Article pubs.acs.org/Biomac
Effect of PEGylation on the Diffusion and Stability of Chitosan−DNA Polyplexes in Collagen Gels Sylvie Lélu,† Sabina P. Strand,‡ Jan Steine,† and Catharina de Lange Davies*,† †
Department of Physics and ‡Department of Biotechnology, The Norwegian University of Science and Technology, 7491 Trondheim, Norway ABSTRACT: Diffusion through the extracellular matrix (ECM) is a critical step for the delivery of nanoparticles and genes. Gene delivery requires a carrier that protects the nucleic acid from degradation and facilitates transport. Chitosan is a promising carrier. To increase the circulation time, PEGylation of the carrier is performed. However, the effect of PEGylation on the transport and stability of gene delivery systems in the ECM has only been studied in solutions containing ECM components. We used polymerized collagen and collagen−hyaluronic acid (HA) gels to study the effects of PEGylation on the diffusion and stability of chitosan−DNA polyplexes. We found that PEGylation of the polyplexes was required for diffusion to occur, and PEGylation increased the dissociation between DNA and chitosan to some extent. The presence of HA had a contradictory role: it decreased the penetration depth of PEGylated polyplexes into the gels and increased the diffusion of the polyplexes being mixed into the gels.
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used to improve the biocompatibility and stability of the polyplexes.11 The PEGylation shields the surface charge of the polyplexes and provides steric stabilization, thereby preventing aggregation. PEGylation of the carrier also reduces the interactions with components of the reticuloendothelial system, which limits opsonization and consequently increases the circulation time in the blood vessels.6,9,12,13 Regardless of whether they are delivered locally or systemically, nonviral vectors and the genes they carry must overcome the ECM. The ECM is a complex environment in which protein fibers are embedded in a hydrophilic gel of glycosaminoglycans (GAG) and proteoglycans. Collagen proteins are the major structural component of the matrix, and it has been suggested that the collagen network has the main responsibility for the transport hindrance presented by the ECM,14 especially for larger molecules,15 whereas GAGs and proteoglycans represent a barrier to smaller molecules 15 and might also increase the transport hindrance presented by the protein network.16 In addition, not only the ECM structure but also its composition affects both transport hindrance17,18 and polyplex stability.19 The ECM is a negatively charged matrix, as the GAG molecules are heavily negatively charged. Collagen fibers, however, have an overall slightly positive charge, but both hydrophilic and hydrophobic patches and positive and negative charges are distributed along the fibers. Lieleg and co-workers 20 showed that the ECM acted as an electrostatic bandpass window, reducing diffusion coefficients dramatically to almost complete immobilization for both positively and negatively
INTRODUCTION The cationic polysaccharide chitosan, which is a copolymer of N-acetylglucosamine and glucosamine, is widely studied as a nonviral gene delivery vector. Chitosan offers several advantages compared with synthetic polycations, such as biocompatibility, biodegradability, versatility, and low toxicity.1−3 Chitosan spontaneously self-assembles with DNA into nanosized complexes. The transfection efficacy of these polyplexes depends on the intrinsic properties of chitosan, such as the degree of polymerization (DPn), fraction of acetylated units (FA), and the ratio between chitosan and DNA (amino groups per phosphate groups, A/P ratio). Whereas the in vitro transfection efficiency of chitosan is encouraging, the in vivo efficiency remains too low for clinical application.2−6 In vitro, monolayers of cells are typically used to optimize the molecular weight, charge density, and A/P ratio necessary to achieve higher transfection rates.4 However, these models do not take into account extracellular barriers to gene delivery, and poor correlation between in vitro and in vivo studies is observed.7 Thus, to improve the efficiency of a gene carrier, it is necessary to focus specifically on the extracellular barriers. The nature of the extracellular barriers depends on the route of administration of the polyplexes. However, in any case, the polyplexes must be capable of penetrating the extracellular matrix (ECM), which represents a major obstacle for the successful delivery of DNA.2 Little is known about the distribution and fate of polyplexes in the ECM. Polycations are generally not suitable for systemic delivery because of excessive aggregation and interactions with blood components.6 Thus, chemical modifications are necessary to reduce aggregation and avoid clearance from the blood, thereby increasing the circulation time.8−10 Grafting of nonionic poly(ethylene glycol) (PEG) to the vector has been widely © 2011 American Chemical Society
Received: June 30, 2011 Revised: August 25, 2011 Published: August 25, 2011 3656
