Article pubs.acs.org/molecularpharmaceutics
Next Generation Carbon Nanoparticles for Efficient Gene Therapy Santosh K. Misra, Ayako Ohoka, Nicholas J. Kolmodin, and Dipanjan Pan* Department of Bioengineering, University of Illinois Urbana−Champaign, Urbana, Illinois 61801, United States Beckman Institute, Biomedical Research Center, Carle Foundation Hospital, Urbana, Illinois 61801, United States S Supporting Information *
ABSTRACT: In a pursuit to develop a commercially exploitable and traceable gene delivery vehicle, here, we develop next generation carbon nanoparticle-DNA complex (CNPLex). CNPLexes were used to transfect green fluorescent protein (GFP) reporter gene containing plasmid DNA (pDNA) pEGFP-N1 targeting breast cancer cells MCF-7 and MDA-MB231. Prepared CNPs were optimized for particle size, surface potential, polymer surface decoration, absorbance efficiency, fluorescence efficiency, IR spectroscopic signatures, and DNA loading and release efficiencies. Rigorous biophysical methods were employed to determine the variations in physicochemical properties of CNPs after surface decoration with polymers followed by complexation with pDNA. Optimized CNPLexes were used to deliver pEGFP-N1 plasmid and efficiency of GFP was followed by fluorescence microscopy and quantified by flow assisted cell sorting. Lipofectamine2000 was used as positive control according to manufacturer’s protocol and found to be comparative in transfection efficiency with one of our novel formulations. Further evaluation of cell toxicity and cell viability was performed by LDH activity and MTT assay, respectively. It was found that cell toxicity furnished by polymer decorated carbon nanoparticles was significantly low compared to the parent polymer (polyethylenimine, PEI). Similarly cell viability was found to be much higher with CNPLexes compared to PEI alone. This established the developed particles as better transfecting agents for reporter gene plasmid pEGFP-N1 compared to PEI and showed similar efficacy to one of the best known commercial transfection agents Liofectamine2000 in breast cancer cells MCF-7 and MDA-MB231. KEYWORDS: gene delivery, nanometer sized agents, carbon particles, breast cancer, gene therapy of PEI on cell membranes inducing necrosis,12 and its undesired interaction with blood components,13,14 make it inappropriate for clinical translation. Added to this pharmacokinetic and organ distribution data demonstrates rapid unfavorable clearance of positively charged polyplexes from circulation.15−17 To decrease the cytotoxicity and extend the blood circulation times of nonviral vectors, various strategies have been used with partial success. A few of these approaches include covalent modification of the complexes with polymers such as poly(ethylene glycol) (PEG) or poly(N-(2hydroxypropyl)methacrylamide) (pHPMA) coupled to poly-
1. INTRODUCTION Gene therapy brings great promise to treatment of various diseases. The use of genes as therapeutic agent opens potential for the treatment of yet incurable diseases, like cancer.1 Myriad advancement has been made to approach efficient gene delivery. The examples of gene delivery vehicles are dominated mostly by viral vectors, which are known to be associated with complications from lethal nature of viruses and possible mutations.2−4 Plethora of nonviral-based strategies for gene delivery have been proposed and demonstrated their efficiency in numerous in vitro and in vivo studies.5 For successful gene therapy, it is necessary to design and develop vectors with low toxicity, sufficient systemic stability, and prolonged circulation time in the bloodstream. A common strategy to deliver genes is to use cationic polymers such as polyethylenimine (PEI).6−10 Although PEI−DNA complexes have shown to have excellent cell transfection capabilities, the intrinsic nature of the material is highly cytotoxic.11 Furthermore, aggregation of huge clusters © XXXX American Chemical Society
Special Issue: Next Generation Gene Delivery Approaches: Recent Progress and Hurdles Received: November 7, 2014 Revised: December 9, 2014 Accepted: December 16, 2014
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Molecular Pharmaceutics cations.18−20 Covalent grafting of lipophilic polymers generates a protective “stealth” shielding similar to PEGylated liposomes.21 These PEG-modified polycations have shown desired neutral zeta potential, very low cytotoxicity, and little or no tendency for aggregation on complexation with pDNA.18,20,22 However, the desired properties can only be achieved if the molecular weight of the parent cationic polymer, chain length, and grafting density of PEG are well-defined, which greatly influence the polyplex size and long-term colloidal stability.23 A superior yet simpler and commercially viable chemical approach is long desired and therefore the focus of our present study. We hypothesize that the introduction of a solid core with ability of traceable fluorescence, strong and reversible noncovalent association with PEI, high DNA loading and release ability, high serum stability, and improved gene transfection can offer a better solution. In a pursuit to develop such translatable gene delivery vehicle, here, we present a method of preparing PEI coated luminescent carbon nanoparticles (CNPEIs). Physicochemically characterized CNPs, PEI decorated CNPs, and CNPLexes were compared for chemical and physical variations with their biological properties. PEI decorated CNPs could produce DNA complexes (CNPLexes) for reporter gene delivery as evident from the in vitro transfection studies in breast cancer cell lines of different estrogen receptor status. We have shown that the prepared CNPLexes would induce improved efficacy and low cell-toxicity in gene delivery compared to parent PEI. Cell internalization and cytoplasmic distribution of CNPLexes were observed by fluorescence microscopic studies.
