Bioconjugate Chem. 2003, 14, 311−319
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Nanoparticulate DNA Packaging Using Terpolymers of Poly(lysine-g-(lactide-b-ethylene glycol)) Susan Park†,‡ and Kevin E. Healy*,† University of California at Berkeley, Departments of Bioengineering and Materials Science and Engineering, 459 Evans Hall, Berkeley, California 94270-1762, and Northwestern University, Department of Biomedical Engineering, McCormick School of Engineering and Applied Science, 2145 Sheridan Rd., Tech E310, Evanston, Illinois 60208. Received October 8, 2002; Revised Manuscript Received January 9, 2003
Terpolymers of poly(lysine-g-(lactide-b-ethylene glycol)) (pK-pLL-pEG) were synthesized by using ring-opening polymerization and functional end-group grafting. Synthesis was characterized with gel permeation chromatography, proton nuclear magnetic resonance spectroscopy, and a trinitrobenzene sulfonic acid binding assay. Polymer association behavior with DNA was investigated using an ethidium bromide exclusion assay, static light scattering, and scanning electron microscopy. Polylactide molecular weight was varied to investigate its impact on DNA association and resulting complex characteristics. Polylysine (Mw ) 8800, DP ) 42) modified with either 7400 or 10 870 Mw pLL-pEG reduced the minimum amount of primary amines necessary for complete condensation by 23% and 48%, respectively, compared to unmodified polylysine (pK42). Complexes formed with the highest molecular weight terpolymer demonstrated significantly (p < 0.1) greater resistance to DNase I than lyophilized pK42-DNA particles. This study suggests that modification of pK42 with pLL-pEG diblock copolymers impacts polylysine’s associative and binding behavior to DNA and resulting particle characteristics. Modulation of terpolymer composition in complexes can enable control over intracellular plasmid dissociation rates to improve transfection efficiency.
INTRODUCTION
Gene therapy is one field that has exploited spontaneous polycation assembly with DNA as an alternative to packaging plasmids within either viral vectors or liposomes. Some of these systems include those based on polyamidoamines (dendrimer), polyacrylates, polyamino acids, and polyethyleneimine (1-4). Recent investigations have found that the gross morphology of condensates is surprisingly insensitive to large variances in polymer microarchitecture as well as DNA size, within 400 to 1 million base pairs (1, 5, 6). However, there is some evidence that polycation associative behavior can vary according to the form of DNA present (7). Free DNA that has a hydrodynamic radius on the order of microns can be condensed into complexes by highly branched polymers (e.g., polyethyleneimine) as well as more simple multivalent cations (e.g., Co(NH3)63+). The resulting particles are consistently on the order of 50-300 nm in diameter and can often appear as torroids, rods, and large aggregates; however, the predominance of each is affected by condensation parameters as well as polymer composition (1, 8). The type of polycation used can have a dramatic effect on cytotoxicity, cellular response, and intracellular trafficking of the complexes (9-11). Two polymers that have generated the most publications in the literature are polyethyleneimine (pEI) and polylysine (pK). Polyethyleneimine is a polymer that can be found in linear or in hyperbranched form. The latter contains 1°, 2°, and 3° amines at a respective ratio of 1:2:1. The different pKa’s * To whom correspondence should be addressed. kehealy@ socrates.berkeley.edu. † University of California at Berkeley. ‡ Northwestern University.
of the amines on branched pEI give it an usually high buffering capacity across a wide pH range as well as the ability to effectively condense DNA employing its protonated amines (1). Both forms of pEI have been shown to exhibit high levels of transfection (4, 11-14). It has been hypothesized that pEI’s high transfection efficiency can be attributed to its large buffering capacity that neutralizes lysosomal pH and induces lysis via the resulting change in ionic osmolarity (14, 15). Unfortunately, pEI’s membrane permeablizing ability has raised concern about its cytotoxicity under conditions necessary for transfection (16, 17). Even though pEI is considerably less toxic when associated with DNA, it is not degradable, and its intracellular fate, as well as any other long-term implications on normal cellular transcriptive processes, is not yet known. Polylysine (pK) is a polyamino acid that possesses side chains with terminal amines, which are protonated at physiological pH. It is an attractive option for gene therapy techniques since it is synthesized from a noncytotoxic, naturally occurring monomer. The molecular weight of pK has some effect on particle diameter, with the smallest pK (DP ) 19) forming particles of about 30 nm in diameter and the largest (DP ) 1075) forming complexes about 120-300 nm (18). However, timeresolved static light scattering investigation of high molecular weight pK-DNA complexes revealed that they can aggregate into supramolecular particles of several micrometers in diameter (19). Furthermore, the molecular weight of polylysine used can have a significant effect on cell toxicity, transfection efficiency, and biodistribution (20). The tenacity of electrostatic association between DNA and polycations should be considered when developing synthetic vectors for gene therapy. It has been shown that
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312 Bioconjugate Chem., Vol. 14, No. 2, 2003