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molar C/N ratio of 10.3, which corresponds to the degree of substitution of ∼2%. Chitosans were labeled with fluorescein isothiocyanate (FITC, Sigma-Aldrich) at a ratio of 1 FITC molecule to every 50 glucosamine groups. Briefly, freeze-dried chitosan oligomers were first dissolved in Milli-Q (MQ) water and then in an equal amount of 0.2 M sodium acetate buffer (pH 5.5) to obtain a chitosan concentration equal to 2.0 mg/mL. The pH of the chitosan solution was adjusted to a minimum of 6 using a 0.1 M sodium hydroxide solution, and 2 mg/mL FITC in ethanol was then added to the chitosan. The labeling reaction was allowed to proceed for 1 h in the dark at room temperature, before unbound dye molecules were removed by overnight dialysis (SpectraPor, cutoff 12−14 kDa) of the chitosan solution against water. The labeled chitosan was then freeze-dried. Preparation of the Polyplexes. Chitosan diluted in MQ water was slowly added under mild vortexing to an equal volume of Cy5 or YOYO-labeled DNA. To obtain a final A/P of 10, the mass ratio between chitosan and DNA was 11.65 mg chitosan to 2 mg DNA. The polyplexes were incubated in the dark for 30 min to allow complexation. The polyplexes were applied to the gels in two different ways: either 125 μL of polyplex solution containing 13.3 mg/mL DNA was mixed with an equal volume of collagen or collagen−HA solution before polymerization, or 75 μL of polyplexes in water containing 26.6 mg/mL DNA was added on top of 170 μL of collagen or collagen− HA gels and incubated at room temperature for 3 h in the dark to allow penetration into the gels. Final A/P ratios of 10 were used for the colocalization analysis and DLS experiments. For FCS measurements, final A/P ratios of 5 and 10 were use for PEGylated and nonPEGylated polyplexes, respectively. Preparation of the Gels. Collagen gels were made from rat-tail collagen type I high concentration (8−11 mg/mL), provided by BD Biosciences. Collagen solutions were prepared following the manufacturer’s protocol by mixing solutions of 10× concentrated phosphate-buffered saline (PBS, Sigma-Aldrich), MQ water, 1 M sodium hydroxide, and concentrated collagen on ice. The preparation of collagen−HA matrices was performed using HA sodium salt from Streptococcus equi (Sigma-Aldrich, MW 1.63 MDa). HA was hydrated with MQ water for at least 12 h prior to the preparation of the gels. Collagen−HA solutions were prepared using the HA solutions instead of MQ water. Both collagen and collagen−HA solutions were vortexed, and their final pH was adjusted to 7.4 ± 0.2 by the addition of 0.1 M sodium hydroxide or 0.1 M hydrochloric acid solution. Collagen and collagen−HA solutions were incubated at 37 °C for 1 h for polymerization in 8-well plates (IBIDI). 170 or 250 μL of gel was added to each well when the polyplexes were added to the top or mixed into the gel, respectively. The final concentrations of collagen and HA in the gels were 2.5 and 1.0 mg/mL, respectively. Dynamic Light Scattering and Zeta Potential Measurements. The size of the chitosan−DNA complexes was determined at room temperature by dynamic light scattering using a Zetasizer Nano ZS apparatus (Malvern Instruments). The z-average radii of the complexes were obtained by a cumulative analysis of the correlation function using the viscosity and refractive index of water in the calculations. The zeta potential of the complexes was determined by laser Doppler velocimetry using the same Zetasizer Nano ZS instrument. Polyplex solutions containing 20 mg/mL DNA were prepared at an A/P of 10. Each sample was analyzed three times and prepared in triplicate in MQ water or PBS. One volume of 5× PBS was added to 4 volumes of polyplex in MQ water. Confocal Laser Scanning Microscopy (CLSM). The distribution of the polyplexes in the gels was observed using an inverted LSM 510 microscope (Carl Zeiss). The objectives C-Apochromat 40X/1.2 NA W or C-Achroplan 40X/0.8 W were used for the imaging of polyplexes mixed into the gels or added to the top of the gels, respectively. The 40X/0.8 objective has a working distance of up to 1.75 mm; thus, it could image polyplexes applied on the top of the gel. FITC-labeled chitosans and Cy5-labeled DNA were excited using wavelengths of 488 and 633 nm, respectively. The emitted light was collected from FITC-chitosans using a BP 500-530 IR filter and from Cy5-DNA using a meta-detector with a spectral interval 651 to 694
charged nanoparticles that had a surface charge outside the window. Previous studies of interactions between polyplexes and specific molecules of the ECM in solution have observed a dissociation of polyplexes in contact with sulfated GAG. 21,22 Furthermore, collagen has been used as a potential nonviral vector,23,24 and the capacity of chitosan to self-assemble with collagen and hyaluronic acid (HA) has been used for the formation of polyelectrolyte films and biomaterials. 24−26 Altogether, these results show that decreasing or shielding the cationic charge presented by the polyplexes, such as by PEGylation, could improve the transport of polyplexes through the ECM and increase their probability of reaching the cells. However, despite the positive role that PEGylation plays in overcoming the barriers posed by capillaries and the ECM, the PEGylation of polyplexes might also present some drawbacks. Recent studies observed a decreased stability of PEGylated PEI−DNA polyplexes, not only in contact with sulfated GAGs but also in the presence of collagen.21 Such premature unpacking of the polyplexes in the ECM would expose the DNA to enzymatic attack and compromise its transport through the ECM and the cell membrane and eventually its transfection. The purpose of this study was to compare the mobility and stability of non-PEGylated and PEGylated chitosan−DNA polyplexes in an artificial ECM gel of collagen fibers and HA. HA was used because it is the most abundant GAG in the ECM, and HA is reported to bind to chitosan.24,25 Doublestranded plasmid DNA, which is the most common type of DNA used in gene therapy,27 was complexed with low molecular weight chitosan. Confocal laser scanning microscopy (CLSM) and fluorescence correlation spectroscopy (FCS) were used to study the diffusion, aggregation, and unpacking of the chitosan−DNA polyplexes. The unpacking of DNA was studied by estimating the colocalization between fluorescently labeled chitosan and DNA in the gels.
