STABILITY OF POLYCATION-DNA COMPLEXES: COMPARISON OF ... · STABILITY OF POLYCATION-DNA COMPLEXES: COMPARISON OF COMPUTER MODEL AND EXPERIMENTAL DATA J. Dybal1, K. Huml1, M. Kabeláè2,

STABILITY OF POLYCATION-DNA COMPLEXES: COMPARISON OF COMPUTER MODEL AND EXPERIMENTAL DATA

J. Dybal1, K. Huml1, M. Kabeláè2, T. Reschel1, K. Ulbrich1

1Institute of Macromolecular Chem is try, Acad emy of Sci ences of the Czech Re pub lic, HeyrovskýSq. 2, 162 06 Prague 6, Czech Re pub lic

2J. Heyrovský In sti tute of Phys i cal Chem is try, Acad emy of Sci ences of the Czech Re pub lic, andCen ter for Com plex Mo lec u lar Sys tems and Biomolecules, Dolejškova 3, 182 23 Prague 8, Czech

Republic

Keywords:polycation-DNA com plexes, gene de liv ery, quan tum me -chan i cal cal cu la tions, mo lec u lar mod el ing

Ab stractPolycations con tain ing pri mary and ter tiary amino andqua ter nary am mo nium groups formed sta ble com plexeswith DNA only in pure wa ter. In creas ing con cen tra tion ofso dium chlo ride in the sa line so lu tion re sulted in dis so ci a -tion, de com po si tion of the polyplexes and re lease of in cor -po rated DNA. Sta bil ity of the com plexes de creasedde pend ing on polycation substituent as fol lows: pri maryamine > sec ond ary amine > ter tiary amine > qua ter naryam mo nium group. Re sults of com puter mod el ing of the in -ter ac tion of the oligocations with a model DNA chain werein good agree ment with the ex per i men tal data.

In tro duc tion

Polycation-DNA com plexes (polyplexes) have been de -signed as po ten tial vec tors for in vivo gene de liv ery. Meth -ods and con di tions used for prep a ra tion of polyplexes,chem i cal struc ture of the polycations and their mo lec u larweight are the most im por tant fac tors in flu enc ing sta bil ityof the com plexes in aque ous so lu tions, DNA-plasmid re -lease and ef fi ciency of gene transfection. One of the tasksof gene ther apy is suc cess ful de liv ery of genes to the tar getcell and pro duc tion of a ther a peu tic pro tein by the cell.How ever, a ma jor ob sta cle in ex ploit ing the full po ten tialof gene ther apy is the lack of a safe and ef fi cient gene de liv -ery sys tem. Non-vi ral gene de liv ery sys tems based on com -plexes of cationic poly mers with DNA or spe cific plasmids of fer a prom is ing ap proach to the so lu tion of safe and ef -fec tive gene de liv ery. For sys temic de liv ery, the vec tor and its com po nents should be non-toxic, non-immunogenic,sta ble in the blood cir cu la tion and small enough for ef fec -tive extravasation. Ef fi cient gene de liv ery sys tem shoulden ter the cell via re cep tor-me di ated endocytosis, avoiddeg ra da tion of DNA by lysosomal en zymes, en sure cy to -plas mic mo bil ity, nu clear traf fick ing and fi nallyunpackaging in the cell nu cleus [1]. In our pre vi ous pa perswe de scribed prep a ra tion and study of prop er ties of a va ri -ety of polycation-DNA com plexes deal ing with par tic u larprob lems of their use for gene de liv ery, e.g. self-as sem blyand com plex for ma tion [2-4], sta bil ity of the com plexes [5, 6] or their tar get ing [7, 8].

The aim of this work is an at tempt to con trib ute to theelu ci da tion of the in flu ence of the polycation struc ture oninterpolyelectrolyte in ter ac tions in the com plexes ofplasmid DNA with var i ous polycations, pro posed as

nonviral vec tors for the de liv ery of genes into the nu cleusof the tar get cells and ver ify pos si bil i ties of com puter mod -el ing in de sign and prog nos tics of properties of thecomplexes.