resuspended in aqueous medium in desired concentrations and stored in germ-free conditions. Postsynthetic coating of CNPs with various amounts of branched polyethylenimine (PEI-10K) were carried out by vortex-incubation method relying on columbic interactions. Prepared suspensions were isolated from unbound PEI by serial centrifugation method. CNPlexes were prepared by coincubating PEI decorated CNPs and pBR322 vector DNA or pEGFP-N1 plasmid DNA at various weight ratios with PEI decorated CNPs for physicochemical and biological experiments (Table 1). Table 1. Composition of Various Formulations Used in Different Experimental Procedures sample name CNPLex-2 CNPLex-1 CNPLex-0.5 CNPLex0.25 CNPLex0.125
DNA (μg; pBR322/pEGFPN1)
PEI (mg per 50 μL of CNP, CNP-M-P)b
agave nectar (g/ mL, CNP-M)a
0.2−3.2 0.2−3.2 0.2−3.2 0.2−3.2
1 1 1 1
2 1 0.5 0.25
0.2−3.2
1
0.125
a
Amount of agave nectar to start the preparation of CNPs and description as sample name where M varies from 2 to 0.125. bAmount of PEI coated on CNP-M samples in achieving CNP-M-P where P is 1 in optimized cases.
2.2. Determination of Hydrodynamic Diameter. Hydrodynamic diameter distribution and distribution averages for the CNPs, PEI decorated CNPs, and CNPLexes in aqueous solutions were determined by dynamic light scattering. Hydrodynamic diameters were determined using a Malvern Zetasizer ZS90 particle size analyzer, while scattered light was collected at a fixed angle of 90°. A photomultiplier aperture of 400 mm was used, with the incident laser power adjusted to obtain a photon counting rate between 200 and 300 kcps. Measurements for which the measured and calculated baselines of the intensity autocorrelation function agreed to within +0.1% were used to calculate hydrodynamic diameter values. Hydrodynamic diameter was analyzed using number distribution in accordance with various previous reports.24 All determinations were made in multiples of 3 consecutive measurements with 15 runs each. 2.3. Stability of CNPLexes in the Presence of Anionic Surfactant and Low pH. CNP-2-1, CNP-1P, CNP-0.5P, CNP-0.25P, and CNP-0.125P (10 μL each) were admixed with 200 ng of pBR322 vector DNA, and the complex was allowed to settle at room temperature for 1 h. To this mixture, 990 μL of water was added followed by the DLS measurements (0 h time point). Subsequently, more DLS measurements were taken at 1, 3, and 6 h time points in solution of pH 4.6 and anionic surfactant SDS in two different sets of the same formulations. 2.4. Determination of Surface Zeta Potential. Zeta potential (ζ) values for the CNPs, PEI decorated CNPs, and CNPLexes in aqueous solutions were determined with a nano series Malvern Zetasizer zeta potential analyzer. Data were acquired in the phase analysis light scattering (PALS) mode following solution equilibration at 25 °C when calculation of ζ from the measured electrophoretic mobility (μ) employed the Smoluchowski equation: μ = εζ/η (where ε and η are the dielectric constant and the absolute viscosity of the medium,
2. MATERIALS AND METHODS CNPs were prepared from nectar (HoneyTree’s Organic Agave Nectar, Onsted, MI). CNPs were sonicated at required parameters (Q700, Qsonica Sonicators, Newtown, CT). Prepared CNPs were filtered by 0.2 μm filter (Millex, Merck Millipore Ltd., Tullagreen, Carrigtwohill, County Cork, Ireland). DLS measurements were taken by Zetasizer Nano S, Malvern Instruments, Malvern, PA). Polyethylenimine (PEI) (Sigma-Aldrich, St. Louis, MO) was used for coating and free PEI removed by centrifugation (Optima MAX-XP Ultracentrifuge, Beckman-Coulter, Brea, CA and 5424 R, Eppendorf, Hauppauge, NY). Gel electrophoresis was performed using pBR322 vector DNA (New England Biolabs, Ipswich, MA) and imaged under Universal Hood III, Bio-Rad, Hercules, CA. Stability of CNPLexes were studied under the influence of heparan sulfate (Sigma-Aldrich). Transfection of pEGFP-N1 followed under fluorescent microscope DMI3000 B, Leica Microsystems, Buffalo Grove, IL. Cytotoxicity was measured by MTT reduction using plate reader (Synergy HT, Bio-Tek) and Pierce LDH Cytotoxicity Kit (Thermo Scientific, Rockford, IL). Biological experiments were performed in MDA-MB-231 and MCF-7 (ATCC) cells. 