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MATERIALS AND METHODS
Plasmid DNA and Labeling. Plasmid DNA (gWizLuc, 6.7 kbp) was purchased from Aldevron and labeled with Cy5 (Mirus BIO LLC) according to the manufacturer’s protocol. Cy5 binding covalently to DNA was used in colocalization analysis between DNA and chitosan. FCS measurements were performed using DNA labeled with YOYO-1 (Molecular Probes, Invitrogen) at a ratio of one dye molecule per 50 base pairs. YOYO-1 was chosen over Cy5 for FCS because of its higher photostability. Chitosan Preparation. Chitosan with a number-average degree of polymerization (DPn ) of ∼200 was prepared by nitrous depolymerization of fully de-N-acetylated chitosan (FA < 0.002, Mw 146 kDa) as described earlier.28 The depolymerized sample was dialyzed (SpectraPor, cutoff 12−14 kDa), lyophilized, and analyzed by size-exclusion chromatography with refractive index (RI, Dawn Optilab 903, Wyatt Technology) and a multiangle laser light scattering detector (MALLS, Dawn DSP, Wyatt Technology).29 The molecular weight (MW) of the depolymerized sample was 76 kDa, and the polydispersity index (Mw/Mn) was 2.0. PEGylation and FITC-Labeling. Chitosan was PEGylated by Nsuccinimidyl-activated succinyl-PEG (NOF Corp.) with a MW of 5 kDa. The activated PEG was added to the chitosan solution (1 mg/ mL, 50 mM MOPS, pH 6.0) in the amount corresponding to a theoretical degree of substitution of 5% of PEG-substituted Nacetylglucosamine. The conjugation was performed overnight at room temperature. PEG−chitosan copolymers were extensively dialyzed to remove the nonreacted PEG (SpectraPor, cutoff 12−14 kDa). The PEGylated chitosan was analyzed by SEC-MALLS as described above. MW of the pegylated chitosan was 99 kDa, and the polydispersity index was 1.8. The C/N analysis of the PEGylated chitosan showed a 3657
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Table 1. Characterization of the Polyplexesa z-average radius (nm) non-PEGylated polyplexes PEGylated polyplexes
zeta potential (mV)
in water
in PBS
in water
in PBS
in HA
100 ± 18 101 ± 14
970 ± 271 114 ± 8
38 ± 6 28 ± 4
1±3 0±1
−59 ± 3 −52 ± 1
a
The z-average radius and zeta potential of unlabeled PEGylated and non-PEGylated polyplexes measured in water, in PBS (I = 0.05), and in 0.5 mg/mL HA by DLS. Measurements in PBS were performed immediately after addition of PBS to the polyplexes. making new gels and polyplexes each time. Diffusion times τD were extracted by fitting the two-component model in eq 1 to the experimental autocorrelated curves obtained for every single intensity recording:
nm. Collagen fibers were imaged with confocal reflection microscopy (CRM) using a 543 nm excitation wavelength and a BP 500-550 IR filter. The pinhole aperture was set to 90 μm for all channels. Colocalization and Particle Size Analysis. The colocalization between DNA and chitosan was estimated using ImageJ software and the JACoP plugin developed by Bolte and Cordelières.30 The plugin allows a colocalization analysis based on different methods. There are several colocalization coefficients available, each with different limitations; the most common are Pearson’s and Manders’ coefficients. Pearson’s coefficient is an indicator of the dependency of pixel intensity in two channels (here, green and red). It is sensitive to noise and to intensity differences between the channels. Manders’ coefficients M1 and M2 are less dependent on fluorescence intensity differences, and they have the advantage of providing the specific colocalization of one channel with the other.31 The colocalization between chitosan and DNA was therefore estimated using Manders’ coefficients. To calculate M1, the intensity of the green pixels colocalizing with red pixels was summed and divided by the total intensity emitted by the green pixels. M2 was calculated similarly by inversing the channels. Thresholding of the images was necessary before colocalization analysis. Various algorithms as well as manual thresholding were tested and resulted in somewhat different colocalization coefficients. However, the differences in the coefficients between the samples were the same, independent of the thresholding used. Thus, we determined the optimal thresholding manually based on the criteria of eliminating background and blurry objects out of focus and still keeping the small objects. The same threshold was used to calculate the sizes of the polyplexes. After segmentation, the green and red channels were merged so that only colocalized objects appeared, whereas naked DNA and free chitosan were excluded from the resulting image. The sizes of the polyplexes in the merged images were calculated using the particle analyzer function of ImageJ. The average radii of the particles were calculated under the assumption that the polyplexes appeared as circular objects in the CLSM images. Fluorescence Correlation Spectroscopy (FCS). The polyplexes were mixed with the collagen