig.1C). Both Mgpe9 and Mgpe10 exhibit stability up to a high CS:peptide wt/wt ratio of 100:1 which is indicated by the absence of the band for free plasmid DNA in the gel. To confirm our findings we used an even more sensitive EtBr exclusion assay where increase in fluorescence intensity upon binding of EtBr to even the partially relaxed plasmid DNA can be evaluated (Fig.1D). It was observed that Mgpe9 showed presence of plasmid DNA starting from CS:peptide wt/wt ratio of 0.5:1 and showed a maximum fluorescence recovery of 30% - 40% at higher CS:peptide wt/wt ratios. In case of Mgpe10, on the other hand, the fluorescence recovery was less than 30% even at the highest concentration of CS:peptide (10:1). Comparing the two assays, we could conclude that while the DNA may not be completely released at high amount of GAGs (as indicated by the gel assay), there could be partial relaxation of the nanocomplexes (as indicated by the EtBr assay). Often early release of plasmid DNA may also lead to its degradation by extracellular nucleases. To assess the possible loss of plasmid DNA from the nanocomplex treated with CS at CS:peptide wt/wt ratio of


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0.25, we challenged these nanocomplexes with DNaseI enzyme under in vitro conditions (Fig.S1B). Eventually, the enzyme was inactivated and amount of plasmid DNA within the nanocomplex was retrieved using heparin and trypsin treatment (as described in Materials and Methods). We argued that if the DNA in the nanocomplexes remained protected, the entire DNA would be released and would show similar band as in case of free DNA (control). However, if there is degradation of the DNA due to less protection, less DNA would be observed in the gel with respect to control. It was observed that both Mgpe9 and Mgpe10 were able to protect DNA from the nuclease degradation since there was recovery of the plasmid DNA even after the nuclease treatment. However, in case of Mgpe9, the presence of CS was not offering complete protection from degradation as indicated by low intensity of the DNA band on the gel for coated nanocomplexes (lane7) in comparison to control DNA (lane1) and uncoated ones (lane 6). For Mgpe10 nanocomplexes, the presence of CS imparted complete protection as indicated by high and comparable intensity of the DNA band for coated nanocomplexes (lane7) as compared to uncoated (lane6) and control DNA (lane1). Hence overall addition of exogenous CS at this ratio led to increased size of nanocomplexes along with reduced surface charge and the nature of the peptide controlled the stability. Since nanocomplexes coated with CS at CS:peptide wt/wt ratio of 0.25 seemed to be most appropriate for subsequent cellular studies, we further analyzed the morphological alterations at this condition by visualizing them through Atomic Force Microscopy. As seen from Fig.1E, the nanocomplexes were still completely condensed even in presence of exogenous CS at CS:peptide wt/wt ratio of 0.25. However, lower number of nanocomplexes was observed on the mica surface. This could be because of the decrease in overall surface positive charge resulting in less binding on the negatively charged mica surface.

Low concentrations of exogenous CS increases the transfection efficiency in vitro Since results from the previous section indicate that the nanocomplexes are stable at CS:peptide wt/wt ratio of 0.25, we next explored the effect of coating of the nanocomplexes with this amount of CS, on cellular uptake as well as DNA delivery efficiency of the peptides in CHO-K1 cells.