2.1. General Methods. In order to produce the CNPs, a simple hydrothermal method was employed at 300 °C by burning the solutions for ∼1 h and resuspended with volumes of water to produce CNP-2, CNP-1, CNP-0.5, CNP-0.25, and CNP-0.125 having nectar concentrations of 2, 1, 0.5, 0.25, and 0.125 g/mL, respectively. The samples were probe sonicated (Q700, Qsonica Sonicators, Newtown, CT) for 2 min (Pulsed Amp, 1; on, 2 s; off, 1 s). The solutions were filtered with a 0.2 μm filter (Millex, Merck Millipore Ltd., Tullagreen, Carrigtwohill, County Cork, Ireland). As-synthesized CNPs were B
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Molecular Pharmaceutics respectively). Measurements of ζ were reproducible to within ±3 mV of the mean value given by 3 determinations of 15 data accumulations. 2.5. UV−vis Spectroscopic Evaluation of CNPLexes. The absorption spectra for all sets of the CNPs, PEI decorated CNPs, and CNPLexes (10 μL each of CNP-2P, CNP-1P, CNP0.5P, CNP-0.25P, and CNP-0.125P with 200 ng of pBR322 vector DNA) were acquired following the incubation of samples at room temperature for >6 h (Genesys 10S UV−vis Spectrophotometer, Thermo Scientific, Rockford, IL). Additionally, the absorption spectra for CNP-2, CNP-1, CNP-0.5, CNP-0.25, CNP-0.125, CNP-2P, CNP-1P, CNP-0.5P, CNP0.25P, and CNP-0.125P were taken with 2000× diluted concentrations. 2.6. Fluorescence Spectroscopic Evaluation of CNPLexes. The fluorescence spectra for all sets of the CNPs, PEI decorated CNPs, and CNPLexes (10 μL each of CNP-2P, CNP-1P, CNP-0.5P, CNP-0.25P, and CNP-0.125P with 200 ng of pBR322 vector DNA) were acquired after the samples were incubated at room temperature for >6 h (Genesys 10S UV−vis Spectrophotometer, Thermo Scientific, Rockford, IL). Additionally, the absorption spectra for CNP-2, CNP-1, CNP-0.5, CNP-0.25, CNP-0.125, CNP-2P, CNP-1P, CNP0.5P, CNP-0.25P, and CNP-0.125P were taken with 2000× diluted concentrations. 2.7. Preparation of CNPLexes from CNPEIs for Gel Electrophoresis. Plasmid DNA and pBR322 vector DNA (New England Biolabs, Ipswich, MA) was used for preparation of CNPLexes using CNP-2, CNP-1, CNP-2P, and CNP-1P samples. These formulations were complexed with 200 ng of DNA at room temperature for 1 h. The samples were run on a 1% agarose gel at 100 V for 30 min. The gel was then stained in 3% ethidium bromide solution (10 mg/mL) for 5 min, and washed in ethidium bromide solution for 5 min before being imaged (Universal Hood III, Bio-Rad, Hercules, CA). 2.8. Heparin Mediated Destabilization of CNPLexes. Using 20 μL of CNP-2, CNP-1, CNP-2P, and CNP-1P, the particles were allowed to complex with 200 ng of pBR322 vector DNA for 1 h at room temperature. To this mixture, 3 nmol of heparin sulfate (Sigma-Aldrich) was added to each sample and incubated for 1 h at room temperature. A 1% agarose gel was run for 30 min at 100 V. The gel was stained, washed, and imaged using the same methods as mentioned above. 2.9. SDS-Mediated DNA Release from CNPLexes. Using 10 μL of CNP-2, CNP-1, CNP-2P, and CNP-1P, the particles were complexed with 200 ng of pBR322 vector DNA for 1 h at room temperature. Then 1 mM of SDS (Fisher Scientific, Pittsburgh, PA) was added to each sample and incubated for 1 h at room temperature. A 1% agarose gel was run for 30 min at 100 V. The gel was stained, washed, and imaged using the same methods as mentioned previously. 2.10. Human Transformed Cancer Cell Culture. MCF-7 cells (ER (+) breast cancer cells) and MD-MB231 cells (ER (−) breast cancer cells) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS) in T25 culture flasks (Cellstar; Germany) and were incubated at 37 °C in a 99% humidified atmosphere containing 5% CO2. Cells were regularly passaged by trypsinization with 0.1% trypsin (EDTA 0.02%, dextrose 0.05%, and trypsin 0.1%) in DPBS (pH 7.4). Nonsynchronized cells were used for all the in vitro experiments.
