Protein Based-DNA Gel

Article pubs.acs.org/Biomac

Sustainable DNA Release from Chitosan/Protein Based-DNA Gel Particles M. Carmen Morán,*,†,‡ Andreia F. Jorge,§ and M. Pilar Vinardell†,‡ †

Departament de Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, Avda. Joan XXIII, 08028 Barcelona, Spain Interaction of Surfactants with Cell Membranes, Unit Associated with CSIC, Facultat de Farmàcia, Universitat de Barcelona, Avda. Joan XXII, 08028 Barcelona, Spain § Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal ‡

S Supporting Information *

ABSTRACT: Chitosan lactate (CL) alone and in combination with protamine sulfate (PS) was used as an intrinsic biocompatible carrier to form DNA gel particles by interfacial diffusion. Protamine sulfate is highly positively charged, arginine-rich protein, which has been previosly used in the formation of mixed carriers for modulating DNA release. In view of the promising properties of oligosaccharides and the well-known cell-penetrating and nuclear localization capabilities of protamines, we presume that both structures could play a critical role in DNA delivery. The purpose of this study was to evaluate the capability of water-soluble, low molecular weight chitosan lactate to form DNA gel particles alone (binary system) and in combination with the protein protamine sulfate (ternary system). The particles were characterized with respect to the degree of DNA entrapment, the swelling and dissolution behavior, the secondary structure of DNA in the particles, and the kinetics and mechanisms of DNA release. We controlled the magnitude of DNA release and achieved controlled release by using mixed systems and changing the CL/PS ratio in the solution where the particles were formed. The Rose Bengal partition assay was applied for the first time to estimate the surface hydrophobicity of DNA gel particles. Both CL alone and in combination with PS promotes the formation of DNA gel particles that have an acute hydrophilic character, which may govern the posterior adsorption of plasma proteins and influence the bioavailability of the systems. The lack of hemolytic effect of these DNA gel particles suggests their potential application as long-term blood-contacting medical devices.

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INTRODUCTION The main aim of gene therapy is to transfer genetic material into cells to treat diseases through the expression of certain proteins. Despite significant advances in the past couple of decades, gene therapy is still in the clinical trial stage, mainly due to the lack of safe and efficient delivery vehicles for therapeutic nucleic acids. Deoxyribonucleic acid (DNA) is a negatively charged biomacromolecule that is degraded in the bloodstream by endogenous nucleases.1 Moreover, it is too large to cross cellular membranes. The most common strategy employed for “packaging” DNA is based on electrostatic interaction between the anionic nucleic acid and the positive charges of the synthetic vector, which can complex and condense the nucleic acid.2 Thus, DNA molecules are formulated into synthetic delivery systems to enhance cellular transformation efficiencies. Research on colloidal delivery systems in genetic therapeutics is based on the molecular level, focusing on the interdisciplinary development of pharmaceutical DNA delivery approaches. Colloidal delivery systems modify many physicochemical properties and are designed to protect DNA from degradation, minimize DNA loss, prevent harmful side effects, enhance DNA targeting, increase drug bioavailability, and stimulate the immune system.3−5 Synthetic and natural physically cross-linked hydrogels have led to the concept of reversible or degradable hydrogels that undergo a transition from the three-dimensionally stable © XXXX American Chemical Society

structure to a polymer solution. Most often these hydrogels have been used to encapsulate proteins6 or cells7 and then release them through the dissolution of the hydrogel structure. In many applications, it is important to control the degradation rate of the gel. The formation and degradation of the gel can be achieved by exploiting the association between surfactants and polyelectrolytes. Oppositely charged surfactants and polyelectrolytes have a strong tendency to bind to one another. When the surfactant/ polyelectrolyte attraction overcomes their solubility in the solvent, associative phase separation occurs.8 This result in the formation of concentrated liquid, gel or precipitate phases in equilibrium with a dilute liquid. The polyelectrolyte chains can adopt two types of conformations, either expanded, as in a solution or a hydrogel, or collapsed, such as around a surfactant aggregate as a precipitate. Control over the transitions between these states allows the exploitation of surfactant and polyelectrolyte mixtures in a wide array of commercial applications, such as drug delivery, cosmetic formulations, and rheological modification.9 A general understanding of the interactions between DNA and oppositely charged agents has given us a basis for developing novel DNA-based materials, including gels, membranes and gel Received: July 17, 2014 Revised: September 23, 2014

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dx.doi.org/10.1021/bm501039g | Biomacromolecules XXXX, XXX, XXX−XXX

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particles.10 We have recently prepared novel DNA gel particles based on associative phase separation and interfacial diffusion. By mixing solutions of either single- (ssDNA) or double-stranded (dsDNA) DNA with solutions of different cationic agents, such as surfactants, proteins, and polysaccharides, we confirmed that DNA gel particles could be formed without adding any kind of cross-linker or organic solvent.11−13 Studies involving the interaction of ss-DNA and ds-DNA with surfactants and polycations have demonstrated the importance of the charge density, chain flexibility, and hydrophobicity in these systems.10 The difference between ss-DNA and ds-DNA is larger when surfactants are used with the other investigated cosolutes. Both a higher flexibility of ssDNA and a higher hydrophobicity due to the exposed bases are found to play an important role in the interaction of DNA. This is strong evidence for the importance of hydrophobic interactions in these systems.11 Our current study focuses on the preparation of DNA gel particles with cationic compounds that have much improved intrinsic biocompatibility. These include cationic proteins such as lysozyme14,15 and protamine sulfate15 and polysaccharides such as chitosan.16 When lysozyme is used in combination with protamine sulfate as a DNA carrier, a large degree of control over the release profile is achieved. Both proteins can promote an increased level of charge matching and strengthened hydrophobic interaction, favored by their discrepancy in size, shape, and positioning of charges.15 Furthermore, recent studies have demonstrated that the protein composition and the preparation method are controlling parameters for the in vitro biocompatibility of these protein−DNA gel particles.17 With regard to polysaccharides, we have demonstrated that DNA gel particles formed when DNA is mixed with chitosan of different molecular weights. DNA was effectively entrapped in the chitosan solutions, protecting its secondary structure. Chitosan molecular weight was found to be a good controlling parameter: when we decreased the molecular weight of chitosan, we significantly extended the effective release of DNA. However, the application of the native polysaccharide was limited by its high molecular weight, resulting in its low solubility in acid-free aqueous media. To overcome this problem, many soluble chitosan derivatives have been developed by modifying the reactive functional groups of chitosan or by depolymerizing it.18 Chitosan can also form water-soluble salts, such as chitosan acetate and chitosan lactate, with some aqueous inorganic or organic salts.19 The major underlying challenges in the development of a carrier system are to ensure the safe and efficient delivery of DNA to the target site, followed by cellular uptake, internalization and processing, and the production of the therapeutic level of gene products for a desired period. In the absence of cell division, an additional limiting step is the translocation of DNA through the nuclear envelope (NE). Nuclear pore complexes (NPCs) correspond to large protein transport complexes responsible for selective nucleocytoplasmic exchange. The NPCs contain an aqueous channel that allows passive diffusion of small molecules. For larger molecules, an energy and signal-dependent mechanism is required. This active transport is mediated by nuclear localization signals (NLSs). Endogenous nuclear proteins are interesting candidates for mediating nuclear translocation, as they have one or more NLSs. They include proteins from the high mobility group, histones and protamines.20−22 Recent studies demonstrated that glycoproteins lacking NLSs can enter the nucleus. Oligosaccharides can be recognized by lectins present on the NPCs. If bound to DNA, these oligosaccharides can facilitate transport through the NE.23

