Imidazole groups on a linear, cyclodextrin-containing polycationproduce enhanced gene delivery via multiple processes
Swaroop Mishra a,1, Jeremy D. Heidel a,2, Paul Webster b, Mark E. Davis a,⁎
a Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USAb Ahmanson EM and Advanced Imaging Center, House Ear Institute, Los Angeles, CA 90057, USA
Received 1 April 2006; accepted 21 June 2006Available online 27 June 2006
The delivery efficiency of non-viral vectors must be improvedif these materials are to achieve their therapeutic potential. A bevyof materials have been identified that are capable of condensingnucleic acids into small particles and delivering them to cells.Despite extensive uptake of exogenous nucleic acids by cells,non-viral vectors do not generate the levels of gene expressionthat are possible with viral vectors. Further development of non-viral vectors has been hindered by poor understanding of theintracellular fate of the gene delivery particles. This is especiallytrue for particle formulations that provide for in vivo use.
Once internalized by cells, non-viral gene delivery particles aregenerally deposited into the endocytic pathway and must escape inorder to avoid degradation by the lysosomal system [1–6].
1 Current address: Department of Bioengineering, University of Washington,Seattle, WA, USA.2 Current address: Calando Pharmaceuticals, Inc., Duarte, CA, USA.
Numerous investigations have focused on the concept that escapefrom the endocytic pathway may be facilitated by exploiting theprogressive acidification of its compartments [7,8]. This approachhas been inspired in part by the influenza virus, for which acidic pHtriggers reorganization of the hemagglutinin protein and facilitatesviral escape from the endocytic pathway. The intracellular pH-buffering activity of some polymers, particularly polyethylenei-mine (PEI), has also been linked to increases in transfectionefficiency [5,9], and the utility of pH-buffering activity in non-viralgene transfer has been supported by increases in transfectionefficiency in the presence of chloroquine, a small molecule that canbuffer the vesicles of the endocytic pathway [10–14].
Several non-viral vectors, including poly-L-lysine and thelinear, cyclodextrin-containing polycation (CDP), do not containany pH-buffering elements [15,16]. However, the positive aspectsof these non-buffering materials (such as the low cytotoxicity ofCDP) have motivated efforts to improve their gene deliveryperformance through rational modification. The relative effec-tiveness of pH-buffering vectors, most notably PEI, has promptedincorporation of buffering elements in both existing and new, non-buffering delivery vectors; these modified materials often exhibitimprovements in transfection efficiency [16–23].
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Modification of CDP to contain imidazole groups has beenshown to improve its transfection efficiency at low charge ratios(of most interest for systemic administration) without concom-itant increases in toxicity [17]. The imidazole-containing variant(CDPim) buffers the pH experienced by delivered nucleic acids,whereas CDP does not [23]. Recent reports have shown thatpH-buffering activity does not always correlate with improvedtransfection efficiency, raising questions about the value ornecessity of this feature in non-viral vectors [8,23–26]. Differ-ences in delivery behavior between CDP and its pH-buffering,imidazole-containing variant (CDPim) are investigated here inorder to elucidate mechanism(s) by which these two relatedmaterials exhibit differences in gene delivery. The goal of thiswork is to obtain experimental evidence necessary to evaluatethe value and necessity of pH-buffering components in non-viralgene delivery systems.We investigate the behaviors of polyplexesand their sterically-stabilized versions that are appropriate for usein animals in order to draw conclusions that are applicable to invitro and in vivo settings.
2. Materials and methods
2.1. Polycations, plasmid DNA, and polyplexes
A β-cyclodextrin-containing polycation (CDP) and itsimidazole-containing variant (CDPim) were synthesized asdescribed previously [17] (Fig. 1). The plasmid DNA (pDNA)pGL3-CV (Promega, Madison, WI), containing the luciferasegene under the control of the SV40 promoter, and the pDNApT7Luc, containing the luciferase gene under the control of theT7 promoter, were amplified by E. coli strain DH5α and purifiedusing the Ultramobius 1000 plasmid kit (Novagen, San Diego,CA). Polyplexes were formulated at a charge ratio of 5± byadding solutions of polycation (CDP or CDPim) (1.03 mg/mL indH2O) to equal volumes of nucleic acids (0.1 mg/mL in dH2O)
Fig. 1. Reaction of CDP to give CDPim. An imidazole end group can be directly cresulting imidazole-containing polycation is denoted as CDPim.
and incubating for 30 min. For surface modification with poly(ethylene glycol) (PEGylation), 35 μg adamantane-PEG (AD-PEG, 100 mg/mL in dH2O) per μg DNAwas added to CDP orCDPim polyplexes after formulation, and solutions wereincubated a further 30 min [27]. 25-kDa branched polyethyle-nimine (PEI) (Sigma, St. Louis, MO) was used to formulatepolyplexes as previously described [28].
For measurements of the effective diameter and zeta potentialof polyplexes, 100 μL solutions of polyplexes were diluted inwater to a final volume of 1.4 mL. These solutions were added tosquare cuvettes (1 cm pathlength) and analyzed using theZetaPALS particle analyzer (Brookhaven Instruments Corp.,Holtsville, NY). For each sample, the effective diameter wasrecorded as the average of 10 consecutive measurements of 30 seach. The zeta potential was measured using the Smoluchowskimethod and recorded as the average of 10 consecutive measure-ments having a target residual of 0.05. The results reported arethe mean and standard deviation of triplicate (n=3) measure-ments for each condition.