2.11. Cell Transfection Studies. Transfection experiments were performed to demonstrate the efficiency of CNPlexes in delivering reporter gene plasmid pEGFP-N1 in cultured monolayers of MCF-7 and MDA-MB231. Experiment was performed by plating cells at a cell density of 60,000 cells/well in media (DMEM with 10% FBS by volume) 24 h before performing the transfection experiment. CNPlexes were prepared using CNP-1P and pEGFP-N1 in different weight ratios ranging from 0.4 to 2 (w/w) using either 0.8 or 1.6 μg of pDNA for each well and was allowed to complex for 30 min. The complexation was performed in 200 μL of plain DMEM solution. After incubation period, CNPLexes were further diluted with 200 μL of plain DMEM. CNPLexes were added to cell monolayer in duplicates as 200 μL per well. Cells were incubated with CNPLexes for 12 h before replacing the medium with 10% FBS containing DMEM for the next 36 h followed by the flow assisted cell sorting (FACS) analysis. At the end of the incubation period, cells were imaged using a fluorescent microscope (DMI3000 B, Leica Microsystems, Buffalo Grove, IL). Further cells were washed with 1× DPBS and incubated with 1× trypsin for 3 min at 37 °C. At the end, 400 μL of 0.2% FBS containing DPBS was added to each well, and all the cells were collected before the FACS analysis. 2.12. Flow Assisted Cell Sorting (FACS) Analysis. Triplicate wells per sample from the 96-well plate or duplicates from 24-well plates were pooled to acquire the GFP expression by FACS. The culture medium was pipetted out, and the cells were washed with DPBS. After pipetting out the DPBS, 50 μL of 1× trypsin was added to each well and immediately pipetted out. The plate was incubated for 3 min at 37 °C and 5% CO2. Two hundred microliters of a collection buffer (0.2% FBS in DPBS) was added to each well, and the mixture was collected in 1.5 mL centrifuge tubes for FACS analysis. 2.13. Fluorescence Microscopy. All the pEGFP-N1 transfection experiments were followed by fluorescence microscopy at different time points. Microscopy results to characterize GFP expression at the end of 48 h posttransfection period were compared for different formulations. GFP expression was observed under fluorescence microscope at 20× magnification. 2.14. MTT Cell Viability Assay. The cell viability of CNP formulations were investigated by using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay in the presence of 10% FBS in antibiotic free media. The yellow tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) is cleaved by dehydrogenases to its purple formazan derivative (MTT-formazan) with maximum absorbance at 560−570 nm.25 The intensity of purple formazans indirectly reveals the mammalian cell survival and proliferation.26,27 Experiment was performed in 96 well plates (Cellstar; Germany) growing 15,000 breast cancer cells per well 24 h before treatments. Experiments were performed for various concentrations of CNPs, CNPEIs, and CNPLexes ranging from 100 to 6.25 mg/mL of CNPs present as free or in the form of CNPEIs. Cells were incubated for 48 h before performing the MTT assay. After incubation period, cells were treated with MTT (20 μL, 5 mg/mL) per well and further incubated for 4 h. At the end of the incubation the entire medium was removed from the wells and 200 μL of DMSO was added to dissolve blue colored formazan crystals. The percentage cell viability was obtained from plate reader and was calculated using the formula %viability = {[A630(treated C
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Molecular Pharmaceutics cells) − (background)]/[A630(untreated cells) −background]} × 100. 2.15. Colorimetric Cytotoxicity Assay for Quantitative Measurement of Lactate Dehydrogenase (LDH Assay). The LDH assay measures the oxidation of P-NADH to p-NAD + when LDH reduces pyruvate to lactate. Conversion of PNADH to P-NADf is measured as a decrease in absorbance (340 nm). Percent cell death/lysis is determined by comparing LDH activity released into the media to the total LDH activity in the media and cells. Experiment was performed in 96 well plates (Cellstar; Germany) growing 8000 cells per well 24 h before treatments. Following 24 h of incubation, the cells were treated with various concentrations of CNPs, CNPEIs, and CNPLexes ranging from 100 to 6.25 mg/mL of CNPs present as free or in the form of CNPEIs. Cells were incubated for 48 h before performing the LDH Assay. After 48 h of incubation at 37 °C and 5% CO2, LDH activity was determined using the Pierce LDH Cytotoxicity Kit (Thermo Scientific, Rockford, IL) following manufacturer’s protocol.
3. RESULTS AND DISCUSSION In the early phase of gene therapy research, natural viruses were used as gene transporters.23 However, the initial excitement was Figure 3. Morphological variations on surface decoration of CNP with PEI and FT-IR spectroscopic signatures. TEM image of (A) CNP-1 (inset scale bar, 50 nm) and (B) CNP-1P (inset scale bar, 50 nm). (C) FTIR spectroscopic variations show shifts at 3340 and 1620 cm−1 for hydroxyl and carbonyl functional groups, respectively. Figure 1. Schematic representation of surface decorated CNP and microscopic image of green fluorescence protein expressing cells as a result of transfected pEGFP-N1 reporter gene plasmid in breast cancer cell MDA-MB231.
dampened due to the concern of high risk of infection or adverse immunogenicity.28 Consequently the field of gene therapy research experienced a great amount of work being
Figure 2. Physicospectroscopic characterization of CNPs and progressive surface decorations. (A) Increment in hydrodynamic diameter of CNP-1 on PEI surface decoration (CNP-1P) and complexation with plasmid DNA pEGFP-N1 (CNPLex-1). (B) Variation in zeta potential distribution of CNP on surface decoration and complexation. (C) Integral fluorescence efficiency of CNPs and response to surface decoration and sequential complexation. (D) CNPLex response to external stimuli of low pH and high anionic surfactant content. D
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Figure 4. DNA loading and unloading efficiency of CNP-1P formulation. (A) Strategy of EtBr fluorescence variation as signal to DNA loading and unloading efficiency. (B) EtBr exclusion and drop in EtBr fluorescence as a factor of increasing CNP-1P per unit weight of pDNA indicating DNA loading efficiency. (C) SDS mediated reinclusion of EtBr and improvement in fluorescence as a factor of increasing CNP-1P per unit weight of pDNA, indicating DNA unloading efficiency.
