Supplemental Information:
Formulation, Characterization, and Antitumor Properties of Transand Cis-Citral in the 4T1 Breast Cancer Xenograft Mouse Model
San Zeng,1 Arvinder Kapur,2 Manish S. Patankar,2 May P. Xiong1, *
1
School of Pharmacy, University of Wisconsin–Madison, Madison, Wisconsin 53705-2222, USA
2
Department of Obstetrics and Gynecology, University of Wisconsin–Madison, Madison,
Wisconsin 53705-2222, USA
*Corresponding author, telephone: +1 608 890 0699, email: [email protected]
Additional data:

1
H-NMR of geranial, neral, and commercial citral (Fig. 1S)
 Table of size and PDI of drug-loaded micelles (Table 1S)
 Release kinetics of drugs from micelles (Fig. 2S)
 Relative LC3B and Atg5 signal intensity on 4T1 cells (in vitro western blot) (Fig. 3S)
 Flow cytometry assay for apoptosis in 4T1 cells treated with geranial/NP (Fig. 4S)
 pH degradation studies for neral, geranial, and citral at pH 7.4 (Fig. 5S)
 Complete image of tumors isolated from all animals (Fig. 6S)
1
H-NMR of geranial, neral, and commercial citral
A
B
C
Fig. 1S 1H-NMR of synthesized geranial (A), synthesized neral (B), and commercially purchased
citral (C) in CDCl3 at 25 oC. A major difference in the geranial spectrum compared to neral
spectrum is the presence of the aldehyde proton (peak 8, ca. 10.0 ppm) being more low-field shifted
compared to neral’s (peak 8’, ca. 9.9 ppm). Peaks 4 and 5 also overlap, ca. 2.25 ppm, in geranial
whereas peak 4 (2.25 ppm) and peak 5 (2.6 ppm) are better separated in neral. The NMR of
commercially purchased citral (C) reveals a ca. 2:1 ratio geranial to neral (see peaks 8 and 8’ at
around 9.9 and 10.0 ppm) in this particular batch.
Size and PDI of drug-loaded micelles by DLS
Table IS. Diameter and PDI of PEG-b-PCL micelle formulations of geranial, neral, and citral
at 5-40% drug to polymer loading (w/w)
Release kinetics of drugs from micelles
Fig. 2S The release kinetics of citral, geranial or neral from PEG-b-PCL micelles was conducted
by incubating the formulations in dialysis cassettes at 37 ºC and dialyzing against ddH2O for 48 h.
Drug was formulated into all micelles at the 5% (w/w) drug to polymer ratio to ensure all drug
molecules would be preferentially encapsulated into the hydrophobic PCL core. When citral was
formulated into the micelles (citral/NP), we obtained a t1/2 of ca. 8.5 h to 50% drug release. Geranial
formulated into PEG-b-PCL micelles (geranial/NP) revealed a t1/2 of ca. 8.3 h and neral formulated
into the micelles (neral/NP) yielded a t1/2 of ca. 8.9 h.
Relative LC3B and Atg5 signal intensity on 4T1 cells (in vitro)
A
B
Fig. 3S In terms of signal strength, geranial/NP expressed more LC3B (A) and Atg5 (B) than
citral/NP*, and neral/NP**. A * means p<0.01 and ** means p<0.001
Flow cytometry assay for apoptosis in 4T1 cells treated with geranial/NP
Fig. 4S Apoptosis assay by flow cytometry for geranial (25 M) on 4T1 cells. There were no
significant differences between the control groups and the treatment groups after 24 or 48 hrs
incubation with geranial/NP, indicating that the cell death mechanism induced by geranial was not
apoptosis.
pH degradation studies for geranial, neral, and citral at pH 7.4
A
B
Fig. 5S At pH 7.4, the HPLC profiles of citral/NP (VI), citral (V), neral/NP (IV), neral (V),
geranial/NP (II) and geranial (I) at 0h (A) were very similar to their profiles at 24h incubation (B).
This indicates there was none or very little evidence of drug degradation at pH 7.4 up to 24 h
incubation. Peak 1 at ca. 5.9 min corresponds to neral and peak 2 at ca. 6.1 min corresponds to
geranial.
Complete picture of tumors isolated from all animals
Fig. 6S Picture of tumors excised from all animals at the end of the in vivo study, prior to western
blot analysis of tumor tissues.