Discrete, Oxidized Multi-Walled Carbon Nanotubes for Efficient Plasmid DNA Transfection

Discrete, Oxidized Multi-Walled Carbon Nanotubes for Efficient Plasmid DNA Transfection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discrete, Oxidized Multi-Walled Carbon Nanotubes for Efficient Plasmid DNA Transfection Conner Quinlan, Aaron Foote, Kevin Castillo, Clive Bosnyak, Kurt Swogger This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8108596/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Carbon nanotubes have been identified as a possible plasmid and drug delivery method but are not developed enough to be as effective compared to other commercial methods such as lipid nanoparticles, electroporation and viral particles due to cytotoxicity and non-competitive transfection rates. Results Here, a stable dispersion of discrete, oxidized multi-walled carbon nanotubes (Fig. 1) was developed (do-MWCNT) using a lipid-like surfactant, DSPE-PEG-NH 2 2000 MW, and a cationic polymer 270k MW bPEI, that demonstrate better transfection-related gene expression with less cytotoxicity than commercially available lipid nanoparticles. The stable dispersion was then used to deliver a plasmid of 8.78kb with expression measured via qPCR that was comparable to Lipofectamine 3000. Analogous studies with discrete functionalized single wall carbon nanotubes showed further improvements with cell health. Conclusion Carbon nanotubes are capable of comparable transfection to lipid nanoparticles only after plasmid DNA is incubated with a dispersion for an extended period of time compared to lipid nanoparticles, 96 hours. This implies the limiting mechanism of nanotube transfection is pDNA binding. The novelty of single walled carbon nanotubes is also demonstrated by matching the surface surfactant conditions between the single and multi-walled carbon nanotube dispersions. It is further implied that the optimal geometry of carbon nanotube for transfection has yet to be explored. Single Wall Multi Wall Carbon Nanotube Transfection Plasmid DNA Lipofectamine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Transfection of mammalian cells has led to several key advances in medicine and biotechnology, including innovative therapeutics ( 1 ), new research tools ( 2 ), next generation vaccines ( 3 ), and improved biomanufacturing ( 4 ). Common transfection methods fall into three general categories: lipid nanoparticles (LNPs) ( 5 ), electroporation ( 6 ), and viral vectors ( 7 ). The market share for each of these methods reflects their unique advantages and disadvantages. Viral Vectors provide excellent transfection efficiency ( 8 ) and occupy a market space around 2.45 billion USD between 2022 and 2024 ( 9 ). This relatively large market share is due to recent approvals for AAVs in a clinical setting for gene therapy ( 10 ) but this method is limited by the size of their genetic cargo ( 11 ) and can cause a severe immune reaction in their host organism ( 12 ). Scalability is also a concern for viral vectors, since it requires different vectors to be used for different scenarios which are dependent on keeping extensive libraries of plasmids, bioreactors for production, and the use of bacteria ( 13 ). Due to these downsides, there are many transfection applications in vitro and in vivo that are not well served by viral vectors. Despite efforts being made to increase the shelf-life, yields and purity of production in viral vectors ( 14 ), there is a strong incentive to develop a non-viral transfection method that is comparable to viral vectors in transfection efficiency that is more accessible and can deliver a greater variety of cargo. One alternate solution would be electroporation, which occupies the smallest market share because of high cell cytotoxicity ( 15 ) with an estimated value of between 232–739 million USD in 2022 ( 16 , 17 , 18 ). It requires the use of specialty equipment that restricts its therapeutic abilities to ex vivo and in vitro applications ( 19 ) but provides a reliable method of intracellular delivery of a wide range of biomolecules ( 20 ). Electroporation’s use in therapies is limited by the necessity of secondary safety measures such as heat sinks, requiring secondary drug treatments to be effective, and some electroporation techniques lack clear mechanisms of action generating skepticism on their efficacy ( 21 ). In contrast to viral vectors and electroporation, lipid nanoparticles are a commonly used transfection method in the lab ( 22 ) and clinical applications ( 23 ). LNPs have the largest market share with the two leading LNP product producers, Pfizer and Moderna, reported sales of 37 and 17.7 billion USD respectively for LNP products in 2022 ( 23 ). This relatively large market share is due to recent developments in LNP technology generating cheaper ( 24 ), effective ( 24 , 25 , 26 ) and easier to use LNP formulations ( 27 ) with lower immunogenic potential ( 28 ). LNPs are limited in what biomolecules they can carry ( 29 ), decrease cell viability ( 30 ) and transfect with decreased efficiency when delivering large plasmids (> 5 Kb) ( 31 , 32 ). LNPs are unstable without ideal storage conditions ( 33 ) and have a limited shelf life once formed ( 34 ). The limitations of LNPs demonstrate a need for an alternative transfection method that can provide a wide range of biomolecule delivery, low toxicity, and ease of use. One approach that has shown promise is the use of discrete, functionalized, biocompatible carbon nanotubes as a transfection reagent. Since their discovery in 1991 ( 35 ), carbon nanotubes have radically altered diverse fields such as energy storage ( 36 ) and robust, durable polymers ( 37 ) while showing potential in biotechnology ( 38 ). Despite their significant potential, carbon nanotubes have not been commercialized at scale for biotechnology. The full value of carbon nanotubes in biology can only be unlocked when using functionalized, individual nanotubes that are cleaned of residual catalyst and separated from their aggregated bundles, making their high aspect ratio, large surface area, and customizable surface chemistry accessible. Carbon nanotubes have been observed inducing cell proliferation ( 39 ), delivering proteins in vitro ( 40 ), acting as a transfection agent ( 41 ), detecting different cell properties and analytes as biosensors ( 42 ), and releasing loaded drugs on demand in hydrogel systems ( 43 ). Despite the variety of unique phenomenon carbon nanotubes have shown, we believe their full potential for transfection is yet to be proven. Since 2004, a variety of methods of surface functionalization and types of carbon nanotubes have been used as transfection agents in research contexts, but to our knowledge, they are not commonly used for transfection commercially. As made, unfunctionalized carbon nanotubes are poor reagents for transfection due to their potential to aggregate and inability to effectively load DNA in a reversible manner. Carbon nanotubes can however be covalently functionalized with different functional groups and noncovalently dispersed with a range of surfactants to improve dispersibility and enable favorable binding to biomolecules ( 44 , 45 , 46 ). The size of nanotubes that have been able to cross cellular membranes are reported to be between 20 nm and 1 µm in length and between 1 nm to 60nm in diameter, with different mechanisms of uptake theorized ( 47 ). The cell internalization efficiency is dependent on the surface chemistry and surfactants utilized ( 47 , 48 ). In recent years, functionalized carbon nanotubes have been shown to bind to a variety of biomolecules including mRNA ( 49 ), DNA ( 50 ), proteins ( 51 ), and small molecule drugs ( 52 ). The demonstrated binding range suggests the capabilities of carbon nanotubes as a multifaceted transfection tool, though most of the carbon nanotube transfection literature lacks a direct comparison between carbon nanotubes and other transfection methods. This work introduces a novel formulation of discrete oxidized multiwalled carbon nanotubes (do-MWCNTs) as a safe and effective transfection agent. This formulation was designed to minimize toxicity and maximize transfection efficiency through dual surfactant surface chemistry. Hydroxyl DSPE-PEG-NH 2 , an amphiphilic surfactant, is non-covalently bound to the carbon nanotube surface to obtain stable dispersions followed by physisorption of polyethylene-imine (PEI) acting as a surfactant and to stimulate DNA binding. This dual-surfactant carbon nanotube formulation is evaluated for delivering plasmid DNA, the subsequent gene expression and its resulting toxicity profile compared to the leading standard of in vitro transfection, Lipofectamine 3000. Materials and Methods MWCNTs The MWCNTs were functionalized by Molecular Rebar Design LLC using a combination of concentrated nitric acid and high energy dispersion methods. After extensive washing to remove potentially toxic residual catalysts from the MWCNT synthesis the MWCNT was provided in the form of a wet cake of ~ 6% weight solid nanotube content. MWCNT Dispersion The 6% wt. MWCNT wet cake was processed in water with a solids percent not exceeding 1% wt. The primary surfactant, DSPE-PEG-Amine (2000 MW manufactured by Laysan Bio) was added to this dilute wet cake and vortex mixed. This mixture was then bath sonicated for 30 minutes at room temperature to disperse the oxidized carbon nanotubes before the addition of branched polyethylene-imine (bPEI, 270k MW manufactured by Sigma Aldrich). Bath sonication was then performed again for 90 minutes at room temperature. The dispersion was then filtered via dialysis to remove unbound surfactants. This dialysis is done with a 1,000,000 MW cutoff. It is assumed that this dialysis removes a majority of the unbound surfactant. The filtered dispersions were then autoclaved to sterilize the solutions and subsequently re-sonicated for another total of 120 minutes and centrifuged at 20,000 gs for 10 minutes to remove any larger particles. SWCNTs The Single-Wall Carbon Nanotubes were functionalized by Molecular Rebar Design LLC and was provided to BioPact in a wet cake of ~ 10% wt. solid nanotube content. SWCNT Dispersion This dispersion was composed of the same surfactants as do-MWCNTs. The calculations for this dispersion were based on the theoretical surface density of surfactant per individual nanotube for only the exterior wall. This is because the interior of do-MWCNTs (about 5nm) and the space between walls (0.34 nm) is inaccessible to large molecules or micelle-like surfactants. SWCNTs have interior diameters in the range of about 0.7 to 1.5 nm but have a strong tendency to rope together which can reduce their effective surface area. To generate a working solution for SWCNT dispersion, a more intensive probe sonication was used with SWCNT and water added prior to the addition of surfactants to increase the available surface area of the SWCNT for the surfactants, when added, to adhere to. Further information on these calculations and assumptions made are available in supplementary information. Plasmids Used In transfection studies that did not investigate the loading of large plasmids, pTRIOZ_hIgG1 (Invivogen: ptrioz-higg1) was used with HIV-specific antibody variable regions ( 53 ) and tagged with an IL-2 secretion tag provided by Invivogen. The total plasmid size was 8.78 