The main purpose of this study was to evaluate the capability of water-soluble, low molecular weight chitosan lactate to form DNA gel particles (binary system). Protamine sulfate is highly positively charged, arginine-rich protein, which has been previosly used in the formation of mixed carriers for modulating DNA release.15 For this reason, the combination of chitosan lactate with the protein protamine sulfate (ternary system) has been also proposed. In view of the promising properties of oligosaccharides and the well-known cell-penetrating and nuclear localization capabilities of protamines, we presume that both structures could play a critical role in DNA delivery. Here, we report on the physicochemical properties (particle morphology, swelling/dissolution behavior, degree of DNA entrapment and DNA release responses) of these chitosan lactate-based DNA gel particles as a function of the imposed compositions. To understand the mechanisms and kinetics of DNA release, several mathematical models were applied to the in vitro release data curves. As the surface properties determine the physicochemical characteristics and fate in blood circulation, the hydrophobicity of the DNA gel particles was characterized. The hemocompatibility of the DNA gel particles was also determined by means of a hemolysis assay.

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MATERIALS Sodium salt of deoxyribonucleic acid (DNA) from salmon testes with an average degree of polymerization of ≈2000 base pairs (bp) was purchased from Sigma and used as received. We determined DNA concentrations spectrophotometrically, and assumed that for an absorbance of 1 at 260 nm, a solution of dsDNA has a concentration of 50 μg mL−1. Chitosan oligosaccharide lactate (CL) with a molecular mass of 5 kDa and protamine from salmon in the sulfate salt form (PS) with a molecular mass of 5.1 kDa were purchased from Sigma and used as received. N,N,N′,N′-Tetramethylacridine-3,6-diamine (acridine orange AO) was supplied by Molecular Probes (Invitrogen). The 4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein disodium salt (Rose Bengal sodium salt, RB) was supplied by Sigma.

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METHODS

Particle Preparation. DNA stock solutions were prepared in 10 mM NaBr to stabilize the DNA secondary structure in its native B-form conformation. CL and PS were dissolved in a PBS buffer pH 7.4 (unless stated otherwise). In the case of binary systems, particles were prepared at different ratio R, where R = [DNA]/ [CL], and [DNA] and [CL] were expressed in % (w/v). R values varied between 39.6 and 1. In the case of the ternary systems, particles were prepared at a ratio R equal to 1, where R = [DNA]/[C+], and [C+] is the concentration of the corresponding cationic system, expressed in % (w/v). The composition of the mixed systems varied between 15 and 85% PS. In all cases, [DNA] was equal to 2% (w/v). DNA solutions were added dropwise via a 22-gauge needle into gently agitated cationic solutions (2 mL). Under optimal conditions, droplets from DNA solutions instantaneously gelled into discrete particles on contact with the cationic solution. Thereafter, the particles were equilibrated in the solutions for a period of 2 h at room temperature. After this period, particles were separated and washed with PBS to remove excess salt. Fluorescence Microscopy Imaging. Particle integrity and the DNA conformational state were determined by the acridine orange (AO) fluorescence assay. Freshly prepared particles were stained for 10 min with AO (0.3 mg mL−1) and washed in distilled water. The stained samples were immediately examined with an Olympus BX41 microscope equipped with a UV-mercury lamp (100 W Ushio Olympus) and a U-N51004v2- FlTC/TRITC type filter set (FITC: BP480-495, DM500-545, BA515-535 and TRITC: BP550-570, DM575-, BA590621). Images were digitized on a computer through a video camera B


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(Olympus digital camera XC50) and were analyzed with an image processor (Cell B analysis). Determination of Degree of DNA Entrapment. The degree of DNA entrapment in the DNA gel particles was determined by quantifying the free DNA in the supernatant solution. The entire quantity of supernatant solution containing the free DNA was removed for quantification by spectrophotometry at 260 nm. The concentration of DNA bound in the DNA gel particles was calculated as the difference between the concentration of DNA added into the cationic solutions and the DNA that remain free in the solutions, after the particle formation:

