Interaction of liposome encapsulated cisplatin with biomolecules

Bharat Baruah*, Alexandr Surin

Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, GA 30144-

5591

Table S1. Dynamic light scattering data representing the hydrodynamic diameters (d, nm) of

liposomes at 37ºC in PBS (isoosmotic) solution at pH 7.6.

Sample Solutions Average d (nm) Average PDI Empty liposome 92.0 ± 3.0 0.063 Cisplatin in liposome 105 ± 5.0 0.058 5'-GMP in liposome 130 ± 5.0 0.101 9-EtG with liposome 93.0 ± 5.0 0.067 Cisplatin in liposome + 5'-GMP in liposome 107 ± 6.0 0.102 Cisplatin in liposome + 9-EtG with liposome 91.0 ± 4.0 0.055

Table S2. Zeta Potential measurements on liposomes in 10 mM PBS at 37ºC.

Sample Solutions Zeta potential (mV) Empty liposome 6.1 ± 2.0 Cisplatin in liposome 17.2 ± 1.0 5'-GMP in liposome 6.8 ± 2.0 9-EtG with liposome 7.8 ± 3.0

Scheme S1. Hydrolysis of cisplatin in water at 37ºC to form positively charged aquated species.

Scheme S2. Adduct formation due to the reaction of mono aqua species of cisplatin with 5’-

GMP and 9-EtG.

Fig. S1. Hydrodynamic diameter (d) were systematically plotted against empty liposomes

(DOPC), cisplatin-containing liposome (DOPC + A), 5’-GMP containing-liposome (DOPC + B),

9-EtG-containing liposome (DOPC + C), 1:1 mixture of cisplatin-containing liposome (DOPC +

A) and 5’-GMP-containing liposome (DOPC + B) after 48 hours of mixing and 1:1 mixture of

cisplatin-containing liposome (DOPC + A) and 9-EtG-containing liposome (DOPC + C) after 48

hours of mixing. All measurements were performed at 37ºC with isoosmotic PBS (filled squares

and continuous line) and hypoosmotic PBS (open squares and dashed line). In these samples 50

µM cisplatin, 200 µM 5’-GMP, 200 µM 9-EtG and 200 µM DOPC were used.

Figure S2. 1H NMR spectra of the reaction between 5 mM 5’-GMP and 1.5 mM cispaltin in

D2O at 37ºC in 10 mM PBS at pH 7.6 from 10 min to 48 hours. H8’ and H1’’ are the signals

from the cisplatin-5’-GMP adduct. Progress of the reaction of 5’-GMP (5.0 mM) with

[PtII(NH3)2(Cl)(H2O)]+ (1.5 mM) followed by 1H NMR spectroscopy.

Fig. S3. 1H NMR spectra of the reaction between 5 mM 5’-GMP in DOPC liposomes and 1.5

mM cisplatin in DOPC liposomes in D2O at 37ºC in 10 mM PBS at pH 7.6 from 10 min to 10

days. H8’ and H1’’ are the signals from the cisplatin-5’-GMP adduct.

Fig. S4. 1H NMR spectra of the reaction between 5 mM 5’-GMP in D2O and 1.5 mM cisplatin in

DOPC liposomes in D2O at 37ºC in 10 mM PBS at pH 7.6 from 10 min to 10 days. H8’ and H1’’

are the signals from the cisplatin-5’-GMP adduct.

Fig. S5. 1H NMR spectra of the reaction between 5 mM 9-EtG and 1.5 mM cisplatin in D2O at

37ºC in 10 mM PBS at pH 7.6 from 10 min to 10 days. CH’ is the signal from the cisplatin-9-

EtG adduct. The signals marked with asterisks (*) are intermediate species formed.

Fig. S6. 1H NMR spectra of the reaction between 5 mM 9-EtG in DOPC liposomes and 1.5 mM

cisplatin in DOPC liposomes in D2O at 37ºC in 10 mM PBS at pH 7.6 from 10 min to 10 days.

CH’ is the signal from the cisplatin-9-EtG adduct. The signals marked with asterisks (*) are

intermediate species formed.

Fig. S7. Particle size distribution of empty liposomes (_____, continuous line), cisplatin-

incorporated liposomes ( , dashed line), 5’-GMP-incorporated liposomes (……., dotted line)

and after 48 hours of reaction between cisplatin-incorporated liposomes and 5’-GMP-

incorporated liposomes (, dashed dot dot dashed line) in 10 mM PBS. Empty liposomes are

nearly monodispersed with a mean hydrodynamic diameter of 101 ± 4.0 nm; incorporation of

cisplatin and 5’-GMP causes the formation of stable particle size with a mean hydrodynamic

diameter of 104 ± 5.0 and 126 ± 7.0 nm, respectively. After 48 hours of reaction between

cisplatin-incorporated liposomes and 5’-GMP-incorporated liposomes yields a stable particle

size with a mean hydrodynamic diameter of 113 ± 5.0 nm.

Fig. S8. Particle size distribution of empty liposomes (_____, continuous line), cisplatin-

incorporated liposomes ( , dashed line), liposomes in the presence of 9-EtG (……., dotted

line) and after 48 hours of reaction between cisplatin-incorporated liposomes and liposomes in

presence of 9-EtG (, dashed dot dot dashed line) in PBS. Empty liposomes are nearly

monodispersed with mean hydrodynamic diameter of 101 ± 4.0 nm; Liposomes prepared with

cisplatin and 9-EtG result in the formation of stable particle size with a mean hydrodynamic

diameter of 104 ± 5.0 and 97 ± 4.0 nm, respectively. After 48 hours of reaction between

cisplatin-incorporated liposomes and liposomes in presence of 9-EtG gives a stable particle size

with a mean hydrodynamic diameter of 100 ± 5.0 nm.

Fig. S9. TEM images of (A) empty DOPC liposome, (B) cisplatin-encapsulated DOPC

liposomes, (c) 5’-GMP-incorporated DOPC liposomes and (d) 9-EtG containing DOPC

liposomes. Images were taken with a Zeiss EM-10 TEM at an acceleration voltage of 60 kV.

Samples were prepared by spreading a drop of sample on an ultrathin 300 mesh Formvar/carbon-

coated copper grid, dried in air, and then negatively stained with PTA for contrast.

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Fig. S10. Zeta potential of (a) empty DOPC liposome, (b) cisplatin-incorporated DOPC

liposomes, (c) 5’-GMP-incorporated DOPC liposomes and (d) 9-EtG containing DOPC

liposomes. In these samples the concentrations of DOPC, cisplatin, 5’-GMP and 9-ETG were 8

M, 2 M, 8 M and 8 M, respectively in 10 mM PBS.