We studied the internalization of the nanocomplexes in CHO-K1 cells at 37°C by treating them for 4h with nanocomplexes containing rhodamine labeled plasmid DNA and FITC labeled CS followed by visualization through confocal microscopy. It was observed that for both the peptides, CS (depicted by green fluorescence inside the cells) internalized along with the nanocomplexes (depicted by red fluorescence inside the cells) (Fig.2A). Merged image shows the co-localization (depicted by yellow color in the image) of nanocomplexes with CS inside the cells. The visualization of images indicate that Mgpe10 nanocomplexes exhibited higher co-localization with CS as compared to that observed in Mgpe9 nanocomplexes. For quantification of cellular uptake, the nanocomplexes prepared using FITC labeled plasmid DNA and either uncoated or coated with unlabeled CS (CS:peptide wt/wt ratio of 0.25) were added to the cells and incubated for 4h followed by assessment of percentage FITC positive cells and mean fluorescence intensity using flow cytometry (as described in the Materials and Methods section). It was seen that Mgpe9 nanocomplexes did not show any change in percentage positive cells and exhibited low mean fluorescence intensity in presence of CS. However addition of CS in case of Mgpe10 nanocomplexes exhibited 20% increase in the percentage FITC positive cells with a similar trend of mean fluorescence intensity (Fig.2B) indicating increased cellular uptake. We next performed transfection efficiency analysis (Fig.2C) to check whether the plasmid DNA delivery of CS coated nanocomplexes would exhibit similar effects as the cellular uptake. CHO-K1 cells were treated with CS coated and uncoated nanocomplexes formed using plasmid DNA encoding for firefly Luciferase enzyme and enzyme activity was assessed at 24h. It was observed that at 0.25 CS:peptide wt/wt ratio, both Mgpe9 and Mgpe10 exhibited increase in transfection efficiency. Moreover further increase in CS led to decreased transfection efficiency for both the peptides. Mgpe10 nanocomplexes showed higher increase in transfection efficiency in presence of exogenous CS with ~2 orders increase in delivery efficiency. On the other hand, there is only slight increase in case of the Mgpe9 nanocomplexes. Moreover, transfection efficiency of nanocomplexes coated with CS was comparable to or higher than the commercial agents used.


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We further evaluated the effect of exogenous addition of CS on the toxicity of the nanocomplexes using Celltitre Glo assay as explained in the Materials and Methods section. It was observed that for both the peptides, >90% cell viability is maintained upon addition of CS to nanocomplexes at CS:peptide wt/wt ratio of 0.25:1 (Fig.S2A). For Mgpe9 nanocomplexes, cell viability remained same irrespective of presence of CS coat even up to 24h (Fig.S2B). It is to be noted that LipofectamineTM2000, whose transfection efficiency was similar to that of CS-coated nanocomplexes, exhibited only 40-50% cell viability at 24h (Fig.S2B) indicating the superiority of CS-coated nanocomplexes over this commercial agent. Another interesting observation was that when nanocomplexes were prepared at a higher charge with Mgpe10 peptide, the presence of CS coating significantly improved the cell viability at 4h and 24h as compared to native nanocomplexes which were toxic to the cells at this charge ratio (Fig.S2C).

CS helps in active transport of Mgpe10 nanocomplexes and better nuclear accumulation Exogenous GAGs are also known to play a crucial role in determining the intracellular trafficking of carrier systems which in turn might alter their delivery characteristics. Therefore we next studied how the presence of exogenous CS can alter the cellular entry pathway of amphipathic nanocomplexes as well as their trafficking inside the cells. In order to check this, we first explored the entry of CS coated nanocomplexes at two different temperatures of 37°C and 4°C. It was observed that CS coated Mgpe10 nanocomplexes had more dependence on active transport as depicted by 60-80% decrease in percentage positive cells (which denotes cellular uptake) at low temperature. On the other hand, there is only 20% decrease in percentage positive cells in case of Mgpe9 nanocomplexes. A similar trend is observed for mean fluorescence intensity as well (Fig.3A) indicating that the coated Mgpe9 nanocomplexes can also adopt energy-independent routes of entry. We further checked the cellular entry of these nanocomplexes in presence of different endocytosis pathway inhibitors to delineate the exact pathway when energydependent route like endocytosis is operative. It was observed that in case of Mgpe9, the CS coated nanocomplexes showed similar trend as the native nanocomplexes with maximum decrease in cellular uptake in presence of Genistein indicating a preference towards caveolae mediated entry pathway.