or the collagen−HA solutions prior to the formation of the collagen fibers, and the FCS measurements were performed shortly after the polymerization of the collagen or collagen−HA solutions mixed with polyplexes. To assess the diffusion of polyplexes, we chose to focus only on the diffusion of DNA and to ignore the large number of free chitosan molecules in the polyplex solution by using unlabeled chitosan and YOYO-1-labeled DNA. On the basis of the reported high affinity between free DNA and collagen,32,33 we assumed that we mainly measured the diffusion of DNA polyplexed with chitosan and not any free DNA. In support of this, preliminary work performed with the plasmids mixed with collagen solutions prior to their polymerization confirmed colocalization between DNA and collagen fibers (data not shown). FCS was performed on an LSM 510 microscope equipped with Confocor 2 hardware and a C-Apochromat 40X/1.2NA W objective. The confocal volume created by the 488 nm laser focal point was placed randomly in the gel. The signal emitted by the excited YOYO-1 dye in the confocal volume passed through a BP 500-530 filter and was collected by a single photon avalanche photodiode detector. The pinhole diameter was 70 μm. The autocorrelation curves were obtained using software provided by Carl Zeiss, based on a Marquardt algorithm. Each measurement consisted of a minimum of seven intensity recordings performed for 10 s and was repeated at minimum eight locations in the gel. The experiments were repeated three times,
(1) where α is the triplet fraction, τT is the triplet time, F1 is the fraction of the fast diffusing component, and S is the structure parameter that characterizes the ratio of the height of the confocal volume to its width. The autocorrelated curves that could not be fitted by a twocomponent model were discarded. The distribution of the diffusion times was assessed by plotting their log values into a frequency histogram, which allowed the fitting of a three-parameter Gaussian regression function to the frequency histogram: (2) x0
The distribution of the average diffusion times τD = 10 was defined as 10x0−sd ≤ 10x0 ≤ 10x0+sd μs, where x0 is the mean and sd is the standard deviation of the samples. The diffusion coefficients D of the polyplexes were estimated from the diffusion times using the following equation:
(3) where ω is the radius of the laser beam. The average hydrodynamic radius Rh of the polyplexes in MQ water was calculated from the diffusion coefficients using the Stokes−Einstein equation: (4) where k is the Boltzmann constant, T the temperature, and η the viscosity of water. Statistical Analysis. Statistical analysis of the radius of polyplexes in the various gels were performed using the Mann−Whitney test and the software Minitab (Minitab Inc.). The radii were not normal distributed; thus, the median values of the radii are given. The significance criterion was p ≤ 0.05.
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RESULTS
Characterization of the Polyplexes in Solution. The zeta potential and the radius of unlabeled non-PEGylated and PEGylated chitosan−DNA polyplexes were measured in water and PBS, as the polyplexes were formed in water, and the gels consisted of PBS. The results are presented in Table 1. There
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was no difference in the z-average radius measured by DLS when the two types of polyplexes were in water. However, immediately after the addition of PBS, the radius of the nonPEGylated polyplexes increased ∼9 times, indicating aggregation, which we have also reported previously.4 No significant change in size was observed for PEGylated polyplexes in PBS. Labeling chitosan with FITC did not affect the radius of nonPEGylated polyplexes, whereas the radius of PEGylated polyplexes increased approximately to 150 nm. The zeta potential was positive for non-PEGylated and PEGylated polyplexes in water, and PEGylation reduced the zeta potential by ∼30%. In PBS, the zeta potential was ∼0 for both polyplexes because of reduced charge density of the chitosan due to shielding by the added salt ions. Adding HA to polyplexes in water reduced the zeta potential in a dosedependent manner. 