kb. Cloning and manufacturing was done by VectorBuilder USA. Loading Experiments To evaluate the DNA loading capability of different dispersions under a constant separatory force, we used gel electrophoresis. do-MWCNT is extremely large compared to DNA, thus making it difficult for it to traverse as quickly as a large DNA construct. A do-MWCNT dispersion is loaded with DNA for four hours before being placed in a 1% wt. Agarose gel (Thomas Scientific: J66501.30) and run at 100 V for 30 mins. This concentration of agarose gel provides a pore size that allows DNA to move through the gel while the larger carbon nanotubes are unable to move. The amount of DNA offloading can be visualized as a separate band from the dark dispersion band. do-MWCNT Dosing A standard dose of do-MWCNTs consists of an approximate ratio of 20 µg of discrete MWCNT dispersion to 1 µg of pDNA in a total of 100 µL of DEPC treated water. This was stored in 4°C or 48 hours prior to use unless otherwise stated in studies relating to incubation time. Cell Line and Plating CHO-K1 cells were purchased from ATCC (ATCC, CHO-K1 CCL-61). CHO-K1 cells were grown, maintained and plated in F-12K + 10% FBS. CHO-K1 cells were plated in all subsequent experiments at 2x10 5 cells/mL in 12-Well plates. Cell Incubation Time After the cells were allowed to settle to the bottom of the plate, each was given a total volume of 100 µL of their respective treatment group described above. After this, the cells were allowed to incubate for 72 hours prior to subsequent processing for qPCR. In the lipofectamine 3000 containing study, we extended the post-treatment incubation period to the highest recommended time of 96 hours prior to analysis as per the lipofectamine 3000 protocol to provide optimal conditions. Cells were maintained at consistent temperature and humidity of 37°C and 90+% with 5% CO2. Cell Growth Data Cells were counted using a Countess II cell counter with Trypan Blue stain (Included in ThermoFisher: T10282). do-MWCNTs interferes with trypan blue staining by causing cells to appear darker, thus preventing an accurate live/dead ratio. A total cell count was taken instead. To avoid biasing cell counts with dead cells, all groups on a 12-well plate had their media removed and were washed with 1X PBS (ThermoFisher: 10010023) to remove any non-adherent cells before the addition of Trypsin (ThermoFisher: 25200056) and inactivation in the cell-appropriate media, and then resuspended in 333 µL of 1X PBS and mixed via pipetting. 20 µL of the PBS cell solution was taken and 20 µL of trypan blue was added. Cell RNA Extractions and RT After the cells have been incubated for the appropriate amount of time and cell counts have been taken, the cells are centrifuged and the excess media is removed. 500 µL of TriZol (ThermoFisher: 15596026) and 50 µL of Chloroform are added to the cell pellet to begin the extraction of RNA which is performed according to the TriZol protocol scaled for the volumes reported. Once an RNA pellet is produced, the pellet is resolubilized in 20 µL of DEPC treated water. Concentrations are taken and normalized to an end concentration of 1 µg per 10 µL for cDNA production. This normalization prevents biasing results downstream due to differences in the cell count. This method is established in literature for studying transfection between transfection agents ( 54 ). cDNA is produced with the High-Capacity cDNA reverse transcription kit with RNase inhibitor (ThermoFisher: 4374966) using 1 µg of RNA per reaction for each respective group. Each of the reactions is performed in an Applied Biosstems MiniAmp Plus and diluted to an appropriate volume for the qPCR process. qPCR The genes screened by qPCR are Beta-Actin (ActB), Aldo-Keto-Reductase (AKR), Human IgG constant region heavy chain, Human IgG constant region light chain, VRC01 HIV-Specific Antibody Light Chain region (VRC01), and PGDM HIV-Specific Antibody Heavy Chain region (PGDM). ActB and AKR are the two endogenous controls, VRC01 is our experimental group. PGDM is used as the negative control due to the similarities in characteristics to VRC01, as they are both antibody-encoding for a variable region, approximately the same size and are both as specific to a sequence. The qPCR reagent used in this study is SYBR green master mix (ThermoFisher: 4309155) and any dilutions performed are with DEPC treated water. The qPCR reactions are performed as specified by the Comparative Ct Protocol in the Applied Biosystems Quantstudio 3. All data is interpreted through QuantStudio Design and Analysis software. In early studies, Human IgG constant regions were analyzed due to the commercial availability of these primers but in subsequent studies, for increased confidence in the specificity of our studies, we used VRC01 light-chain sequences. Data for multi-plate studies was done using appropriate inter-run controls and, where possible, multiple inter-run controls. qPCR methods and practices were based on suggestions from literature ( 55 ). Results Loading Conditions To be both non-cytotoxic and favorably interact with plasmid DNA, a carbon nanotube formulation with dual-surfactant surface chemistry was developed. First MWCNTs are dispersed using DSPE-PEG-Amine which has been shown to yield discrete and non-cytotoxic nanotubes at a range of concentrations in vitro ( 56 ). A secondary surfactant, branched PEI, was added for its high positive charge density to encourage DNA-nanotube complexing, promote cellular internalization, and facilitate endosomal release ( 57 ). The optimum relative mass ratio of do-MWCNTs, DSPE-PEG-NH 2 , and PEI was determined by testing a range of ratios and selecting the formulation that yielded the dispersion with no aggregates under brightfield microscopy. Once the relative masses of surfactants were chosen, the plasmid DNA binding was optimized by using agarose gel electrophoresis to evaluate two formulation parameters: the molecular weight of the PEI and mass ratio of do-MWCNTs to plasmid. The molecular weight of branched PEI plays an important role in the conformations available to the pDNA-do-MWCNT complex, and some molecular weights could be preferred over others. Additionally, the relative amounts of pDNA and do-MWCNT complex played a significant role in surface charge density and stability in solution, impacting downstream cellular internalization. Three different molecular weights of PEI were tested for their binding efficiency with plasmid DNA, 10 kDa, 25 kDa, and 270 kDa branched PEI. In this study, 1% agarose gel electrophoresis allows for the larger do-MWCNT + pDNA complexes to remain at the start of the gel while unbound pDNA or loosely bound pDNA will travel further from the well. A complexed dispersion with a lack of offloaded DNA in the gel environment can indicate a stronger association between the pDNA and do-MWCNT, which is believed to be important for transfection strength. This method also simulates a constant force and charged environment that could encourage weak complexes to break down. With 100 ng of plasmid DNA, do-MWCNT was added in increasing ratios from 2.5:1 to 20:1 do-MWCNT to DNA mass ratios. After the dispersion had incubated with the pDNA for three hours at 4°C they were loaded on the gel. In this simulated offloading environment, low brightness in an individual lane indicates high pDNA association with do-MWCNT, as do-MWCNT quenches the fluorescence while a higher brightness indicates low pDNA association with do-MWCNT, indicative of pDNA offloading. By comparing the relative brightness of each well offloaded pDNA (Fig. 2 ), we can determine that of the three molecular weights of PEI, 270 kDa was able to inhibit pDNA migration the most. In all the molecular weights of PEI, 20:1 of do-MWCNT to pDNA was able to inhibit pDNA migration greater than the other mass ratios. Subsequent testing will be performed using the 270 kDa PEI to optimize the performance of the mass-loading ratio for transfection with 20:1 as the reference ratio of do-MWCNT to pDNA. Loading qPCR In order to validate the loading ratio 20:1 that was observed to create the highest association between do-MWCNT and pDNA, we needed to observe ratios above and below 20:1 in a transfection study. 1 µg of pDNA was loaded at three ratios, 10:1, 20:1 and 25:1 do-MWCNT to pDNA in DEPC water. These solutions were loaded at 4°C for 7 hours. CHO-K1 cells were treated with their respective do-MWCNT-pDNA complexes and left to incubate for 72 hours. We utilized qPCR to determine which of the mass-loading ratios would cause greater expression of genes unique to our plasmid and thus, function as a better transfection agent. In Fig. 3 , the 20:1 Mass-loading group significantly outperformed both groups (**** both 1:10 and 1:25) loaded at higher and lower ratios, indicating that subsequent optimization experiments should use this loading ratio. More information on the proposed significance of this mass loading ratio is available in the supplementary information section. Effect of DNA Loading Temperature To characterize the incubation conditions of do-MWCNT + pDNA, we chose three relevant temperatures to form the complex in. Refrigeration temperature, 4°C, Room temperature, 27°C, and Biologically relevant temperature, 37°C. These solutions were left to load for 7 hours in their respective environment. CHO-K1 cells were treated with their respective groups and allowed to incubate for three days. qPCR was performed to determine which group maximized gene expression. Tests were begun for the variable chain region of our plasmid due to the increased confidence that a highly specific primer set would afford. Figure 4 shows the relative expression of pDNA was clearly negatively affected by temperature with a significant (**) drop observed between 4°C and 37°C. Subsequent experiments used 4°C for incubation of do-MWCNT + pDNA. Time of DNA Loading To determine the amount of time do-MWCNT and pDNA should be incubated together prior to transfection, loadings of do-MWCNT and pDNA at intervals of 1 hour, 3 hours, 7 hours, 24 hours, and 32 hours were made. CHO-K1 cells were treated with their respective groups and allowed to incubate for three days. This experiment was split between two qPCR studies with appropriate inter-run controls for normalization. In Fig. 5 , there was a significant increase in expression of the plasmid as the complexing time of do-MWCNT and pDNA was increased. Based on these findings, a subsequent study was conducted to observe the peak of the transfection efficiency. do-MWCNT was loaded at 32 hours, 48 hours, 96 hours, and 168 hours. In Fig. 6 , a peak in transfection efficiency was observed at 96 hours of pDNA and do-MWCNT incubation with significance relative to 48 hours and 168 hours (**** and *** respectively). To determine if this increase in transfection efficiency would increase cytotoxicity, cell counts were conducted of the longer incubation period groups. In transfection, it is often observed that increased transfection results in an increase in cytotoxicity ( 58 ). In contrast, as seen in Fig. 7 , the extension of incubation time of the do-MWCNT + pDNA increased in transfection-related gene expression without a decrease in cell population. A significant drop-off in the average cell population was however observed at the 168-hour time point in reference to the 48-hour time point. This decrease in