Figure 6. Fluorescence microscopic visualization of GFP expression in MDA-MB231 cells transfected with CNPLex-1 containing pEGFP-N1 plasmid complexed to CNP-1P. Experiment was performed for 12 h incubation of CNPLex-1 with cells and 36 h after transfection before performing the fluorescence microscopy. CNPLex-1 was prepared at wt ratio of 2 of CNP-1P/pDNA using 0.8 μg pDNA per well.
immunogenic reactions. Examples of such nonviral gene transfer agents include novel polymers29,30 and dendrimers,31,32 among others.33,34 However, the search continued to design a delivery approach with improved gene transfection integrated with critical characteristics such as strong affinity for nucleic acids with reversible noncovalent association. Some of the other prerequisites include high biocompatibility and trackability without the need of any additional use of fluorophores. A facile synthetic route to reach these nanoparticles with multifunctionality is also highly desirable to ensure an eventual commercial translation. At this end we propose a carbon nanoparticle-based approach with tunable luminescent characteristics, offering strong affinity for DNA with reversible noncovalent association with PEI and overall excellent gene transfection efficacy. Our strategy involved the preparation of five new classes of carbon nanoparticles (CNPs) from natural sweetener nectar using a
Figure 5. Gel electrophoretic pattern of pDNA in free or complexed form with CNP-1P formulation and under the influence of SDS, heparin, and blood serum. Gel retardation assay for qualitative expression of (A) pDNA binding to CNP-1P and CNP-2P at ratio of 1:0.5 (w/w). (B) pDNA release after incubation with SDS at ratio of 1:200 for 60 min. (C) Stability of CNPLexes while incubated with heparin at molar ratio of 10:1 (heparin/pDNA) for 60 min. (D) Stability of CNPlexes in the presence of 10% fetal bovine serum for 60 min.
devoted to the design and syntheses of nonviral agents with the anticipation that they will not typically elicit significant E
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Figure 7. Quantification of cell population expressing green fluorescence protein after transfection with CNPLex-1 at various CNP-1P/pDNA ratios using 0.8 and 1.6 μg pDNA per well. Experiment was performed for 12 h incubation of CNPLex-1 with MCF-7 (A,B) and MDA-MB231 (C,D) cells and 36 h after transfection before performing the flow assisted cell sorting. CNPLex-1 was prepared at ratios ranging from 0.4 to 2.0.
negative ranging from −10 to −30 mV (Figure S2A, Supporting Information). Among the formulations of CNP-1 with further surface decoration with PEI (CNP-1P) and complexation (CNPLex-1), values were found to be changing from ∼−10 to +20 and again dropping back to ∼−20 mV, respectively (Figure 2B). This observation rationally supports the sequential layerby-layer patterning of carbon nanoparticles with cationic PEI followed by complexataion with pDNA. In the case of CNP-2, CNP-2P, and CNPLex-2, representative zeta values changed from −15 to +40 mV and dropping back to −15 mV (Figure S2B, Supporting Information) pointing toward the presence of higher negative functionalities on surface. This allowed very high loading of PEI with not so significant optimal packing of pDNA. 3.2. Spectroscopic Properties of CNPs. Prepared CNPs were found to be significantly efficient in absorbing and emitting photons at particular λmax. CNPs exhibited strong absorbance with UV−vis spectroscopy at λ ranging from 280 to 375 nm with varying λmax = 295 nm with no significant variance across CNP-2, CNP-1, CNP-0.5, CNP-0.25, and CNP-0.125 (Figure S3A, Supporting Information). Interestingly, surface coating of PEI on CNPs reduces the absorbance efficiency drastically in CNP-2P, CNP-1P, CNP-0.5P, CNP-0.25P, and CNP-0.125P (Figure S3B, Supporting Information), while complexation with pDNA did not influence any change for CNPlex-2, CNPLex-1, CNPLex-0.5, CNPLex-0.25, and CNPLex-0.125 (Figure S3C, Supporting Information). For fluorescence properties, when excited at λmax = 360 nm, it was also found that CNPs were significantly fluorescent emitting at λmax = 490 nm across all the CNPs. Relative fluorescence unit (RFU) showed maximum fluorescence furnished by CNP-1 (Figures 2C and S4A, Supporting Information). Similar to their absorbance results, surface