0.5 mg/mL HA, which is less than the concentration used in the gel, resulted in a negative zeta potential of the polyplexes. This demonstrates that the negatively charged HA interacts with the positive amino groups of chitosan, changing the positive zeta potential to a negative potential. Distribution and Diffusion of the Polyplexes in Gels. To characterize the transport of polyplexes into collagen-based matrices, we first assessed the ability of PEGylated and nonPEGylated chitosan−DNA polyplexes to penetrate collagen and collagen−HA gels. Polyplexes in water were applied to the surfaces of the gels. Three hours later, FITC-labeled chitosan and Cy5-labeled DNA were imaged from the surfaces and into the gels. Because Cy5 was strongly photobleached, the kinetics of aggregation could not be studied, and only the penetration into the gels was measured. Collagen fibers were visualized by CRM. The brightness of the fibers depends on their inclination from the imaging plane, i.e., fibers aligned parallel to the imaging plane appear to be bright on CRM images, whereas fibers aligned vertically are not detectable.34 Therefore, collagen fibers were only used to identify the surface of the gels, and no quantitative measurements, such as fiber diameters or pore sizes, were performed. PEGylated and non-PEGylated polyplexes were differently distributed on the surfaces of the gels (Figure 1). For nonPEGylated polyplexes, a broad range of sizes was observed, and some very large polyplexes were present. The PEGylated polyplexes were apparently smaller and present in greater numbers, i.e., approximately 600 and 1000 non-PEGylated and PEGylated polyplexes per image, respectively. Indeed, nonPEGylated polyplexes first seemed to aggregate on collagen fibers, and then the polyplexes deposited on the top of each other (data not shown) to form large, blurry objects. PEGylated polyplexes behaved somewhat similarly, but their initial distribution on the surface of the gel was more homogeneous, with more and apparently smaller objects. The addition of HA to collagen did not seem to modify the polyplex aggregation to a great extent. The median radius of the polyplexes, the range of sizes indicated by the minimum and maximum radius, and the first and third quartiles are shown in Figure 2. The median radii of PEGylated and non-PEGylated polyplexes on the surfaces of the collagen gels were not statistically different because of the large variations in radii, especially for nonPEGylated polyplexes (Figure 2a). For collagen−HA gels, PEGylated polyplexes were significantly larger than nonPEGylated polyplexes. However, although statistically significant, the differences between the measured radii were only ∼10% (Figure 2b).
Figure 1. Distribution of (a, c) non-PEGylated polyplexes and (b, d) PEGylated polyplexes on the surface of the matrixes (green: chitosan; red: DNA; white: collagen fibers). (a, b) 2.5 mg/mL collagen gels; (c, d) 2.5 mg/mL collagen and 1.0 mg/mL HA.
The distribution of green and red fluorescent signals on top of the gel seemed to be different for PEGylated and nonPEGylated polyplexes (Figure 1), which might indicate that the two types of polyplexes dissociate differently. For nonPEGylated polyplexes, the green emission from FITC−chitosan and the red emission from Cy5−DNA appeared to be relatively well superimposed, as indicated by the yellow aggregates. For PEGylated polyplexes, red fluorescence was dominant, which might indicate that Cy5−DNA is mostly free of FITC−PEG− chitosan. Non-PEGylated polyplexes were never observed below the surface of the collagen or collagen−HA gels, indicating that they were immobilized at the surface. Neither DNA nor chitosan, free or polyplexed, was observed below the surface of the gel. Free and polyplexed PEG−chitosan and DNA, however, were imaged under the surfaces of both collagen and collagen−HA gels. PEGylated chitosan was more abundant than DNA because of the excess of chitosan and was occasionally observed even at the bottoms of the wells. The average size of the polyplexes at any location inside the gel was less than the size of polyplexes on top of the gel (Figure 2 a,b). The median radii of the polyplexes were reduced respectively 30% and 65% in collagen and collagen−HA gels. The penetration depth of DNA seemed to be limited in collagen− HA gels compared to collagen gels. DNA was able to penetrate 30−300 μm into collagen gels and 15 to at least 150 μm into collagen−HA gels. Although the penetration of PEGylated polyplexes varied between experiments, which prevented us from calculating representative distances, the penetration depth observed in collagen−HA gels was always less than the depth in pure collagen gels. Additionally, fewer Cy5−DNA particles were observed inside collagen−HA matrices compared with collagen gels (data not shown). Mixing chitosan−DNA polyplexes together with collagen and HA before the polymerization of the gels resulted in different distributions and sizes of the polyplexes compared with the application of the polyplexes on the top of the gels (Figures 2 and 3). A rather homogeneous distribution of the polyplexes 3659
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Figure 2. Polyplexes average radii (minimum, median, maximum, gray box delimited by the first and third quartiles) measured from CLSM pictures of polyplexes deposited on the top of the collagen gels (a) and collagen−HA gels (b) and mixed inside the collagen gels (c) and collagen−HA gels (d). The darker box plots in (a) and (b) indicate the radii of the polyplexes that penetrated below the surface of the gels. The pictures were analyzed using the command “particle average size” of Image J.
