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Dual-Sensitive Micellar Nanoparticles Regulate DNAUnpacking and Enhance Gene-Delivery Efficiency

By Xuan Jiang, Yiran Zheng, Hunter H. Chen, Kam W. Leong,

Tza-Huei Wang, and Hai-Quan Mao*

Polymer-based gene carriers have been increasingly proposed as asafer alternative to viral vectors, due to their ease of synthesis,flexibility in the size of the transgene to be delivered, andminimalhost immune responses. A major challenge to apply polycation/DNA nanoparticles in vivo, however, is the poor colloidal stabilityand serum stability, which lead to rapid aggregation followed bymacrophage uptake, and premature dissociation of nanoparticlesand release of DNA payload, respectively. Together they conspireto yield extremely low gene delivery efficiency throughintravenous injection. To improve efficiency, an ideal polyca-tion/DNA delivery system should satisfy the conflicting require-ments of high stability in extracellular environment andendolysosomal compartments, so that the nanoparticles maintaintheir small size and integrity in circulation and endosomalsequestration, followed by low stability in cytosol and nucleus soas to allow for DNA release and transcription.

Recently, we have synthesized a series of polyethyleneglycol-block-polyphosphoramidate (PEG-b-PPA)/DNA micellarnanoparticles for liver-targeted gene delivery.[1] The PEG coronaof the micelles around the PPA/DNA core effectively reducedprotein adsorption in physiological media and led to improved

[*] Prof. H.-Q. MaoDepartment of Materials Science and Engineering andWhitaker Biomedical Engineering InstituteJohns Hopkins University206 Maryland Hall, Baltimore, MD 21218 (USA)E-mail: hmao@jhu.edu

X. JiangDepartment of Materials Science and EngineeringJohns Hopkins UniversityBaltimore, MD 21218 (USA)

Y. ZhengDepartment of Chemical and Biomolecular EngineeringJohns Hopkins UniversityBaltimore, MD 21218 (USA)

Dr. H. H. Chen, Prof. T.-H. WangDepartment of Biomedical EngineeringJohns Hopkins University, School of Medicine720 Rutland Avenue, Baltimore, MD 21231 (USA)

Prof. K. W. LeongDepartment of Biomedical EngineeringDuke UniversityDurham, North Carolina 27708 (USA)

Prof. T.-H. WangDepartment of Mechanical EngineeringJohns Hopkins UniversityBaltimore, MD 21218 (USA)

DOI: 10.1002/adma.200903933

� 2010 WILEY-VCH Verlag Gmb

serum stability and enhanced gene transfection efficiency in therat liver after infusion through the tail vein or bile duct, ascompared with PPA/DNA nanoparticles and PEI/DNA com-plexes. In a series of PEG-b-PPA carriers prepared with 12KDaPEG, we observed increasing DNA binding affinity as themolecular weight of PPA block increased from 3KDa to 80KDa.Interestingly however, micelles formed with PEG12K-b-PPA128K

(the molecular weight of PPA block is 128KDa) exhibitedinstability in solution with physiological salt concentration andreleasedmost of encapsulated plasmid DNA 4h after transfectionin HEK293 cells.[2] The quick DNA unpacking ability of thesemicelles as a result of salt instability could be advantageous forintracellular unpacking of DNA cargo upon delivery to the cytosoland nucleus. To avoid the premature release of encapsulatedplasmid DNA in extracellular milieu or in endocytic compart-ments, we have introduced reversible reduction-sensitive cross-links to themicelles. The resultingmicellar nanoparticles showeddual sensitivity that affords stability of the nanoparticles inextracellular and endocytic environments and DNA unpackingability in cytosols and nuclei (Fig. 1a). As reported previously,[3,4]

disulfide crosslinking is readily reduced in the cytosol andnucleus where the L-glutathione concentration is two orders ofmagnitude higher than that in the more oxidizing endocyticcompartments and in extracellular environment. We havedemonstrated that these disulfide-crosslinked PEG12K-b-PPA128K/DNA micelles have significantly improved stability in serum andsalt solutions. Moreover, different from other reported disul-fide-crosslinked complex or nanoparticle systems,[5–7] uponreduction of disulfide crosslinks in cytosol and nucleus, themicelles became unstable and hence the release of the DNA cargocan be regulatedmore effectively, due to the lowDNA compactionability of PEG12K-b-PPA128K in physiological ionic strength. Moreimportantly, we showed that the dual-sensitive micellar nano-particles mediated enhanced and prolonged gene transfectionefficiency in vitro.

