(PEG-PE) CONJUGATES
Li Mu, Tamer A. Elbayoumi, Aruna Karkala, Suna Erdogan, Rupa D. Dabholkar, Tatyana S. Levchenko, Dmitry A. Mongayt,
                                                Vladimir P. Torchilin
                Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, USA
                                             E-mail: v.torchilin@neu.edu


INTRODUCTION                                                      various concentrations of the free TPP and TPP in PEG-
                                                                  PE micelles in the darkness for 6 and 18h. After the
Poor water solubility of many anticancer agents (such as          incubation, the cells were washed to remove the non-cell-
paclitaxel, PCT; camptothecin, CPT; and certain                   associated drug, and the plates were photo-irradiated for
porphyrins like meso-tetraphenylporphine, TPP, used in            30 min with the light source (LC-122) 630 nm at 12 mm
photodynamic therapy, PDT) hinders their application and          spot. The cells were (as incubated for another 24 h, and
complicates direct parenteral administration. Various             the viability of irradiated and non-irradiated cells control)
formulation strategies based on the use of drug carrier           was evaluated using the MTT [3-(4,5-dimethylthiazol-2-
systems have been suggested to overcome their poor                yl)-2,5-diphenyltetrazolium bromide] assay.
solubility, low stability, and toxic side effects (1,2).
Among such systems, polymeric micelles have drawn                 RESULTS AND DISCUSSION
much attention owing to their easily controlled properties
and good pharmacological characteristics (3). Micelles            Drug-loaded micelles of various compositions have been
prepared from PEG-diacyllipids conjugates, such as PEG-           prepared (PEG-PE micelles with variable MW of PEG,
PE, are of particular interest (4).                               2,000 and 5,000 Da; and mixed micelles of PEG-PE and
Here, we describe the preparation, properties, and activity       TPGS). The size of all micelles was within the 5-to-30 nm
against cancer cells in vitro of PCT-, CPT-, and TPP-             interval. The methods used allowed for the samples
loaded PEG-PE micelles as well as mixed micelles made             containing 15-to-200 µg of a drug per mL of the micelle
of PEG-PE and D-α-tocopheryl polyetheyene glycol 1000             suspension depending on the drug and micelle
succinate (TPGS).                                                 composition (CPT is the least solubilized, while TPP is
                                                                  the best solubilized drug; mixed micelles provide better
EXPERIMENTAL METHODS                                              solubilization as well as micelles made of PEG-PE with
                                                                  “longer” PEG block). Micelles are rather stable, since the
Drug-loaded micelles were prepared by dispersing dry              storage of drug-loaded micelles at either room
film of the mixture of micelle-forming material (PEG-PE           temperature or at 4°C for over a month did not cause any
or/and TPGS) and drug in an aqueous buffer solution. The          changes in micelle size. They firmly retain the
content of the drug incorporated into the micelles was            incorporated drug – some initial burst drug release was
assayed by HPLC for PCT and CPT and by fluorescence               observed (about 15% of incorporated drug – probably, the
spectroscopy for TPP. The particle size analysis was done         drug loosely associated with micelle corona) with no
by the dynamic light scattering. The in vitro drug release        further drug loss over weeks.
from various micelles was investigated in an aqueous              In vitro antiproliferative activity of various preparations
solution containing sodium salicylate in order to create          (typical data for CPT- and TPP-loaded micelles are shown
pseudo-sink conditions.                                           in Figs 1 through 4) indicates that the cytotoxicity of
Murine LLC1 Lewis lung carcinoma and human HT-29                  drug-loaded PEG-PE-based micelles was markedly higher
colorectal adenocarcinoma and MCF-7 breast cancer cells           compared to the free drug (empty micelles do not show
were maintained in Dulbecco's Modified Eagle Medium               any cytotoxicity). Thus, micellar preparations of CPT
(DMEM) and Eagle’s Minimum Essential Medium                       were cytotoxic against LLC1 and HT-29 cells even at
(EMEM) supplemented with 10% fetal bovine serum and               CPT concentrations below 50 ng/ml (Figs 1 and 2).
penicillin/streptomycin at 37°C in 5% CO2. Standard in            A representative cytotoxicity for 6 and 18 hrs incubation
vitro cytotoxicity tests were with PCT- and CPT-loaded            with TPP-loaded PEG-PE micelles before and after the
PEG-PE micelles.                                                  light treatment are shown in Figs. 3 and 4 for LLC and
For phototoxicity studies, cells were grown in a 96-well          MCF-7 cells. In both cases, there was a dramatic increase
microplate overnight and then incubated at 37°C with              in the post-light-irradiation cytotoxicity (PDT conditions)

           15th International Symposium on MICROENCAPSULATION, Parma (Italy), September 18-21, 2005

of TPP-containing PEG-PE-micelles when compared to
not light-irradiated TPP-containing PEG-PE-micelles.
The incubation time of the cells with TPP-micelles prior                          100
to light irradiation was essential for the treatment                               80

                                                                     % survival
outcome. Cells incubation with TPP-containing micelles                             60
for 18 h resulted in better cell killing than 6 h incubation.
Naturally, the cell killing was TPP concentration-                                 40
dependent.                                                                         20
                                                                                        0   5   10   15   20   25 30 35     40   45
                                                                                                TPP concentration (µg/ml)

                                                                    Figure 4. Phototoxic effects of TPP encapsulated in PEG-
                                                                    PE micelles with and without light on LLC cells ( no
                                                                    light, 18 hrs exposure; with light:  6 hrs,  18 hrs drug

Figure 1. Cytotoxicity of CPT and CPT-loaded PEG-PE                 Poorly soluble anticancer drugs for different treatment
micelles against LLC1 cells.                                        modalities (PCT, CPT, and TPP) can be stably solubilized
                                                                    using PEG-PE-based micelles, in particular mixed
                                                                    micelles from PEG-PE and TPGS. Antiproliferative
                                                                    activity in vitro against various cancer cells was
                                                                    significantly higher for micellar drugs than for free drugs.
                                                                    PEG-PE-based micelles can represent a promising
                                                                    opportunity for increasing the drug efficiency.


                                                                    This work was supported by the NIH grant R01-
                                                                    EB001961 to Vladimir Torchilin.
Figure 2. Cytotoxicity of CPT and CPT-loaded PEG-PE
micelles against HT-29 cells.                                       REFERENCES

                                                                    1. Garcia-Carbonero, R., and Supko, J.G., (2002),
                                                                    “Current perspectives on the clinical experience,
                                                                    pharmacology, and continued development of the
                                                                    camptothecins”, Clinical Cancer Research, 8, 641-661.
                                                                    2. Singla, A.K., Garg, A., and Aggarwal, D., (2002),
                                                                    “Paclitaxel and its formulation”, International Journal of
                                                                    Pharmaceutics, 235, 179-192.
                                                                    3. Torchilin, V.P., (2001), “Structure and design of
                                                                    polymeric surfactant-based drug delivery systems”,
                                                                    Journal of Controlled Release 73, 137–172.
Figure 3. Phototoxic effects of TPP encapsulated in PEG-            4. Lukyanov, A.N., and Torchilin, V.P., (2004), “Micelles
PE micelles with and without light on MCF-7cells ( no              from lipid derivatives of water-soluble polymers as
light, 18 hrs exposure; with light:  6 hrs,  18 hrs drug          delivery systems for poorly soluble drugs”, Advanced
exposure).                                                          Drug Delivery Reviews, 56, 1273-1289.

           15th International Symposium on MICROENCAPSULATION, Parma (Italy), September 18-21, 2005

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