polyplexes inside the gel was smaller than that for polyplexes on top of the gel (Figure 2), indicating less aggregation. Furthermore, the range of sizes was smaller. Significant differences among all of the radii in Figure 2c,d were found, although there was a rather large variation in the radii. In collagen gels in particular, non-PEGylated polyplexes aggregated significantly more than PEGylated polyplexes, with a 50% increase in radii. Similar to what was observed for the polyplexes on top of the gel, more PEGylated than nonPEGylated polyplexes were observed, approximately 120 and 30 per image, respectively (Figure 2c,d). The lower number of non-PEGylated polyplexes probably reflected increased aggregation. The diffusion of single particles in the gels was measured by FCS, which provides the diffusion constants of small and fluorescently labeled particles moving into the confocal volume of the microscope. The fluorescence intensity of the particles was also recorded in MQ water. Representative intensity traces in water and in gels are presented in Figure 4. Whereas the intensity signals showed peaks of up to a few thousands of kHz for all the polyplexes in water (Figure 4a,b), no peaks were observed in the gels except for PEGylated chitosan−DNA complexes in collagen−HA matrices (Figure 4f), demonstrating mobility of PEGylated polyplexes only in collagen−HA gels. In the other gels, the initial intensity signals at around a few hundred kHz slowly decayed over time to reach minimum intensity values of 40 to 60 kHz after 40 s (Figure 4c−e). These minimum intensity signals cannot be attributed to the background intensity emitted by collagen or collagen−HA gels, which has been measured to be below 7 kHz for both collagen or HA gels. On the basis of the bleached fluorescence
Figure 3. Distribution of (a, c) non-PEGylated polyplexes and (b, d) PEGylated polyplexes on the top layer of the matrixes (green: chitosan; red: DNA; white: collagen fibers). (a, b) 2.5 mg/mL collagen gels; (c, d), 2.5 mg/mL collagen and 1 mg/mL HA.
was observed, and the DNA was either free or bound to chitosan, as shown by the red and yellow particles, respectively. Collagen fibers were clearly visible in all of the images, and the fibers appeared longer in the collagen gels than in the collagen−HA gels (Figure 3). The median radius of the 3660
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Figure 4. Representative fluorescence intensity signals emitted by YOYO-1 labeled DNA in MQ water (a, b), in collagen gels (c, d), and collagen− HA gels (e, f). Prior to FCS measurements DNA was polyplexed with non-PEGylated chitosan (a, c, e) and PEGylated chitosan (b, d, f).
Figure 5. Logarithmic distribution of the diffusion times (microseconds) obtained by FCS of non-PEGylated (a) and PEGylated polyplexes (b, c) in MQ water (a, b) and in collagen−HA gels (c).
intensity level in Figure 4c−e, a count per molecules close to 0 kHz was measured, which indicates that PEGylated chitosan− DNA polyplexes in collagen and non-PEGylated chitosan− DNA in collagen and collagen−HA gels were present, but not moving in the confocal volume. The diffusion times showed a broad distribution and varied 4−5 decades (Figure 5). From the diffusion times, the average diffusion coefficients for PEGylated chitosan−DNA polyplexes in collagen−HA matrices were calculated and compared with the diffusion coefficients in solution (Table 2). In water, polyplexes without PEG diffused slightly faster than PEGylated chitosan−DNA complexes. No differences were observed between the diffusion coefficients of PEGylated chitosan− DNA particles in solution and those in collagen−HA gels. Estimation of the hydrodynamic radii (eq 4) of the polyplexes in water, based on FCS diffusion coefficients obtained in solution, indicated that the PEGylated polyplexes were larger than non-PEGylated polyplexes, with mean Rh values of 46 < 234 < 1180 nm and 58 < 106 < 194 nm, respectively. This increase in size was probably caused by the large PEG molecules surrounding the polyplexes. Comparison of particle sizes obtained from FCS with the sizes measured from
Table 2. Diffusion Coefficients of Non-PEGylated and PEGylated Polyplexesa diffusion coefficients (μm2 s−1) in water in collagen gel in collagen−HA gel
chitosan−DNA
PEG chitosan−DNA
1.1 < 2.0 < 3.7* NA NA
0.2 < 0.9 < 4.6 NA 0.1 < 0.7 < 4.8
a
The diffusion coefficients were estimated from the average diffusion times measured by FCS. The asterisk indicates statistical significant differences between the diffusion coefficients of non-PEGylated and PEGylated polyplexes. No diffusion was observed for non-PEGylated polyplexes in gels or for PEGylated polyplexes in collagen gels; hence, no diffusion coefficients were estimated for these systems (NA).
microscopy images (Figure 2d) showed similar median sizes of PEGylated polyplexes in water and in collagen−HA gels. Stability of the Polyplexes in the Matrices. The colocalization between FITC−chitosan and Cy5−DNA was assessed for polyplexes applied to the tops of the gels and after the complexes were mixed into the matrices, and Manders’ correlation coefficients were determined. Manders’ coefficients were chosen because we wanted to separate the colocalization 3661
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Figure 6. Manders coefficients estimated for FITC−chitosan polyplexed with Cy5−DNA. Significant differences were observed between PEGylated (gray bars) and non-PEGylated (black bars) polyplexes (∗∗) as well as between collagen and collagen−HA gels (∗).