To introduce the disulfide crosslinking, the PPA block ofPEG12K-b-PPA128K polymer was modified with Traut’s reagent atdifferent thiolation degrees (see the Supporting Information,Fig. S1a).[8] Traut’s reagent was chosen because the thiolation ofPPA block does not change the positive charge density on the PPAblock.[9] The crosslinked micellar nanoparticles exhibited similarparticle size as the noncrosslinked micelles. The difference inparticle sizes between micelles in water and in 0.15 M NaClsolution was used as an indicator of complex stability of themicelles (Fig. S1b). The complex stability of the crosslinkedmicelles increased with the thiolation degree until it reached18.8%, at which point there was no difference in particles size

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Figure 1. a) Preparation of dual-sensitive micelles. Micelles are preparedin distilled water to yield compact nanoparticles, then oxided in thepresence of DMSO. The disulfide-crosslinked micelles are stable in blood,extracellular melieu, and the endolysosomal compartment where theglutathione (GSH) concentration is in the micromolar range. Thesecrosslinks can be reduced when they reach the cytosol and nucleus whereGSH concentration is in the range of 1–10mM; the reduced micellesbecome unstable due to the salt-sensitive nature of the PEG12K-b-PPA128Kcarrier, thus releasing unpacked DNA. b,c) TEM images of dual-sensitivemicelles prepared with 18.8% thiolated copolymer in deionized water(b) and after incubation with 0.15 M NaCl for 30min (c).

between the two media. The PEG12K-b-PPA128K with a thiolationdegree of 18.8% was used for all the following experiments and ishereby referred to as the crosslinked micellar nanoparticles.

Transmission electron microscopy (TEM) images showed thatthese crosslinked micellar nanoparticles were mostly sphericalwith diameters ranging from 100 to 150 nm (Fig. 1b), corrobo-rated well with the size (123.4� 8.7 nm) measured by dynamiclight scattering (DLS) method. There were less than 10% ofmicelles assuming elongated morphology with a diameter of60–80 nm and length of about 200 nm. Consistent with the sizemeasurement by DLS, we did not observe any change in size forthe crosslinked nanoparticles after incubation in 0.15 M NaClsolution under TEM (Fig. 1c), suggesting the robust complexstability in salt containing medium. In contrast, the size of thenoncrosslinked micelles increased from 90 nm to around 220 nmrapidly after addition to the 0.15 M NaCl solution.

The salt stability was also confirmed by using decomplexationassay with ethidium bromide. The fluorescence efficiency ofethidium bromide increased dramatically upon binding to doublestranded DNA by intercalation.[10] If the complexation betweenplasmid DNA and polymers is disrupted or relaxed in thepresence of salt, ethidium bromide intercalation with uncom-plexed DNA can result in an increase in fluorescence intensity;

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and the decomplexation degree of micelles can be calculatedusing Equation (1). As the concentration of salt went up graduallyfrom 0 to 0.5 M, increasing amount of DNA was decomplexedfrom the noncrosslinked micelles, as indicated by an increase indecomplexation degree to 44% (Fig. 2a). In contrast, thedecomplexation degree of the crosslinked micelles increasedslightly to 7%. In serum stability test, the noncrosslinked andcrosslinked micelles were challenged by fetal bovine serum(FBS). After incubation with 10% (v/v) FBS for 30min, theparticle size of noncrosslinked micelles increased to around250 nm (Fig. 2b), whereas the average size of the crosslinkedmicelles remained unchanged. These results collectively indicatethat the shielding effect of PEG corona and disulfide crosslinkseffectively enhanced the complex stability in salt solution andcolloidal stability in serum containing medium.

To validate the hypothesis that the crosslinked micelles canrecover its salt sensitivity and unpack plasmid DNA in thereducing environment, the crosslinked micelles were incubatedwith 15mM L-glutathione (a concentration similar to cytosoliclevel) at 37 8C.[3] At different time intervals after the addition ofL-glutathione, themicelles were challenged with 0.15MNaCl solutionand the decomplexation degree was measured. Interestingly,crosslinked micelles regained the salt sensitivity much more slowlythan we had expected. One hour after incubation with L-glutathione,the decomplexation degree of crosslinked micelles increasedslightly by only 3% (data not shown), suggesting that most ofdisulfide crosslinks were still intact. A more significant increasein salt sensitivity was observed at 24 h after incubation, when thedecomplexation degree increased to 28% (Fig. 2c). The decom-plexation degree increased gradually and reached a comparablelevel to that of noncrosslinked micelles by day 7. This observationsuggests that the cleavage of disulfide crosslinks was a relativelyslow process in a reduction microenvironment similar to thatfound in the cytosol and nucleus.[3]