increasing pK molecular weight interfered with in vitro RNA transcription and was associated with a decrease in plasmid unpackaging rates as well as a protracted reporter expression (21, 22). In addition, higher transfection efficiencies were observed when 40% of the -amines on pK were neutralized with sugar residues when compared to unsubstituted pK (23, 24). Overall, pK has been shown to be a less effective transfection vehicle compared to PEI. Observations on intracellular trafficking of pK-plasmid complexes indicate that they are sequestered into endosomes and nuclear localization is not as prevalent when compared to complexes formed with PEI (9, 21). However, functionalization of pK with targeting ligands that induce receptor-mediated endocytosis can improve transfection efficiency significantly (22, 25, 26). In addition, reporter expression levels can be increased dramatically by conjugating fusogenic peptides derived from virus glycoproteins. These pH responsive peptides undergo a conformational change in acidic pH and then can disrupt lysosomal membranes (27-29). Other agents that combat endosomal degradation, such as chloroquine or hemolytic polymers, can be added to abet transfection (2, 30). Block or graft copolymers have also been investigated with respect to their interaction with DNA and have often employed hydrophilic moieties to disguise complexes from recognition and subsequent sequestration by the reticuloendothelial system. When poly(ethylene glycol) (pEG) was bound to various polycations, the resulting complexes demonstrated improved colloidal stability and extended in vivo circulation lifetimes (17, 25, 31-34). The benefit of coupling pEG is not only due to greater particle hydrophilicity, but also diminished protein adsorption that activates phagocytosis. To address some of the aforementioned issues, we have developed a polylysine-based (Mw ) 8800, DP ) 42) terpolymer grafted with diblock polymer components. A terminal pEG segment (Mw ) 3400) was employed to improve complex stability as well as to provide end-group functionality for conjugation of bioactive molecules, such as targeting ligands and fusogenic peptides that increase transfection specificity and efficiency. The intermediate segment of our terpolymer is a hydrolytically labile polyL-lactide (pLL) block, which is intended to aid in protection of DNA from intracellular degradation as well as to promote premature DNA condensation through hydrophobic interactions. We hypothesize that a reduction in the amount of lysine moieties required for complete condensation will increase the rate of DNA unpackaging that is necessary for plasmid transcription. DNA release will also be contingent on the rate of pLL degradation and could possibly enable tailoring of transfection profiles by modulating the molecular weight of the pLL and/or pK segments. This investigation focuses on the synthesis and characterization of pK-pLL-pEG terpolymers as well as its interaction with plasmid DNA. The molecular weight of the pLL segment was varied to assess its impact on terpolymer association and complex characteristics. MATERIALS AND METHODS
A heterofunctional poly(ethylene glycol) (NH2-pEG-OH, Mw ) 3400) bearing a terminal amino and hydroxyl group was purchased from Shearwater Corporation (Huntsville, AL). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were used as received unless otherwise noted. The DNA used was
Park and Healy
supercoiled ΦX174 RF DNA (5386 bp) from Promega (Madison, WI). PEG Functionalization. The amino end of 3400 Mw NH2-pEG-OH was protected by reaction with a 6 molar excess of N-(9-fluorenylmethoxycarbonyloxy)succinimide (osu-fmoc) in dioxane for 4 h. The resulting fmoc-pEG was purified by repeated precipitation into cold ether until the characteristic succinimidyl proton peak at 2.82 ppm (in CDCl3) was undetectable by proton nuclear magnetic resonance spectroscopy (1H NMR, Bruker AMX300). Percent substitution was calculated using the ratio of the fmoc doublet at 7.88-7.90 ppm with respect to the pEG peak at 3.63 ppm (in CDCl3). The polymer was dried under vacuum overnight. Diblock Copolymer Synthesis and Characterization. L-Lactide was purified by recrystallization from toluene and dried under vacuum for 1 h immediately before use. A 0.5M stock solution of stannous octoate in anhydrous toluene was prepared and used at a constant lactide monomer:initiator ratio (M/I) of 300. The L-lactide and fmoc-pEG were reacted at monomer:hydroxyl (M/ OH) ratios of 40 and 75 to control pLL molecular weight. To produce the diblock copolymer of lowest molecular weight, a M/OH of 75 was used and the reaction stopped after 2 h. A 20% w/v solution of the above reactants in anhydrous toluene was prepared in a sealed reaction vessel in a dry nitrogen atmosphere. Polymerization of L-lactide proceeded under refluxing toluene at 100 °C under a gentle flow of dry nitrogen for 6 h. The copolymer (pLL-pEG-fmoc) was purified by repeated dissolution in dioxane and precipitation into cold ether. Molecular weight characterization by size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) was performed using an Agilent 1100 HPLC with 2 Polymer Laboratories mixed E columns in series and methylene chloride as the mobile phase. The detectors used included a multi-angle light scattering detector, refractive interferometer (Wyatt Technology Corporation, Santa Barbara, CA) and a photodiode array detector (Agilent Technologies, Mountainview, CA). The dn/dc values for each copolymer in methylene chloride were determined experimentally using a refractive interferometer (Wyatt). For comparison, proton nuclear magnetic resonance spectroscopy (1H NMR) was also used to calculate molecular weight by using the known molecular weight of pEG as an internal standard. The hydroxyl on the polylactide end of the diblock copolymer was then functionalized to be amine reactive (su-pLL-pEG-fmoc). Disuccinimidyl carbonate (DSC) (0.5M) and 4-dimethyl aminopyridine (DMAP) (0.5M) at a 6 molar excess in anhydrous dimethylformamide was added to the copolymer dissolved in anhydrous dioxane. The solution was stirred for 6 h under a dry nitrogen atmosphere and purified by precipitation into cold ether until the aromatic DMAP protons (in CDCl3) at 6.45 and 8.20 ppm disappeared. Percent substitution was calculated using the ratio of the reappeared succinimidyl peak area at 2.82 ppm to that of pEG at 3.63 ppm (in CDCl3). Terpolymer Synthesis and Characterization. The terpolymer of poly(lysine-g-(lactide-b-ethylene glycol)) was synthesized by coupling of su-pLL-pEG-fmoc to the -amines on poly-L-lysine (pK) (Mw ) 8800, DP 42) at a 1:1 molar ratio in dimethyl sulfoxide. The nomenclature for the resulting terpolymers is pK42-Xk, where pK with a degree of polymerization of 42 has been grafted to a pLL-pEG diblock copolymer with a combined molecular weight of approximately X000.