However, in case of Mgpe10, while the native nanocomplexes seemed to utilize multiple pathways of entry, the CS coated nanocomplexes exhibited maximum decrease in fluorescence positive cells in presence of Dimethyl amilioride indicating macropinocytosis as the preferred entry route (Fig.3B). These results indicated that in case of Mgpe10, the presence of CS alters the pathway of entry of the nanocomplexes. We further checked whether exogenously added CS leads to any changes in the intracellular processing thereby altering the nuclear accumulation of these nanocomplexes inside the cells. To investigate this, we performed a co-localization study to assess the role of CS in determining lysosomal entrapment of these nanocomplexes (Fig.3C). Cellular lysosomes were labeled using Lysotracker Deep Red and localization of CS coated and uncoated Mgpe9 and Mgpe10 nanocomplexes (plasmid labeled with FITC) was visualized through confocal microscopy. It was observed that presence of CS coating reduced the lysosomal localization of Mgpe10 nanocomplexes. However Mgpe9 nanocomplexes did not exhibit any such differences in lysosomal co-localization in presence or absence of CS coating. Calculation of Pearson's coefficient confirmed the extent of co-localization for these nanocomplexes and lysosomal compartments (Fig.3C). In order to eliminate any experimental artifact arising out of alteration in FITC fluorescence in the acidic lysosomal compartment, we also repeated the experiment with Lysotracker Green and nanocomplexes containing Rhodamine-labeled plasmid DNA. The observed trend was similar (data not shown). We further used fluorescence microscopy to analyze the nuclear accumulation of these nanocomplexes. The co-localization of FITC labeled CS coated nanocomplexes and DAPI stained nuclei of cells was visualized and analyzed in this case (Fig.S3A). Although visually we were not able to discern any significant difference in nuclear accumulation of the nanocomplexes, calculation of the Pearson's coefficient of co-localization indicated that Mgpe10 conferred better nuclear accumulation as compared to Mgpe9 in presence of CS (Fig.S3B). Moreover, z-stacking study confirmed the increase accumulation of these nanocomplexes inside the nucleus (Fig.S4-7). These results indicate that exogenous CS increased the cellular uptake of primary amphipathic peptide Mgpe10 based nanocomplexes, altered the route of


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entry, followed by favorable trafficking leading to less lysosomal localization and better nuclear accumulation.

Coating with exogenous CS in small amounts can improve the transfection efficiency of Mgpe10 nanocomplexes even in presence of serum We next wanted to check whether presence of CS coating can alter the transfection efficiency even in presence of serum which is a primary barrier encountered during systemic applications. Therefore transfection was carried out with Mgpe10 nanocomplexes in presence of complete medium having 10% FBS and with increasing concentrations of FBS (i.e.10-50%) (Fig.S8A,B). It was observed that Mgpe10 nanocomplexes coated with CS:peptide wt/wt ratio of 0.25 retained high transfection efficiency in presence of 10% serum and the transfection was comparable to that in absence of serum. Uncoated and nanocomplexes coated with higher CS:peptide wt/wt ratio of 1.0 however showed a drop in presence of serum. Moreover, increasing the concentration of serum further leads to slight decrease (of 1-2 orders) in transfection efficiency of CS-coated nanocomplexes (0.25 wt/wt CS:peptide)-however but it is still higher than the native nanocomplexes at all the serum concentrations, indicating that the CS coating might have beneficial effects in presence of serum. An additional observation that the high transfection is retained even at 48h for CS coated Mgpe10 nanocomplexes (Fig.S9) with only slight decrease in gene expression efficiency as compared to that at 24h is encouraging towards further development of this peptide. DISCUSSION Glycosaminoglycans (GAGs) are ubiquitous molecules that have been primarily considered as impediments towards the success of non-viral vector mediated gene delivery. Cell surface GAGs have extensively been studied for their influence on physico-chemical properties, cellular uptake, intracellular distribution and transfection efficiency of various polymer, lipid and peptide-based cationic carrier systems9-11. These studies provide contradicting evidence on importance of cell surface GAGs in mediating effective internalization or nuclear entry of nanocomplexes. However, the overall conclusions



emphasize that the properties of both GAG and carrier play a crucial role in determining their subsequent interaction as well as the effect on delivery efficiency10. On the other hand, very few reports have explored the contribution of exogenous GAGs in a similar context23-25. In this regard, in a previous study from our laboratory, we reported the effect of exogenous GAGs on cellular uptake, transfection efficiency and intracellular trafficking of arginine-rich cationic peptides13,14. We observed that addition of small amounts of exogenous GAG increased the cellular uptake and transfection efficiency of these nanocomplexes remarkably. Detailed investigation to gain mechanistic insights revealed increased stability of nanocomplexes due to GAG coating and their altered intracellular trafficking as possible reasons for the enhanced effects. In addition to this study, there have been few more reports that validate the importance of exogenous GAGs on the gene delivery efficiency of other cationic systems like lipids and polymers24. However, detailed investigation of the effect of exogenous GAGs on delivery efficiency as well as translocation mechanism of amphipathic peptides still remains relatively less explored. In this study, we have analyzed the influence of exogenously added CS on nanocomplex stability, cellular uptake, transfection efficiency and intracellular trafficking for two amphipathic peptides Mgpe9 and Mgpe10 (which have the same chemical composition and differ in amphipathicity as mentioned before). Our study indicates that there are differences particularly in intracellular processing between the two peptides. Since both these peptides contain same number of arginines and other residues; this differential effect seems to indicate that the amphipathicity of the peptide plays a role in the effect of exogenous chondroitin sulphate on the gene delivery efficiency. The stability of the nanocomplexes was analyzed by a number of assays. The size and zeta potential measurements indicate that both Mgpe9 and Mgpe10 interact with CS and there is increase in size and reduction in charge of the peptide-DNA nanocomplexes at low CS:peptide ratio. The negatively charged CS molecules are likely to be forming a coat on the surface of the nanocomplexes. Similar observation was made in case of cationic peptides earlier14. Mgpe9 exhibits higher increase in size than Mgpe10, which could be attributed to either the differential placement of arginines within the sequence or possible