average cell population began between 48 hours and 96 hours (not significant) and concluded with a significant drop in cell health at 168 hours. Because the difference between 48 hours and 96 hours, and 96 hours and 168 hours is insignificant, it is believed that in conjunction with the peak in transfection, 96 hours is the best transfection when considering toxicity effects. This drop in cell health at 168 hours is believed to be related to the formation of aggregates of the do-MWCNT + pDNA complex that forms accumulations or flocculants that are known to have deleterious effects to cell health ( 59 ). With appropriate Inter-run controls, the data is normalized across the multiple transfection studies in Fig. 8 . Given the dramatic difference in the magnitude of expression observed, 96 hours was used in subsequent transfection studies. Significant (****) differences were seen between 48 hours, 96 hours and 168 hours in the gene expression of transfected pDNA. do-MWCNT vs Lipofectamine 3000 in Cell Cytotoxicity To determine if an optimized do-MWCNT + pDNA complex is capable of transfection comparable to current products, a do-MWCNT dispersion was loaded for 96 hours at 20:1 do-MWCNT:pDNA in 4°C against Lipofectamine 3000 (ThermoFisher L3000001) in CHO-K1 cells. A cell count was made after cells had incubated with their respective transfection agent for 5 days. The bed of adherent cells was washed with PBS to ensure that dead, nonadherent cells would be excluded from this count. Lipofectamine was prepared according to the provided protocol. Half-doses of both transfection reagents were prepared to show a dose-dependent relationship in transfection and cell counts. The population of CHO-K1 cells treated with lipofectamine observed a significant (***) 30% drop in cell population compared to the no treatment group as the baseline as seen in Fig. 9 . do-MWCNT treated groups and the half dose lipofectamine group experienced no significant deviation from the no treatment group, indicating minimal impact to cell health. do-MWCNT vs Lipofectamine 3000 in Transfection After cell counts were completed, qPCR was conducted to measure VRC01 specific sequences in CHO-K1 cells via the described method. With the no treatment group acting as the baseline for normalization, do-MWCNT was able to generate a significant (*) average of 11% higher expression of pDNA than Lipofectamine 3000 in a full dose as observed in Fig. 10 . This method of qPCR testing would prevent an increase in cell population from affecting the ratio of endogenous gene to VRC01 antibody-specific gene sequences. Because of this methodology choice, it is concluded that do-MWCNT is capable of higher rates of gene expression per cell at full dosing conditions and implies a higher transfection rate than Lipofectamine. do-MWCNT’s ability to transfect and effect a higher expression level than Lipofectamine 3000 is unique in its ability to not affect the population of cells and change transfection rates independently of cell population until 168 hours of do-MWCNT + pDNA incubation. It was determined that Lipofectamine outperformed do-MWCNT when the dosage was halved. Single Walled CNT Translation from do-MWCNT To determine the effects of different tube geometries on transfection, we set out on making an oxidized single-wall nanotube dispersion using the same surfactants and methodology of testing as do-MWCNT. This geometry and oxidation was chosen because of the benefits inherent in single-wall nanotubes in biology; Oxidized single wall nanotubes are far more likely to be broken down by cells, single walls are smaller and more likely to passively enter the cell, and have no interior walls, thus no biologically inaccessible surface area than multi-wall nanotubes. Due to structural stability limitations, we were unable to further oxidize the single wall nanotubes beyond half the oxidation of do-MWCNT. The approach used to translate the formulation from do-MWCNT to SWCNT was based on a particle matched ratio of the components of do-MWCNT wherein the surface of both formulations would effectively have the same density of each surfactant component and DNA. The values used to generate these surfactant concentrations per area of the nanotubes are available in the supplementary information section. At the particle matched ratio of 10:27.7:17 nanotubes:DSPE-PEG-NH 2 :270k bPEI, there were no visible flocculants. Transfection of SWCNT The ratio of Nanotubes:DNA when translated based on surface area on a per-particle basis was 0.71:1 which translated to an approximate mass ratio of 2.1:1. This mass ratio SWCNT:pDNA was loaded for 96 hours to compare single walls to the previous do-MWCNT dispersion. Figure 11 demonstrates that transfection still occurred after altering the underlying nanoparticle geometry. Due to the reduced amount of nanomaterial used for this study, it was decided to test if an increased dose would produce higher gene expression without hitting the dosage limit to produce deleterious effects to cell health. Upper Limit of SWCNT Transfection do-MWCNT’s maximum dose was established to be around 20 µg of nanotubes for 1 µg of DNA due to a downturn in cell health. Because of the significant reduction in the mass of SWCNT added to the same amount of DNA, it was surmised that the upper limit of transfection with SWCNT was not reached. Most transfection agents are limited by the transfection efficiency of a dose and the toxicity of that dose. The lipofectamine protocol even includes separate ratios to try for their two-component system to optimize for better transfection which implies that the highest recommended dose is the limit of its transfection abilities in the conditions tested. To find the upper limit of SWCNTs 1x, 2x, and 4x of the initial dose given was examined. do-MWCNT was included at 1x and 2x concentrations to illustrate that it has achieved close to the peak of its ability to transfect with maximum gene expression without interfering with cell health. As observed in Fig. 12 , the increasing dosages of SWCNTs do not show a significant increase in toxicity when compared to do-MWCNT that has a significant decrease in cell count at the 2x dose. Figure 13 shows that as the dosage increases of the single-wall transfection agent, the transfection decreases. It is likely that because the single-wall particle count was based off of the same number of particles present in the maximum dose of the do-MWCNT multi-wall agent, that the limiting factor in carbon nanotube transfection may be related to aggregation of the carbon nanotubes. Even though SWCNTs are far smaller in diameter and are on average for the present tubes, 22% longer than do-MWCNT, the addition of large surfactants like 270k bPEI generates a much larger surface area per nanotube. It may be that the maximum dose for carbon nanotube transfection systems is related to the number of individual carbon nanotubes. In both do-MWCNT and SWCNT systems, both contained approximately 9.04 x 10 10 carbon nanotubes per mL. Advantages of SWCNT over do-MWCNT do-MWCNT has previously been tested with eGFP containing plasmids. It was observed that, despite relatively high gene expression of transfection related sequences, the observed fluorescence was minimal. Figure 14: eGFP plasmid transfection from do-MWCNT after 3 days of incubation post-transfection. A.) Fluorescent view of do-MWCNT treated CHO-K1 cells. B.) Brightfield image of do-MWCNT treated CHO-K1 cells with fluorescent overlay. It is believed that do-MWCNT’s ability to quench fluorescence is amplified by the cells accumulating visible amounts of nanotubes around or inside them as seen in Fig. 14. The visible expression of eGFP by SWCNTs (Fig. 15 ) shows that do-MWCNT and SWCNTs, despite having different transfection efficiencies, might pose different advantages depending on the end-user application. Discussion The use of multi-walled carbon nanotubes as a transfection agent was first identified in 2005 ( 60 ) where carbon nanotubes were functionalized with ammonia to generate a strong positive charge on the surface of the nanotube. The loading ratios tested in that study were from 1:1 to 10:1 f-CNT:DNA with 30 mins of complexing time. There was no comparison for this tested transfection agent against any other commercial transfection agent, so there is no way to directly compare the results of these studies. However, that publication shares most of the same concerns as this one in regard to DNA binding. The long complexing time, 96 hours, and relatively high ratio required to complex do-MWCNT and pDNA (20:1, compared to literature) implies that the concerns of pDNA binding too tightly to the nanotube is less of an issue as otherwise stated in previous studies. One concern brought up in that previous publication’s discussion is that SWCNT modified with ammonia exhibited weaker DNA complexes even in higher loading ratios than multiwall carbon nanotubes. This may be due to the ability of a specific DNA molecule to wrap the respective nanotube geometry compared to the larger MWCNTs. Large plasmid (< 5kb) delivery has been considered as a requirement for use of CRISPR, especially given the search for alternative nonviral strategies that can address the limitations of other transfection methods such as viral vector efficacy and toxicity ( 61 ). It would be beneficial to see if do-MWCNT shares the general limitations of other transfection agents where transfection efficiency begins to drop with the use of larger plasmids. The plasmid DNA used here encodes for VRC01 ( 62 ) Human IgG Anti-HIV Antibodies. The higher expression of these antibody sequences by do-MWCNT transfection could be useful for future therapeutic purposes or biomanufacturing in mammalian systems, lowering the required dose for effective therapeutic efficacy. An advantage that do-MWCNT affords as a transfection reagent is a lower material cost than Lipofectamine 3000. Lipofectamine requires a specific media type, Opti-MEM, as per the ThermoFisher protocol for optimal transfection. do-MWCNT is optimized for complex formation in a sterile water environment and like Lipofectamine 3000, can be treated directly into serum-containing media. do-MWCNT is also a single-component system and does not require the creation of multiple solutions for optimal complex formation, rather, a specific order of addition is necessary for optimal do-MWCNT loading. One of the primary drawbacks of do-MWCNT is the formation time of the complex. The increase in gene expression in relation to loading time is implicative of a slow physical interaction between the surfactant structural configuration and the coiling configuration of the pDNA. Sonication has been theorized as a potential solution that would enable pDNA to form into a more adaptable configuration for do-MWCNT. Solving the high-incubation time barrier would significantly increase adoption of a CNT-based transfection agent. SWCNT Implications The translation of this transfection phenomenon across different carbon nanotube geometries implies that carbon nanoparticles, when dispersed in similar surfactants at nearly identical conditions in the surfactant ratio per surface area unit, will not behave identically. This likely means that there is an optimal geometry of carbon nanotube for pDNA transfection that was not fully examined in this present study. One of the most important