interpreted language intended for numerical computations. It provides capabilities for the numerical solution of linear and nonlinear problems. In nonlinear regression analysis, the Levenberg−Marquardt algorithm minimized the least-squares and the detailed description is documented in a collection of Octave packages.29 The model parameters for the different functions with their standard errors can be obtained based on the calculation results. The selection of the best model was based on the comparisons of the higher determination coefficient (Tables S4 and S5). Rose Bengal Adsorption Assay. Individual DNA gel particles were placed in tubes and their weight was determined. Then, a series of different volumes of the Rose Bengal (RB) solution (1 mg mL−1), ranging from 5 to 40 μL, were placed in the tubes. PBS buffer was added to each tube to a final volume of 1 mL. The tubes were incubated at room temperature for 180 min under shaking conditions using an Atom 190 shaker (Atom). After incubation, the amount of free RB (RB not bound to the particle surface) was determined in the supernatant by UV−vis spectroscopy at 549 nm. The quantity of free RB in the solution was determined by interpolation from a calibration curve. The concentration of RB bound to the particle surface was calculated as the difference between the total concentrations of RB used in the assay and free RB. The data were transformed to the adsorption isotherms (Γ), which correspond to the RB bound as a function of the particle weight, compared to the RB at equilibrium (free RB). Interaction with Erythrocytes. Preparation of Red Blood Cell Suspensions. Red blood cells were obtained in accordance with the procedure described in our laboratory.17 Briefly, blood was obtained from anaesthetized rats by cardiac puncture and drawn into tubes containing EDTA. The serum was removed from the blood by centrifugation and by subsequent suction. The red blood cells were then washed three times at 4 °C by centrifugation at 3000 rpm with isotonic saline PBS solution (pH 7.4). Following the last wash, the cells density was fixed at 8 × 109 cell/mL. Hemolytic Study. The membrane-lytic activity of the systems was examined by a hemolysis assay. First, the hemolytic response to the polysaccharide or protein in solution was tested. A series of different volumes of the cationic solution (10 mg mL−1) were placed in polystyrene tubes and an aliquot of erythrocyte suspension was added to each tube. The tubes were incubated at room temperature for 10 min under shaking conditions using an Atom 190 shaker (Atom). Following incubation, the tubes were centrifuged (5 min at 10000 rpm). The degree of hemolysis was determined by comparing the absorbance (540 nm; Shimadzu UV-160A) of the supernatant with that of the control samples totally hemolysed with distilled water. Positive and negative controls were obtained by adding an aliquot of erythrocyte suspension to distilled water and isotonic PBS solution, respectively. In the case of DNA gel particles, individual DNA gel particles were placed in the tubes and an aliquot of erythrocyte suspension was added to each tube. The tubes were incubated at room temperature for different times (10−180 min) under shaking conditions. At the same defined times, the incubated samples were centrifuged. The degree of hemolysis was determined following the same procedure as described above.

[bound DNA] = [total DNA] − [free DNA] The degree of DNA entrapment is expressed through the loading capacity and loading efficiency values. Loading capacity (LC) takes the amount of DNA entrapped in the particles as a function of their weight. Loading efficiency (LE) is calculated by comparing the amount of DNA included in the particles with the total amount during particle formation. Loading capacity (LC) and loading efficiency (LE) were determined by the following equations:

LC(%) = [bound DNA]/weight of particles × 100

(1)

LE(%) = [bound DNA]/[total DNA] × 100

(2)

Three batches of particles were prepared in each system and the results are given as average and standard deviations. Swelling and Dissolution Behavior of the Particles. The initial weight (Wi) of the particles formed under each condition was measured immediately after they were prepared. They were then separated by filtration and washed with pH 7.4 PBS to remove excess salt. Particles (around 100 mg) were exposed to the initial PBS buffer (4 mL) at an agitation rate of 30 rpm and at room temperature, using the Rocker PMR-30 shaking platform (Grant-bio). Additionally, wet particles were measured at each time point (Wt) immediately after removal of the release solution. Then, fresh solution was added to maintain a clean environment. The swelling ratio of the particles at each time point was calculated accordingly from the following equation:

relative weight(RW) = Wt /Wi

(3)

This value reflects the change in weight of the particles at each time point, with respect to the initial weight of the gel. The highest point indicates the highest swelled points for each hydrogel and the zeroreturned point implies the points where the weight of the hydrogel has returned to their starting point. Complete degradation of a particle sample was noted when the presence of the particles, or fragments of them, were no longer visually apparent. Swelling experiments were performed at room temperature (18−22 °C). To ensure identical environmental conditions for all the hydrogels, particles prepared using binary and ternary systems were prepared at the same time. DNA Release from the Particles. DNA release studies were carried out at the same time as the studies of swelling/dissolution behavior. The release solution was completely removed from the samples and entirely replaced with fresh solution at periodic time points. The amount of DNA in the release solutions was quantified by measuring the absorbance at 260 nm using the NanoPhotometer (Implen). The cumulative DNA release was normalized with respect to the initial particle weight and expressed as a percentage. Evaluation of DNA Release Kinetics. Mathematical models have been used extensively for the parametric representation of dissolution data. The standard models in the dissolution data analysis include the cubic root law,24 square root of time equation,25 and first-order exponential function.26 For the general case of tablets, however, the interaction of disintegration and dissolution is complex and requires models which are applicable for S-shaped dissolution profiles. Weibull distribution27 as well as the logistic model28 are able to describe S-shaped sigmoidal dissolution profiles. The mathematical models, shown in Table S3, were fitted to the entire set of experimental data using GNU Octave software, a high-level

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RESULTS AND DISCUSSION Particle Preparation. Using binary systems, DNA gel particles were prepared by dropwise addition of DNA solutions into the polysaccharide solutions. The mixing of the two solutions is not instantaneous, because of the relatively high viscosity of the DNA solution. Before the two solutions can mix, the polysaccharide diffuses into the polyelectrolyte phase and forms a gel shell at the interface, which stabilizes the particles. This is the general behavior observed for DNA placed in polysaccharide solutions. However, the behavior depends strongly on the R values. In this study, the R values varied from 39.6 to 1. Systems with R values lower than 1 had a fairly viscose CL solution, which made the formation of CL-DNA gel particles difficult. Consequently, 1 was the lowest R value used. Although CL was initially C


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Figure 1. Representative images of the DNA gel particles at different ratios R = [DNA]/[CL] for CL-DNA gel particles (a) and CL/PS-DNA gel particles (b). Evolution of particle size (c) as a function of the composition (binary systems: light gray; ternary systems: dark gray). The values shown in (c) were measured in triplicate and are given as average and the standard deviation.