straightforward hydrothermal method. Prepared CNPs (CNP2, CNP-1, CNP-0.5, CNP-0.25, and CNP-0.125 were surface decorated with PEI to generate CNP-2P, CNP-1P, CNP-0.5P, CNP-0.25P, and CNP-0.125P and finally complexed with plasmid DNA pBR322 and pEGFP-N1, for different experiments as applicable (Figure 1). 3.1. Physical Characterizations. 3.1.1. Dynamic Light Scattering (DLS). DLS data (Figure S1, Supporting Information) show that the hydrodynamic diameters of these CNPs in aqueous media ranged from ∼50−100 nm in diameter. The smallest size ∼50 nm was obtained with CNP-1 and biggest size of ∼100 nm in the case CNP-0.125 and CNP-2, while CNP-0.5 and CNP-0.25 produced intermediate particles of ∼90 and ∼60 nm (Figures 2A and S1, Supporting Information), respectively. Following surface decoration with PEI, the size of the particles grew to a considerable extent with the smallest size of ∼80 nm in CNP-1P, while all others were much bigger ranging from ∼150−280 nm. In the case of CNPLex-1, the complex grew bigger (∼300 nm) presumably due to the high loading of pDNA. The increments in CNPLex-2, CNPLex-0.5, and CNPLex-0.25 were modest, indicating a lesser ability to associate with pDNA. Interestingly, the size of CNPLex-0.125 was found to be ∼90 nm, which was even smaller than the CNP-0.125P. This observation possibly indicates the pealing off of PEI layers from CNP-0.125 after complexation with pDNA leaving CNP-0.125 alone and regaining the approximate size of parent CNP-0.125 formulation. A thorough analyses of particle size distribution thus indicates that CNP-1 (∼50 nm) is the smallest in size, which forms an optimal layer of PEI in CNP-1P (∼80 nm) followed by producing CNPLex-1 (∼300 nm) with a stable, maximally complexed pDNA (Figure 2A). 3.1.2. CNPLex Formation As Followed by Zeta Potential Titrations. Zeta potential of all the CNPs was found to be F
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Figure 8. Histographical presentation of increased green fluorescence content in pEGFP-N1 transfected cells, represented as the M2 region, while M1 represents the region of cell populations with integral fluorescence independent of the pEGFP-N1 transfection. Experiment was performed in MCF-7 cells. Histograms are from cells (A) treated with 0.8 μg pDNA/well with PEI amount (B) 0.4; (C) 0.8; (D) 1.2; (E) 1.6; and (F) 2.0, and from cells treated with 1.6 μg pDNA/well with PEI amount as (G) 0.4; (H) 0.8; (I) 1.2; (J) 1.6; (K) 2.0; and (L) Lipofectamine2000.
decoration of CNPs with PEI decreases their RFU to a significant level, while optimal extent of surface decoration could be seen with maximum decrease in RFU for CNP-1P (Figures 2C and S4B, Supporting Information). A further loss in indigenous fluorescence was seen following complexation of pDNA in CNPlexes (Figure 2C and S4C, Supporting Information). These results clearly support the critical role of surface chemistry and how it influences indigenous optical properties of CNPs. 3.3. Stability of CNPLexes and Likely Mechanism of DNA Release. For delivering pDNA in cytoplasm of cellular system, three major phenomena are required, the stable packing of DNA, subsequent release, and stability during circulatory transportation in between. An efficient carrier should load high % of DNA, while prepared complexes must be stable in circulatory situations and cytoplasmic factors should be able to
trigger the release of DNA. CNPLexes revealed high stability of CNPLex-1 at pH 6.8 (water), 10% FBS, and in the presence of SDS across 0−6 h of time points, while at pH 4.7 a significant change in size was noted at the 3 h time point (Figures 2D and S5C, Supporting Information). Stability in FBS insures the circulatory stability, while no significant response to SDS refutes DNA release by endosomal inner anionic membrane interactions as major pathway. However, instability at acidic environment makes it prone to experience endosomal proton sponge effect, presumably contributed from PEI, decoding the release of DNA from CNPLexes. 3.4. Effect of Surface Decoration on Transmission Electron Microscopy (TEM) and FT-IR Spectroscopic Patterns of CNPs with pDNA Complexation. Transmission electron microscopy was employed to determine anhydrous morphology of CNP-1 and its surface decorated counterpart G
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Figure 9. MTT and LDH release assay for evaluation of cell toxicity on incubation with CNP formulations and compared with parent PEI at various concentrations of PEI in free form or decorated to CNPs. Experiment was performed in MDA-MB231 (A,B) and MCF-7 (C,D) cells after 12 h incubation with formulation and assay performed after 36 h of post-treatment.