features of using SWCNT over do-MWCNT is that even when the mass of nanotubes used was decreased by ~ 15x, we were able to achieve about half the transfection capability of do-MWCNT which implies a greater efficiency per unit of carbon by SWCNT. Not only is it more efficient in terms of carbon, but surfactant used per unit of transfection. To achieve the same level of transfection for do-MWCNT, ~2x the amount of surfactant must be used per dose. This data on translating transfection-optimized multi-walled carbon nanotubes to appropriately surface-area scaled single-walled system implies a significant decrease in associated costs of producing nanotube-based transfection agents. Scaled on the data in Fig. 11 , we can find the price per unit of RQ to find the most cost-effective transfection agent. Lipofectamine scales to 2.3x10 − 6 USD per RQ, do-MWCNT scales to 6.4x10 − 8 USD per RQ and SWCNT scale to 1.2x10 − 7 USD per RQ. Based on this efficiency, do-MWCNT is 36 times cheaper per unit of transfection than Lipofectamine 3000 while achieving the same magnitude of transfection as Lipofectamine 3000. SWCNT are only 18.5 times cheaper than Lipofectamine and do not achieve the same magnitude of transfection but show far less toxicity than both do-MWCNT and Lipofectamine 3000. The prices here are calculated based on the chemical suppliers of surfactants and CNTs for BioPact at scale production. DNA is not included in the cost analysis. On a single dose for a 12-Well plate constituting 1 µg of DNA for the maximum recommended dose of Lipofectamine 3000, Lipofectamine’s retail cost per dose is 2.725 USD including the cost of Opti-MEM. On a material cost provided by Molecular Rebar Design LLC, for 1 µg of DNA transfection, do-MWCNT is 0.084 USD per dose. SWCNT, on the same scale as do-MWCNT, scales to 0.07 USD per dose. A further advantage of SWCNTs is the likelihood of biological systems being able to break down the administered nanotubes. do-MWCNTs have been shown to be degraded in some specific cell types by the use of oxide species such as peroxides ( 63 ). Carbon nanotubes with a higher oxidation state are easier to degrade by enzymes ( 64 ). do-MWCNTs, despite being capable of higher surface oxidation than SWCNTs, have the highest oxidation state on the exterior of the nanotube which makes the exterior wall the easiest to degrade. It is thought that the interior walls are almost entirely without functionalization and are harder to degrade. Once the oxidized outer layer begins to degrade, surfactant may begin to dissociate and cause both aggregation and a lower rate of degradation. Because SWCNTs have only one wall to break down, they are not only more likely to be degraded by natural cell processes, but less likely to aggregate as they are broken down due to surfactant disassociation and hold the advantage of requiring less mass of SWCNTs per treatment than do-MWCNTs. SWCNTs, per unit of mass, have lower absorption than do-MWCNTs. This provides SWCNTs with a significant advantage over do-MWCNTs in transfection applications, wherein do-MWCNTs have been shown to quench fluorescence by reporter genes. SWCNTs have enabled fluorescence applications in a CNT-based system for alternate or expanded detection methodologies such as flow cytometry. Even though the previously mentioned study ( 60 ) did not consider the per-particle ratio changes, they noted that SWCNT showed less binding capabilities for pDNA than multi-walled carbon nanotubes. It is believed that the inclusion of bPEI scaled according to the particle count was crucial to alleviating this binding issue by making the binding activity dependent on the bPEI’s behavior in solution rather than the surface area of the CNT that was exposed to the solution. Overall, this study has shown the promising nature of carbon nanotubes as a commercially viable transfection agent with similar transfection magnitude to commercially available solutions. In its current state, it may pose as another option to explore when other transfection agents have failed to produce desired results. Abbreviations do-MWCNT – Discrete, Oxidized Multi-Walled Carbon Nanotubes SWCNT – Single Walled Carbon Nanotubes CNT – Carbon Nanotubes pDNA – Plasmid DNA RQ – Relative Quantification LNP - Lipid Nanoparticle f-CNT – Functionalized CNT Declarations Competing Interests Conner Quinlan, Aaron Foote, and Kevin Castillo are former employees of Molecular Rebar Design LLC and its subsidiaries. BioPact is a subsidiary of Molecular Rebar Design LLC. Funding Declaration Funding was provided by Molecular Rebar Design LLC and contract number W81XWH22C0090 as part of the DoD SBIR program. Author Contribution Conner Quinlan wrote the main manuscript text under the guidance of Aaron Foote. All figures were prepared by Conner Quinlan. Aaron Foote, Kevin Castillo, and Clive Bosnyak reviewed the manuscript and suggested edits. Kurt Swogger and Clive Bosnyak provided materials from Molecular Rebar Design LLC.Corresponding Author: Conner Quinlan Acknowledgement This material is based on work supported by the US Army Medical Research and Development Command (USAMRDC) or U.S. Army Medical Research Acquisition Activity (USAMRAA) under contract No. W81XWH21P0101 and W81XWH22C0090 from 2021 to 2025. 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Error represents standard error of the mean. Left column of each group represents the IgG constant region heavy chain while the right column of each group represents the IgG constant region light chain.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8108596/v1/f5ab1e2206c6369eae1cc68e.png"},{"id":96445760,"identity":"1e4628d3-4b06-45ff-a719-45476a233ef6","added_by":"auto","created_at":"2025-11-21 07:57:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":118511,"visible":true,"origin":"","legend":"\u003cp\u003eRelative Quantification of expression for VRC01 Light Chain variable region sequences. Error bars represent standard error of the mean. N=3 Technical. 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Brightfield image of do-MWCNT treated CHO-K1 cells with fluorescent overlay.\u003c/p\u003e","description":"","filename":"image14.png","url":"https://assets-eu.researchsquare.com/files/rs-8108596/v1/b33090f70904bbf4d0bb701a.png"},{"id":96445790,"identity":"506b4e9a-0e8a-4185-b471-b57340a44800","added_by":"auto","created_at":"2025-11-21 07:57:19","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":182341,"visible":true,"origin":"","legend":"\u003cp\u003eeGFP plasmid transfection from SWCNT. A.) CHO-K1 cells treated with SWCNT after three days of incubation. B.) CHO-K1 cells treated with SWCNT after five days of incubation.\u003c/p\u003e","description":"","filename":"image15.png","url":"https://assets-eu.researchsquare.com/files/rs-8108596/v1/7cd3e6f9a1695b5223959ca6.png"},{"id":96708352,"identity":"1035b20d-fe56-4072-8fb6-7aba236b7952","added_by":"auto","created_at":"2025-11-25 10:01:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3160852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8108596/v1/ba42ba66-05f9-4c65-ba18-f3ce1aef46ce.pdf"}],"financialInterests":"Competing interest reported. Conner Quinlan, Aaron Foote, and Kevin Castillo are former employees of Molecular Rebar Design LLC and its subsidiaries. BioPact is a subsidiary of Molecular Rebar Design LLC.","formattedTitle":"Discrete, Oxidized Multi-Walled Carbon Nanotubes for Efficient Plasmid DNA Transfection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTransfection of mammalian cells has led to several key advances in medicine and biotechnology, including innovative therapeutics (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), new research tools (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), next generation vaccines (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and improved biomanufacturing (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Common transfection methods fall into three general categories: lipid nanoparticles (LNPs) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), electroporation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), and viral vectors (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The market share for each of these methods reflects their unique advantages and disadvantages.\u003c/p\u003e\u003cp\u003eViral Vectors provide excellent transfection efficiency (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) and occupy a market space around 2.45\u0026nbsp;billion USD between 2022 and 2024 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This relatively large market share is due to recent approvals for AAVs in a clinical setting for gene therapy (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) but this method is limited by the size of their genetic cargo (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) and can cause a severe immune reaction in their host organism (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Scalability is also a concern for viral vectors, since it requires different vectors to be used for different scenarios which are dependent on keeping extensive libraries of plasmids, bioreactors for production, and the use of bacteria (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Due to these downsides, there are many transfection applications \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e that are not well served by viral vectors. Despite efforts being made to increase the shelf-life, yields and purity of production in viral vectors (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), there is a strong incentive to develop a non-viral transfection method that is comparable to viral vectors in transfection efficiency that is more accessible and can deliver a greater variety of cargo.\u003c/p\u003e\u003cp\u003eOne alternate solution would be electroporation, which occupies the smallest market share because of high cell cytotoxicity (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) with an estimated value of between 232\u0026ndash;739\u0026nbsp;million USD in 2022 (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). It requires the use of specialty equipment that restricts its therapeutic abilities to \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e applications (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) but provides a reliable method of intracellular delivery of a wide range of biomolecules (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Electroporation\u0026rsquo;s use in therapies is limited by the necessity of secondary safety measures such as heat sinks, requiring secondary drug treatments to be effective, and some electroporation techniques lack clear mechanisms of action generating skepticism on their efficacy (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast to viral vectors and electroporation, lipid nanoparticles are a commonly used transfection method in the lab (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and clinical applications (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). LNPs have the largest market share with the two leading LNP product producers, Pfizer and Moderna, reported sales of 37 and 17.7\u0026nbsp;billion USD respectively for LNP products in 2022 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This relatively large market share is due to recent developments in LNP technology generating cheaper (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), effective (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and easier to use LNP formulations (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) with lower immunogenic potential (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLNPs are limited in what biomolecules they can carry (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), decrease cell viability (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) and transfect with decreased efficiency when delivering large plasmids (\u0026gt;\u0026thinsp;5 Kb) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). LNPs are unstable without ideal storage conditions (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and have a limited shelf life once formed (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The limitations of LNPs demonstrate a need for an alternative transfection method that can provide a wide range of biomolecule delivery, low toxicity, and ease of use.