increased.30 In the case of ternary systems, the particle size increased as the percentage of PS in the mixtures rose. Morphological Characterization of the DNA Gel Particles. Fluorescence microscopy (FM) using the fluorescence dye acridine orange (AO) was used to confirm the presence of DNA and to assess the secondary structure of the nucleic acid in the particles. AO (excitation: 500 nm/emission: 526 nm) intercalates into double-stranded DNA as a monomer, whereas it binds to single-stranded DNA as an aggregate. On excitation, the monomeric acridine orange bound to double-stranded DNA fluoresces green, with an emission maximum at 530 nm. The aggregated acridine orange on single-stranded DNA fluoresces red, with emission at about 640 nm.31 Figure 2a shows fluorescence micrographs of individual DNA gel particles prepared using binary systems. The fluorescence emission depended strongly on the R values. For the highest R values, the particles were disrupted quite easily during the washing step to eliminate the excess dye. Thus, the particle structure was lost. In the case of the system formed at R = 32, some red emission was visible, as a consequence of DNA denaturation. Systems formed at R values between 15.84 and 1 showed green emission, which suggests that DNA gel particles were formed with the conservation of the secondary structure of the DNA. From visual observation during the particle formation process and information about the secondary structure derived from FM studies, we established that the interval between R = 7.92 and R = 1 was optimal subsequent experiments. FM images of freshly prepared ternary system particles, using AO staining, showed green emission independently of the composition (Figure 2b). This behavior confirms that the secondary structure was conserved in the formation of CL/PS-DNA gel particles. Determination of the Degree of DNA Entrapment. The degree of DNA entrapment is expressed through loading capacity

dissolved in pH 7.4 PBS buffer, limitations to the solubilization of this derivative were found. However, clear solutions were obtained when CL was dissolved in bidistilled water. Stable DNA gel particles were obtained for R values ranging from 7.92 to 1. Systems with R values ranging from 15.84 to 39.6 produced really weak DNA gel particles. Moreover, the size of the final particles depended strongly on the R value. Figure 1a shows a representative image of individual CL-DNA gel particles prepared at different R values. Taking into account the results, particles prepared using the ternary system were at a ratio R equal to 1, where R = [DNA]/ [C+], and [C+] is the concentration of the cationic system, which ranged between pure CL systems and pure PS systems. The composition of the mixed systems varied between 15 and 85% PS. Figure 1b shows representative images of the CL/PS-DNA gel particles. Spherical, regular particles were obtained, regardless of the composition. The effect of the PS content was easily observed during the preparation process. Particles containing large amounts of PS became more visible, and opaque particles were obtained. Particles with PS alone, exhibited a strong tendency to aggregate. Changes from translucent to opaque have also been observed in other family of DNA gel particles15 and attributed to the collapse and shrinkage of the protein−DNA network. Using this analogy, the formation of opaque DNA particles, when the PS content is increased, probably indicates the formation of condensed structures. The isolation of individual particles for each system allowed us to estimate their dimensions. Figure 1c summarizes the size distribution as a function of composition. PS-DNA gel particles exhibited a strong tendency to aggregate, which made it difficult to obtain individual particles. In general, particle size decreased as the R value dropped as a consequence of the higher compaction/ condensation degree as the concentration of the cationic agent D


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Figure 2. Representative fluorescence micrographs of the CL-DNA gel particles (a) and CL/PS-DNA gel particles (b) in the presence of acridine orange fluorescent dye as a function of the composition.

as the concentration of CL rose. Although the viscosity of the CL solutions has not been determined, visual inspection of corresponding solutions supported these differences. These results confirm, the effectiveness of DNA entrapment in these polysaccharide solutions, in contrast to those observed for DNA gel particles obtained with chitosan of higher molecular weight (MW ranged between 50 and 400 kDa).16 The limited solubility of the studied chitosan derivatives confined the optimum R value to 13.3 and restricted the LC to the range between 0.6 and 1.1%. In the case of ternary systems, the loading capacity (LC) and loading efficiency (LE) values depended on the composition. The characteristics of the different systems are summarized in Figure 3b. There was no clear trend in LC values when the composition was varied. LC values varied between 1.9 and 3.5%, for CLPS85 and CLPS50, respectively. LE values varied between 98.1 and 99.9%, for systems ranging from pure CL to pure PS, respectively. These results confirm the effectiveness of DNA entrapment in these solutions. Swelling Kinetics. Gels are thought to have great potential as drug reservoirs. Loaded drugs are released by diffusion from the gels or by erosion. Hence, the release mechanism can be controlled by swelling or dissolution of the gels. Figure 4 shows the

and loading efficiency values. Loading capacity (LC) assesses the amount of DNA entrapped in the particles as a function of their weight. Loading efficiency (LE) is calculated by comparing the amount of DNA included in the particles with the total DNA added during particle formation. Loading capacity (LC) and loading efficiency (LE) depended on the R values. The characteristics of the different systems are summarized in Figure 3. Although the observed differences between these systems were not really pronounced, binary systems containing the highest concentration of CL had the highest LC values (Figure 3a). This trend can be correlated with an increase in the homogeneity of the gelation process. LC values increased progressively as the R values decreased. The LC values varied between 1.0 and 3.3%, when the R value ranged between 7.92 and 1, respectively. LE values varied between 99.8 and 98.1% for R values from 7.92 to 1, respectively. However, this result seems to be opposite of what could be expected for DNAoppositely charged compounds for which a higher compaction/ condensation degree is expected as the concentration of the cationic agent increased.30 This behavior can be correlated with differences in the viscosity of the CL solutions as a function of their concentration. The viscosity of the CL solutions increased E


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Figure 3. Characterization of the CL-DNA gel particles (a) and CL/PS-DNA gel particles (b) with respect to DNA loading efficiency (LE) and loading capacity (LC) as a function of the ratio R values. All values were measured in triplicate and are given as average and the standard deviation.

Figure 4. Time-dependent changes in the relative weight of CL-DNA gel particles (a) and CL/PS-DNA gel particles (b) as a function of the composition, after exposure to PBS pH 7.4 buffer solutions.

swelling, it is well know that molecular interactions in aqueous solution are affected to a greater or lesser extent by the presence of electrolytes, depending on the contribution of electrostatic forces to the binding. Previous studies in our lab demonstrated the contribution of electrostatic interactions in CTAB-DNA binding by monitoring the effect of the medium ionic strength on the DNA release assays.32 The electrostatic attraction is obviously expected to be weaker in the presence of electrolyte, or in the case of buffering salt. A weakening of this association is expected to result in a partial or complete dissolution. DNA Release. The kinetics of DNA release for the different CL-DNA particles depended on the R values (Figure 5a). Generally, the release pattern resembled that observed in the swelling/ dissolution profiles. The percentage of cumulative DNA release from CL-DNA systems could be modulated from 2.15 to 79.80% in the first 2 h by variation of the R values from 1 to 7.92, respectively. In general, the percentage of cumulative DNA release decreased when the R values dropped. At the end of the experiment (120 h), total DNA release from the particles was observed. The kinetics of DNA release for the different CL/PS-DNA particles also depended on the composition (Figure 5b). CLDNA gel particles exhibited fast burst release behavior by a dissolution mechanism. After 24 h, 65% of DNA was released. When the formulation contained either pure PS or, in mixed systems, the initial burst release was absent. The percentage of DNA released in the dissolution media, after 24 h, varied from 0.2 to 8.6% in the protein-containing systems. The absence of a burst effect suggests that minimal amounts of unencapsulated DNA are present on the surface of these particles. Using ternary systems, the release rates could be progressively modulated by the