Terpolymer−DNA Complexes for Gene Therapy
Proton NMR in DMSO-d6 was used to determine the amount of pK present in each terpolymer. For comparison, a binding assay using trinitrobenzyl sulfonic acid (TNBS) was performed using a modified standard protocol (35). In short, the copolymer was dissolved in DMSO (1 or 2 mg/mL) and reacted with an excess of TNBS in an overall reaction solution of 75% DMSO and 25% sodium bicarbonate buffer (pH 8.5). The reaction was performed at 37 °C for 1 h, after which 6M HCl was immediately added. The solution absorbance was then read at 436 nm and the amount of -amines per milligram of polymer was calculated using a calibration curve made with known amounts of pK42. Particle Formation and Characterization. Complex Formation. Complexes were formed by adding a pK or pK-pLL-pEG in DMSO to approximately 5 µg of DNA in ultrapure water (UPW, 18 MΩ cm) in Teflon vials. The total volume of DMSO used was kept below 10% v/v to minimize solvent effects on condensation (8). After 20 min, an additional volume of water was added, and the solutions were lyophilized. Complexes were resuspended in UPW for further characterization unless otherwise noted. Degree of Condensation. Ethidium bromide (EB) was used as an indicator of condensation since it becomes intensely fluorescent upon intercalating with free DNA but cannot bind to condensed DNA (36). Particles were made and resuspended as described above, mixed with 1mM EB, and read in a black 96 well plate using a microplate fluorimeter (Molecular Devices, Sunnyvale, CA) (ex: 339 nm, em: 610 nm, cutoff: 515 nm). The autofluorescence of free EB was subtracted from each sample value and then normalized to the maximum fluorescence obtained for DNA with no polymer added. DNA condensation was considered to be complete when the percent relative fluorescence units (RFU) reached background levels. Determination of complex apparent Mw* and rg*. Static light scattering of complexes was performed using a multiangle light scattering detector (Wyatt) in microbatch mode to assess particle apparent molecular weight (Mw*) and apparent radius of gyration (rg*). The UPW mobile phase was filtered with 0.02 µm syringe filters, and light scattering measurements were obtained every 2 s using a flow rate of 0.2 mL/min. Sample dilutions were carefully made by weight, and the final concentration was considered to be the total amount of DNA plus polymer. The data used for analysis was limited to that from seven low-angle detectors (36-90°), since these low angles are the most critical to obtain the apparent molecular weight and rg* of complexes. The lowest measurable angles at 14° and 26° were excluded since low-angle detectors were the most sensitive to contaminating particles and were easily saturated. Samples were run in triplicate, and each run produced at least 20 data points. Only those data with a Debye fit of less than 10% error were used for further analysis. Complexes were also visualized by cold field emission scanning electron microscopy (Hitachi S-5000) using an accelerating voltage of 5kV. Samples were prepared by depositing drops of resuspended particles onto silicon wafers, freeze-drying and coating with 8-10 nm of carbon. Resistance to DNase Degradation. The ability of particles to resist degradation by bovine pancreatic DNase I was assessed by agarose gel electrophoresis. Particles were made as described above except at a 5-fold increase in concentration and then evenly aliquoted into microvials before lyophilization. Dulbecco’s complete phosphate buffered saline (PBS, Gibco BRL) was used for
Bioconjugate Chem., Vol. 14, No. 2, 2003 313 Scheme 1. Synthesis of pK-pLL-pEG Terpolymers
resuspension, and 2.5 units of DNase I (Pharmacia, Piscataway, NJ) were added for varying amounts of time. The reaction was stopped with 2 or 3 µL of 6N NaOH/ 0.5M EDTA and run on a 0.8% agarose gel for 40 min at 4.0V/cm. The running buffer was an alkaline 20mM trisacetate solution with 1mM EDTA (pH 12). The DNA was visualized by staining with 0.5µg/mL ethidium bromide in 1X TAE buffer for 20 min and subsequent destaining for 15 min. The gels were then stained with 0.1% Coomassie Brilliant Blue in UPW, methanol, glacial acetic acid (5:4:1 v/v/v) for 4 min and destained overnight in a solution mixed at a ratio of 15:4:1, respectively. Scanalytics gel analysis software was used on digital gel images to determine the extent of DNA degradation for each well. Since DNA concentration is proportional to fluorescence intensity, the amount of intact DNA was considered to be the intensity area that did not migrate past the negative control (0 min.). The value was then normalized to the total intensity in the lane (intact + degraded) in order to compensate for unavoidable fluctuations in sample loading concentration. RESULTS
Synthesis Characterization. The synthesis is depicted in Scheme 1, and its progression and purification
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Figure 2. Cold field emission scanning electron microscopy on complexes coated with ∼10 nm carbon. Complexes were made at an amine to phosphate charge ratio (N:P) of 1.0. The scale bars equal 500 nm in all images. Table 1. Diblock Mw Results 1H
pLL-pEG
NMR (g/mol)
MALLS (g/mol)
PDI
5k 7k 11k
3692 7648 11094
5800 7628 10440
1.02 1.02 1.06
Table 2. 1° Amine Content and Ethidium Bromide Assay Results µmol NH2/mg polymer [% subst] TNBS pK42 pK42-5k pK42-7k pK42-11k
Figure 1. Representative proton nuclear magnetic resonance spectra of pK42-7k used to monitor the progression, purification and degree of end-group substitution after each reaction step. Spectra 1-6 correspond to the molecules numbered in Scheme 1. Peak shifts for spectra 1-4 (CDCl2): 1, 2.82 (s, 4H); 2, 4.56 (d, 2H); 3, 4.33 (t, 1H); 4, 7.77 (d, 2H); 5, 7.62 (d, 2H); 6, 7.42 (t, 2H); 7, 7.35 (t, 2H); 8, 3.63 (s, 4H); 9, 1.56 (d, 3H); 10, 5.13 (q, 1H). Peak shifts for spectrum 5 (DMSO-d6): 1, 1.56 (s, 4H) & 1.33 (s, 2H); 2, 2.76 (s, 2H); 3, 8.05 (d, 1H); 4, 4.25 (s, 1H).