of the medium increases. The stability of non-PEGylated chitosan polyplexes relies on electrostatic repulsion mediated by the protonated amino groups of chitosan. Because the charge density of chitosan is greatly reduced at physiological pH (pKa of 6.5), the repulsion forces are reduced, and the polyplexes aggregate. Similarly, aggregation also occurs with an increase in the ionic strength of the medium because of the screening of the electrostatic interactions.37 Influence of PEGylation on Polyplex Transport. The complete immobilization of non-PEGylated polyplexes might be attributed to one of two factors: either the polyplexes are too large compared with the pore size of the gels and are thus entrapped in the matrices, or the polyplexes electrostatically bind to the collagen fibers and/or HA. Previous studies estimated the pore sizes of gels formed by collagen at a concentration of 2.0 mg/mL, which was approximately the same as the one used in this work, to be ∼1.2 μm.15 Such pore sizes would exclude the large polyplexes that rapidly aggregated into particles with diameters up to 10 μm (Figure 2a,b). However, also smaller polyplexes down to 100 nm were observed. Thus, the pore size alone cannot explain the immobilization of non-PEGylated polyplexes on top of the gel, as the size distribution of the polyplexes was broad, and smaller polyplexes should still have been able to penetrate into the gels. This is probably the case for PEGylated polyplexes, and smaller PEGylated polyplexes were observed inside the gels. Regarding the impact of electrostatic interactions, chitosan has been reported to form polyelectrolyte complexes with collagen and HA, where chitosan primary amine groups interact electrostatically with the carboxyl groups in collagen and HA macromolecules.26 Lieleg and co-workers20 demonstrated that a zeta potential in the range of −30 to +10 mV was required for nanoparticles to be able to diffuse in an ECM matrigel. Although the zeta potential measurements depend on experimental conditions, the concept of an electrostatic bandpass filter is of interest, and one can speculate whether our polyplexes were within such a filter. The zeta potential of the polyplexes in water was high, but when the polyplexes came into contact with the gels made with PBS, the potential was likely reduced. Non-PEGylated chitosan polyplexes also possess patches with cationic charges that are capable of interacting with the carboxyl groups of collagen and HA, possibly leading to their binding on gel surfaces. The grafting of relatively long and noncharged PEG chains on chitosan introduces a steric
coefficients for the red and green channels. Because of the excess of free chitosan, Manders M1 coefficient, which characterizes the colocalization of chitosan with DNA, does not indicate the DNA unpacking and PEGylation influences on polyplexes; rather, it reflects the A/P ratio. The M2 coefficient, which represents the colocalization of DNA with chitosan, is a good indicator of DNA packing/unpacking. Although M2 is of the most interest, both coefficients are presented in Figure 6. The colocalization coefficients obtained from aggregates applied at the top of the gels were, in most cases, artificially high because the coefficients were determined between DNA and chitosan immobilized within the aggregates. These correlation coefficients are therefore not shown. A determination of Manders’ correlation coefficients between chitosan and DNA for polyplexes mixed into the gels showed that PEGylation reduced the M2 values in both collagen and collagen−HA gels. Seventy percent of the DNA colocalized with non-PEGylated chitosan, and ∼40% colocalized with PEGylated chitosan. The M1 coefficients, however, showed an opposite effect of PEGylation in collagen−HA gels. This contradictory result is probably because of the excess of free chitosan, which makes M1 less relevant than M2. The matrix composition also had an impact on DNA colocalization with PEGylated chitosan, and less correlation was observed in collagen gels than in collagen−HA gels (Figure 6).
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DISCUSSION The transfection of DNA complexed with nonviral vectors depends on the ability of the complexes to diffuse through the extracellular space without dissociating. Diffusion through the ECM is size- and charge-dependent,14,20,35,36 and these two parameters are strongly affected by surface modifications of nonviral vectors, such as PEGylation, and by the ECM composition and structure. The results presented here show that whereas PEGylated chitosan−DNA polyplexes were able to penetrate collagen and collagen−HA gels, no diffusion was observed for non-PEGylated polyplexes. Influence of PEGylation on Polyplex Aggregation and Zeta Potential. The PEGylation of chitosan reduced the zeta potential of the polyplexes and prevented their aggregation, both in the PBS solution and when they were mixed into the collagen/collagen−HA solutions. This demonstrates the importance of the shielding provided by PEGylation. PEG chains create a steric hindrance around the polyplexes, which prevents their aggregation as the ionic strength and/or the pH 3662
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shielding around PEGylated polyplexes that likely decreases the ionic interaction with the matrix. This steric shielding effect might also explain the capacity of PEGylated polyplexes to diffuse in the collagen−HA gels when the polyplexes were mixed with collagen−HA solution before polymerization. Influence of Matrix Composition on Polyplex Transport. The addition of HA to the collagen gel reduced the penetration of PEGylated polyplexes from the gel surfaces into the gels; however, the presence of HA was necessary to obtain diffusion of PEGylated polyplexes that were mixed into the gels. This apparently contradictory role of HA is probably due to differences in the gel in these two situations. The structure and organization of the collagen fibers and HA might be different when the polymerization occurs in the presence of polyplexes as compared with when the polyplexes are applied on the