2.2. Cell culture and transfections
HeLa, BHK-21, and 293 cells were obtained from ATCC(Manassas, VA) and were maintained in Dulbecco's ModifiedEagle's Medium supplemented with 10% fetal bovine serum,100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 μg/mLamphotericin B. 293-T7 cells, which constitutively express T7RNA polymerase, were maintained in media as above with0.8 mg/mL geneticin (G418) [29]. The media was purchasedfrom Mediatech (Herndon, VA) and supplements were pur-chased from Gibco BRL (Gaithersburg, MD).
For evaluation of transfection efficiency through luciferaseassay, cells were plated in 24-well plates at a density of 5×104
cells/well. Polyplexes were formulated between CDP or CDPimand either the pGL3-CVor pT7Luc plasmid, then diluted 20-fold
onjugated to the termini of the cyclodextrin-containing polycation (CDP). The
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in Opti-MEM serum-free media (Invitrogen). This polyplexsolution (200 μL) was applied to each well 1 day after cells hadbeen plated; for transfections in the presence of chloroquine(CQ), immediately after polyplex-containing media was addedto cells for transfection, an appropriate volume of chloroquinestock solution (5 mM) was added to the media and mixed. After4 h, the polyplex solution was replaced with 1 mL of regularmedia. Two days after transfection, luciferase activity of celllysates was evaluated with the Luciferase Assay System(Promega) and total protein concentration was assessed by theDC Protein Assay (Bio-Rad, Hercules, CA).
2.3. Dye exclusion from polyplexes
The stability of polyplexes against increasing salt concen-tration was evaluated by dye exclusion [30]. After formulation,polyplexes were diluted 5× in dH2O. Separately, 200× dilutionsof the supplied PicoGreen stock solution (Molecular Probes)were prepared in 10 mM HEPES buffer containing variousconcentrations of NaCl. For each sample, 50 μL of polyplexsolution was transferred to a well of an opaque, black 96-wellplate and combined with 50 μL of PicoGreen solution; after10 min, the fluorescence (λexcitation 488 nm; λemission 535 nm) ofthe polyplex–PicoGreen solution was evaluated using a Spec-trafluor Plus plate reader (Tecan, Durham, NC). The percentagedye exclusion was calculated from the ratio ((FDNA−Fsample)/(FDNA−FH2O)), where FDNA is the fluorescence of a sample ofDNA alone (no polycation), Fsample is the sample fluorescence,and FH2O is the fluorescence of a blank (control) sample.
The accessibility of polyplex-compacted DNA in the presenceof chloroquine was evaluated by dye exclusion, through modifi-cation of the above procedure. After formulation, polyplexes werediluted 2.5× in dH2O. For each sample, 25μL of polyplex solutionwas combined with 25 μL of chloroquine-containing solution attwice the concentration of interest, and incubated for 5min at roomtemperature. Subsequently, each 50 μL sample was combinedwith 50 μL of PicoGreen solution and evaluated as above.
2.4.Heparan sulfate displacement of nucleic acids from polyplexes
Displacement of pDNA from polyplexes by heparan sulfatewas evaluated by gel electrophoresis. For each sample, an ap-propriate volume of heparan sulfate (5 mg/mL in dH2O) wasadded to 10 μL of polyplex solution. After 5 min incubation atroom temperature, 1 μL of loading buffer was added to eachsample and the samples were transferred to a 0.5% agarose gel(30 μg ethidium bromide/50 mL TAE buffer), electrophoresed,and visualized under UV illumination.
2.5. Transmission electron microscopy of CDP and CDPimpolyplexes within cells
For transmission electron microscopy, BHK-21 cells weretransfected with CDP/pDNA or CDPim/pDNA polyplexes. Atincreasing timepoints after transfection, the medium was aspi-rated and the cells were rinsed with PBS. Cells were fixed with2% glutaraldehyde in 100 mM sodium cacodylate with 2%
sucrose, collected by scraping, and pelleted by centrifugation.Cell pellets were post-fixed in osmium tetroxide, dehydrated inethanol, and embedded in Epon Spurr resin. Thin sections wereprepared on an Ultracut S ultramicrotome (Leica Microsystems,Deerfield, IL), contrasted with uranyl acetate and lead citrate, andimaged on a BioTwin CM120 transmission election microscope(FEI, Hillsboro, OR) operating at 80 kV.
For immunolabeling experiments, pDNAwas labeled withthe LabelIT biotin-labeling kit (Mirus, Madison, WI) at adensity of ∼10 labels per plasmid and purified by ethanolprecipitation. Polyplexes were formulated with CDP orCDPim and administered to BHK-21 cells. At desiredtimepoints after transfection, the medium was aspirated andthe cells were rinsed with PBS. Cells were fixed with 2%glutaraldehyde in 100 mM sodium cacodylate with 2%sucrose, collected by scraping, and pelleted by centrifugation.Cell pellets were infiltrated in 2.3 M sucrose, frozen byimmersion in liquid nitrogen, freeze substituted at −80 °Cwith dry methanol, and embedded at −50 °C in LowicrylHM20 resin (Electron Microscopy Sciences, Fort Washing-ton, PA). Thin sections were prepared on an Ultracut Sultramicrotome and immunolabeled with a mouse anti-biotinprimary antibody (Sigma), a rabbit anti-mouse secondaryantibody (Organon Technika, Durham, NC), and colloidalgold (10 nm)-labeled protein A (University of Utrecht, TheNetherlands). 1× PBS containing 10% fetal calf serum wasused as the blocking solution, and immunolabeled sectionswere contrasted with uranyl acetate and lead citrate andimaged on a BioTwin CM120 transmission election micro-scope operating at 80 kV. Immunolabeling of control samplesprepared without biotin-labeled pDNAwas used to determineappropriate concentrations for antibody and colloidal goldlabeling. An iterative process was used to minimizebackground labeling from gold-labeled protein A alone, thesecondary antibody and gold-labeled protein A together, andfinally both antibodies and gold-labeled protein A together.