was monitored primarily by proton NMR characterization after each step (Figure 1). The characteristic proton peaks for osu-fmoc are shown in spectrum 1, and after coupling to the terminal amine of NH2-pEG-OH, the characteristic ether peak appeared at 3.63 ppm (spectrum 2, peak #8). The disappearance of the succinimidyl peak at 2.82 ppm indicated that unreacted osu-fmoc was removed and the degree of fmoc substitution on pEG was assessed to be above 85% (data not shown). Varying the lactide monomer:pEG hydroxyl ratio for ring opening polymerization or the reaction time easily enabled control over the pLL segment length. Lactide polymerization was indicated by shifts in its methine (from 5.02 to 5.13 ppm) and methyl (from 1.62 to 1.56 ppm) groups (spectrum 3). Succinimidyl functionalization of pLL-pEG-fmoc resulted in the reappearance of a peak at 2.82 ppm (spectrum 4, peak #1). Spectra of the final terpolymer after grafting su-pLL-pEG to pK revealed all relevant peaks after purification (spectrum 6). There was good corroboration between molecular weight data obtained by 1H NMR using the known molecular weight of pEG as an internal standard and SEC-MALLS
3.55 ( 0.20 1.99 ( 0.15 1.00 ( 0.09
1N
NMR
3.20 [1.8%] 1.81 [4.3%] 1.05 [6.7%]
N:Pmin 0.81 0.91 0.62 0.42
(Table 1). Polydispersity indices for all pLL-pEG diblocks were below 1.1, and a peak corresponding to unreacted pEG-fmoc was not revealed by any of the three detectors (data not shown). The TNBS binding assay, developed to determine the amount of amines present in the pK-pLL-pEG terpolymer, agreed very well with data obtained with 1H NMR (Table 2). Results from 1H NMR were used to calculate the amount of amines added for all subsequent DNA condensation experiments. Minimum Amine:Phosphate Charge Ratio Determination. The progression of DNA condensation was studied by measuring the relative EB fluorescence. The minimum polymer amine:DNA phosphate charge ratio (N:Pmin) necessary for complete condensation is summarized in Table 2. Polylysine (pK42) was able to condense DNA at a N:Pmin of 0.81, while pK42-5k required a slightly higher N:Pmin of 0.91. However, when larger pLL segments were present in pK42-7k and pK42-11k, the N:Pmin decreased substantially to 0.62 and 0.42, respectively. Scanning electron micrographs revealed heterogeneous submicron complexes where the population size varied between 50 and 500 nm (Figure 2). The carbon coating did not seem to severely obstruct morphology since samples with thinner (∼1 nm) platinum coatings showed similar images with a backscattering detector (not shown). Static Light Scattering of Complexes. Static light scattering investigation of complexes provided information about the apparent Mw* and radius of gyration (rg*) by measuring the excess Rayleigh ratio (RΘ) values
Terpolymer−DNA Complexes for Gene Therapy
Bioconjugate Chem., Vol. 14, No. 2, 2003 315
Figure 3. A representative Debye fit to static light scattering data used to calculate apparent Mw* and rg* of complexes. This plot is for pK42-11k complexes (N:P ) 0.5) where Mw* ) (1.216 ( 0.064) × 106 g/mol and rg* ) 112.5 ( 9.3 nm.
scattered from particle solutions and fitting a secondorder Debye curve at zero concentration limits according to the following relation:
Rθ ) MwP(θ) K*c
(1)
where
K* )
4π2n2 dn λ4NA dc
( )
2
The particle scattering function, P(θ), is defined by
(4πλsin θ/2)
P(θ) ) 1 - R1
2
(4πλsin θ/2)
+ R2
4
- ...
(2)
where the coefficient Rx is proportional to ∫r2x dM and is relevant to particle shape. The root-mean-square radius (〈rg2〉 or rms) of the molecule can be derived from
〈r2g〉 ) 3R1 The concentration (c) and angle of measurement (θ) are known parameters, while K* contains constants for incident light wavelength (λ), solvent refractive index (n) and Avogadro’s number (NA). The refractive index increment (dn/dc) for each component in UPW was measured experimentally, except for pLL, which was estimated to have a dn/dc value approximately equal to that of pEG due to their similar indices of refraction (data not shown). The overall dn/dc values for particles of varying compositions were calculated using the relation: dn/dctotal ) Σxi(dn/dc)i where xi denotes the weight percent of species i. When eq 1 is plotted as Rθ/K*c versus sin2(θ/2), the molecular weight (Mw or Mw*) can be calculated by extrapolation of the curve to zero angle, and the radius of gyration (rg or rg*) is determined by the initial slope. Figure 3 illustrates a typical Debye plot for pK42-11k (N:P ) 0.5) used to determine the apparent Mw* and rg*. Analysis of the naked ΦX174 plasmid resulted in values of 135 nm for rg* and 3.57 × 106D for Mw*, which agreed extremely well with the theoretical value of 3.55 × 106 D when using the common conversion of 6.6 × 105 D ) 1 kbp (37) Particles made with pK42 showed an order of magnitude increase in apparent Mw* at low charge ratios (Figure 4a). When condensation was complete, as indicated by the accompanying EB curve (% RFU), the apparent Mw* decreased dramatically. Values for rg* generally remained stable despite the fact that a decrease was expected. Particles made with each of the terpolymers did not show as extreme an increase in
apparent Mw* at N:P < N:Pmin, but showed similar overall trends with respect to the relative degree of condensation (Figure 4b-d). At charge ratios greater than N:Pmin, the Mw* for each of the three terpolymers decreased more significantly than seen for pK42 and stabilized at approximately 1 × 106 g/mol. Only complexes made with pK42-7k and pK42-11k displayed the expected decrease in rg* when DNA condensation was complete (p < 0.001 at N:P ) 1.2, ANOVA, Tukey HSD). The SEM images supported that there were many particles on the order of less than 100 nm; however, the large variance in the rg* is the result of heterogeneity in the particle size distribution. Degradation by DNase I. Figure 5 shows the electrophoretic migration of free DNA and pK42-DNA complexes after exposure to DNase I and subsequent dissociation from polycations. Polymer staining with Coomassie Blue indicated that complex dissociation by NaOH was complete and maintained by the alkaline running buffer, since all staining was localized around the well, apart from the migrating DNA. At a pH of 12, the primary amines were largely deprotonated and, therefore, did not migrate toward the anode. Ethidium bromide staining of DNA revealed that simply making pK42-DNA complexes in the same manner as the triblock copolymer complexes dramatically decreased the extent of degradation by DNase I. Particles made with all pK-pLL-pEG triblock copolymers also showed improved DNA resistance to degradation over free DNA and freshly made pK42 particles (Figure 6)). At 15 min, the percentage of intact DNA from particles made with the highest polylactide segment, pK42-11k, was significantly (p < 0.1, ANOVA, Tukey HSD) higher than that from similarly made (lyophilized) pK42-DNA particles. DISCUSSION