Com puter mod el ing of the self-as sem bly of a plasmidDNA dou ble he lix with a syn thetic polycations is the firststep of our at tempt to de scribe the sys tem based on thepolyelectrolyte com plexes be tween nat u ral polyanions(DNA) and syn thetic polycations. Gene de liv ery vec tors ofthis type are as sumed to pro tect DNA dur ing the trans portin body flu ids and af ter extravasation in the cy to plasm ofthe tar get cell, and to re lease it in the nu cleus to evoke thepro duc tion of a ther a peu tic pro tein [9]. The start ing modelwas based on re sults of our pre vi ous physico-chem i calstudy of sta bil ity and transfection ac tiv ity of DNA com -plexes [10]. In this study, the polycations used for self-as -sem bly of polyplexes in cluded cationic poly mers andco pol y mers con tain ing pri mary and ter tiary amino andqua ter nary am mo nium groups. It was found that the size ofthe com plexes was small enough to en sure ef fec tiveextravasation, it in creased with in creas ing pH of the bufferand in creas ing tem per a ture in the com plex for ma tion [10].By de creas ing the mo lec u lar weight of the re spec tivepolycation the com plexes be came smaller but, at the sametime, less sta ble to com plex dis so ci a tion and co ag u la tion in aque ous so lu tions. DNA com plexes with polycations con -tain ing pri mary amino groups showed the best sta bil ity insa line so lu tions with the sta bil ity of poly(lysine) com -plexes be ing su pe rior to that pre pared with metha cry -loylated aminoamides. Lower sta bil ity was found forcom plexes formed with ter tiary amines and the least re sis -tant to de struc tion and DNA re lease were com plexes pre -pared with poly mers con tain ing qua ter nary am mo niumsalts. In transfection ex per i ments, the high est transfectionac tiv ity was found us ing com plexes pre pared from primary amino group-containing polycations and complexes withquaternary ammonium groups provided the lowesttransfection activity.

Computional ap proach

Bind ing of the CH2-R group to the phos phate. To se lecta polycation of suf fi cient en ergy of bind ing to the DNAphos phate groups, three types of pos si ble mono mers con -tain ing the CH2-R group were tested (cf. Ta ble 1), where Ris pri mary, sec ond ary and ter tiary amino and qua ter naryam mo nium groups. Dimethyl phos phate in its most sta blegauche, gauche con for ma tion [11, 12], was used as amodel rep re sent ing neg a tively charged DNA phos phategroups. To es ti mate the bind ing en ergy, quan tum me chan i -

Ó Krystalografická spoleènost

Materials Structure, vol. 11, number 1 (2004) 3

cal (Gaussi an 98) [13] and mo lec u lar me chan i cal (Dis -cover 2000) [14] cal cu la tions were per formed. Quan tumme chan i cal cal cu la tions were car ried us ing the 6-31G* ba -sis set which in cludes po lar iza tion func tions on all at omsex cept hy dro gens. Ge om e tries have been fully op ti mizedfor the small est sys tems at the sec ond-or der Moller Plesset(MP2) level of the ory and for the re main ing sys tems at theden sity func tional the ory (DFT) level us ing the B3LYPfunc tional [15]. In ac cord with the ex per i men tal data [10],the lysine with pri mary amino group (Fig ure 1) showed thehigh est bind ing en ergy and with qua ter nary am mo niumgroup the low est bind ing en ergy. Sim i lar re sults were ob -tained from the mo lec u lar me chan ics (525 and 361 kJ/molwith pri mary amino and qua ter nary am mo nium group, re -spec tively). Cal cu la tions were also per formed us ing suiteof Biosym/MSI [14] pro grams, par tic u larly the InsightIIand the Dis cover pro grams. The cff91 force field with the

di elec tric con stant e =1 was used in all mo lec u lar me chan -ics cal cu la tions.