2.6. Measure of intracellular nucleic acid unpackaging
To obtain a relative measure of intracellular nucleic acidunpackaging, HeLa cells in 24-well plates were transfectedwith 2 μg of a FITC-labeled DNA oligonucleotide (FITC-oligo) condensed in CDP or CDPim polyplexes. At selectedtimes after transfection, cells were collected by trypsinizationand pelleted by centrifugation (6 min at 2400 rpm). Each pelletwas resuspended in 10 μL 1× cell culture lysis buffer(Promega) and stored at 4 °C. Control samples were preparedas solutions of known amounts of FITC-oligo in 10 μL 1× cellculture lysis buffer. After 30 min incubation, each sample wasmixed with 25 μL of low-melting-point agarose solution (1%in TAE buffer, 37 °C) and transferred immediately to a well ofa 0.5% agarose gel. The agarose gel was subjected toelectrophoresis and then imaged under UV illumination.Detection of FITC-oligo was achieved through its fluorescentsignal. Migration of FITC-oligo from control samples was usedto indicate the expected migration distance of unpackagedFITC-oligo from cell lysates.
Fig. 2. In vitro transfection efficiency of CDP and CDPim. (A) CDP or CDPimwas used to transfect HeLa or BHK-21 cells with pDNA. In each case, CDPimgenerated greater luciferase expression. (B) There was no significant expressionwhen 293 cells were transfected with pT7Luc, suggesting that pT7Lucexpression depends on the presence of the cytoplasmic T7 polymerase. Whenused to transfect 293-T7 cells with pT7Luc, CDPim generated greater luciferaseexpression than CDP. (C) The addition of chloroquine produced a significantincrease in luciferase expression, particularly in transfection by CDP polyplexes.
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2.7. Flow cytometry for evaluation of nucleic acid uptake
For flow cytometry, polyplexes were formulated with a FITC-labeled oligonucleotide (FITC-oligo). Labeled polyplexes wereused to transfect cells as described; 4 h after transfection, thepolyplex-containing medium was aspirated and replaced withregular growth medium. Cells were washed (10 U/mL DNase,3 mM MgCl2 in Hank's buffered saline solution (HBSS)), sus-pended in propidium iodide-containing buffer (HBSS, 2.5mg/mLbovine serum albumin, 0.01 mg/mL propidium iodide), and ana-lyzed on a FACScalibur instrument (Becton Dickinson, FranklinLakes, NJ) with a 488 nm excitation line. Samples were initiallygated by forward and side scatter, and viable cells were assayedfor FITC fluorescence.
3. Results
3.1. Transfection efficiency of CDPim is greater than that ofCDP, for both nuclear and cytoplasmic expression, exceptwhen chloroquine is present
Polyplexes formulated with CDP or with CDPim exhibitsimilar size and zeta potential (Table 1). When used to deliverluciferase-expressing plasmid DNA (pDNA) to cultured cells,CDPim generates greater levels of gene expression than CDP(Fig. 2A). These results were obtained with pGL3-CV, pDNAthat should be transcribed and expressed only when available tothe transcriptional machinery within the cell nucleus.
The pDNA pT7Luc expresses luciferase under control of a T7promoter and can be transcribed and expressed in the cytoplasmof cells constitutively expressing the T7 RNA polymerase [29].Its poor expression when delivered to 293 cells (lacking the T7RNA polymerase) confirms that the polymerase is necessary forpT7Luc expression (Fig. 2B).When pT7Luc is delivered to 293-T7 cells (constitutively expressing the cytoplasmic T7 RNApolymerase), thus allowing for cytoplasmic expression, CDPimagain demonstrates greater transfection efficiency than CDP(Fig. 2B).
Transfection experiments were also conducted in thepresence or absence of chloroquine (CQ), an agent that hasbeen widely reported to give increases in transfection efficiencyin vitro for a variety of non-viral vectors [10–14]. Improvementsin transfection efficiency with CQ were noticeably greater forCDP polyplexes than for CDPim polyplexes, and significanteffects on CDPim were seen only with 0.2 mM CQ (Fig. 2C).While CQ contributed significantly to the transfection efficiency
Table 1Particle sizing and zeta potential of CDP/DNA and CDPim/DNA polyplexes
PlasmidDNAwas used to formulateCDP or CDPimpolyplexes, and dynamic lightscatteringwas used to evaluate the particle size and zeta potential of the polyplexes.Data represent the mean and standard deviation of triplicate samples.
of both CDP and CDPim, the greater enhancements for CDPraised CDP-mediated expression in the presence of CQ to levelsbeyond CDPim-mediated expression in the presence of CQ.
3.2. Exclusion of intercalating dye byCDPandCDPim polyplexes
PicoGreen experiences significant fluorescence enhance-ment upon intercalation in nucleic acids, such that its exclusionindicates inaccessibility of the nucleic acids to intercalation[30]. This characteristic was used to evaluate different types ofpolyplexes for the relative accessibility of pDNA.