Ring-opening polymerization of L-lactide from poly(ethylene glycol) has been widely documented regarding the effect of monomer-to-initiator ratio as well as monomer-to-hydroxyl ratio (38-40). A survey of the literature enabled predictable control over pLL molecular weight in the terpolymer by adjusting the M/OH ratio. There was some difficulty in producing the lowest molecular weight pLL-pEG (5, 800), perhaps due to slight impurities in the system that helped consume the small amount of monomer. However, the polymerization rates were monitored as a function of reaction time (data not shown) and the desirable pLL molecular weight was obtainable simply by adjusting the stop time. Size exclusion chromatography for all copolymers revealed single peaks attributable to pLL-pEG-fmoc diblock copolymers that eluted earlier than pEG-fmoc alone, indicating that there was no unreacted pEG remaining. Polydispersities calculated by multiangle light scattering for all synthesized diblocks were below 1.1, and there was good agreement between molecular weights assessed by 1H NMR and static light scattering. Prior knowledge of the pEG molecular weight (3400) was useful in providing an internal standard for 1H NMR in calculating the degree of polymerization of other polymer segments as well as degree of substitution of functional end groups (Figure 1). In these experiments, the fmoc group limited reactivity to just the hydroxyl end of pEG during pLL polymerization. In the future, a cross-linker will be substituted for fmoc to enable conjugation of bioactive moieties to the end of the pEG chains. The amount of pK present in each terpolymer was assessed by 1H NMR in DMSO-d6 as well as indirectly
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Figure 4. Static light scattering results of complexes made with (A) pK42, (B) pK42-5k, (C) pK42-7k, and (D) pK42-11k. The ethidium bromide fluorescence curves are overlaid as reference to correlate the degree of condensation to Mw* and rg trends. The peak Mw* (indicated on each graph by the solid squares) for pK42 complexes (*) were statistically greater than the peak Mw* for all other samples (p < 0.001, ANOVA).
Figure 5. Alkaline agarose gel electrophoresis of free DNA, fresh pK42 complexes and lyophilized pK42 complexes after incubation with DNase I for varying time points. The top rows show coomassie blue staining of the dissociated polycation while the bottom rows reveal DNA stained by ethidium bromide.
by using a TNBS binding assay. A standard of pK42 was used to create the assay calibration curve since the binding efficiency was anticipated to be similar to that of the terpolymers. The amount of amines per milligram of terpolymer decreased with increasing pLL length since
Figure 6. Quantitative analysis of DNA degradation by DNase I. Complexes were made with pK42-5k (O), pK42-7k (0), pK42-11k (9), pK42 (2) or pK42 without lyophilization (4) at a N:P ) 1. At 15 min, degradation of fresh pK42 complexes (#) was significantly greater (p < 0.05) than all other groups. Degradation of lyophilized pK42 complexes (*) was significantly greater (p < 0.1) than pK42-11k samples. ANOVA statistics and Tukey HSD post-hoc comparisons were used on triplicate samples.
the weight percent of the pLL-pEG diblock became more significant. Although pK and pLL-pEG were reacted at
Terpolymer−DNA Complexes for Gene Therapy
1:1 molar ratios for all terpolymer synthesis, the degree of substitution increased with increasing pLL-pEG Mw possibly due to differences in purification efficiencies. The proton NMR results closely supported that from the TNBS assay (Table 2). SEC-MALLS data were not obtainable for the synthesized terpolymers due to solubility problems. Attempts at using several binary solvent mixtures resulted in very high and inconsistent dn/dc measurements due to preferential solvation of solvent components with the hydrophilic and hydrophobic polymer segments. To obtain accurate measurements, extensive dialysis and preequilibration would have been required, which could have reduced the molecular weight of the hydrolytically degradable copolymer (41, 42). The terpolymers were soluble in dimethyl sulfoxide, but aggregation and adsorption onto the column matrix occurred at the relatively high sample concentrations required for sufficient light scattering signal. Figure 4 shows how the apparent Mw* and apparent rg* changes with respect to the amine to phosphate charge ratio (N:P) can be used to characterize particle formation. For pK42, the Mw* showed an initial dramatic increase at a N:P < 1 and decreased upon complete condensation. When there was excess DNA, each pK strand had the opportunity to interact with many DNA molecules, and the large increases in Mw* most likely reflect that several DNA were linked together. Independent atomic force microscopy investigations have shown that partially condensed DNA on mica surfaces by spermidine, lipospermine, polyethyleneimine, and pK-pEG are multimolecular and possess centralized flowerlike conformations with highly overlapping crossover points (12, 43, 44). These observations would explain why rg* did not increase with corresponding Mw* in our system. At high N:P ratios, condensation is complete and complex Mw* decreases. For complexes made with pK42-7k and pK42-11k, a large decrease rg* was observed (p < 0.001 between N:P ) 0 and N:P ) 1.2, ANOVA, Tukey HSD) despite large variances associated with those data due to the heterogeneous size population (Figure 2). As the electrostatic repulsion is locally neutralized, other factors, including hydration, counterion, and hydrophobic forces, become more significant and are hypothesized to be the cause of complete condensation at less than electroneutral charge (45, 46). It is possible that the pLL segments of pK42-7k and pK42-11k were significant enough to aid condensation by hydrophobic interactions, as reflected in the lower N:Pmin. Our observations are supported by other theoretical and experimental investigations of the effect of surfactant hydrophobicity on its interaction with DNA (47, 48). In both studies, increasing the amount of hydrophobic moieties increased the surfactants’ activity in associating with DNA. Corresponding analysis shows that at low N:P, the initial increase in particle Mw* for pK42-7k and pK42-11k was not as dramatic as that for unmodified pK and was attributed to the presence of pLL and/or pEG. Since pEG is known to possess a large excluded volume, it is possible that it hindered the approach of other DNA molecules and reduced multi-molecular complex formation. In addition, if pLL participated in DNA condensation, it could have also constrained cationic counterion mobility and secondary DNA strand reorganization through hydrophobic interactions with neutralized pK and/or DNA (49, 50). The complexes developed in this system deviated slightly from those predicted by traditional charge inversion and reentrance models (51, 52). The hydrophilic pEG