\u003c/p\u003e\u003cp\u003eOne approach that has shown promise is the use of discrete, functionalized, biocompatible carbon nanotubes as a transfection reagent. Since their discovery in 1991 (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), carbon nanotubes have radically altered diverse fields such as energy storage (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and robust, durable polymers (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) while showing potential in biotechnology (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Despite their significant potential, carbon nanotubes have not been commercialized at scale for biotechnology. The full value of carbon nanotubes in biology can only be unlocked when using functionalized, individual nanotubes that are cleaned of residual catalyst and separated from their aggregated bundles, making their high aspect ratio, large surface area, and customizable surface chemistry accessible.\u003c/p\u003e\u003cp\u003eCarbon nanotubes have been observed inducing cell proliferation (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), delivering proteins \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), acting as a transfection agent (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), detecting different cell properties and analytes as biosensors (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), and releasing loaded drugs on demand in hydrogel systems (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Despite the variety of unique phenomenon carbon nanotubes have shown, we believe their full potential for transfection is yet to be proven. Since 2004, a variety of methods of surface functionalization and types of carbon nanotubes have been used as transfection agents in research contexts, but to our knowledge, they are not commonly used for transfection commercially.\u003c/p\u003e\u003cp\u003eAs made, unfunctionalized carbon nanotubes are poor reagents for transfection due to their potential to aggregate and inability to effectively load DNA in a reversible manner. Carbon nanotubes can however be covalently functionalized with different functional groups and noncovalently dispersed with a range of surfactants to improve dispersibility and enable favorable binding to biomolecules (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The size of nanotubes that have been able to cross cellular membranes are reported to be between 20 nm and 1 \u0026micro;m in length and between 1 nm to 60nm in diameter, with different mechanisms of uptake theorized (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). The cell internalization efficiency is dependent on the surface chemistry and surfactants utilized (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent years, functionalized carbon nanotubes have been shown to bind to a variety of biomolecules including mRNA (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), DNA (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), proteins (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), and small molecule drugs (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). The demonstrated binding range suggests the capabilities of carbon nanotubes as a multifaceted transfection tool, though most of the carbon nanotube transfection literature lacks a direct comparison between carbon nanotubes and other transfection methods.\u003c/p\u003e\u003cp\u003eThis work introduces a novel formulation of discrete oxidized multiwalled carbon nanotubes (do-MWCNTs) as a safe and effective transfection agent. This formulation was designed to minimize toxicity and maximize transfection efficiency through dual surfactant surface chemistry. Hydroxyl DSPE-PEG-NH\u003csub\u003e2\u003c/sub\u003e, an amphiphilic surfactant, is non-covalently bound to the carbon nanotube surface to obtain stable dispersions followed by physisorption of polyethylene-imine (PEI) acting as a surfactant and to stimulate DNA binding. This dual-surfactant carbon nanotube formulation is evaluated for delivering plasmid DNA, the subsequent gene expression and its resulting toxicity profile compared to the leading standard of \u003cem\u003ein vitro\u003c/em\u003e transfection, Lipofectamine 3000.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMWCNTs\u003c/h2\u003e\u003cp\u003eThe MWCNTs were functionalized by Molecular Rebar Design LLC using a combination of concentrated nitric acid and high energy dispersion methods. After extensive washing to remove potentially toxic residual catalysts from the MWCNT synthesis the MWCNT was provided in the form of a wet cake of ~\u0026thinsp;6% weight solid nanotube content.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMWCNT Dispersion\u003c/h3\u003e\n\u003cp\u003eThe 6% wt. MWCNT wet cake was processed in water with a solids percent not exceeding 1% wt. The primary surfactant, DSPE-PEG-Amine (2000 MW manufactured by Laysan Bio) was added to this dilute wet cake and vortex mixed. This mixture was then bath sonicated for 30 minutes at room temperature to disperse the oxidized carbon nanotubes before the addition of branched polyethylene-imine (bPEI, 270k MW manufactured by Sigma Aldrich). Bath sonication was then performed again for 90 minutes at room temperature. The dispersion was then filtered via dialysis to remove unbound surfactants. This dialysis is done with a 1,000,000 MW cutoff. It is assumed that this dialysis removes a majority of the unbound surfactant. The filtered dispersions were then autoclaved to sterilize the solutions and subsequently re-sonicated for another total of 120 minutes and centrifuged at 20,000 gs for 10 minutes to remove any larger particles.\u003c/p\u003e\n\u003ch3\u003eSWCNTs\u003c/h3\u003e\n\u003cp\u003eThe Single-Wall Carbon Nanotubes were functionalized by Molecular Rebar Design LLC and was provided to BioPact in a wet cake of ~\u0026thinsp;10% wt. solid nanotube content.\u003c/p\u003e\n\u003ch3\u003eSWCNT Dispersion\u003c/h3\u003e\n\u003cp\u003eThis dispersion was composed of the same surfactants as do-MWCNTs. The calculations for this dispersion were based on the theoretical surface density of surfactant per individual nanotube for only the exterior wall. This is because the interior of do-MWCNTs (about 5nm) and the space between walls (0.34 nm) is inaccessible to large molecules or micelle-like surfactants. SWCNTs have interior diameters in the range of about 0.7 to 1.5 nm but have a strong tendency to rope together which can reduce their effective surface area. To generate a working solution for SWCNT dispersion, a more intensive probe sonication was used with SWCNT and water added prior to the addition of surfactants to increase the available surface area of the SWCNT for the surfactants, when added, to adhere to. Further information on these calculations and assumptions made are available in supplementary information.\u003c/p\u003e\n\u003ch3\u003ePlasmids Used\u003c/h3\u003e\n\u003cp\u003eIn transfection studies that did not investigate the loading of large plasmids, pTRIOZ_hIgG1 (Invivogen: ptrioz-higg1) was used with HIV-specific antibody variable regions (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) and tagged with an IL-2 secretion tag provided by Invivogen. The total plasmid size was 8.78 kb. Cloning and manufacturing was done by VectorBuilder USA.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eLoading Experiments\u003c/h2\u003e\u003cp\u003eTo evaluate the DNA loading capability of different dispersions under a constant separatory force, we used gel electrophoresis. do-MWCNT is extremely large compared to DNA, thus making it difficult for it to traverse as quickly as a large DNA construct. A do-MWCNT dispersion is loaded with DNA for four hours before being placed in a 1% wt. Agarose gel (Thomas Scientific: J66501.30) and run at 100 V for 30 mins. This concentration of agarose gel provides a pore size that allows DNA to move through the gel while the larger carbon nanotubes are unable to move. The amount of DNA offloading can be visualized as a separate band from the dark dispersion band.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003edo-MWCNT Dosing\u003c/h3\u003e\n\u003cp\u003eA standard dose of do-MWCNTs consists of an approximate ratio of 20 \u0026micro;g of discrete MWCNT dispersion to 1 \u0026micro;g of pDNA in a total of 100 \u0026micro;L of DEPC treated water. This was stored in 4\u0026deg;C or 48 hours prior to use unless otherwise stated in studies relating to incubation time.\u003c/p\u003e\n\u003ch3\u003eCell Line and Plating\u003c/h3\u003e\n\u003cp\u003eCHO-K1 cells were purchased from ATCC (ATCC, CHO-K1 CCL-61). CHO-K1 cells were grown, maintained and plated in F-12K\u0026thinsp;+\u0026thinsp;10% FBS. CHO-K1 cells were plated in all subsequent experiments at 2x10\u003csup\u003e5\u003c/sup\u003e cells/mL in 12-Well plates.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCell Incubation Time\u003c/h2\u003e\u003cp\u003eAfter the cells were allowed to settle to the bottom of the plate, each was given a total volume of 100 \u0026micro;L of their respective treatment group described above. After this, the cells were allowed to incubate for 72 hours prior to subsequent processing for qPCR. In the lipofectamine 3000 containing study, we extended the post-treatment incubation period to the highest recommended time of 96 hours prior to analysis as per the lipofectamine 3000 protocol to provide optimal conditions. Cells were maintained at consistent temperature and humidity of 37\u0026deg;C and 90+% with 5% CO2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCell Growth Data\u003c/h2\u003e\u003cp\u003eCells were counted using a Countess II cell counter with Trypan Blue stain (Included in ThermoFisher: T10282). do-MWCNTs interferes with trypan blue staining by causing cells to appear darker, thus preventing an accurate live/dead ratio. A total cell count was taken instead. To avoid biasing cell counts with dead cells, all groups on a 12-well plate had their media removed and were washed with 1X PBS (ThermoFisher: 10010023) to remove any non-adherent cells before the addition of Trypsin (ThermoFisher: 25200056) and inactivation in the cell-appropriate media, and then resuspended in 333 \u0026micro;L of 1X PBS and mixed via pipetting. 20 \u0026micro;L of the PBS cell solution was taken and 20 \u0026micro;L of trypan blue was added.