(Figures 4B and S6A, Supporting Information). A ∼2:1 (w/w) ratio of SDS/CNP-1P induced the release of pDNA from the CNPLex-1 to an extent of ∼80% (Figures 4C and S6B, Supporting Information). An optimized and significantly high release of pDNA from CNPLexes compared to the loading % of ∼50% makes it a better transfecting carrier and delivery agent. 3.5.2. Gel Electrophoresis. To establish pDNA binding and release efficiency, gel electrophoresis experiment was performed on 1% agarose gel under 1× TAE buffer at 100 V. It was found that CNP-2 and CNP-1 did not show any interaction and retardation of pDNA. However, CNP-1P was found to be very similar to CNP-2P when CNPLex-1 and CNPLex-2 were prepared at a PEI to pDNA ratio of 5000:1 (Figure 5A). We further established the SDS mediated release where CNPLex-1 prepared from CNP-1P and pDNA was found to be much better in releasing the pDNA compared to CNPLex-2 prepared from CNP-2P and pDNA under the influence of electrophoresis at ratios of SDS/CNP-1P or SDS/CNP-2P (Figure 5B). Heparin is a known anticoagulant that resides in blood circulation and plays an important role in destabilizing the carrier−DNA complexes in circulatory path during delivery of DNA. CNPLex-1 and CNPLex-2 were incubated with heparin (3 mM) and were subjected to gel electrophoresis for 60 min. We could establish that CNPLex-1 prepared from CNP-1P was able to control the destabilizing effect of heparin in a much better fashion compared to CNP-2P complex with pDNA (Figure 5C). Serum proteins are notoriously known to play a role in decomplexing the DNA complexes from carrier particles. We performed a similar gel electrophoresis experiment with 10% FBS incubated with CNPLex-1 and CNPLex-2. Our results
CNP-1P. Anhydrous morphological distributions were found to vary from 50 ± 10 nm for CNP-1 (Figure 3A) to 80 ± 15 nm in the case of CNP-1P (Figure 3B). Increment in size justifies the surface decoration of CNP-1 with PEI during preparation of CNP-1P. IR spectroscopic analysis of CNP-1 showed signature bands at 3340 and 1620 cm−1 for hydroxyl and carbonyl functional groups, respectively. In CNP-1P, signature peaks at 3400 and ∼1600 cm−1 disappeared probably due to the suppressed hydroxyl and carbonyl groups under surface decorated PEI. Interestingly, the complexation with pDNA allowed the signature peak at ∼1600 cm−1 to reappear in CNPLex-1 (Figure 3C). 3.5. Interaction with pDNA. As stated above, to prepare an efficient DNA carrier, its DNA loading and release efficiency must be optimized such that DNA release plays the major role of efficiency limiting step. It signifies the fact that the efficiency of a carrier could be optimized for better DNA release at lower DNA loading efficiency. We performed EtBr exclusion and gel electrophoresis assays to determine DNA interaction with CNP-1P. 3.5.1. EtBr Exclusion and Reintercalation Assay. It is wellknown that EtBr exclusion and reintercalation assay works via increased fluorescence of EtBr in DNA intercalated form and decrease in fluorescence in its free form. Competitive interaction between carrier and DNA would decide the extent of exclusion and in turn revealing the complexation ability or DNA binding ability of the carrier (Figure 4A). The addition of an anionic surfactant initiates a competitive decomplexation of DNA and allows reintercalation of EtBr to increase the fluorescence decomplexation. From the EtBr exclusion assay, it was found that the CNP-1P bind the pDNA to the extent of ∼50% by the wt ratio of ∼1:1 H
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transfecting agent to the commercially sold transfecting agent Lipofectamine2000 along with superior cell viability and significantly low cell toxicity compared to parent PEI.
clearly indicated high stability of CNPLex-1 compared to CNPLex-2 prepared from CNP-1P and CNP-2P, respectively (Figure 5D). pDNA interaction studies in the absence and presence of major influencers of DNA delivery pathways established CNP-1P as a better carrier for pDNA. 3.6. Biological Experiments. We employed various physicochemical and biophysical experiments to elucidate the fate of carbon-based carrier−DNA complexes and determine the overall suitability as transfecting agents. As investigated above, CNP-1P was found to be significantly better compared to the other transfecting agent of the series, CNP-2P. The rigorous analytical characterization allowed us to narrow down to a preferred CNPlex for conducting the biological experiments in vitro. 3.6.1. Gene Transfection Efficiency of CNPlexes. Gene transfection efficiency was established by transfecting green fluorescence protein (GFP) expressing gene containing plasmid pEGFP-N1. Efficiency was followed by fluorescence microscopy and visualizing green fluorescence protein in cytoplasm of the transfected cells. Extent of transfection was correlated with GFP content in cell population (mean fluorescence intensity, MFI) and % GFP positive cell population. Transfection of pEGFP-N1 in MCF-7 and MDA-MB231 using CNP-1P showed around equally expressed GFPs in MDA-MB231 cells compared to commercially available Lipofectamine2000 (Figure 6B,D). The bright field images were compared to establish the scenario of cell growth density and morphology (Figure 6A,C) in MDA-MB231 cells. Fluorescence-activated cell sorting (FACS) quantification was employed to determine % GFP positive cells and MFI among transfected cell population (Figure 7) as represented by M2 region in histograms of Figures 8 and S7, Supporting Information. The intrinsic fluorescence due to the presence of tryptophan amino acid in cellular proteins and autofluorescence from internalized CNPs were set as negative controls as represented by the M1 region in the histograms (Figures 8A and S7A, Supporting Information). FACS results showed that a maximum of ∼80% GFP positive cells were enriched with MFI of ∼60 in the case of PEI/pDNA 2.5 (wt ratio) with pDNA of 0.8 μg/well. However, ∼70% GFP positive cells with ∼60 MFI in MCF-7 cells varied to ∼60% GFP positive cells with ∼50 MFI by CNPlex-1 compared to ∼65% GFP positive cells with MFI of ∼50 by Lipofectamine2000 in MDA-MB231. This pattern corroborated well for both the cell lines as evident from representative histograms of MCF-7 (Figure 8) and MDA-MB231 (Figure S7, Supporting Information). 