relative weight ratio of the different gel particles after exposure to PBS pH 7.4 buffer solutions. A general trend is that CL-DNA gel particles initially swelled and then dissolved (Figure 4a). The time in the swollen state before dissolution depended on the R values. Table S1 summarized the values of maximum swelling points for all compositions. In general, the relative weight ratio became larger as the polysaccharide concentration increased. From the maximum weight ratio to the full dissolution point, it could be seen that there is a period of stability before dissolution. The stabilized time would be longer as the polymer concentration increases. After the onset point, the relative weight ratio would be 1.0 (defined as the zero-returned point), and then the hydrogel would monotonously dissolve. When the profile is recalculated from the maximum relative weight to the full dissolution point, the zero-returned point can be estimated (Table S1). Nearly, it was found that the degree of swelling and the zeroreturned point increased as the R values decreased. Swelling experiments carried out with the different CL/ PS-DNA particles demonstrated that the relative weight depended strongly on the composition (Figure 4b). Table S1 summarized the values of maximum swelling points for all compositions. DNA gel particles prepared in the presence of the protein in either the pure PS systems or the mixed systems did not exhibit dissolution kinetics. Particles show water uptake from the medium and swelling could be observed. The swelling continues during the entire time interval studied. Only in the case of the CL and CLPS30 systems was there a return to the original particle weight (Table S2). Osmotic pressure forces, electrostatic forces and viscoelastic restoring forces are the three main forces governing the swelling behavior of hydrogels. Concerning the mechanism for the F


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Figure 5. Time-dependent changes in DNA release profiles for the CL-DNA gel particles (a) and CL/PS-DNA gel particles (b) as a function of the composition, after exposure to PBS pH 7.4 buffer solutions.

Figure 6. Experimental release profile of DNA (symbols) and best-fit curves for the Weibull function (lines) from CL-DNA gel particles (a) and from CL/PS-DNA gel particles (b).

As the cylinder is the most common geometrical shape of the devices used in pharmaceutics, the analysis of one-dimensional radial release33 and axial and radial release from cylinder according to Case II drug transport have been performed.34 Under these conditions, the value of the exponent b is an indicator of the drug transport mechanism through the polymer matrix. Estimates for b < 0.75 indicate Fickian diffusion while a combined mechanism (Fickian diffusion and Case II transport) is associated with b values in the range 0.75 < b < 1. For values of b higher than 1, the drug transport follows a complex release mechanism.35 Studies carried out on the modeling rehydration of food particulates demonstrated that the Weibull b-shape parameter varied with geometry and the mechanism of water uptake.36 The derived values for spheres, cylinders, and slabs were for diffusion, 0.67, 0.72, and 0.81, respectively; for internal resistance, 1.00, 0.98, and 0.97, respectively; and for relaxation, 1.21, 1.32, and 1.60, respectively. Regardless of the geometry, the estimates for b lie very close to the described range for Fickian diffusion (b < 0.75), combined mechanism (0.75 < b < 1), and super Case II (b > 1). The model parameters derived from the Weibull function, fitting the DNA release profiles from CL-DNA gel particles suggest that the release mechanism changes as a function of the R values (Table S6). The b exponent systematically changes to values lower than 1 for R between 7.92 and 2, to values close to 1 when R = 1.3 and above 1, for the R = 1 system. Figure 6a shows the curve fitting, using the Weibull model, to the experimental DNA release profiles corresponding to the different systems. According to this model, the shape parameter (b) characterizes the curves as either exponential (b = 1), sigmoid, S-shaped, with upward curvature followed by a turning point (b > 1), or parabolic, with a higher initial slope, followed by a curve consistent with the exponential (b < 1).

incorporation of a small fraction of protein PS in the mixtures (CLPS15 and CLPS30 systems). However, when the PS content was increased, the DNA release profiles showed slower release rates than those observed in pure systems. It is also noteworthy that the final release percentage was largely dependent on the CL/PS ratio. The cumulative DNA release at the end of the experiment (900 h) varied from 2.13, 40, and 100% for the CLPS50, PS, and CL systems, respectively. Evaluation of DNA Release Kinetics. The modeling of drug release from delivery systems helps us to understand and elucidate the transport mechanisms. To ascertain the DNA release kinetics, the release profiles were fitted to different mathematical models that include least-squares fit, zero-order, firstorder, Higuchi, Hixson-Crowell, Weibull, Logistic, KorsmeyerPeppas, and Hopfenberg models. Tables S3−S5 summarizes the model functions used in this study and the corresponding coefficient of determination (r2) quantifies goodness of fit using both linear and nonlinear regression for the analysis of the entire set of experimental data. The fitting of the profiles obtained from these DNA gel particles involves models that can describe S-shaped dissolution profiles. These include the Weibull model. Recent studies based on Monte Carlo simulations indicate that Fickian drug release can be described by the Weibull function. These observations suggested that the Weibull function could be used to analyze the entire set of experimental data for controlled release formulations, instead of the classical analysis based on the Korsemeyer-Peppas model, which is usually applied to the first 60% of the release curve. Although the Weibull function has been used empirically to analyze release kinetics, the results provide a link between the values of b and the diffusion mechanism of the release. The linear relationship that was established indicates not only the mathematical relevance of b and n exponents, but also the physical connection between the model parameters and the release mechanism. G