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCell RNA Extractions and RT\u003c/h2\u003e\u003cp\u003eAfter the cells have been incubated for the appropriate amount of time and cell counts have been taken, the cells are centrifuged and the excess media is removed. 500 \u0026micro;L of TriZol (ThermoFisher: 15596026) and 50 \u0026micro;L of Chloroform are added to the cell pellet to begin the extraction of RNA which is performed according to the TriZol protocol scaled for the volumes reported. Once an RNA pellet is produced, the pellet is resolubilized in 20 \u0026micro;L of DEPC treated water. Concentrations are taken and normalized to an end concentration of 1 \u0026micro;g per 10 \u0026micro;L for cDNA production. This normalization prevents biasing results downstream due to differences in the cell count. This method is established in literature for studying transfection between transfection agents (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). cDNA is produced with the High-Capacity cDNA reverse transcription kit with RNase inhibitor (ThermoFisher: 4374966) using 1 \u0026micro;g of RNA per reaction for each respective group. Each of the reactions is performed in an Applied Biosstems MiniAmp Plus and diluted to an appropriate volume for the qPCR process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eqPCR\u003c/h2\u003e\u003cp\u003eThe genes screened by qPCR are Beta-Actin (ActB), Aldo-Keto-Reductase (AKR), Human IgG constant region heavy chain, Human IgG constant region light chain, VRC01 HIV-Specific Antibody Light Chain region (VRC01), and PGDM HIV-Specific Antibody Heavy Chain region (PGDM). ActB and AKR are the two endogenous controls, VRC01 is our experimental group. PGDM is used as the negative control due to the similarities in characteristics to VRC01, as they are both antibody-encoding for a variable region, approximately the same size and are both as specific to a sequence. The qPCR reagent used in this study is SYBR green master mix (ThermoFisher: 4309155) and any dilutions performed are with DEPC treated water. The qPCR reactions are performed as specified by the Comparative Ct Protocol in the Applied Biosystems Quantstudio 3. All data is interpreted through QuantStudio Design and Analysis software. In early studies, Human IgG constant regions were analyzed due to the commercial availability of these primers but in subsequent studies, for increased confidence in the specificity of our studies, we used VRC01 light-chain sequences. Data for multi-plate studies was done using appropriate inter-run controls and, where possible, multiple inter-run controls. qPCR methods and practices were based on suggestions from literature (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eLoading Conditions\u003c/h2\u003e\u003cp\u003eTo be both non-cytotoxic and favorably interact with plasmid DNA, a carbon nanotube formulation with dual-surfactant surface chemistry was developed. First MWCNTs are dispersed using DSPE-PEG-Amine which has been shown to yield discrete and non-cytotoxic nanotubes at a range of concentrations \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). A secondary surfactant, branched PEI, was added for its high positive charge density to encourage DNA-nanotube complexing, promote cellular internalization, and facilitate endosomal release (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). The optimum relative mass ratio of do-MWCNTs, DSPE-PEG-NH\u003csub\u003e2\u003c/sub\u003e, and PEI was determined by testing a range of ratios and selecting the formulation that yielded the dispersion with no aggregates under brightfield microscopy.\u003c/p\u003e\u003cp\u003eOnce the relative masses of surfactants were chosen, the plasmid DNA binding was optimized by using agarose gel electrophoresis to evaluate two formulation parameters: the molecular weight of the PEI and mass ratio of do-MWCNTs to plasmid. The molecular weight of branched PEI plays an important role in the conformations available to the pDNA-do-MWCNT complex, and some molecular weights could be preferred over others. Additionally, the relative amounts of pDNA and do-MWCNT complex played a significant role in surface charge density and stability in solution, impacting downstream cellular internalization. Three different molecular weights of PEI were tested for their binding efficiency with plasmid DNA, 10 kDa, 25 kDa, and 270 kDa branched PEI.\u003c/p\u003e\u003cp\u003eIn this study, 1% agarose gel electrophoresis allows for the larger do-MWCNT\u0026thinsp;+\u0026thinsp;pDNA complexes to remain at the start of the gel while unbound pDNA or loosely bound pDNA will travel further from the well. A complexed dispersion with a lack of offloaded DNA in the gel environment can indicate a stronger association between the pDNA and do-MWCNT, which is believed to be important for transfection strength. This method also simulates a constant force and charged environment that could encourage weak complexes to break down. With 100 ng of plasmid DNA, do-MWCNT was added in increasing ratios from 2.5:1 to 20:1 do-MWCNT to DNA mass ratios. After the dispersion had incubated with the pDNA for three hours at 4\u0026deg;C they were loaded on the gel.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn this simulated offloading environment, low brightness in an individual lane indicates high pDNA association with do-MWCNT, as do-MWCNT quenches the fluorescence while a higher brightness indicates low pDNA association with do-MWCNT, indicative of pDNA offloading. By comparing the relative brightness of each well offloaded pDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we can determine that of the three molecular weights of PEI, 270 kDa was able to inhibit pDNA migration the most. In all the molecular weights of PEI, 20:1 of do-MWCNT to pDNA was able to inhibit pDNA migration greater than the other mass ratios. Subsequent testing will be performed using the 270 kDa PEI to optimize the performance of the mass-loading ratio for transfection with 20:1 as the reference ratio of do-MWCNT to pDNA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLoading qPCR\u003c/h2\u003e\u003cp\u003eIn order to validate the loading ratio 20:1 that was observed to create the highest association between do-MWCNT and pDNA, we needed to observe ratios above and below 20:1 in a transfection study. 1 \u0026micro;g of pDNA was loaded at three ratios, 10:1, 20:1 and 25:1 do-MWCNT to pDNA in DEPC water. These solutions were loaded at 4\u0026deg;C for 7 hours. CHO-K1 cells were treated with their respective do-MWCNT-pDNA complexes and left to incubate for 72 hours. We utilized qPCR to determine which of the mass-loading ratios would cause greater expression of genes unique to our plasmid and thus, function as a better transfection agent.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the 20:1 Mass-loading group significantly outperformed both groups (**** both 1:10 and 1:25) loaded at higher and lower ratios, indicating that subsequent optimization experiments should use this loading ratio. More information on the proposed significance of this mass loading ratio is available in the supplementary information section.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEffect of DNA Loading Temperature\u003c/h2\u003e\u003cp\u003eTo characterize the incubation conditions of do-MWCNT\u0026thinsp;+\u0026thinsp;pDNA, we chose three relevant temperatures to form the complex in. Refrigeration temperature, 4\u0026deg;C, Room temperature, 27\u0026deg;C, and Biologically relevant temperature, 37\u0026deg;C. These solutions were left to load for 7 hours in their respective environment. CHO-K1 cells were treated with their respective groups and allowed to incubate for three days. qPCR was performed to determine which group maximized gene expression. Tests were begun for the variable chain region of our plasmid due to the increased confidence that a highly specific primer set would afford.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the relative expression of pDNA was clearly negatively affected by temperature with a significant (**) drop observed between 4\u0026deg;C and 37\u0026deg;C. Subsequent experiments used 4\u0026deg;C for incubation of do-MWCNT\u0026thinsp;+\u0026thinsp;pDNA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eTime of DNA Loading\u003c/h2\u003e\u003cp\u003eTo determine the amount of time do-MWCNT and pDNA should be incubated together prior to transfection, loadings of do-MWCNT and pDNA at intervals of 1 hour, 3 hours, 7 hours, 24 hours, and 32 hours were made. CHO-K1 cells were treated with their respective groups and allowed to incubate for three days. This experiment was split between two qPCR studies with appropriate inter-run controls for normalization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, there was a significant increase in expression of the plasmid as the complexing time of do-MWCNT and pDNA was increased. Based on these findings, a subsequent study was conducted to observe the peak of the transfection efficiency. do-MWCNT was loaded at 32 hours, 48 hours, 96 hours, and 168 hours.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, a peak in transfection efficiency was observed at 96 hours of pDNA and do-MWCNT incubation with significance relative to 48 hours and 168 hours (**** and *** respectively). To determine if this increase in transfection efficiency would increase cytotoxicity, cell counts were conducted of the longer incubation period groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn transfection, it is often observed that increased transfection results in an increase in cytotoxicity (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). In contrast, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the extension of incubation time of the do-MWCNT\u0026thinsp;+\u0026thinsp;pDNA increased in transfection-related gene expression without a decrease in cell population. A significant drop-off in the average cell population was however observed at the 168-hour time point in reference to the 48-hour time point. This decrease in average cell population began between 48 hours and 96 hours (not significant) and concluded with a significant drop in cell health at 168 hours. Because the difference between 48 hours and 96 hours, and 96 hours and 168 hours is insignificant, it is believed that in conjunction with the peak in transfection, 96 hours is the best transfection when considering toxicity effects. This drop in cell health at 168 hours is believed to be related to the formation of aggregates of the do-MWCNT\u0026thinsp;+\u0026thinsp;pDNA complex that forms accumulations or flocculants that are known to have deleterious effects to cell health (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). With appropriate Inter-run controls, the data is normalized across the multiple transfection studies in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven the dramatic difference in the magnitude of expression observed, 96 hours was used in subsequent transfection studies. Significant (****) differences were seen between 48 hours, 96 hours and 168 hours in the gene expression of transfected pDNA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003edo-MWCNT vs Lipofectamine 3000 in Cell Cytotoxicity\u003c/h2\u003e\u003cp\u003eTo determine if an optimized do-MWCNT\u0026thinsp;+\u0026thinsp;pDNA complex is capable of transfection comparable to current products, a do-MWCNT dispersion was loaded for 96 hours at 20:1 do-MWCNT:pDNA in 4\u0026deg;C against Lipofectamine 3000 (ThermoFisher L3000001) in CHO-K1 cells. A cell count was made after cells had incubated with their respective transfection agent for 5 days. The bed of adherent cells was washed with PBS to ensure that dead, nonadherent cells would be excluded from this count. Lipofectamine was prepared according to the provided protocol. Half-doses of both transfection reagents were prepared to show a dose-dependent relationship in transfection and cell counts.