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contribution of the partial alpha helical conformation of the peptide as discussed later. The larger increase in size and drop in surface charge in case of Mgpe9 indicates less compaction of the nanocomplexes as compared to Mgpe10. This could be the reason for their lower stability towards nuclease mediated degradation. In contrast, CS retains the stability of the nanocomplexes completely in case of Mgpe10 and the nanocomplexes are better protected from nuclease degradation. At higher amount of CS, although there is a drop in size in both the cases, the Mgpe10 nanocomplexes are still larger in size as compared to the native nanocomplexes while in case of Mgpe9, the sizes are similar to the native nanocomplexes. Since the negative charges are further higher in both the cases, it indicates that large amounts of CS are accommodated in both the nanocomplexes, though the complexation state may be different in the two cases. There could be stronger interaction between CS and the secondary amphipathic peptide as compared to the primary amphipathic peptide at this ratio. Previous studies have shown that the presence of secondary structures in peptides allows better interaction with negatively charged membrane molecules like phospholipids or GAGs which further leads to high membrane traversing ability as compared to primary amphipathic peptides17-19. We explored whether the cellular entry of the nanocomplexes was altered by enhanced interaction of Mgpe9 nanocomplexes with CS (over its primary counterpart Mgpe10). To our surprise, we observe that in presence of exogenous CS, the increase is more pronounced for Mgpe10 nanocomplexes as compared to Mgpe9 nanocomplexes. To rationalize this, we further looked into the possible intracellular entry and intracellular routing mechanisms and whether these processes control the differential entry between the two types of peptides. There is literature evidence to suggest that GAGs can alter the route of entry for non-viral gene delivery vectors thereby affecting the downstream events inside the cells. Moreover GAGs are also known to play a crucial role in mediating cellular entry of cationic carriers alone or as nanocomplexes either through direct translocation or macropinocytosis mediated endocytosis pathways23-28. The direct translocation primarily relies on the clustering ability of GAGs in case of cell penetrating peptides whereby it may or may not affect the cellular uptake. However few other reports mention the ability of arginine-rich peptides



to interact with cell surface proteoglycans which eventually leads to actin re-organization and macropinocytosis mediated endocytosis29,30. We studied the cellular uptake of coated and uncoated nanocomplexes at different temperatures and in presence of chemical inhibitors for the commonly observed endocytic uptake routes. CS coated Mgpe10 nanocomplexes enter the cells through active processes and there is a transition of their entry route from multiple pathways towards macropinocytosis in absence and presence of CS respectively. In case of Mgpe9 the cellular uptake of nanocomplexes coated with CS is high at 4°C. This could be attributed to direct translocation ability of the secondary amphipathic peptide Mgpe9 owing to presence of partial alpha helical conformation which imparts better membrane traversing ability and thereby allows passive transport into the cells. However, cell penetrating peptides are often reported as responsible for GAGs clustering events at cell surface thereby mediating their entry through caveolae mediated endocytosis as well31-35. Our results show that caveolae mediated endocytosis is also a prominent route for Mgpe9 nanocomplexes since genistein almost stops the cellular entry of the nanocomplexes. Such a drastic reduction indicates that the direct route of entry is operative only when energy-dependent routes are nonoperative. In case of Mgpe10 nanocomplexes, however, the difference in amphipathicity might be responsible for the low uptake at low temperatures, indicating that uptake is mainly through endocytosis. In spite of multiple cellular entry pathways, Mgpe9 nanocomplexes do not exhibit high transfection efficiency as compared to Mgpe10 possibly due to intracellular lysosomal degradation. This is particularly interesting because nanocomplexes going through macropinocytosis pathway may avoid the lysosomal entrapment or degradation through lysosomal enzymes which in turn might result in overall enhancement of their cellular bioavailability as well as transfection efficiency36. Our results indicate that there is less lysosomal co-localization in case of the CS coated Mgpe10 unlike the native ones. This was not observed in case of the Mgpe9 nanocomplexes. We observe increased accumulation of CS coated Mgpe10 nanocomplexes inside and around the nuclear periphery of cells unlike CS coated Mgpe9 or native nanocomplexes. CS thus remains associated with the nanocomplexes up to the nucleus and aids in nuclear accumulation of Mgpe10 nanocomplexes better than the Mgpe9 nanocomplexes.