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influences protein binding, the surface properties (charge and hydrophobicity) are likely to be more important. The question arises as to how materials can be designed to specifically adsorb certain proteins or avoid their adsorption.43 Masking the particle surface with polyethylene glycol (PEG) is the best-known strategy to diminish protein adsorption. The protective properties of PEG as a function of PEG chain length and PEG surface density have been reviewed.44 Nevertheless, PEG cannot fully prevent protein adsorption or opsonization. The Rose Bengal (RB) partitioning method was developed to estimate the surface properties of uncoated polymer particles. The measurements can be made using a UV−vis spectrophotometer. The advantage of measuring in the visible spectrum is that impurities like surfactants or monomers released by the colloidal particles over time do not disturb the spectrum of the dye. RB is a xanthene dye used as a photosensitizer, fluorescent label, and an adsorption marker.45 We studied the RB adsorption on the DNA gel particles. The quantity of free RB in the solution was determined by interpolation from a calibration curve. The concentration of RB bound to the particle surface was calculated as the difference between the total concentrations of RB used in the assay and free RB. The data were transformed to the adsorption isotherms (Γ), which correspond to the RB bound as a function of the particle weight, compared to the RB at equilibrium (free RB). The absorption spectra of RB were perturbed in the presence of the DNA gel particles. Like other dyes of this class, it was confirmed that the color and spectroscopic characteristics mainly depended on the environment red shift of RB absorption from 549 nm in PBS solution46 to 554.5 and 564.5 nm in the presence of individual CL-DNA particles and CL/PS-DNA gel particles, respectively. The evolution of the adsorption isotherms of RB in the presence of CL-DNA gel particles as a function of the R values is shown in Figure 7a. Due to the relative fragility of the CL-DNA particles prepared at R = 7.92, no RB adsorption experiments were carried out with this system. In general, the maxima of RB adsorption increased when the R values decreased. The shape of the isotherms is not sophisticated at all, and shows the decay of adsorption at higher equilibrium concentrations. This decay was mainly observed for the systems prepared at lower R values, for which we expected a higher concentration of CL on the particles. The observed decay on the adsorption isotherms can be correlated with the aggregate formation of the dye, as observed in the case of RB with cationic surfactant molecules.47 Similar experiments were carried out with the CL/PS-DNA gel particles prepared at different compositions (Figure 7b). As the particles formed with pure PS exhibited a strong tendency to aggregate (see Figure 1), no RB adsorption experiments were carried out with this pure protein system. In general, the RB adsorption increased when the percentage of PS in the composition are increased. The decay of RB adsorption at higher equilibrium concentrations became less important when the percentage of PS in the composition increased. Taking into account the Γmax value, we established the relative hydrophobicity of the DNA gel particles (Figure 7c). We expected that the higher the Γmax value, the higher is the relative hydrophobicity. The Γmax values ranged between 9.1 × 10−5 and 5.2 × 10−4 mmol g−1 when the R values decreased (binary system) and 1.2 × 10−3 and 2.8 × 10−3 mmol g−1 when the percentage of PS in the mixtures increased (ternary system). In order to grade the relative hydrophobicity of the particles, these values have to be compared with others described in the

The release mechanism of DNA from the CL-DNA gel particles can be described as pure Fickian diffusion for R values between 7.92 and 2. The mechanism of DNA release from CLDNA gel particles prepared at a ratio of R = 1.33 is compatible with first-order release, where the concentration gradient in the dissolution media drives the rate of release. For CL-DNA gel particles prepared at a ratio of R = 1, a complex mechanism governs the release process. In this kind of transport, there are two simultaneous fluxes. The first flux is the rate at which the diffusing material is released at an interface by relaxation of the polymer matrix. In the glassy state, the matrix has a finite relaxation time, associated with the length of the polymers in relation to the entanglement network.37 The second flux is the rate at which the material diffuses away from the interface. At this point, the polymer is in the rubbery state and, it swells, making the relaxation time almost instantaneous. The parameters governing the release of the dissolved material are thus the rate at which the interface moves, the diffusivity of the dissolved material in the rubbery polymer, and the total length of the diffusional path.38 In this kind of transport, polymer relaxation is the rate-limiting step to water transport. The relevance of the diffusion mechanism of DNA release from CL-DNA gel particles was demonstrated when a first-order equation was fitted to the experimental DNA release (Table S4). The results can be compared with those observed in the DNA release from chitosan-DNA gel particles using chitosan of different molecular weights.16 From model fitting studies and the statistical treatment, we verified that the modified Weibull model was the one that best described the DNA release kinetics from the chitosan-DNA particles. The results listed in Table S7 suggest that the release mechanism from CL/PS-DNA gel particles changes as a function of the composition. In the case of mixed systems, the b values always remain lower than 1. The b exponent systematically changes to values higher than 1 for the pure CL and pure PS systems. Consequently, the release mechanism in the mixed systems can be described as pure Fickian diffusion and a combined mechanism (Fickian diffusion and Case II transport). However, for CL-DNA gel particles and PS-DNA gel particles, the super Case II situation describes the release profiles, and two simultaneous fluxes are responsible for this kind of transport.37 The results can be compared with those observed in the DNA release from mixed protein DNA gel particles containing lysozyme and protamine sulfate.15 The analysis of release data corresponding to particles with high PS content revealed a dualstage release mechanism. During the first stage of the release, a zero-order profile indicating a relaxation-controlled mechanism was found in most cases. After this period, the fitting of the obtained profile required models that are applicable to S-shaped profiles, such as the Weibull function. We only observed a sigmoidal profile without any apparent change in mechanism for the PS-DNA particles, and we again resorted to the Weibull model for its description. Evaluation of the Surface Hydrophobicity of DNA Gel Particles. Along with other factors, the surface properties of the carriers determine their physicochemical characteristics, fate and blood circulation.39 The hydrophobicity of the particle surface governs the adsorption of plasma proteins40 and subsequently the rate of clearance from systemic circulation. Generally, hydrophobic particles are opsonized more quickly than hydrophilic particles due to the enhanced absorbability of plasma proteins onto the surface of hydrophobic particles.41,42 Though particle composition (base material type, shape, and size) clearly H


Biomacromolecules

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Figure 7. Adsorption isotherms of Rose Bengal on CL-DNA gel particles (a), adsorption isotherms of Rose Bengal on CL/PS-DNA gel particles (b) and Γmax values of Rose Bengal (c) for the CL-DNA gel particles and CL/PS-DNA gel particles as a function of the composition (binary systems: light gray; ternary systems: dark gray).