L-lysine oligocation. Pos i tively charged oligo(L-lysine) was cho sen as a proper can di date [16] to form a polycationin a com plex with B-DNA un der phys i o log i cal con di tions(pKa 10.80) [17]. To show an op ti mal ge om e try of the pos i -tively charged oligo(L-lysine) frag ment, mo lec u lar mod el -ing of a free dodecamer of the frag ment men tioned wasper formed. In po ten tial en ergy minimization, in con trast tothe un charged oligo(L-lysine), the pos i tively chargedoligo(L-lysine) con verged to a he lix of sim i lar con for ma -

tion in de pend ently of the start ing model (b-strand, a-he lix,

p-he lix, 310-he lix). The rise per amino acid res i due was al -ways higher than 300 pm and the he li cal twist more than

100° in de pend ence on the start ing model. The he lix con -for ma tion shows the po ten tial en ergy per one res i due lower

by 544 kJ/mol than the b-strand con for ma tion.

B-DNA frag ment. The eleven-base-pair B-DNA du plexof 5'-(ATATATATATA)-3' was ar bi trarily cho sen fromthe stan dard struc tures of InsightII pro gram [14] to sim u -late part of the plasmid. It can be shown that po si tions ofthe B-DNA-OPO3- groups are prop erly ori ented to meetthe pos i tively charged oligo(L-lysine) -NH3

+ groups of thein ter act ing oligocation. The de gree of the charge com pen -sa tion N/P close to unity sup ports the model where the flex -i ble pos i tively charged oligo(L-lysine) b-strand em bedsinto the ma jor groove of the B-DNA.

Dock ing of oligo(L-lysine) in the B-DNA. A sys tem ofeleven-base-pair B-DNA rigid du plex frag ment and pos i -tively charged flex i ble hexamer of L-lysine was in tro duced into the fi nal at tempt of dock ing pro ce dure sim u la tion. Theaim of this at tempt was to draw a qual i ta tive model only.Suite of Biosym/MSI [14] pro grams was uti lized un der the same con di tions as men tioned pre vi ously. A sim pli fied ap -proach was cho sen in all cal cu la tions. Both mol e cules were as sumed to be in vacuo in the ab sence of other mol e cules(wa ter, ions, etc.). Ge neric dis tances be tween six -NH3

+

and -OPO3- groups were in tro duced with an up per bound

of 500 pm. Be low the 500 pm limit, the ad di tional at trac -tion was set to zero. The steep est de scent (22,000 it er a -tions) and con ju gate gra di ents (10,000 it er a tions) ofpo ten tial en ergy minimization were au to mat i cally stoppedwhen the gra di ent of po ten tial en ergy was lower than 0.42kJ/mol.nm.

In our model we as sume that wa ter mol e cules, smallcat ions and an ions are pushed away of the B-DNA sur faceand, there fore, they do not play a sig nif i cant role in shield -ing the phos phate groups. Our pro posed model (Fig. 2)shows the pos i tively charged oligo(L-lysine) flat strandem bed ded in the ma jor groove of the B-DNA mol e cule.The -NH3

+ groups are point ing to the -OPO3- groups with a

rather long av er age dis tance be tween ox y gen and ni tro genat oms of 381(5) pm. For a re li able quan ti ta tive model, acom plex cal cu la tion, in clud ing flex i ble and lon ger B-DNA du plex, would be nec es sary.

Ó Krystalografická spoleènost

4 STABILITY OF POLYCATION-DNA COMPLEXES

203202

196156 241

242

178174 162 174

NNN

N

N

P

O

PP

P

OO

O

O

N

O

OOO

O

C

OO

O OOO

CC

C

CCC

CC

C

C

C

CC

CC

C C

C

C CC

Figure1. MP2/6-31G* op ti mized model struc tures of com plexesof CH3CH2-R with the phos phate group. Dis tances are in pm.

Fig ure 2. B-DNA - pos i tively charged oligo(L-lysine) in ter ac -tion. Hexamer of the pos i tively charged oligo(L-lysine), balls of0.5 vdW ra dius are em bed ded in the ma jor groove of B-DNA(spa ghetti model).