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe population of CHO-K1 cells treated with lipofectamine observed a significant (***) 30% drop in cell population compared to the no treatment group as the baseline as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. do-MWCNT treated groups and the half dose lipofectamine group experienced no significant deviation from the no treatment group, indicating minimal impact to cell health.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003edo-MWCNT vs Lipofectamine 3000 in Transfection\u003c/h2\u003e\u003cp\u003eAfter cell counts were completed, qPCR was conducted to measure VRC01 specific sequences in CHO-K1 cells via the described method. With the no treatment group acting as the baseline for normalization, do-MWCNT was able to generate a significant (*) average of 11% higher expression of pDNA than Lipofectamine 3000 in a full dose as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis method of qPCR testing would prevent an increase in cell population from affecting the ratio of endogenous gene to VRC01 antibody-specific gene sequences. Because of this methodology choice, it is concluded that do-MWCNT is capable of higher rates of gene expression per cell at full dosing conditions and implies a higher transfection rate than Lipofectamine. do-MWCNT\u0026rsquo;s ability to transfect and effect a higher expression level than Lipofectamine 3000 is unique in its ability to not affect the population of cells and change transfection rates independently of cell population until 168 hours of do-MWCNT\u0026thinsp;+\u0026thinsp;pDNA incubation. It was determined that Lipofectamine outperformed do-MWCNT when the dosage was halved.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eSingle Walled CNT Translation from do-MWCNT\u003c/h2\u003e\u003cp\u003eTo determine the effects of different tube geometries on transfection, we set out on making an oxidized single-wall nanotube dispersion using the same surfactants and methodology of testing as do-MWCNT. This geometry and oxidation was chosen because of the benefits inherent in single-wall nanotubes in biology; Oxidized single wall nanotubes are far more likely to be broken down by cells, single walls are smaller and more likely to passively enter the cell, and have no interior walls, thus no biologically inaccessible surface area than multi-wall nanotubes. Due to structural stability limitations, we were unable to further oxidize the single wall nanotubes beyond half the oxidation of do-MWCNT.\u003c/p\u003e\u003cp\u003eThe approach used to translate the formulation from do-MWCNT to SWCNT was based on a particle matched ratio of the components of do-MWCNT wherein the surface of both formulations would effectively have the same density of each surfactant component and DNA. The values used to generate these surfactant concentrations per area of the nanotubes are available in the supplementary information section. At the particle matched ratio of 10:27.7:17 nanotubes:DSPE-PEG-NH\u003csub\u003e2\u003c/sub\u003e:270k bPEI, there were no visible flocculants.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eTransfection of SWCNT\u003c/h2\u003e\u003cp\u003eThe ratio of Nanotubes:DNA when translated based on surface area on a per-particle basis was 0.71:1 which translated to an approximate mass ratio of 2.1:1. This mass ratio SWCNT:pDNA was loaded for 96 hours to compare single walls to the previous do-MWCNT dispersion.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e demonstrates that transfection still occurred after altering the underlying nanoparticle geometry. Due to the reduced amount of nanomaterial used for this study, it was decided to test if an increased dose would produce higher gene expression without hitting the dosage limit to produce deleterious effects to cell health.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eUpper Limit of SWCNT Transfection\u003c/h2\u003e\u003cp\u003edo-MWCNT\u0026rsquo;s maximum dose was established to be around 20 \u0026micro;g of nanotubes for 1 \u0026micro;g of DNA due to a downturn in cell health. Because of the significant reduction in the mass of SWCNT added to the same amount of DNA, it was surmised that the upper limit of transfection with SWCNT was not reached. Most transfection agents are limited by the transfection efficiency of a dose and the toxicity of that dose. The lipofectamine protocol even includes separate ratios to try for their two-component system to optimize for better transfection which implies that the highest recommended dose is the limit of its transfection abilities in the conditions tested. To find the upper limit of SWCNTs 1x, 2x, and 4x of the initial dose given was examined.\u003c/p\u003e\u003cp\u003edo-MWCNT was included at 1x and 2x concentrations to illustrate that it has achieved close to the peak of its ability to transfect with maximum gene expression without interfering with cell health.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e, the increasing dosages of SWCNTs do not show a significant increase in toxicity when compared to do-MWCNT that has a significant decrease in cell count at the 2x dose.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e shows that as the dosage increases of the single-wall transfection agent, the transfection decreases. It is likely that because the single-wall particle count was based off of the same number of particles present in the maximum dose of the do-MWCNT multi-wall agent, that the limiting factor in carbon nanotube transfection may be related to aggregation of the carbon nanotubes. Even though SWCNTs are far smaller in diameter and are on average for the present tubes, 22% longer than do-MWCNT, the addition of large surfactants like 270k bPEI generates a much larger surface area per nanotube. It may be that the maximum dose for carbon nanotube transfection systems is related to the number of individual carbon nanotubes. In both do-MWCNT and SWCNT systems, both contained approximately 9.04 x 10\u003csup\u003e10\u003c/sup\u003e carbon nanotubes per mL.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdvantages of SWCNT over do-MWCNT\u003c/b\u003e\u003c/p\u003e\u003cp\u003edo-MWCNT has previously been tested with eGFP containing plasmids. It was observed that, despite relatively high gene expression of transfection related sequences, the observed fluorescence was minimal.\u003c/p\u003e\u003cp\u003eFigure 14: eGFP plasmid transfection from do-MWCNT after 3 days of incubation post-transfection. A.) Fluorescent view of do-MWCNT treated CHO-K1 cells. B.) Brightfield image of do-MWCNT treated CHO-K1 cells with fluorescent overlay.\u003c/p\u003e\u003cp\u003eIt is believed that do-MWCNT\u0026rsquo;s ability to quench fluorescence is amplified by the cells accumulating visible amounts of nanotubes around or inside them as seen in Fig.\u0026nbsp;14.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe visible expression of eGFP by SWCNTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e15\u003c/span\u003e) shows that do-MWCNT and SWCNTs, despite having different transfection efficiencies, might pose different advantages depending on the end-user application.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe use of multi-walled carbon nanotubes as a transfection agent was first identified in 2005 (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) where carbon nanotubes were functionalized with ammonia to generate a strong positive charge on the surface of the nanotube. The loading ratios tested in that study were from 1:1 to 10:1 f-CNT:DNA with 30 mins of complexing time. There was no comparison for this tested transfection agent against any other commercial transfection agent, so there is no way to directly compare the results of these studies. However, that publication shares most of the same concerns as this one in regard to DNA binding.\u003c/p\u003e\u003cp\u003eThe long complexing time, 96 hours, and relatively high ratio required to complex do-MWCNT and pDNA (20:1, compared to literature) implies that the concerns of pDNA binding too tightly to the nanotube is less of an issue as otherwise stated in previous studies. One concern brought up in that previous publication\u0026rsquo;s discussion is that SWCNT modified with ammonia exhibited weaker DNA complexes even in higher loading ratios than multiwall carbon nanotubes. This may be due to the ability of a specific DNA molecule to wrap the respective nanotube geometry compared to the larger MWCNTs.\u003c/p\u003e\u003cp\u003eLarge plasmid (\u0026lt;\u0026thinsp;5kb) delivery has been considered as a requirement for use of CRISPR, especially given the search for alternative nonviral strategies that can address the limitations of other transfection methods such as viral vector efficacy and toxicity (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). It would be beneficial to see if do-MWCNT shares the general limitations of other transfection agents where transfection efficiency begins to drop with the use of larger plasmids.\u003c/p\u003e\u003cp\u003eThe plasmid DNA used here encodes for VRC01 (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) Human IgG Anti-HIV Antibodies. The higher expression of these antibody sequences by do-MWCNT transfection could be useful for future therapeutic purposes or biomanufacturing in mammalian systems, lowering the required dose for effective therapeutic efficacy.\u003c/p\u003e\u003cp\u003eAn advantage that do-MWCNT affords as a transfection reagent is a lower material cost than Lipofectamine 3000. Lipofectamine requires a specific media type, Opti-MEM, as per the ThermoFisher protocol for optimal transfection. do-MWCNT is optimized for complex formation in a sterile water environment and like Lipofectamine 3000, can be treated directly into serum-containing media. do-MWCNT is also a single-component system and does not require the creation of multiple solutions for optimal complex formation, rather, a specific order of addition is necessary for optimal do-MWCNT loading.\u003c/p\u003e\u003cp\u003eOne of the primary drawbacks of do-MWCNT is the formation time of the complex. The increase in gene expression in relation to loading time is implicative of a slow physical interaction between the surfactant structural configuration and the coiling configuration of the pDNA. Sonication has been theorized as a potential solution that would enable pDNA to form into a more adaptable configuration for do-MWCNT. Solving the high-incubation time barrier would significantly increase adoption of a CNT-based transfection agent.