The transfection efficiency of noncrosslinkedmicelles was firstscreened by transfecting HEK293 cells with micelles prepared atdifferent N/P ratios (themolar ratios of nitrogen atoms in PPAs tophosphorus atoms in DNA) from 1 to 15. Micelles prepared withan N/P ratio of 8 showed the highest transfection efficiency at twodays after transfection (see the Supporting Information, Fig. S2).Therefore, both noncrosslinked and crosslinked micelles at theN/P ratio of 8 were used to evaluate the effect of dual sensitivityon transfection efficiency. As shown in Figure 3, the PEI/DNAcomplexes mediated the highest transfection efficiency on day 1.The transgene expression decreased slightly on day 3, anddropped by 90% on day 5 and then to the background level on day7. Transfection efficiency of the noncrosslinked micelles (e.g.,salt-sensitive) was much lower than that of PEI/DNA nanopar-ticles. The transgene expression level mediated by noncross-linked micelles peaked on day 3 and decreased to the backgroundlevel on day 7. In contrast, the transfection efficiency of thecrosslinked micelles (e.g., dual sensitive) was 2- and 4-fold higherthan noncrosslinked micelles on days 1 and 3, respectively, likelydue to the protective and stabilization effect of the crosslinking.Transgene expression maintained for four days at the peak level,which was only 6-fold lower than the peak expression level by PEI/DNA nanoparticles. By day 10, 35% of the peak expression levelwas still observed in cells transfected by the dual-sensitivemicelles.

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Figure 3. Transgene expression level mediated by dual-sensitive micellesand noncrosslinkedmicelles in HEK293 cells over time, in comparison withPEI/DNA nanoparticles. Each data point represents average� standarddivision (n¼ 4).

Figure 2. Stability of noncrosslinked and dual-sensitive micelles in thepresence of NaCl solution of different concentrations (a) or 10% fetalbovine serum (b). c) Degree of de-complexation of DNA after incubatingthe dual-sensitive micelles with 15mM L-glutathione for different intervalsat 37 8C.

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The fact that the crosslinked dual-sensitive micelles enhancedand significantly prolonged transgene expression compared withnoncrosslinked micelles can be related to the different DNAunpacking mechanism from reversibly crosslinked micelles. As

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demonstrated in Figure 2c, the restoration of salt sensitivity for thecrosslinked micelles was a slow process. Therefore, the unpackingof crosslinked micelles may be significantly delayed compared withthat of noncrosslinkedmicelles. Based on this hypothesis, we used arecently developed quantum dot (QD)-FRET technique to study theintracellular DNA unpacking kinetics of both noncrosslinked andcrosslinked micelles. PEG12K-b-PPA128K or thiolated PEG12K-b-PPA128K

block polymers and plasmid DNA were labeled with Cy5 and QD605,respectively. After cell uptake of Cy5 and QD-labeled micelles, thehigh signal-to-noise ratio in QD-mediated FRETenabled sensitivedetection of discrete changes in micelle stability. From confocalimage stacks obtained at each time point, the distributions of intactand unpackedmicelles in the cytosol, endolysosomal compartment,and nucleus were quantified with a particle-specific voxel-basedmethod adapted from a previous report using a pixel-basedalgorithm.[11] In this experiment, thiolated PEG12K-b-PPA128K

polymer with a thiolation degree of 8.8% was used becausecrosslinked micelles formed with block copolymers with higherthiolation degrees induced strong fluorescence at 505 nm uponexcitation of QDs, which interfered seriously with the signal fromLysotracker Green (for the localization of endo/lysosomes). InFigure 4, histograms were generated to compare the fractions ofcohorts of cells having specific volume fractions of unpackedDNA ( fU) at 2 and 4 h post-transfection. There is a markeddifference in the overall unpacking rate and unpacked plasmidDNA fraction between noncrosslinked and crosslinked micellesfrom 2 to 4 h time points. At 2 h after transfection, the majority(�88%) of cells transfected with noncrosslinked micelles had anfU> 0.5, which indicated that more than 50% of the internalizedDNA was unpacked among these cells. In contrast, nearly 50% ofcells transfected with dual-sensitive micelles showed an fU> 0.5at 2 h time point. Among all the cells, an average of 37% ofinternalized DNA was unpacked as compared to 73% fromnoncrosslinked micelles. At 4 h, the histogram for noncross-linked micelles was significantly shifted toward higher fU values.All cells had an fU value of higher than 0.6 and, on average, 80% ofall internalized DNA was unpacked, whereas dual-sensitive

