New potential antitumoral fluorescent tetracyclic thieno[3,2-b]pyridine derivatives: interaction with DNA and nanosized liposomes
© Castanheira et al; licensee Springer. 2011
Received: 28 September 2010
Accepted: 12 May 2011
Published: 12 May 2011
Fluorescence properties of two new potential antitumoral tetracyclic thieno[3,2-b]pyridine derivatives were studied in solution and in liposomes of DPPC (dipalmitoyl phosphatidylcholine), egg lecithin (phosphatidylcholine from egg yolk; Egg-PC) and DODAB (dioctadecyldimethylammonium bromide). Compound 1, pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, exhibits reasonably high fluorescence quantum yields in all solvents studied (0.20 ≤ ΦF ≤ 0.30), while for compound 2, 3-[(p-methoxyphenyl)ethynyl]pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, the values are much lower (0.01 ≤ ΦF ≤ 0.05). The interaction of these compounds with salmon sperm DNA was studied using spectroscopic methods, allowing the determination of intrinsic binding constants, K i = (8.7 ± 0.9) × 103 M-1 for compound 1 and K i = (5.9 ± 0.6) × 103 M-1 for 2, and binding site sizes of n = 11 ± 3 and n = 7 ± 2 base pairs, respectively. Compound 2 is the most intercalative compound in salmon sperm DNA (35%), while for compound 1 only 11% of the molecules are intercalated. Studies of incorporation of both compounds in liposomes of DPPC, Egg-PC and DODAB revealed that compound 2 is mainly located in the hydrophobic region of the lipid bilayer, while compound 1 prefers a hydrated and fluid environment.
Liposomes are among technological delivery developments for chemotherapeutic drugs in the treatment of cancer. This technique can potentially overcome many common pharmacologic problems, such as those involving solubility, pharmacokinetics, in vivo stability and toxicity [1–3]. Liposomes are closed spherical vesicles consisting of a lipid bilayer that encapsulates an aqueous phase in which hydrophilic drugs can be stored, while water insoluble compounds can be incorporated in the hydrophobic region of the lipid bilayer .
Due to the antitumoral potential of the two compounds 1 and 2, related with their possible intercalation between the DNA base pairs, interactions with natural double-stranded salmon sperm DNA were studied. These interactions can be assessed using spectroscopic measurements, which are important tools for monitoring DNA-binding processes. The investigation based on DNA interactions has a key importance in order to understand the mechanisms of action of antitumor and antiviral drugs and to design new DNA-targeted drugs [7, 8]. Small molecules are stabilized on groove binding and intercalation with DNA through a series of associative interactions such as π-stacking, hydrogen bonding, attractive van der Waals and hydrophobic interactions . The occurrence of intercalation seems to be an essential (but not sufficient) step for antitumoral activity . Fluorescence quenching experiments using external quenchers are also very useful to distinguish between DNA binding modes  since intercalated molecules are less accessible to anionic quenchers due to electrostatic repulsion with negatively charged DNA .
Salmon sperm DNA from Invitrogen (Carlsbad, CA, USA) and compounds stock solutions were prepared in 10 mM Tris-HCl buffer (pH = 7.4), with 1 mM EDTA. The DNA concentration in number of bases was determined from the molar absorption coefficient, ε = 6600 M-1 cm-1 at 260 nm . Fluorescence spectra of several solutions with different [DNA]/[compound] ratios and constant compound concentration (5 × 10-6 M) were recorded. The solutions were left several hours to stabilize.
Dipalmitoyl phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (Egg-PC), from Sigma-Aldrich (St. Louis, Missouri, USA), and dioctadecyldimethylammonium bromide (DODAB), from Tokyo Kasei (Tokyo, Japan), were used as received. Liposomes were prepared by the ethanolic injection method, previously used for the preparation of Egg-PC and DPPC liposomes [12–15] and DODAB vesicles [16, 17]. An ethanolic solution of a lipid/compound mixture was injected in an aqueous buffer solution under vigorous stirring, above the melting transition temperature of the lipid (approx. 41°C for DPPC  and 45°C for DODAB ). The final lipid concentration was 1 mM, with a compound/lipid molar ratio of 1:500. One millilitre solutions of liposome dispersions were placed in 3 mL disposable polystyrene cuvettes for dynamic light scattering (DLS) measurements in a Malvern ZetaSizer Nano ZS particle analyzer (Worcestershire, UK). Five independent measurements were performed for each sample. Malvern Dispersion Technology Software (DTS) (Worcestershire, UK) was used with multiple narrow mode (high resolution) data processing, and mean size (nm) and error values were considered.
Absorption spectra were recorded in a Shimadzu UV-3101PC UV-Vis-NIR spectrophotometer (Kyoto, Japan) and fluorescence measurements were obtained in a Fluorolog 3 spectrofluorimeter (HORIBA Scientific, Kyoto, Japan) equipped with Glan-Thompson polarizers. Fluorescence spectra were corrected for the instrumental response of the system. The fluorescence quantum yields were determined by the standard method [20, 21], using 9,10-diphenylanthracene in ethanol as reference, Φr = 0.95 . The solutions were previously bubbled for 20 min with ultrapure nitrogen.