Dis cus sion

Three types of mono mers men tioned in Ta ble 1 show thesame trend in bind ing en er gies. This sup ports the idea thatthe ligand struc ture (con tain ing a dou ble bond, sin gle bond, short frag ment only) has no de ci sive in flu ence on the bind -ing pro cess. All three struc tures mod el ing var i ous cationicmono mer units in cor po rated in the polycation struc turedem on strate the high est en ergy of in ter ac tion with phos -phate group of the DNA chain for methacryloyl-basedunits con tain ing pri mary amino group. The bind ing en ergyis de creas ing in the or der: pri mary amine > sec ond aryamine > ter tiary amine > qua ter nary am mo nium group.This find ing is in a very good agree ment with our ex per i -men tal data [10] de scrib ing the sta bil ity of poly -cation/DNA com plexes in sa line so lu tions. Here, the NaClcon cen tra tions at which 50 % of DNA is re leased from thecom plex (cs

50) was used as the dis so ci a tion sta bil ity char ac -ter is tics. In agree ment with the cal cu lated bind ing en ergy,the ex per i men tal data show the high est sta bil ity for DNAcom plexes formed by poly(L-lysine) and poly(AEMA)(pri mary amines, cs

50 ~ 1.3 mol/l), lower for com plexescon tain ing poly(DMAEMA) (sec ond ary amine, cs

50 ~ 0.7mol/l) and the low est for poly(TMAEM) (qua ter nary am -mo nium group, cs

50 ~ 0.5 mol/l) [10]. The alphameric sym -bols of the com pounds were cho sen in ac cord with thedef i ni tion given in pre vi ous pa per [10]. For their chem i calstruc ture and ex pla na tion of the sym bols, see Ta ble 1. Theagree ment be tween the o ret i cal and ex per i men tal data ver i -fies the use of ap prox i ma tions in model cal cu la tions and, atthe same time, it dem on strates the po ten tial of a com putermod el ing in de sign ing polyplexes as vec tors for ef fi cientgene de liv ery.

Con clu sions

De spite the rather rough ap prox i ma tion used in this mod el -ing, a rea son ably sim ple model of B-DNA - pos i tivelycharged poly(L-lysine) com plex was de signed, which ap -pears to be in agree ment with pre vi ous physicochemicalex per i men tal re sults. Also the bind ing en ergy cal cu latedfor in ter ac tion of var i ous oligocations with the phos phategroups in model DNA chain is in good agree ment with ex -per i men tal data ob tained ear lier, dem on strat ing a drop inthe polycation-DNA com plex sta bil ity in de pend ence onthe polycation struc ture in the fol low ing or der: pri maryamines > sec ond ary amines > ter tiary amines > qua ter naryam mo nium group. The agree ment of cal cu lated and ex per i -men tal data ver i fied the fea si bil ity of com puter mod el ingand cal cu la tion in de sign ing polycation-based gene de liv -ery sys tems.

Ac knowl edge mentThis work was sup ported by the Grant of the Eu ro pean Un -ion Pro gram (QLK-2000-00280), and by the Acad emy ofSci ences of the Czech Re pub lic (pro jects No. AVOZ4050913 and KSK 4055109).

Re fe ren ces

1. S.J. Hwang & M.E. Da vis, Curr. Opin. Mol. Ther., 3(2001) 183-191.

2. M.A. Wol fert, E. H. Schacht, V. Ton che va, K. Ulb rich,O.Na za ro va & L.W. Sey mour, Hu man Gene Ther., 7(1966) 2123-2133.

3. È. Koòák, L. Mrk viè ko vá, O. Na za ro va, K. Ulb rich & L. W. Sey mour, Supra mol. Sci., 5 (1998) 67-74.

4. D. Ou pic ký, È. Koòák & K. Ulb rich, J. Bi o med. Sci. Po -lym. Ed., 10 (1999) 573-590.

5. D. Ou pic ký, K. A. Ho ward, È. Koòák, P.R. Dash, K. Ulb rich & L.W. Sey mour, Bi o con ju ga te Chem., 11(2000) 492-501.