3.6.2. Cell Toxicity. Successful clinical translation of gene delivery carriers has been jolted by their high toxicity during in vitro and in vivo experiments. We tried to establish the % cell viability and % cytotoxicity in both the cell lines under incubation of CNP-1P and CNP-2P by MTT assay (Figure 9B,D) and LDH release assay (Figure 9A,C). In MTT assay, % drop in cell viability correlates with the toxicity of the samples, while LDH release assay does just the reverse where an increase in % LHS release correlates with cellular toxicity inculcated by samples. Studies with MTT assay showed more than ∼80% cell viability by CNP-2P and CNP-1P compared to merely ∼10% in the case of PEI at10 μg/mL across both the used cells. Similarly, LDH release assay expressed the cell death of ∼90% in the case of PEI and ∼10% in the case of CNP-2P and CNP1P at 10 μg/mL across both the used cells. These point to the remarkable cellular level safety with these agents. Thus, prepared CNP-1P has been established as a highly comparable
4. CONCLUSIONS A product of natural sweetener, nectar, was used to produce CNPs of defined sizes. PEI (10 kDa) was used for the surface decoration and was optimized to successfully complex pEGFPN1 for transfection in MCF-7 and MDA-MB231 monolayers. CNP-2, CNP-1, CNP-0.5, CNP-0.25, and CNP-0.125 were synthesized and optimally surface decorated with PEI to produce CNP-2P, CNP-0.5P, CNP-0.25P, and CNP-0.125P. Surface decorated CNPs were used to achieve CNPLexes, CNPlex-2, CNPlex-1, CNPlex-0.5, CNPlex-0.25, and CNPlex0.125 with pDNA. CNP-1 was an ideal candidate with an optimum hydrodynamic diameter of 50 nm, electrophoretic potential of −10 mV, and strong optical absorbance and fluorescence emission, which drops the absorbance and fluorescence intensities. Further complexation with pDNA increases the size of complexes and negative surface potential −20 mV with decreased absorbance and fluorescence intensities. CNPLex-1 was responsive to low pH indicating the fate of DNA release in the intracellular endosomal compartment. TEM images revealed the increment in anhydrous diameter of CNP-1 and variation in vibrational bands on surface protection with PEI, while CNPLex-1 showed changes supporting the involvement of CNP-1P with pDNA in complexation. EtBr exclusion assay showed around ∼50% binding of pDNA to CNP-1P, while reinclusion assay concluded a highly significant extent of ∼80% release in the presence of anionic surfactant. Gel electrophoresis confirmed that CNP-1P was not only a better pDNA complexing agent but an excellent release agent. Studies in the presence of FBS and heparin revealed the higher stability of CNPLex-1, which would decide a better fate in circulatory system during systemic delivery. Transfection of pEGFP-N1 in MCF-7 and MDAMB231 using CNP-1P showed equally expressed GFPs in MDA-MB231 cells compared to Lipofectamine2000. FACS quantification showed % GFP positive cells of ∼80% with MFI of ∼60 compared to ∼70% GFP positive cells with ∼60 MFI in MCF-7 cells. It varied to ∼60% GFP positive cells with ∼50 MFI by CNPlex-1 compared to ∼65% GFP positive cells with MFI of ∼50 by Lipofectamine2000 in MDA-MB231. Further studies with MTT assay showed more than ∼80% cell viability by CNP-2-1 and CNP-1P compared to merely ∼10% in the case of PEI at 10 μg/mL across both the used cells. Similarly, LDH release assay revealed the cell death of ∼90% in the case of PEI and ∼10% in the case of CNP-2-1 and CNP-1P at 10 μg/mL across both the used cells. Thus, prepared and optimized CNP-1P has been established as a highly comparable transfecting agent compared to one of the best commercially sold transfecting agents, Lipofectamine2000, with much better cell viability and significantly low cell toxicity compared to the parent PEI. Our results with these next generation carbon particles presents a novel approach for gene delivery. With these promising in vitro results, further in depth in vivo studies are warranted to fully explore the potential of these agents.
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ASSOCIATED CONTENT
* Supporting Information S
Additional methods and figures. This material is available free of charge via the Internet at http://pubs.acs.org. I
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AUTHOR INFORMATION
Corresponding Author
*Phone: 217-244-2938. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We acknowledge University of Illinois at Urbana−Champaign and Children’s Discovery Institute for financial assistance. We thank Dr. Tor Jensen for help with the FACS analysis. TEM and zeta potential were carried out at Frederick Seitz Materials Research Laboratory Central research facilities, UIUC.
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