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Figure 4. Overall distribution of unpacked DNA inside transfected cells. The overall intracellu-larly unpacked DNA from noncrosslinked micelles (a) and dual-sensitive micelles (b) at 2 and4 h post-transfection. At each time point, histograms show the fraction of an analyzed cohort ofcells (30–34 cells) with a particular binned volume fraction of unpacked DNA ( fU).

micelles showed amuch delayed unpacking of plasmid DNAwithnearly the same fU histogram as that at 2 h.

In addition to the overall distribution of unpacked plasmidDNA in the cells transfected with micelles, the unpacking ofmicelles in three subcellular compartments (endo/lysosomes,cytosol, and nucleus) was also evaluated. Among cells transfectedwith noncrosslinked and crosslinked micelles, low volumefractions of unpacked DNA were localized in endolysosomalcompartments at 2 and 4 h time points (Fig. S3), indicating thatmost of the noncrosslinked and crosslinked micelles found in theendo/lysosomes remained unpacked at these time points. Inaddition, the majority of cells had an f0U (endo/lyso) value< 0.2(Fig. S4), confirming that most of the unpacked DNA was notfound in this compartment. The majority of unpacked DNA fromnoncrosslinked and crosslinked micelles was localized in thecytosolic compartment and nucleus (Fig. S4). Interestingly, whenmore noncrosslinked micelles unpacked at 4 h, most unpackinghappened in cytosolic compartment, since there was a significantupshift of fU (cyto) (Fig. S3) and f0U (cyto) (Fig. S4), and downshiftof fU (nuc) and f0U (nuc) values in the histogram of noncrosslinkedmicelles. As for the crosslinked micelles, the distribution patternof unpacked plasmid DNA in cytosolic compartment and nucleusdid not change significantly over these two hours. Moreimportantly, there was no appreciable increase of DNAunpackingfrom the dual-sensitive micelles from 2h to 4 h in the cytosol andnucleus compartments. Especially, at 4 h, an average of 83% ofintracellular dual-sensitive micelles remained unpacked, ascompared to 31% for the noncrosslinked micelles. This

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difference confirms our hypothesis that thedual-sensitive micelles yielded a significantlydelayed unpacking of DNA compared withnoncrosslinked micelles at early time points.Monitoring longer term DNAunpacking eventshas been challenging due to the fluorescencequenching of the Cy5 signal.

In this report, we have prepared a reversiblycrosslinked micellar nanoparticles that are cap-able of releasing encapsulated DNA in responseto the concentration of reduction agent (e.g.,L-glutathione) and ionic strength (e.g., at physio-logical salt concentration). Such a property wasused to prevent extracellular DNA release andregulate intracellular DNAunpacking. Comparedwith noncrosslinked micelles, the dual-sensitivemicelles showed significantly enhanced complexstability and colloidal stability in salt and serumcontaining media, and more sustained DNArelease at cytosolic salt and glutathione concen-trations. Using a more quantitative QD-FRETmethod, we demonstrated that the dual-sensitivemicelles unpacked DNA primarily in the cytosoland nucleus, and the DNA unpacking wassignificantly delayed compared with noncros-slinked micelles at 4 h after transfection. Due tothe unique DNA unpacking behavior, thedual-sensitive micelles mediated enhanced andmuch prolonged gene expression for at least10 days in HEK 293 cells.

Experimental

Preparation and Characterization of Dual-Sensitive Micelles: The synth-esis of PEG12K-b-PPA128K/DNA micelles with VR1255 plasmid DNA, andcharacterization of size and surface charge of micelles by DLS and TEMimaging were conducted according to the methods we reported previously[1]. For crosslinked micelles, plasmid DNA was incubated with thiolatedcopolymer similarly; then the micelle solution was transferred to a dialysisbag with MWCO of 3,500Da (Spectrapor, Rancho Dominguez, CA) anddialyzed against 0.5% (v/v) DMSO for 24 h to oxidize thiol groups.Complete oxidization and removal of residual DMSO was then achieved bydialyzing the micelle solution against DI water for additional 48 h.