Results and discussion
Maximum absorption (λabs) and emission (λem) wavelengths, molar absorption coefficients (ε) and fluorescence quantum yields of compounds 1 and 2 in several solvents
λabs (nm) (ε/104 M-1 cm-1)
398 (0.84); 377 (1.24); 360 (1.27); 305 (0.95); 258 (3.93)
411 sh (0.33); 354 (2.19); 347 (2.37); 308 (1.25); 291 (1.12); 270 (1.40)
402; 426; 452 sh
398 (0.76); 377 (1.18); 359 (1.20); 305 (1.17); 258 (3.60)
411 sh (0.66); 356 (5.36); 346 (5.40); 309 (3.23); 291 (2.98); 272 (3.33)
407; 428; 455 sh
397 (0.58); 377 (0.91); 360 (0.93); 305 (0.97); 259 (2.70)
410 sh (0.55); 357 (4.37); 311 (2.28); 290 (2.29); 273 (2.78)
395 (0.68); 376 (1.06); 358 (1.06); 304 (1.09); 256 (3.32)
409 sh (0.66); 355 (5.76); 308 (3.41); 289 (3.20); 271 (3.67)
397 (0.78); 377 (1.19); 360 (1.16); 305 (1.19)
411 sh (0.69); 356 (5.52); 311 (3.11); 290 (2.86)
397 (0.77); 378 (1.17); 361 (1.14); 305 (1.17)
412 sh (0.61); 357 (4.70); 313 (2.52)
396 (0.69); 375 (1.13); 358 (1.17); 304 (1.40); 256 (3.59)
408 sh (0.72); 355 (5.50); 311 (2.95); 272 (3.69)
395 (0.67); 374 (1.08); 358 (1.10); 304 (1.34); 256 (3.43)
408 sh (0.62); 354 (5.00); 311 (2.80); 272 (3.41)
394 (0.41); 374 (0.57); 361 (0.58); 303 (0.93); 256 (2.07)
420 sh (0.26); 358 (0.87); 314 (0.94); 278 (0.97)
413 sh; 433
Values of the intrinsic binding constants (K i) and binding site sizes (n) and fraction of compound molecules accessible to external quenchers (f a ) for interaction with salmon sperm DNA
(8.7 ± 0.9) × 103
11 ± 3
(5.9 ± 0.6) × 103
7 ± 2
being I F,0 the fluorescence intensity of the free compound and I F,b the fluorescence intensity of the bound compound at total binding. The binding constants (Table 2) are moderately low, with a large number of base pairs between consecutive intercalated compound molecules (n).
where I 0 is the fluorescence intensity in the absence of quencher, ΔI = I 0 - I, K SV the Stern-Volmer constant, [Q] the quencher concentration and f a the fraction of molecules accessible to quencher.
The representations of the modified Stern-Volmer plot are reasonably linear (Figure 7B) and the f a values are in Table 2. Both compounds exhibit some intercalation in DNA, compound 2 being the more intercalative one, with a lower fraction (65%) of molecules accessible to anionic quencher. The higher hydrophobic character of compound 2, promoted by the functionalization of the pyridine with a triple bond linked to a p-methoxyphenyl group, may justify this behaviour. As both compounds 1 and 2 are neutral molecules (and electrostatic interaction with the negatively charged DNA molecule is not expected), the high f a values indicate that the main type of interaction with the nucleic acid must be the binding to DNA grooves .
Steady-state fluorescence anisotropy (r) values and maximum emission wavelengths (λem) of compounds 1 and 2 incorporated in liposomes
λ em /nm
λ em /nm
Fluorescence anisotropy (r) measurements (Table 3) can give relevant information about the location of the compounds in liposomes, as r increases with the rotational correlation time of the fluorescent molecule (and, thus, with the viscosity of the fluorophore environment) . Anisotropy values in a viscous solvent (glycerol) were also determined, for comparison. Anisotropy results (Table 3) allow to conclude that compound 2 is mainly located in the inner region of the lipid bilayer, feeling the penetration of some water molecules. The transition from the rigid gel phase to the liquid-crystalline phase is clearly detected by a significant decrease in anisotropy at 55°C observed in DPPC and DODAB liposomes. Compound 1 exhibits a different behaviour and anisotropy is very low in all types of liposomes (and much lower than in glycerol, Table 3). Overall, the results indicate that compound 1 prefers a hydrated and fluid environment and the transition from the gel phase to the liquid-crystalline phase is not detected. To further clarify the location of compound 1, the solutions of liposomes with incorporated compound were passed through filters of 0.05 μm diameter. The fluorescence emission of the filtered solutions was negligible, indicating that compound 1 is mainly in the liposome aqueous interior or located at the interfaces, with a very hydrated environment. This behaviour is similar to the observed previously for a benzothienopyridopyrimidone in lipid vesicles . The encapsulation assays performed here may be important for future drug delivery applications of these potential antitumoral compounds using liposomes as drug carriers.
The interaction with DNA of two new potential antitumoral fluorescent planar thieno[3,2-b]pyridine derivatives was studied using spectroscopic methods. Compound 2 was shown to be the most intercalative compound in salmon sperm DNA (35%). The binding to DNA grooves seems to be the main type of interaction with the nucleic acid. Studies of incorporation of both compounds in liposomes of DPPC, Egg-PC and DODAB revealed that compound 2 is mainly located in the hydrophobic region of the lipid bilayer, while compound 1 prefers a hydrated and fluid environment. Our data thus suggest that both potential antitumoral compounds may be transported in liposomes for drug delivery applications.
dynamic light scattering
Dispersion Technology Software
egg yolk phosphatidylcholine
giant unilamellar vesicles
small unilamellar vesicles.
This work was funded by FCT-Portugal through CFUM, CQ-UM, Project PTDC/QUI/81238/2006 (cofinanced by program FEDER/COMPETE, ref. FCOMP-01-0124-FEDER-007467) and PhD grants of M.S.D. Carvalho (SFRH/BD/47052/2008) and R.C. Calhelha (SFRH/BD/29274/2006).
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