Polyplexes were subjected to disruption by increasing con-centrations of NaCl, then combined with solutions of PicoGreenand evaluated for fluorescence intensity. Unlike 25-kDabranched polyethylenimine (PEI), which showed near-completedye exclusion without added NaCl, CDP and CDPim gave onlymoderate exclusion of PicoGreen without NaCl andminimal dyeexclusion at higher salt concentration (Fig. 3A). Furtherexperiments in the range of 0–0.2 M NaCl consistently revealed
Fig. 3. PicoGreen dye exclusion. (A) The exclusion of the DNA-intercalatingdye PicoGreen was used as a measure of polyplex stability against salt-induceddissociation [30]. CDP and CDPim did not exclude PicoGreen to the sameextent as 25-kDa branched polyethylenimine (PEI). (B) The fluorescenceenhancement resulting from PicoGreen–pDNA interactions is ∼2000 times theenhancement that is seen with PicoGreen–CDP or PicoGreen–CDPiminteractions, indicating that the incomplete dye exclusion observed for CDPand CDPim polyplexes does not result from PicoGreen interactions with thepolycation. (C) PicoGreen exclusion was used to assess the accessibility ofpDNA in CDP or CDPim polyplexes to chloroquine (CQ). Both types ofpolyplexes exhibit an increase in dye exclusion in the presence of CQ, indicatingthat CQ interacts with the pDNA therein.
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a slightly greater dye exclusion from CDPim/pDNA polyplexesthan from CDP/pDNA polyplexes (data not shown).
Inclusion complex formation between the PicoGreen dyeand the cyclodextrin cups of CDP or CDPim may impart somefluorescence to these materials. Significant fluorescence of thistype would give a false impression of reduced dye exclusion,consistent with the observations of CDP and CDPim polyplexesin the PicoGreen salt-dissociation assay. To investigate thispossibility, CDP and CDPim were incubated with PicoGreen inthe absence of any pDNA. The fluorescence generated by thesecombinations represents only 0.05% of the fluorescence en-hancement seen with PicoGreen interactions with uncomplexedpDNA (Fig. 3B). Thus, the incomplete dye exclusion (mea-surable fluorescence) observed with CDP and CDPim poly-
plexes in the absence of NaCl is unlikely to be the result ofPicoGreen–cyclodextrin or PicoGreen–polycation interactions.
CQ contributes significantly to the transfection efficiency ofboth CDP and CDPim. Dye exclusion was used to compare therelative accessibility to CQ of pDNA in CDP or CDPim poly-plexes. As-formulated CDP and CDPim polyplexes do notexhibit full exclusion of the DNA intercalating dye PicoGreen,but increasing concentrations of CQ can restrict this access andgive increased dye exclusion (Fig. 3C). Trends in dye exclusionwere similar for CDP/pDNA and CDPim/pDNA polyplexes,reflecting similar accessibility of pDNA therein to CQ.
3.3. CDPim polyplexes are more resistant to heparan sulfatethan CDP polyplexes
Heparan sulfate was employed to evaluate the ability of apolyanion to cause CDP or CDPim to displace from DNA. Poly-plexeswere formulated, incubatedwith various amounts of heparansulfate for 5min at room temperature, and then loaded to an agarosegel and electrophoresed. Polycation-boundDNAwill notmigrate inthe gel. At higher heparan sulfate concentrations, “free” nucleicacids could be observed due to their movement in the gel. Forpolyplexes formulated with a DNA oligonucleotide (Fig. 4A) orwith pDNA (Fig. 4B–C), an equivalent dose of heparan sulfategenerated greater displacement with CDP than with CDPim.
Polyplexes are exposed to acidification when trafficking inthe endocytic pathway; to test whether acidic conditions alterthe relative susceptibility of CDP and CDPim to heparan sul-fate, polyplexes were acidified to pH 3.0 by addition of 0.1 NHCl prior to heparan sulfate incubation. Acidified polyplexesdisplayed an equal response to heparan sulfate as their as-for-mulated counterparts (Fig. 4C).
3.4. Transmission electron microscopy shows CDPim givesenhanced unpackaging and accessibility of delivered DNArelative to CDP
CDPim polyplexes were delivered to cultured cells and vi-sualized using transmission electron microscopy (Fig. 5), andthese results were compared with observations of intracellularCDP polyplexes by the same method (Fig. 5, [31]). Intact poly-plexes are easily identifiable, in each case appearing as irregularaggregates roughly 300–500 nm in diameter. Some aggregatesare composed of smaller rounded components, likely the as-formulated polyplexes readily observable by light scattering inwater (Table 1).
Most intracellular vesicles containing CDP polyplexes con-formed to the aggregates' shape and contained little to no voidspace or other material (Fig. 5A–B, [31]). In contrast, vesiclescontaining CDPim polyplexes occasionally enclosed portions ofvoid space (Fig. 5F–I) or large amounts of material separate fromthe aggregates (Fig. 5F–J). It is unclear if this accompanyingmaterial is derived from or is independent of the polyplexaggregates.