\u003c/p\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003eSWCNT Implications\u003c/h2\u003e\u003cp\u003eThe translation of this transfection phenomenon across different carbon nanotube geometries implies that carbon nanoparticles, when dispersed in similar surfactants at nearly identical conditions in the surfactant ratio per surface area unit, will not behave identically. This likely means that there is an optimal geometry of carbon nanotube for pDNA transfection that was not fully examined in this present study.\u003c/p\u003e\u003cp\u003eOne of the most important features of using SWCNT over do-MWCNT is that even when the mass of nanotubes used was decreased by ~\u0026thinsp;15x, we were able to achieve about half the transfection capability of do-MWCNT which implies a greater efficiency per unit of carbon by SWCNT. Not only is it more efficient in terms of carbon, but surfactant used per unit of transfection. To achieve the same level of transfection for do-MWCNT, ~2x the amount of surfactant must be used per dose. This data on translating transfection-optimized multi-walled carbon nanotubes to appropriately surface-area scaled single-walled system implies a significant decrease in associated costs of producing nanotube-based transfection agents. Scaled on the data in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, we can find the price per unit of RQ to find the most cost-effective transfection agent. Lipofectamine scales to 2.3x10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e USD per RQ, do-MWCNT scales to 6.4x10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e USD per RQ and SWCNT scale to 1.2x10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e USD per RQ. Based on this efficiency, do-MWCNT is 36 times cheaper per unit of transfection than Lipofectamine 3000 while achieving the same magnitude of transfection as Lipofectamine 3000. SWCNT are only 18.5 times cheaper than Lipofectamine and do not achieve the same magnitude of transfection but show far less toxicity than both do-MWCNT and Lipofectamine 3000.\u003c/p\u003e\u003cp\u003eThe prices here are calculated based on the chemical suppliers of surfactants and CNTs for BioPact at scale production. DNA is not included in the cost analysis. On a single dose for a 12-Well plate constituting 1 \u0026micro;g of DNA for the maximum recommended dose of Lipofectamine 3000, Lipofectamine\u0026rsquo;s retail cost per dose is 2.725 USD including the cost of Opti-MEM. On a material cost provided by Molecular Rebar Design LLC, for 1 \u0026micro;g of DNA transfection, do-MWCNT is 0.084 USD per dose. SWCNT, on the same scale as do-MWCNT, scales to 0.07 USD per dose.\u003c/p\u003e\u003cp\u003eA further advantage of SWCNTs is the likelihood of biological systems being able to break down the administered nanotubes. do-MWCNTs have been shown to be degraded in some specific cell types by the use of oxide species such as peroxides (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Carbon nanotubes with a higher oxidation state are easier to degrade by enzymes (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). do-MWCNTs, despite being capable of higher surface oxidation than SWCNTs, have the highest oxidation state on the exterior of the nanotube which makes the exterior wall the easiest to degrade. It is thought that the interior walls are almost entirely without functionalization and are harder to degrade. Once the oxidized outer layer begins to degrade, surfactant may begin to dissociate and cause both aggregation and a lower rate of degradation. Because SWCNTs have only one wall to break down, they are not only more likely to be degraded by natural cell processes, but less likely to aggregate as they are broken down due to surfactant disassociation and hold the advantage of requiring less mass of SWCNTs per treatment than do-MWCNTs.\u003c/p\u003e\u003cp\u003eSWCNTs, per unit of mass, have lower absorption than do-MWCNTs. This provides SWCNTs with a significant advantage over do-MWCNTs in transfection applications, wherein do-MWCNTs have been shown to quench fluorescence by reporter genes. SWCNTs have enabled fluorescence applications in a CNT-based system for alternate or expanded detection methodologies such as flow cytometry.\u003c/p\u003e\u003cp\u003eEven though the previously mentioned study (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) did not consider the per-particle ratio changes, they noted that SWCNT showed less binding capabilities for pDNA than multi-walled carbon nanotubes. It is believed that the inclusion of bPEI scaled according to the particle count was crucial to alleviating this binding issue by making the binding activity dependent on the bPEI\u0026rsquo;s behavior in solution rather than the surface area of the CNT that was exposed to the solution.\u003c/p\u003e\u003cp\u003eOverall, this study has shown the promising nature of carbon nanotubes as a commercially viable transfection agent with similar transfection magnitude to commercially available solutions. In its current state, it may pose as another option to explore when other transfection agents have failed to produce desired results.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003edo-MWCNT \u0026ndash; Discrete, Oxidized Multi-Walled Carbon Nanotubes\u003c/p\u003e\n\u003cp\u003eSWCNT \u0026ndash; Single Walled Carbon Nanotubes\u003c/p\u003e\n\u003cp\u003eCNT \u0026ndash; Carbon Nanotubes\u003c/p\u003e\n\u003cp\u003epDNA \u0026ndash; Plasmid DNA\u003c/p\u003e\n\u003cp\u003eRQ \u0026ndash; Relative Quantification\u003c/p\u003e\n\u003cp\u003eLNP - Lipid Nanoparticle\u003c/p\u003e\n\u003cp\u003ef-CNT \u0026ndash; Functionalized CNT\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConner Quinlan, Aaron Foote, and Kevin Castillo are former employees of Molecular Rebar Design LLC and its subsidiaries. BioPact is a subsidiary of Molecular Rebar Design LLC.\u003c/p\u003e\n\u003ch3\u003eFunding Declaration\u003c/h3\u003e\n\u003cp\u003eFunding was provided by Molecular Rebar Design LLC and contract number W81XWH22C0090 as part of the DoD SBIR program.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConner Quinlan wrote the main manuscript text under the guidance of Aaron Foote. All figures were prepared by Conner Quinlan. Aaron Foote, Kevin Castillo, and Clive Bosnyak reviewed the manuscript and suggested edits. Kurt Swogger and Clive Bosnyak provided materials from Molecular Rebar Design LLC.Corresponding Author: Conner Quinlan\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis material is based on work supported by the US Army Medical Research and Development Command (USAMRDC) or U.S. Army Medical Research Acquisition Activity (USAMRAA) under contract No. W81XWH21P0101 and W81XWH22C0090 from 2021 to 2025. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the US Army Medical Research and Development Command (USAMRDC) or U.S. Army Medical Research Acquisition Activity (USAMRAA).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eNormalized data from individual replicates of associated qPCR studies is available in supplementary information. Cell counts with individual replicates are available in supplementary information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKim TK, Eberwine JH. Mammalian Cell transfection: the Present and the Future. Analytical and Bioanalytical Chemistry. 2010;397(8):3173\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFajrial AK, He QQ, Wirusanti NI, Slansky JE, Ding X. A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing. 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Nanoscale. 2011;3(3):893\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioscience-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioscience Nanotechnology](https://biosciencenanotechnology.biomedcentral.com/)","snPcode":"44331","submissionUrl":"https://submission.springernature.com/new-submission/44331/3","title":"Bioscience Nanotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Single Wall, Multi Wall, Carbon Nanotube, Transfection, Plasmid DNA, Lipofectamine","lastPublishedDoi":"10.21203/rs.3.rs-8108596/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8108596/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eCarbon nanotubes have been identified as a possible plasmid and drug delivery method but are not developed enough to be as effective compared to other commercial methods such as lipid nanoparticles, electroporation and viral particles due to cytotoxicity and non-competitive transfection rates.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eHere, a stable dispersion of discrete, oxidized multi-walled carbon nanotubes (Fig.\u0026nbsp;1) was developed (do-MWCNT) using a lipid-like surfactant, DSPE-PEG-NH\u003csub\u003e2\u003c/sub\u003e 2000 MW, and a cationic polymer 270k MW bPEI, that demonstrate better transfection-related gene expression with less cytotoxicity than commercially available lipid nanoparticles. The stable dispersion was then used to deliver a plasmid of 8.78kb with expression measured via qPCR that was comparable to Lipofectamine 3000. Analogous studies with discrete functionalized single wall carbon nanotubes showed further improvements with cell health.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eCarbon nanotubes are capable of comparable transfection to lipid nanoparticles only after plasmid DNA is incubated with a dispersion for an extended period of time compared to lipid nanoparticles, 96 hours. This implies the limiting mechanism of nanotube transfection is pDNA binding. The novelty of single walled carbon nanotubes is also demonstrated by matching the surface surfactant conditions between the single and multi-walled carbon nanotube dispersions. It is further implied that the optimal geometry of carbon nanotube for transfection has yet to be explored.\u003c/p\u003e","manuscriptTitle":"Discrete, Oxidized Multi-Walled Carbon Nanotubes for Efficient Plasmid DNA Transfection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-21 07:57:14","doi":"10.21203/rs.3.rs-8108596/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-28T15:05:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-27T12:15:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18500525939368043037121364747833763576","date":"2026-01-27T12:06:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-26T17:40:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129426195249103503269801479664953293196","date":"2026-01-08T13:12:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-01T10:05:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-25T00:07:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-25T00:07:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioscience Nanotechnology","date":"2025-11-13T19:13:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioscience-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioscience Nanotechnology](https://biosciencenanotechnology.biomedcentral.com/)","snPcode":"44331","submissionUrl":"https://submission.springernature.com/new-submission/44331/3","title":"Bioscience Nanotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"51b7dadf-6eda-4769-b2fe-c15ecf5267af","owner":[],"postedDate":"November 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:40:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-21 07:57:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8108596","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8108596","identity":"rs-8108596","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}