6. V. Ton che va, M. A. Wol fert, P. R. Dash, D. Ou pic ký, K. Ulb rich, L.W. Sey mour & E. Schacht, Bi o chim. Bi o -phys. Acta, 1380 (1998) 354-368.

7. P. R. Dash, M. Read, K. Fisher, K. Ho ward, M. Wol fert, D. Ou pic ký, V. Šubr, J. Stro halm, K. Ulb rich & L.W. Sey -mour, J. Biol. Chem., 275 (2000) 3793-3802.

8. C.M. Ward, M. Pe char, D. Ou pic ký, K. Ulb rich & L.W. Sey mour, J. Gene Med., 4 (2002) 536-547.

9. C.W. Pou ton & L.W Sey mour, Adv. Drug De li ve ry Rev.,46 (2001) 187-203.

10. T. Reschel, È. Koòák, D. Ou pic ký, L.W. Sey mour & K. Ulb rich, J. Con trol led Re le a se, 81 (2002) 201-217.

11. B. Schne i der, M. Ka be láè, P. Hob za, J. Am. Chem. Soc.118 (1996) 12207-12217.

12. M. D. New ton, J. Am. Chem. Soc. 95 (1973) 256-258.

13. M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria,M.A. Robb, J.R. Cheeseman, V.G. Zakrzewski, J.A. Mont -gom ery, Jr., R.E. Stratmann, J.C. Burant, S. Dapprich, J.M. Millam, A.D. Daniels, K.N. Kudin, M.C. Strain, O. Farkas, J. Tomasi, V. Barone, M. Cossi, R. Cammi, B. Mennucci,C. Pomelli, C. Adamo, S. Clif ford, J. Ochterski, G.A.Petersson, P.Y. Ayala, Q. Cui, K. Morokuma, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.Cioslowski, J.V. Ortiz, A.G. Baboul, B.B. Stefanov, G.Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. Gomperts,R.L. Mar tin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y.Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B.John son, W. Chen, M.W. Wong, J.L. Andres, C. Gon za lez, M. Head-Gordon, E.S. Replogle & J.A. Pople, Gaussi an98, Re vi sion A.9, Gaussi an, Inc., Pitts burgh PA, 1998

14. Bi osym/MSI Man ual (2000). San Diego, CA 92121-3752,USA.

15. A.D. Bec ke, J. Chem. Phys., 98 (1993) 1372-1377.

16. J.F. Ken nedy & J.M.S. Cabral, Bio tech nol ogy, H.J. Rehm,G.J.F. Reed, Eds., VCH, Weinheim 1987.

17. J.D. Rawn, Ed., Bio chem is try, Neil Patterson Pub lish ers,N. Carolina 1989.

Ó Krystalografická spoleènost

J. Dybal, K. Huml, M. Kabeláè, T. Reschel, K. Ulbrich 5

Ó Krystalografická spoleènost

6 Materials Structure, vol. 11, number 1 (2004)

R

Model compound

NH3

+

primary amino group

N+

H

H

CH3

secondary

amino group

N+

CH3

CH3

H

tertiary

amino group

N+

CH3

CH3

CH3

quaternary ammonium

group

CH2 C

CH3

CO NH CH2 CH2 R

B3LYP/6-31G*

AEMA 464

MAEMA

455

DMAEMA

433

TMAEMA

390

CH3

C

CH3

CO NH CH2

CH2

R

CH3

B3LYP/6-31G*

AEPA

453

MAEPA

448

DMAEPA

423

TMAEPA

390

CH3

CH2

R

MP2/6-31G*

AE

531

MAE

515

DMAE 501

TMAE

409

Ta ble 1. Bin ding ener gy (kJ/mol). The al pha me ric sym bols of the com pounds were cho sen in ac cord with the de fi ni ti on gi ven in[10]: AEMA N-(2-ami no ethyl)me ta cry la mide, AEPA N-(2-ami no ethyl)pi va la mide, AE ami no etha ne, M me thyl, DM di me thyl, TM tri me thyl.