As the time between the initial exposure to CDP polyplexesand the cells' fixation was increased from 0.5 to 24 h, aggre-gates of CDP polyplexes increasingly appeared in the vicinity of
Fig. 4. Heparan sulfate release of oligonucleotide from polyplexes. (A) Polyplexes were prepared with a 25-mer DNA oligonucleotide (FITC-oligo) and either CDP(lanes 2–8) or CDPim (lanes 9–15). The resulting polyplexes were incubated with increasing amounts of heparan sulfate (hep. su.) to induce oligonucleotide release.Samples were then loaded to an agarose gel and electrophoresed. With equivalent heparan sulfate loading, CDP polyplexes showed more extensive oligonucleotiderelease than CDPim polyplexes. (B, C) CDP/pDNA (top row) and CDPim/pDNA (bottom row) polyplexes (Poly.) were incubated with increasing amounts of heparansulfate (hep. su.) to induce pDNA release. Samples were then loaded to an agarose gel and electrophoresed. (B) With equivalent heparan sulfate loading, CDPpolyplexes showed more extensive pDNA release than CDPim polyplexes. (C) Equivalent results were obtained for as-formulated polyplexes incubated with heparansulfate (left) or polyplexes acidified to pH 3.0 and then incubated with heparan sulfate (right).
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the cell nucleus (Fig 5B–D, [31]). The larger size of aggregatescloser to the nucleus suggested an aggregation of polyplex aggre-gates was occurring.Only aminority ofCDPpolyplexes appearedto be disassembling [31]. These degrading polyplexes, with lessdistinct and defined borders than most aggregates, were foundmore commonly within vesicles than free in cytoplasm.
In contrast, CDPim polyplexes were observed undergoingsignificant unraveling in intracellular vesicles at 4 or 8 h afterinitial transfection (Fig. 5F–L).Various loops and swirls extendfrom the central mass of some CDPim polyplexes (Fig. 5I–K).Also, some cells contain swirls or ring structures in the absence oflarger aggregates; these features may be found either within largervesicles (Fig. 5F–H) or filling vesicles entirely (Fig. 5K–L). A
fuzzy or hairy surface characterizes many aggregates (Fig. 5H–J)and many of the loops, swirls, and rings (Fig. 5I–L).
Both CDP and CDPim polyplexes could be observed in theabsence of surrounding membranes. Aggregates were visualizedeither in direct contact with the surrounding cytoplasm (Fig. 5C–D,H) or bordering void spaces. Intact polyplexes or theiraggregates were not observed within any cell nucleus.
In order to visualize unpackaged intracellular pDNA, CDP orCDPim was used to deliver biotin-labeled pDNA to cells. Subse-quently, TEM samples were prepared using anti-biotin immuno-labeling with colloidal gold [31]. Intracellular CDP and CDPimpolyplexes are both labeled by this method, but the numbers anddensity of gold particles per polyplex or aggregate are generally
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less with CDP than with CDPim (Fig. 6A, G–J). The CDPimpolyplexes can be seen in cells as large, heavily gold-labeledentities.
Immunolabeling of unpackaged pDNAwithin cells generatesclusters of colloidal gold separate from polyplexes. For both
Fig. 5. TEMs of CDP and CDPim polyplexes. BHK-21 cells were exposed to CDP polpolyplexes appear as large aggregates within vesicles that generally conform to the swithout surrounding membranes (C–D). Later timepoints show an accumulation ofCDPim polyplexes appear as large aggregates within vesicles (E–G). The vesicles convesicles containing polyplex aggregates also appear to have a variety of other contencontained within vesicles (H). To a much greater extent than CDP polyplexes, CDPimobserved emanating from the aggregates (I–K). In other cases, circular or oval ring struwithin larger vesicles (F–H) or filling vesicles entirely (K–L). As with the aggregates
CDP and CDPim, these clusters are found both in the cytoplasm(Fig. 6B–D, K) and in the cell nucleus (Fig. 6E–F, J, L). Quali-tative differences were not apparent among the colloidal goldclusters for the two types of polyplexes, either in their intra-cellular distribution or overall prevalence.
yplexes (A–D) or CDPim polyplexes (E–L) and prepared for TEM imaging. CDPhape of the aggregates (A–B). Some polyplexes are observed in the cytoplasmpolyplexes in a perinuclear region. (See also [31].) As with CDP, intracellulartaining these aggregates do not necessarily conform to the aggregates’ shape, andts (F–J). Some aggregates have a fuzzy exterior (H–J) and not all aggregates arepolyplexes appear to be degrading within vesicles. Various loops and swirls arectures or swirls are observed in the absence of larger aggregates, either contained, many of the loops, swirls, and rings appear fuzzy or hairy (I–L). Bars, 200 nm.
Fig. 5 (continued).
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Cells transfected with unlabeled pDNA and subjected toequivalent preparation and immunolabeling do not reveal col-loidal gold labeling of polyplexes or colloidal gold clustering (notshown).
3.5. CDPim produces more unpackaged intracellular DNAthan CDP
The comet assay is a standard method of evaluating DNAdamage [32,33]. In this assay, cells are gently lysed, embeddedin an agarose gel on a glass slide or coverslip, and exposed toelectrophoresis. Only damaged cellular DNA is reduced to asize that permits migration within the gel, and this damagedDNA will generate a characteristic “comet” pattern upon sub-sequent intra-gel labeling of nucleic acids by a fluorescentmarker. The intensity of the comet reflects the extent of DNAdamage. By this method, individual cells may be visuallyevaluated for DNA damage.