Determination of the Decomplexation Degree of Micelles in the Presence ofSalts: One hundred mL of micelle or DNA solution containing 1 mgplasmid DNA was mixed with an equal volume of ethidium bromidesolution (1mg mL�1). Concentrated NaCl solution (5 M) was added to themixed solution to bring the salt concentration to a desired value. Afterincubation at room temperature for 10min, the fluorescence intensity (I) ofthe micelle and DNA solution in the absence of salts (Im and IDNA,respectively) and in the presence of salts (Im,s and IDNA,s, respectively),were measured using a microplate reader (SpectraMax, Molecular Devices,Sunnyvale, CA, USA) with excitation and emission wavelengths of 310 and590 nm, respectively. The decomplexation degree of the micelles wascalculated according to

Decomplexation Degree ð%Þ ¼Im;s

IDNA;s� Im

IDNA

� �

1� ImIDNA

� � � 100% (1)

In Vitro Gene Transfection: Transfection of HEK293 cells (AmericanType Culture Collection, Manassas, VA) with nanoparticles and

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characterization of transfection efficiency were conducted according to thesame method we described previously [1]. To evaluate the transfectionefficiency on days 3, 5, 7 and 10, another set of cells transfected withnanoparticles were split at 1:5 ratio on day 3 to prevent over-confluence ofcells. On day 5, the cells were split again 1:5 ratio and 80% of the cells wereharvested for the measurement of luciferase activity. On day 10, all the cellswere harvested for the measurement of luciferase activity. To compare thetotal transgene expression level on days 5, 7, and 10 with that at earlier timepoints, the total luciferase activity obtained on days 5, 7, and 10 weremultiplied by factors of 5, 25, and 125, respectively (see the SupportingInformation for details).

Live Cell Imaging and Quantifying Distribution of Complexed or UnpackedDNA: The preparation of QD605-labeled plasmid DNA and Cy5-labeledpolymers and the analysis of intracellular trafficking study was conductedaccording to amethod we reported previously [11]. Detailed procedures areprovided in Supporting Information.

Acknowledgements

The authors thank Prof. Michael McCaffery at the Integrated ImagingCenter at Johns Hopkins University for his assistance with TEM imaging.We also thank Dr. Yi-Ping Ho and Dr. Yong Ren for their technicalassistance. This study was supported by grants from the National Institutesof Health, the National Institute of General Medical Sciences GM073937(HQM), the National Institute of Biomedical Imaging and BioengineeringHL089764 (KWL), and the National Science Foundation ECCS-0725528

� 2010 WILEY-VCH Verlag Gmb

and CBET-0546012 (THW). Supporting Information is available onlinefrom Wiley InterScience or from the author.

Received: November 17, 2009

Revised: December 10, 2009

Published online: May 3, 2010

[1] X. Jiang, H. Dai, C. Y. Ke, X. Mo, M. S. Torbenson, Z. P. Li, H. Q. Mao,

J. Controlled Release 2007, 122, 297.

[2] H. H. Chen, Y. P. Ho, X. Jiang, H. Q. Mao, T. H. Wang, K. W. Leong, Nano

Today 2009, 4, 125.

[3] S. Bauhuber, C. Hozsa, M. Breunig, A. Gopferich, Adv. Mater. 2009, 21,

3286.

[4] D. Soundara Manickam, D. Oupicky, J. Drug Target. 2006, 14, 519.

[5] Y. Kakizawa, A. Harada, K. Kataoka, J. Am. Chem. Soc. 1999, 121, 11247.

[6] D. Oupicky, R. C. Carlisle, L. W. Seymour, Gene Ther. 2001, 8, 713.

[7] M. Neu, O. Germershaus, S. Mao, K. H. Voigt, M. Behe, T. Kissel,

J. Controlled Release 2007, 118, 370.

[8] H. Q. Mao, K. Roy, V. L. Troung-Le, K. A. Janes, K. Y. Lin, Y. Wang,

J. T. August, K. W. Leong, J. Controlled Release 2001, 70, 399.

[9] K. Miyata, Y. Kakizawa, N. Nishiyama, A. Harada, Y. Yamasaki, H. Koyama,

K. Kataoka, J. Am. Chem. Soc. 2004, 126, 2355.

[10] J. B. Lepecq, C. Paoletti, J. Mol. Biol. 1967, 27, 87.

[11] H. H. Chen, Y. P. Ho, X. Jiang, H. Q. Mao, T. H. Wang, K. W. Leong, Mol.

Ther. 2008, 16, 324.

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