Bioconjugate Chem., Vol. 14, No. 2, 2003 317 Table 3. Effect of Concentration Assumption on Mw
pK42 pK42-5k pK42-7k pK42-11k
DNA+polymer (×10-6 g/mol)
DNA (×10-6 g/mol)
2.51 ( 0.80 1.03 ( 0.60 1.04 ( 0.54 0.64 ( 0.28
5.13 ( 1.56 2.71 ( 1.36 3.47 ( 1.66 2.88 ( 1.17
segment aids in solubilizing complexes in aqueous media, thereby circumventing precipitation of charge neutralized particles. In addition, the relatively small variances in rg* at low N:P indicates that the polycation was distributed throughout the DNA instead of generating a mixed population of condensed and free DNA. The coexistence of condensed complexes and free DNA has been shown to be at least partially contingent on a large polycation charge density. Polyethyleneimine at low N:P ratios was shown to produce a mixed population of free and condensed DNA at a pH of 5; however, at pH 7 only partially condensed DNA was formed due to the reduced ionization of its amines (7). Another point of departure from theory was observed regarding Mw* trends. It was expected that complex Mw* should increase as the N:P ratio approached 1 since, electrostatic repulsion between partially neutralized DNA decreased. At N:P ratios greater than 1, excess polycation imparts a net positive charge that discourages intercomplex interactions and reduces complex Mw*. While this trend on a similar system of pK and DNA was reported previously, our samples demonstrated a peak Mw* at approximately 1/2 the N:Pmin instead of a N:P ratio near 1 (19). For all complex systems, the Mw* unexpectedly decreased below that of free DNA at high N:P ratios. This result is attributable to the fact the calculations of Mw* and rg* are apparent rather than true values. At high N:P ratios, the condensing polycation is present in a large weight fraction but is small in size relative to DNA. Therefore, the assumption that sample concentration is a summation of both terpolymer and DNA overestimates sample concentrations at high N:P ratios. Equation 1 shows that RΘ is proportional to Mw* and c, and if other parameters are held constant, then an overestimation in concentration would result in an artificially low Mw* for a given RΘ. However, concentration did not affect calculations for rg* since they were determined from angledependent scattering and not overall light scattering signal. Table 3 illustrates this point by showing the Mw* dependence using both concentration assumptions for each complex system at an N:P of 1.2. If it is assumed that DNA is the predominant scattering center due to its considerably larger size, then the concentration of condensing polymer is negligible with respect to its light scattering contribution (the effect on dn/dc is still considered significant), the recalculated Mw* increases. For pK42, the difference in Mw* is minimized, since pK42 contributes a small weight fraction. For pK42-11k, the adjustment for concentration is the largest due to the large weight fraction of pLL-pEG, which results in the largest percent increase in Mw*. However, even after the concentration adjustment, the Mw* of pK42 complexes remains statistically different from those made with the terpolymers (p < 0.001). With such a system, it is difficult to definitively conclude which of the two assumptions to use for calculations. At high N:P charge ratios, the polymer contribution to the light scattering signal becomes significant and should be factored into the overall concentration in some capacity. The impact of system complexity, polydispersity, and particle inhomogeneity on other parameters, such as dn/dc, needs to be consid-
318 Bioconjugate Chem., Vol. 14, No. 2, 2003
ered before specific physical interpretation can be extracted from these values (53, 54). It has been shown that intracellular trafficking of plasmids condensed with polylysine involves endosomal compartmentalization and so plasmid degradation by nucleases is a concern (9). Condensation of DNA causes gross conformational changes that can hinder binding of nucleases, such as DNase I, whose activity is dependent on the insertion of its active loop into the minor groove. Figure 5 shows that freshly made pK42-DNA complexes slightly improve resistance to DNase I compared to naked DNA. However, lyophilized pK42-DNA complexes displayed even greater resistance to DNase I over those made and used immediately. DNase I has been shown to act as a catalyst that enables water to initiate a nucleophilic attack on the phosphodiester backbone (55). Lyophilization helped protect DNA in these complexes most likely by disrupting molecularly bound water that was not completely restored by resuspension. Since other differences in resistance to DNase I were not definitive by casual inspection of the electrophoresis gels, image analysis elucidated differences in the degree of plasmid degradation found in each lane. Figure 6 is a plot of the percentage of DNA remaining intact after various incubation times with DNase I. Complexes made with pK42-11k showed the greatest amount of intact DNA after 15 min of exposure. This observation could be the result of pLL interfering with DNase I binding either by further altering the backbone structure of condensed DNA or physically blocking binding sites. The ability of pLL to protect against nuclease degradation could extend the intracellular lifetime of plasmids and increase the likelihood of transcription. Ideally, a system can be developed where modulation over the pLL and pK molecular weights can be used to optimize DNA unpackaging rates and, perhaps, temporal expression profiles. Future work will be performed in this direction using complexes functionalized with moieties for cell-specific targeting and endosomal escape. CONCLUSIONS
We have described the synthesis and characterization of poly(lysine-g-(lactide-b-ethylene glycol)) terpolymers that are capable of self-assembling with plasmid DNA. Static light scattering of complexed DNA was useful in characterizing differences in apparent Mw* and rg* trends, which suggested that there were subtle changes in molecular association dynamics when pK was modified with pLL-pEG. In addition, terpolymers of pK-pLL-pEG demonstrated the ability to fully condense DNA at lower amine-to-phosphate ratios when compared to unmodified pK, while also improving upon pK’s poor ability to protect condensed DNA from intracellular nucleases. These efforts to minimize the amount of pK necessary for complete plasmid condensation may improve transfection efficiency and reduce overall cytotoxicity. ACKNOWLEDGMENT
We are grateful for the support from NIDR T32 DE07042-25. LITERATURE CITED (1) Tang, M. X., and Szoka, F. C. (1997) The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther. 4, 823-832.