literature, in which the Γmax values of several polymer lattices have been shown.45 Particles derived from polystyrene, which is strongly hydrophobic, showed Γmax values close to 0.072 mmol g−1. On the other hand, Γmax values for poly(methyl methacrylate) particles, which are hydrophilic, were found to be 0.017 and 0.037 mmol g−1, depending on the ionic character. Therefore, we can conclude that both binary and ternary systems promote the formation of DNA gel particles with a well-defined hydrophilic character. The acute hydrophilic character of the DNA gel particles may govern the adsorption of plasma proteins and influence the overall bioavailability of the systems, as an alternative to any posterior polymer coating step. Hemolytic Assessments of DNA Gel Particles. One important factor in the development of particulate systems for parenteral administration is to determine their ability to cause hemolysis by interaction with the cell membrane. The potential uses of colloidal self-assemblies as drug delivery systems make hemolysis evaluation essential. To this end, we examined this interaction by using erythrocytes as a model biological membrane system, since erythrocytes have been used as a suitable model for studying the interaction of amphiphiles and other molecules with biological membranes.48,49 Hemolysis Induced by CL and PS in Solution. The hemolytic activity of the polysaccharide and protein compounds was assessed at different concentrations. In these experiments, hemolysis was determined at a fixed time (after 10 min of incubation) in the presence of cationic compounds in the range of 200 to 2000 μg mL−1. The hemolysis assay showed that the chitosan derivative and the protein were nonhemolytic in nature (Figure 8a,b). The hemolytic potential of a material is defined as a measure of the extent of hemolysis that may be caused by the system when it comes into contact with blood. For all the concentrations assayed, the extent of hemolysis was lower than the permissible level of 5%.50 In these experiments, hemolysis

was determined in the presence of a range of concentrations which allows us to confirm that the polysaccharide and protein compounds are not hemolytic at 2000 μg mL−1. Hemolysis Induced by DNA Gel Particles. We studied the hemolytic response of the erythrocytes to the DNA gel particles. In order to evaluate the effect of DNA complexation on the hemolytic response, the corresponding DNA gel particles were incubated with the erythrocyte suspensions for different times (ranging from 10 to 180 min). We considered that long incubation periods could affect the stability of the DNA gel particles, promoting the release of the polysaccharide or protein into the solution and altering the hemolytic response. The hemolysis response was not time-dependent when individual CL-DNA gel particles were incubated in the erythrocyte dispersion for periods of time ranging between 10 and 180 min. The maximum percentage of hemolysis was 8%. It is interesting to note that the CL concentrations required to prepare the corresponding CL-DNA gel particles ranged between 2500 and 20000 μg mL−1, which was 1 order of magnitude higher than the highest concentration tested with the polymer in solution. Similar experiments were carried out with DNA gel particles prepared using ternary systems (Figure 8d). The maximum percentage of hemolysis was lower than the permissible 5%. In this case, the corresponding CL and PS concentrations required to prepare CL/PS-DNA gel particles were around 20000 μg mL−1, which was 1 order of magnitude higher than the highest concentration tested with the pure compounds in solution. These results can be compared with those observed in mixed protein DNA gel particles containing lysozyme and protamine sulfate.17 The hemolysis response was not time-dependent in individual protein−DNA gel particles incubated in the erythrocyte dispersion. In all cases, the percentages of hemolysis were lower than 3%. Nevertheless, this behavior differed strongly from that observed for surfactant−DNA gel particles.51 One drawback I


Biomacromolecules

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Figure 8. Dependence of rat erythrocyte hemolysis on polysaccharide (a) and protein (b) concentration. Erythrocytes were incubated for 10 min at room temperature at different concentrations, and the amount of released was determined. The time-course of the hemolytic response of rat erythrocytes after incubation with CL-DNA gel particles (c) and CL/PS-DNA gel particles (d).

slightly hemolytic character of the CL-DNA gel particles, with a maximum percentage of hemolysis close to 8%, can be modulated when they are administered in the ternary CL/PSDNA system, in which the hemolytic responses were lower than the permissible level of 5%.

of surfactant−DNA gel particles, in toxicological terms, is the need for a cationic surfactant, which may cause cell damage. However, our results indicate that the effect of the surfactant can be modulated when it is administered in the DNA system, unlike an aqueous solution. This modulation is due to the strong interaction between the surfactant and the biopolymer, which leads to very slow release of the surfactant from the vehicle. Accordingly, although the HC50 (concentration inducing 50% hemolysis) values for the surfactants in an aqueous solution were very close, strong differences were found when the hemolysis kinetics of the corresponding surfactant−DNA gel particles were determined. The differences found in the hemolysis responses induced by the different surfactants are related to the capacity to form weaker or stronger surfactant−DNA complexes. Most in vitro studies of compound-induced hemolysis spectrophotometrically detect plasma-free hemoglobin derivatives after incubation of these components with blood, followed by separation of undamaged cells by centrifugation. However, these protocols may be altered by the presence of particles. Previous studies in our laboratory have verified the absorption of the released hemoglobin by the particles when erythrocytes were incubated in the presence of some surfactant−DNA gel particles. In this case, the hemoglobin absorption increased with time, yielding false negative results (results not published). Other authors have reported some particle interference due to hemoglobin precipitates adsorbed with the particles on centrifugation.52 Thus, it is difficult to interpret the results of these studies, due to variability in experimental approaches and a lack of universally accepted criteria for determining the test-result validity. In this work, although the dimensions of the DNA gel particles make them unsuitable for injection into the bloodstream, the study demonstrates that their physicochemical properties did not affect their hemolytic characterization under standard protocols. The results obtained using the hemolysis assay indicate that the

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CONCLUSIONS

Chitosan lactate (CL) alone (binary systems) and in combination with protamine sulfate (PS) (ternary systems) was used to form DNA gel particles by interfacial diffusion. Unlike most polysaccharides, CL has positive charges resulted following removal of acetyl units from D-glucosamine residues. This chemical feature allows CL to bind strongly to negatively charged surfaces and responsible for many of observed biological activities. In addition to that, nontoxicity, biodegradability, and biocompatibility of CL promotes their pharmaceutical applications compared to other synthetic polymers. Furthermore, PS is highly positively charged, arginine-rich protein, which has been previosly used in the formation of mixed carriers for modulating DNA release. This property, in addition to its longtime use in pharmaceutical formulations, makes protamine a promising candidate in gene delivery formulations. When CL is used in combination with PS as a DNA carrier, a large degree of control over the release profile is achieved, in comparison to those obtained in single systems. From a physicochemical standpoint, we controlled the magnitude of the DNA release and obtained controlled release in ternary systems by changing the CL/PS ratio in the solution in which the particles were formed. We applied several mathematical functions to the release profiles and obtained the best overall correlation coefficients with the Weibull model. The results indicate a mechanism for drug release known as super case II transport. The Rose Bengal partition assay was applied for the first time to estimate the surface hydrophobicity of DNA gel particles. Even though the DNA gel particles obtained using binary and ternary systems showed strong J