We have modified this assay to produce an aggregate sep-aration and visualization of delivered and unpackaged nucleicacids. Rather than evaluating samples on an individual-cellbasis, cells are collected by centrifugation, lysed, embedded inan agarose gel, and subjected to electrophoresis in order togenerate a relative measure of DNA unpackaging for a particularsample. Also, rather than adding a fluorescent label afterelectrophoresis, we transfect cells using nucleic acids that have
been covalently modified with a fluorescent marker. Thus, weare able to distinguish exogenous (delivered) nucleic acids fromcellular DNA. The “unpackaged” oligonucleotide measured bythis assay is that which migrates through the agarose gel to thesame extent as in the case of direct gel loading of theoligonucleotide. Polyplexes suspended in 1× lysis buffer alonedo not generate this migrating DNA, suggesting that the DNAunpackaging that allows for electrophoretic migration hasoccurred within cells.
As measured by this assay, CDPim consistently generatesgreater amounts of unpackaged nucleic acids (Fig. 7). This isseen for a variety of timepoints ranging from 6 to 44 h after theinitial transfection, and is seenwhether the polycations are used todeliver a DNA oligonucleotide (Fig. 7A, C) or siRNA (Fig. 7B).PEGylation of polyplexes does not alter this effect, as PEGylatedCDPim particles generate greater levels of migrating DNA thanCDP polyplexes or their PEGylated variant (Fig. 7C). PEI wasalso examined in siRNA delivery and did not produce a signalrepresenting unpackaged nucleic acid (Fig. 7B).
3.6. CDP and CDPim polyplexes exhibit similar uptake to cells
Rather than indicating differences in intracellular processing,differences in the amounts of unpackaged intracellular DNAwith CDP and CDPim could simply reflect differences in thetotal amount of DNA delivered to cells. Flow cytometry was
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used to evaluate the cellular uptake and retention of a FITC-labeled DNA oligo delivered to HeLa cells with CDP or CDPimpolyplexes or with their PEGylated variants (Table 2). All par-ticles showed strong uptake to cells, with over 98% of cellsregistering positive at both examined timepoints. CDPim samplesgenerated greater fluorescence than CDP samples. However,CDPim buffers the pH experienced by delivered nucleic acidswhereas CDP does not, irrespective of PEGylation [23], and thefluorescence of FITC is quenched at lower pH. Thus, the greaterfluorescence observedwithCDPim does not necessarily representenhanced uptake.
4. Discussion
Many non-viral gene delivery vectors display improvementsin transfection efficiency upon incorporation of pH-bufferingelements. The “proton sponge” hypothesis proposes a mecha-nism for this behavior, suggesting that the buffering activitygenerates osmotic swelling and lysis of endocytic compart-
Fig. 6. Immunolabeling to detect intracellular DNA delivered by CDP or by CDPim.polyplexes (G–L), and TEM samples were prepared using anti-biotin immunolabelinoutside of polyplexes (C–F), an indication of unpackaged intracellular pDNA. Somelarge, heavily gold-labeled entities (polyplexes or their aggregates) are readily observCDPim polyplexes typically exceeds that observed with CDP. As with CDP polyplepDNA that has unpackaged from polyplexes. Such gold clusters are found in the cy
ments. Reduced lysosomal degradation and greater cytoplasmicavailability of delivered DNAwould then give improvements intransfection efficiency [5,9]. We and others have observed andquantified the vector-dependent intracellular buffering of the pHexperienced by polyplexes [4,23,34,35], and it has been shownthat the pH-buffering vector PEI gives increases in the chlorideaccumulation, volume, and lysis of endosomes [5].
Modification of CDP to contain imidazole groups success-fully introduces intracellular pH-buffering activity to this non-viral gene delivery vector [23]. The new polycation, CDPim,displays DNA uptake comparable to CDP (Table 2) and im-proved transfection efficiency in cultured cells, including cellswith a cytoplasmic expression system (Fig. 2). The results fromthe cytoplasmic expression system indicate that improvementsin transfection efficiency with CDPim result from intracellularphenomena that occur prior to delivery into the cell nucleus.These data are consistent with the hypothesis that the improvedperformance of the pH-buffering CDPim results from enhancedescape and release of the nucleic acid from endocytic vesicles.
Biotin-labeled pDNAwas delivered to cells in CDP polyplexes (A–F) or CDPimg with colloidal gold. With CDP, clusters of colloidal gold (arrows) can be foundof the intracellular gold clusters are within a cell nucleus (E–F). With CDPim,ed (G–J), and the intensity of polyplex labeling (numbers of gold particles) forxes, clusters of colloidal gold (arrows) outside of polyplexes reveal intracellulartoplasm (K) or the cell nucleus (J, L). Bars, 200 nm. Cell nucleus, nu.
Fig. 6 (continued).
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Intracellular observations of CDP polyplexes by transmis-sion electron microscopy (TEM) show vesicle membranesgenerally conform closely to the shape of polyplexes (and theiraggregates) [31]. In contrast, TEM observations of intracellularCDPim polyplexes in cells often reveal some space betweenpolyplexes and vesicle membranes (Fig. 5). It is possible thatthis space within vesicles containing CDPim polyplexesindicates osmotic swelling of the vesicles, suggesting that a“proton sponge” hypothesis may be relevant to CDPim.