Park and Healy (2) Kyriakides, T. R., Cheung, C. Y., Murthy, N., Bornstein, P., Stayton, P. S., and Hoffman, A. S. (2002) pH-sensitive polymers that enhance intracellular drug delivery in vivo. J. Controlled Release 78, 295-303. (3) Dekie, L., Toncheva, V., Dubruel, P., Schacht, E. H., Barrett, L., and Seymour, L. W. (2000) Poly-L-glutamic acid derivatives as vectors for gene therapy. J. Controlled Release 65, 187-202. (4) Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Poly(ethylenimine) and its role in gene delivery. J. Controlled Release 60, 149-160. (5) Chen, Q. R., Zhang, L., Stass, S. A., and Mixson, A. J. (2001) Branched copolymers of histidine and lysine are efficient carriers of plasmids. Nucleic Acids Res. 29, 1334-1340. (6) Widom, J., and Baldwin, R. L. (1980) Cation-induced toroidal condensation of DNA studies with Co3+(NH3)6. J. Mol. Biol. 144, 431-453. (7) Bronich, T. K., Nguyen, H. K., Eisenberg, A., and Kabanov, A. V. (2000) Recognition of DNA topology in reactions between plasmid DNA and cationic copolymers. J. Am. Chem. Soc. 122, 8339-8343. (8) Arscott, P. G., Ma, C., Wenner, J. R., and Bloomfield, V. A. (1995) DNA condensation by cobalt hexaammine (III) in alcohol-water mixtures: dielectric constant and other solvent effects. Biopolymers 36, 345-364. (9) Godbey, W. T., Barry, M. A., Saggau, P., Wu, K. K., and Mikos, A. G. (2000) Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery. J. Biomed. Mater. Res. 51, 321-328. (10) Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl. Acad. Sci. U.S.A. 96, 5177-5181. (11) Pollard, H., Remy, J. S., Loussouarn, G., Demolombe, S., Behr, J. P., and Escande, D. (1998) Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J. Biol. Chem. 273, 7507-7511. (12) Dunlap, D. D., Maggi, A., Soria, M. R., and Monaco, L. (1997) Nanoscopic structure of DNA condensed for gene delivery. Nucleic Acids Res. 25, 3095-3101. (13) Boussif, O., Lezoualch, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301. (14) Erbacher, P., Remy, J. S., and Behr, J. P. (1999) Gene transfer with synthetic virus-like particles via the integrinmediated endocytosis pathway. Gene Ther. 6, 138-145. (15) Remy, J. S., Abdallah, B., Zanta, M. A., Boussif, O., Behr, J. P., and Demeneix, B. (1998) Gene transfer with lipospermines and polyethyleneimines. Adv. Drug Delivery Rev. 30, 85-95. (16) Godbey, W. T., Wu, K. K., and Mikos, A. G. (2001) Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability. Biomaterials 22, 471-480. (17) Ogris, M., Brunner, S., Schuller, S., Kircheis, R., and Wagner, E. (1999) PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 6, 595-605. (18) Wolfert, M. A., and Seymour, L. W. (1996) Atomic force microscopic analysis of the influence of the molecular weight of poly(L)lysine on the size of polyelectrolyte complexes formed with DNA. Gene Ther. 3, 269-273. (19) Lai, E., and van Zanten, J. H. (2001) Monitoring DNA/ poly-L-lysine polyplex formation with time-resolved multiangle laser light scattering. Biophys. J. 80, 864-873. (20) Ward, C. M., Read, M. L., and Seymour, L. W. (2001) Systemic circulation of poly(L-lysine)/DNA vectors is influenced by polycation molecular weight and type of DNA: differential circulation in mice and rats and the implications for human gene therapy. Blood 97, 2221-2229. (21) Schaffer, D. V., Fidelman, N. A., Dan, N., and Lauffenburger, D. A. (2000) Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery. Biotechnol. Bioengineering 67, 598-606.