top of the gels. The addition of HA macromolecules to a network of collagen fibers might decrease the pore size38−40 and might increase the overall negative charge of the ECM gel, while the collagen fibers probably reduce the mobility of the HA macromolecules and their ability to interact with the polyplexes. These factors might explain the lesser penetration depths obtained by PEGylated polyplexes deposited on top of the collagen−HA gel when compared with the pure collagen gel. In the case of polyplexes that are mixed into the gels before polymerization, the addition of HA was necessary for diffusion to take place. This might be because of a redistribution of the electrostatic interactions when the mobility of the HA macromolecules is not restricted by the collagen fibers, i.e., before the polymerization of collagen takes place. The presence of carboxyl groups along the HA macromolecule will introduce attractive forces between HA and cationic polyplexes, demonstrated by the negative zeta potential of polyplexes obtained when HA was added to the solution of polyplexes. For PEGylated polyplexes, however, the attractive forces might be reduced, but interactions between the hydrophilic PEG and hydrophilic HA might facilitate the diffusion of the PEGylated polyplexes in the gels. HA has also been reported to interact with collagen fibers,39−42 which might lead to the shielding of the positive charges on the fibers, thereby reducing the repulsive forces. Influence of PEGylation and Matrix Composition on Polyplex Dissociation. PEGylation reduced the Manders M2 correlation coefficient, which describes DNA colocalization with chitosan, demonstrating that PEGylation caused dissociation between DNA and chitosan. Such dissociation was also supported by the increase in free chitosan seen on top of gels with PEGylated polyplexes compared with non-PEGylated polyplexes. The grafting of PEG onto the chitosan molecule utilizes the amino groups, thereby decreasing the electrostatic interaction between chitosan and DNA, whereas the bulky PEG groups sterically obstruct the interaction between chitosan and DNA. These findings are in agreement with previously reported data showing that PEGylation affected the condensation of DNA and reduced the physical stability of PEI−DNA polyplexes.43 Polyplex dissociation might also be caused by the interactions between DNA and collagen fibers or by chitosan binding to HA. Collagen and DNA spontaneously self-assemble in an aqueous environment as a result of the electrostatic interaction between the strong dipole moment of collagen and the negatively charged DNA, and hydrogen bonds are formed between the donor groups on collagen and the acceptor groups on DNA.32 The formation of DNA−collagen complexes is
reported to depend on the structure of DNA, and fibrillogenesis of collagen and plasmid DNA is slower in the presence of linear DNA.33 On the basis of DNA−collagen interactions, collagen has been investigated as a DNA vector.23,33,44 In the event of dissociation, unpacked DNA is thus expected to be immobilized in the matrix because of its binding to collagen fibers. The capacity of DNA to interact with collagen also causes a competition between collagen and PEG−chitosan in binding to DNA, which might lead to the unpacking of polyplexes. The addition of HA to collagen gels increased the colocalization coefficient between PEGylated chitosan and DNA, which indicates reduced dissociation. HA might reduce DNA unpacking by screening electrostatic interactions between collagen and PEGylated polyplexes. Also, other studies have shown that the polymerization of collagen in the presence of HA resulted in the deposition of GAG on collagen fibers and a decrease in fiber diameters.39−42 Adding HA to the solution of polyplexes resulted in a negative zeta potential, and in agreement with other reports,45 this might indicate that HA binds to PEG−chitosan and provides a kind of coating around PEG−chitosan−DNA polyplexes, which might reduce their dissociation. It should be noted that, consistent with our results, increased dissociation rates have been reported for PEGylated PEI−DNA polyplexes in contact with collagen, but not in contact with HA.21
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CONCLUSION This work demonstrates the importance of shielding charges, specifically via PEGylation, to achieve the diffusion of nanoparticles in a charged matrix. Non-PEGylated chitosan− DNA polyplexes were not capable of diffusing through collagen and collagen−HA gels, whereas diffusion was observed when using PEGylated polyplexes. The steric stabilization provided by the PEG macromolecules prevented the aggregation of chitosan−DNA polyplexes. Additionally, the steric hindrance created by PEG around the polyplexes likely reduced the electrostatic interactions with the ECM components, thereby increasing the capacity of the polyplexes to diffuse. Furthermore, PEGylation was observed to increase the dissociation of the polyplexes, as measured by a reduction in the Manders correlation coefficient between DNA and chitosan. HA was found to play a contradictory role by reducing the penetration depth into the gels and also increasing the diffusion. These contradictory findings probably were because HA reduced the pore size of the gels and also shielded the interactions of collagen fibers with the PEGylated polyplexes. Thus, this study indicates a protective role of HA in polyplex stability in the presence of collagen. Most of the previous work dedicated to polyplex interactions with ECM components focused on polyplex stability in solutions of ECM components rather than in ECM gels. The present study accounts for the pore sizes of the gels, the electrostatic interactions with the ECM constituents, and the stability of the chitosan−DNA polyplexes.
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ACKNOWLEDGMENTS This work was supported by The Norwegian University of Science and Technology and The Research Council of Norway. We are grateful to Gjertrud Maurstad for her comments on the manuscript. 3663
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