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These detailed mechanistic insights reveal the enhanced extracellular stability, ability to escape lysosomal degradation and enhanced nuclear accumulation as the possible reasons for increased cellular uptake and transfection efficiency for nanocomplexes containing the primary amphipathic peptide Mgpe10. Optimum use of primary amphipathic peptide for gene delivery is often impeded owing to their toxic effects on mammalian cells. Here we also observe improved cell viability in presence of CS-coated Mgpe10 nanocomplexes at higher charge ratios (and in comparison to cytotoxic effects of LipofectamineTM2000) which could also be a contributing feature towards its enhanced cellular uptake as well as transfection efficiency37-40. Moreover, transfection efficiencies can often be impeded in the presence of serum, which is one of the major barriers towards in vivo gene delivery. We observe that CS coating of Mgpe10 nanocomplexes improves transfection efficiency in presence of serum which is maintained even at higher concentrations of serum proteins. Higher stability owing to presence of protective CS shield on the nanocomplexes possibly helps in reducing surface interactions with serum proteins and allows controlled release of plasmid DNA to allow sustainable expression.

CONCLUSIONS Overall this study highlights the importance of addition of exogenous chondroitin sulphate in improving delivery efficiency of amphipathic peptide-based nanocomplexes without any chemical conjugation. It also demonstrates the differential role of CS in controlling the delivery efficiency depending upon the nature of the peptide. Further mechanistic studies on peptide-CS interaction is currently being explored in our laboratory in detail and with more peptides. In addition, we are also exploring the in vivo application of CS in gene delivery to skin.

SUPPORTING INFORMATION AVAILABLE The following files are available free of charge: Polydispersity index values of coated and uncoated Mgpe9/Mgpe10 nanocomplexes



DNA protection ability of coated and uncoated Mgpe9/Mgpe10 nanocomplexes Time dependent and charge ratio dependent cell viability analysis in presence of coated and uncoated Mgpe9/Mgpe10 nanocomplexes Nuclear co-localization studies and z-stacking analysis using Mgpe9/10 coated and uncoated nanocomplexes. Transfection efficiency in presence of 10% serum, different serum concentrations and 48h.

ACKNOWLEDGEMENT We acknowledge the Council of Scientific and Industrial Research (CSIR), New Delhi, India (project code CSC0302) and Department of Biotechnology (GAP0101) for providing financial support. We also thank Manish Kumar (CSIR-IGIB) and funding from BSC0403 for the confocal studies.

CONFLICT OF INTEREST Authors do not hold any conflict of interest.

FUNDING This work was supported by Council of Scientific and Industrial Research (CSIR), New Delhi, India (Project:CSC0302) and Department of Biotechnology (GAP0101), New Delhi, India.


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GRAPHIC FOR MANUSCRIPT

Figure 1: Stability of peptide-DNA nanocomplexes in presence of low concentration of exogenously added Chondroitin Sulphate A. (A) Size of nanocomplexes at charge ratio 5 as measured through dynamic light scattering upon addition of Chondroitin Sulphate A (CS) to peptide-DNA nanocomplexes at CS:peptide wt/wt ratio of 0, 0.25, 1. Three independent experiments were performed (in duplicates) and p value significance was calculated between native



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and coated nanocomplexes for both the peptides (Mgpe9 and Mgpe10). p-value

Deciphering the Role of Chondroitin Sulfate in Increasing the

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