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(10) Costa, D.; Morán, M. C.; Miguel, M. G.; Lindman, B. Cross-Linked DNA Gels and Gel Particles, in DNA Interactions with Polymers and Surfactants; Dias, R. S., Lindman, B., Eds.; Wiley Interscience: NJ, 2008. (11) Lindman, B.; Dias, R. S.; Miguel, M. G.; Morán, M. C.; Costa, D. Manipulation of DNA by Surfactants, in Highlights in Colloid Science; Platikanov, D., Exerowa, D., Eds.; Wiley-VCH: Weinheim, 2009. (12) Morán, M. C.; Miguel, M. G.; Lindman, B. DNA gel particles. Soft Matter 2010, 6, 3143−3156. (13) Dias, R.; Morán, M. C.; Costa, D.; Miguel, M.; Lindman, B. DNASurfactant Systems: Particles, Gels and Nanostructures, in Nano-Science: Colloidal and Interfacial Aspects; Starov, V. M, Ed.; Taylor & Francis: U.K.,2010. (14) Morán, M. C.; Miguel, M. G.; Lindman, B. DNA gel particles: Particle preparation and release characteristics. Langmuir 2007, 23, 6478−6481. (15) Morán, M. C.; Pais, A. A. C. C.; Ramalho, A.; Miguel, M. G.; Lindman, B. Mixed protein carriers for modulating DNA release. Langmuir 2009, 25, 10263−10270. (16) Morán, M. C.; Laranjeira, T.; Ribeiro, A.; Miguel, M. G.; Lindman, B. Chitosan-DNA particles for DNA delivery: Effect of chitosan molecular weight on formation and release characteristics. J. Dispers. Sci. Technol. 2009, 30, 1494−1499. (17) Morán, M. C.; Nogueira, D. R.; Vinardell, M. P.; Miguel, M. G.; Lindman, B. Mixed protein-DNA gel particles for DNA delivery: Role of protein composition and preparation method on biocompatibility. Int. J. Pharm. 2013, 454, 192−203. (18) Harish Prashanth, K. V.; Tharanathan, R. N. Depolymerized products of chitosan as potent inhibitors of tumor-induced angiogenesis. Biochim. Biophys. Acta 2005, 1722, 22−29. (19) Pillai, C. K. S.; Paul, W.; Sharma, C. P. Chitin and chitosan polymers: Chemistry, solubility and fiber formation. Prog. Polym. Sci. 2009, 34, 641−678. (20) Hebert, E. Improvement of exogenous DNA nuclear importation by nuclear localization signal-bearing vectors: A promising way for nonviral gene therapy. Biol. Cell. 2003, 95, 59−68. (21) Wienhues, U.; Hosokawa, K.; Hoveler, A.; Siegmann, B.; Doerfler, W. A novel method for transfection and expression of reconstituted DNA-protein complexes in eukaryotic cells. DNA 1987, 6, 81−89. ́ V. I. (22) Bottger, M.; Zaitsev, S. V.; Otto, A.; Haberland, A.; Vorobev, Acid nuclear extracts as mediators of gene transfer and expression. Biochim. Biophys. Acta 1998, 1395, 78−87. (23) Raspaud, E.; Toma, A. C.; Livolant, F.; Rädler, J. Interaction of DNA with cationic polymers, in DNA Interactions with Polymers and Surfactants; Dias, R. S., Lindman, B., Eds.; Wiley Interscience: NJ, 2008. (24) Hixson, A. W.; Crowell, J. H. Dependence of reaction velocity upon surface and agitation I- theoretical consideration. Ind. Eng. Chem. 1931, 23, 923−931. (25) Higuchi, T. Mechanism of sustained-action medication: theoretical analysis of rate or release of solid drug dispersed in solid matrices. J. Pharm. Sci. 1963, 52, 1145−1149. (26) Gibaldi, M.; Feldman, S. Establishment of sink conditions in dissolution rate determinations. Theoretical considerations and applications to non-disintegrating dosage forms. J. Pharm. Sci. 1967, 56, 1238−1242. (27) Langenbucher, F. Linearization of dissolution rate curves by the Weibull distribution. J. Pharm. Pharmacol. 1972, 24, 979−981. (28) Rawlings, J. O. Applied Regression Analysis: A Research Tool; Wadsworth: Belmont, 1988. (29) Eaton, J. W. GNU Octave Software, Version 3.2.3, 2009, http:// octave.sourceforge.net/optim/function/leasqr.html. (30) Gaweda, S.; Morán, M. C.; Pais, A. A. C. C.; Dias, R. S.; Schillen, K.; Lindman, B.; Miguel, M. G. Cationic agents for DNA compaction. J. Colloid Interface Sci. 2008, 323, 75−83. (31) Peacocke, A. R. The interaction of acridines with nucleic acids. In Acheson; Acridines, R. M., Ed.; Interscience Publishers: New York, 1973; pp 723−754. (32) Morán, M. C.; Miguel, M. G.; Lindman, B. Surfactant-DNA gel particles: Formation and release characteristics. Biomacromolecules 2007, 8, 3886−3892.

differences in swelling and release behavior, both binary and ternary systems promoted the formation of DNA gel particles with an acute hydrophilic character, without any posterior polymer coating step. The lack of hemolytic effect of the DNA gel particles suggests that they could be used as long-term bloodcontacting medical devices. From a biocompatible and economic point of view, these DNA gel particles could be considered as potential candidates for development of new nonviral vectors for the delivery of therapeutic DNA. The significance of the obtained results in combination with the well-know immunoadjuvant properties of some chitosan derivatives make these particles a conceptual step in the design and development for new protein and antigen delivery systems. Current studies are focused on the scaled-down production of these biocompatible DNA gel particles.

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ASSOCIATED CONTENT

* Supporting Information S

This section includes information concerning changes in the relative weight of DNA gel particles and as a function of the composition, mathematical models, and the corresponding coefficient of correlation used for the study of the DNA release profiles from DNA gel particles and model parameters derived from the Weibull function after fitting the DNA release profiles from DNA gel particles. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

M.C.M. acknowledges the support of the MICINN (Ramon y Cajal Contract RyC 2009-04683). This study was financially supported by Project MAT2012-38047-C02-01 from the Spanish Ministry of Science and Innovation. The authors would like to thank E. Pérez for her support of this project. The authors would like to thank A.A.C.C. Pais for their assistance in the evaluation of DNA release kinetics.

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Protein Based-DNA Gel

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