Recent reports have questioned the general applicability ofthe “proton sponge” hypothesis [8,23–26]. We investigated thegene delivery behavior of CDP and CDPim in an effort to revealany alternative mechanism(s) by which CDPimmay give greatergene expression than CDP. CDP has a relatively low degree ofpolymerization (∼5), such that its modification can significantlyaffect its chemical and biological properties and the properties ofensuing polyplexes. CDP and CDPim generate polyplexes ofsimilar size (∼100 nm diameter) and zeta potential (Table 1) thatgive similar levels of DNA uptake to cells (Table 2). In cell-freeassays, CDP appears bound more weakly to nucleic acids thanCDPim: CDP polyplexes display slightly weaker dye exclusionthan CDPim polyplexes (Fig. 3A) and are more susceptible toheparan sulfate-induced DNA release (Fig. 4). Despite theseobservations, CDPim appears to facilitate greater intracellularrelease of delivered nucleic acids than CDP. CDP polyplexeswithin cells generally appear in TEM as large, well-defined aggre-gates that grow larger (due to collection or further aggregation of
existing aggregates) over the initial 24 h after transfection (Fig. 5,[31]). In contrast, extensive unraveling and disassembly of intra-cellular CDPim polyplexes are observed 4 or 8 h after initialtransfection (Fig. 5). Immunolabeling of delivered pDNA showsthat CDP and CDPim both produce unpackaged pDNA, but theintensity with which CDPim polyplexes are labeled (under equiv-alent conditions) suggests that the pDNAwithin these polyplexesmay be more accessible to the labeling than that of CDP poly-plexes (Fig. 6). Further, results from a modified comet assayindicate that CDPim transfection produces greater amounts ofintracellular, unpackaged nucleic acids than CDP transfection,irrespective of whether or not the polyplexes are PEGylated(Fig. 7). These data taken together suggest that intracellular factorsplay a significant role in the differences observed between thebehavior of CDP and CDPim in cells.
The mechanism(s) behind the enhanced intracellularunpackaging observed with CDPim is unclear. Dye exclusionindicates that the imidazole groups do not inhibit polycation–DNA interactions (Fig. 3A). Polyplexes may undergo changesin the binding between polycation and nucleic acid as they trafficin the endocytic pathway, particularly as the imidazole groupsbecome protonated, thereby buffering vesicular pH. The observeddisassembly of CDPim polyplexes in intracellular vesicles isconsistent with this hypothesis. Although no changes in DNArelease are observed for polyplexes acidified prior to heparansulfate treatment (Fig. 4C), immersion in the electrophoresisbuffer could mask differences if the DNA release induced by
Fig. 7. Unpackaging of nucleic acids following delivery to HeLa cells. (A) Cellstransfected using CDP or CDPim were evaluated after 6, 12, or 18 h forunpackaged, FITC-labeled DNA oligonucleotide. At all timepoints examined,CDPim samples showed more unpackaged DNA than CDP samples. (B) Cellstransfected using CDP, CDPim, or 25-kDa branched polyethylenimine (PEI) wereevaluated after 16 h for unpackaged, FITC-labeled siRNA. CDPim samplesshowed more unpackaged nucleic acid than CDP samples, while PEI samples didnot generate an unpackaged siRNA signal. (C) CDP, CDPim, or their PEGylatedvariants (PEGyl.) were used to transfect cells, whichwere evaluated after 14, 20, or44 h for the levels of unpackaged, FITC-labeled DNA oligonucleotide. At alltimepoints examined, the greatest amount of unpackaged DNA was seen withCDPim samples, followed by samples transfected with PEGylated CDPimparticles. CDP and PEGylated CDP particles also show unpackaged DNA, but inlesser amounts. For each experiment, “Control” indicates direct gel loading ofFITC-labeled oligonucleotides in the absence of polycations or cells.
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heparan sulfate is reversible. Efforts to conduct the gelelectrophoresis at acidified pH were unsuccessful. Another factorthat may contribute to the differences in unpackaging betweenCDP and CDPim polyplexes is the intracellular distribution ofpolyplexes and polyplex-disrupting factors. If the pH buffering ofCDPim facilitates endosomal escape and the polyplex disruptionoccurs primarily in the cytoplasm, CDPim would generate moreunpackaged DNA. However, this hypothesis is not supported bythe TEMobservations of CDPim polyplexes in that they appear tobe disassembling within intracellular vesicles.
Additional information about the intracellular behavior ofCDP and CDPim polyplexes arises from their differing responsesto treatment with chloroquine (CQ). CQ contributes significantlyto the transfection efficiency of non-viral vectors, although itshows stronger effects on CDP than on CDPim (Fig. 2C). Theenhancements likely result from the ability of CQ to buffer the pHof the endocytic pathway and facilitate unpackaging of the DNA[13]. Because CDPim and CQ independently display similarintracellular pH-buffering activity [23], it is likely that CQcontributes to the transfection efficiency of CDPim (and CDP) byimproving unpackaging of the DNA. CQ may give greaterdisplacement of and be better able to bind DNA unpackaged fromCDP than CDPim due to the spatiotemporal intracellular distri-bution of CQ and of the polyplexes. Protonation of CQ isbelieved to drive its accumulation in endocytic vesicles [36],where it can interact with endocytosed polyplexes. CQ-mediatedpolyplex disruption and CQ interaction with unpackaged nucleicacids may very well occur within these vesicles. If the pHbuffering by CDPim limits the supply of protons necessary todrive accumulation of CQ in endocytic vesicles, or if CDPimpolyplexes more readily escape the endocytic pathway andrelease their DNA cargo, there may be reduced opportunity forinteractions between CQ and the polyplexes and/or between CQand the delivered DNA. Thus, CQ would not produce the sameenhancements in transfection efficiency with CDPim that areobserved with CDP.