Terpolymer−DNA Complexes for Gene Therapy (22) Ziady, A. G., Ferkol, T., Dawson, D. V., Perlmutter, D. H., and Davis, P. B. (1999) Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo. J. Biol. Chem. 274, 4908-4916. (23) Erbacher, P., Roche, A. C., Monsigny, M., and Midoux, P. (1995) Glycosylated polylysine/DNA complexes: gene transfer efficiency in relation with the size and the sugar substitution level of glycosylated polylysines and with the plasmid size. Bioconjugate Chem. 6, 401-410. (24) Erbacher, P., Roche, A. C., Monsigny, M., and Midoux, P. (1997) The reduction of the positive charges of polylysine by partial gluconoylation increases the transfection efficiency of polylysine/DNA complexes. Biochim. Biophys. Acta 1324, 2736. (25) Choi, Y. H., Liu, F., Park, J. S., and Kim, S. W. (1998) Lactose-poly(ethylene glycol)-grafted poly-L-lysine as hepatoma cell-targeted gene carrier. Bioconjugate Chem. 9, 708718. (26) Schaffer, D. V., and Lauffenburger, D. A. (1998) Optimization of cell surface binding enhances efficiency and specificity of molecular conjugate gene delivery. J. Biol. Chem. 273, 28004-28009. (27) Midoux, P., Kichler, A., Boutin, V., Maurizot, J. C., and Monsigny, M. (1998) Membrane permeabilization and efficient gene transfer by a peptide containing several histidines. Bioconjugate Chem. 9, 260-267. (28) Duguid, J. G., Li, C., Shi, M., Logan, M. J., Alila, H., Rolland, A., Tomlinson, E., Sparrow, J. T., and Smith, L. C. (1998) A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy. Biophys. J. 74, 2802-2814. (29) Wagner, E., Plank, C., Zatloukal, K., Cotten, M., and Birnstiel, M. L. (1992) Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc. Natl. Acad. Sci. U.S.A. 89, 7934-7938. (30) Erbacher, P., Roche, A. C., Monsigny, M., and Midoux, P. (1996) Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes. Exp. Cell Res. 225, 186-194. (31) Toncheva, V., Wolfert, M. A., Dash, P. R., Oupicky, D., Ulbrich, K., Seymour, L. W., and Schacht, E. H. (1998) Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers. Biochim. Biophys. Acta 1380, 354-368. (32) Katayose, S., and Kataoka, K. (1997) Water-soluble polyion complex associates of DNA and poly(ethylene glycol)-poly(Llysine) block copolymer. Bioconjugate Chem. 8, 702-707. (33) Blessing, T., Kursa, M., Holzhauser, R., Kircheis, R., and Wagner, E. (2001) Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery. Bioconjugate Chem. 12, 529-537. (34) Kircheis, R., Blessing, T., Brunner, S., Wightman, L., and Wagner, E. (2001) Tumor targeting with surface-shielded ligand-polycation DNA complexes. J. Controlled Release 72, 165-170. (35) Hermanson, G. T. (1996) Bioconjugate Techniques, San Diego, Academic Press. (36) Waring, M. J. (1965) Complex formation between ethidium bromide and nucleic acids. J. Mol. Biol. 13, 269-282. (37) Sambrook, J., and Russell, D. W. (2001) Molecular Cloning. A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Bioconjugate Chem., Vol. 14, No. 2, 2003 319 (38) Quellec, P., Gref, R., Perrin, l., Dellacherie, E., Sommer, F., Verbavatz, J., and Alonso, M. (1998) Protein encapsulation within poly(ethylene glycol)-coated nanospheres. I. Physicochemical characterization. J. Biomed. Mater. Res. 42, 4554. (39) Stevels, W., Bernard, A., Witte, P. V. d., Dijkstra, P., and Feijen, J. (1996) Block copolymers of poly(l-lactide) and poly(e-caprolactone) or poly(ethylene glycol) prepared by reactive extrusion. J. Appl. Polym. Sci. 62, 1295-1301. (40) Zhang, X., Macdonald, D. A., Goosen, M. F. A., and Mcauley, K. B. (1994) Mechanism of lactide polymerization in the presence of stannous octoate: the effect of hydroxy and carboxylic acid substances. J. Polym. Sci., Part A: Polym. Chem. 32, 2965-2970. (41) Nordmeier, E., and Lechner, M. D. (1991) Light scattering from polymer-mixed solvent systems. 1. Selective adsorption phenomena of poly(N-vinylpyrrolidone). Macromolecules 24, 2529-2537. (42) Tuzar, Z., and Kratochvil, P. (1977) Extremely high refractive index increments in polymer/mixed solvent systems. Macromolecules 10, 1108-1110. (43) Fang, Y., and Hoh, J. H. (1998) Early intermediates in spermidine-induced DNA condensation on the surface of mica. J. Am. Chem. Soc. 120, 8903-8909. (44) Wolfert, M. A., Schacht, E. H., Toncheva, V., Ulbrich, K., Nazarova, O., and Seymour, L. W. (1996) Characterization of vectors for gene therapy formed by self-assembly of DNA with synthetic block copolymers. Hum. Genet. Ther. 7, 21232133. (45) Bloomfield, V. A. (1997) DNA condensation by multivalent cations. Biopolymers 44, 269-282. (46) Rau, D. C., and Parsegian, A. (1992) Direct measurement of the intermolecular forces between counterion-condensed DNA double helices. Biophys. J. 61, 246-259. (47) Bhattacharya, S., and Mandal, S. S. (1997) Interaction of surfactants with DNA. Role of hydrophobicity and surface charge on intercalation and DNA melting. Biochim. Biophys. Acta 1323, 29-44. (48) Kuhn, P. S., Barbosa, M. C., and Levin, Y. (2000) Effects of hydrophobicity in DNA surfactant complexation. Physica A 283, 113-118. (49) Ono, M. Y., and Spain, E. M. (1999) Dynamics of DNA condensates at the solid-liquid interface by atomic force microscopy. J. Am. Chem. Soc. 121, 7330-7334. (50) Fenley, M. O., Manning, G. S., and Olson, W. K. (1990) Approach to the limit of counterion condensation. Biopolymers 30, 1191-1203. (51) Nguyen, T. T., Rouzina, I., and Shklovskii, B. I. (2000) Reentrant condensation of DNA induced by multivalent counterions. J. Chem. Physi. 112, 2562-2568. (52) Nguyen, T. T., and Shklovskii, B. I. (2001) Complexation of DNA with positive spheres: Phase diagram of charge inversion and reentrant condensation. J. Chem. Phys. 115, 7298-7308. (53) Huglin, M. B. (1972) Light Scattering from Polymer Solutions; Academic Press, London. (54) Talingting, M. R., Voigt, U., Munk, P., and Webber, S. E. (2000) Observation of massive overcompensation in the complexation of sodium poly(styrenesulfonate) with cationic polymer micelles. Macromolecules 33, 9612-9619. (55) Suck, D., and Oefner, C. (1986) Structure of DNase I at 2.0 A resolution suggests a mechanism for binding to and cutting DNA. Nature 321, 620-625.
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