Table 2Summary of flow cytometry analysis
Nucleicacid
Deliveryvector
20 h 44 h
% cellsFITC-positive
Meanfluorescence
% cellsFITC-positive
Meanfluorescence
– – 0.50 3.41 0.05 2.83F I TC -oligo
CDP 99.33 3109.80 99.11 1291.04
F I TC -oligo
CDP,PEGylated
99.56 1398.83 98.12 579.79
F I TC -oligo
CDPim 99.67 6920.65 99.41 4465.41
F I TC -oligo
CDPim,PEGylated
99.79 6770.88 99.87 3829.96
A FITC-labeled DNA oligonucleotide (FITC-oligo) was used to formulate CDPand CDPim polyplexes and their PEGylated variants. HeLa cells were transfectedwith the particles and evaluated after 20 h or 44 h for FITC fluorescence by flowcytometry. All types of particles gave rise to strong fluorescence at the timepointsexamined, although CDP particles did not generate as much fluorescence asCDPim particles. Data represent the mean of duplicate samples.
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A similar hypothesis could help to explain why PEI bothdemonstrates pH-buffering activity [23,34] and shows little to notransfection efficiency enhancement in the presence of CQ[28,30]. That is, PEI polyplexes may have reduced opportunityfor interaction with CQ because the pH buffering of PEI eitherlimits the accumulation of CQ in intracellular vesicles or facili-tates polyplex escape from the vesicles (in which polyplexesmust remain in order to encounter high concentrations of accu-mulated CQ). Given that CDPim and PEI show similar bufferingactivity [23] but CDPim shows transfection efficiency enhance-ment with high CQ (Fig. 2C) while PEI does not [28,30], it isreasonable to suggest that CQ enhancing effects with CDPim aremainly due to displacement of the DNA from CDPim. This isbecause it would take a higher level of CQ to displace the muchhigher molecular weight PEI from DNA than to displace theshort chains of CDPim. Consistent with this hypothesis are thefacts that DNA in PEI polyplexes is less accessible than inCDPim polyplexes (Fig. 3A) and PEI polyplexes do not give thesignificant levels of unpackaged intracellular nucleic acids thatare seen with CDPim in the modified comet assay (Fig. 7B).
The differences observed between the cell-free experimentsand those using cells suggest that intracellular factors may beresponsible for polyplex disassembly. If so, the response ofCDP and CDPim polyplexes to heparan sulfate treatment maynot reflect their susceptibility to the relevant intracellular factors.Also, polyplexes are observed to undergo significant aggrega-tion in physiological salt concentrations, prior to uptake by cells(Fig. 5, [31]). The performance of as-formulated polyplexes incell-free assays may not be representative of the behavior ofpolyplex aggregates within cells. For in vivo application, polyplexaggregation can be inhibited by approaches such as PEGylation.We have previously shown significant differences in particlemorphology and resulting gene expression between unPEGylatedpolyplexes and their PEGylated variants [31]. It is unclear howcertain characteristics can be significantly altered by PEGylationwhen other characteristics are seemingly unaffected. For example,PEGylated CDPim particles have far different intracellular mor-phology (no aggregation) than the unPEGylated variant and givemarkedly reduced gene expression, yet the levels of DNA uptakeand unpackaged intracellular DNA are observed to be similar(Table 2, Fig. 7).
Significant levels of reporter gene expression (Fig. 2) andindications of pDNA (or pDNA fragments) in the cell nucleus(Fig. 6) demonstrate that CDP and CDPim polyplexes candeliver DNA to the cell nucleus, yet it is unclear how the DNAarrives there. Given our observations of polyplexes unravelingin the cytoplasm and our inability to visualize intact polyplexes,aggregates, or polyplex fragments in any cell nucleus (Fig. 5,[31]), it appears that pDNA delivered to cells by CDP or CDPimis unpackaged prior to nuclear delivery and that unpackagedDNAenters the cell nucleus for transcription. Despite the metabolicinstability and negligible diffusion of unpackaged DNA incytoplasm [37,38], CDP and CDPim give nuclear delivery that issubstantial enough to produce significant levels of gene expres-sion. The cultured cell lines utilized here were not growth-arrested, so it is possible that cell division may be the key factorallowing for nuclear delivery [39,40].
We have used a variety of methods to investigate differencesin the gene delivery behavior of CDP and its imidazole-con-taining variant, CDPim. Consistent with the “proton sponge”hypothesis, the imidazole modification of CDP confers intra-cellular pH-buffering activity that is correlated with an increasein transfection efficiency. However, it is unclear if the improvedtransfection efficiency of CDPim is a result of enhanced endo-somal escape, as it was also observed that CDPim generatesgreater amounts of unpackaged intracellular nucleic acids thanCDP. The increase in unpackaged intracellular DNA withCDPim over CDP is not consistent with cell-free measures andemphasizes the need to evaluate the behavior of non-viral genedelivery vectors within cellular environments. The character-ization of gene delivery behavior with CDP and CDPimreinforces the notion that non-viral gene delivery involvescomplex systems in which simple modifications can impartmultiple effects. It is clear that CDPim does provide pHbuffering but this condition alone is not sufficient to explain theenhanced transfection. Phenomena in addition to pH bufferingare necessary for enabling enhanced gene delivery with non-viral vectors.
Acknowledgments
We are grateful to Patrick Midoux for his kind gift of the293-T7 cells and the plasmid pT7Luc, and we thank InsertTherapeutics, Inc. for partial support of this work.
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