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PAMAM-Camptothecin Conjugate Inhibits Proliferation and Induces Nuclear Fragmentation in Colorectal Carcinoma Cells

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ABSTRACT

Purpose

To synthesize and characterize a poly (amido amine) dendrimer-camptothecin (PAMAM-CPT) conjugate and evaluate its activity on human colorectal carcinoma cells (HCT-116).

Methods

The attachment of CPT to amine-terminated PAMAM was facilitated through a succinic acid-glycine linker. The conjugate was characterized for absence of small molecular weight impurities, size and drug content. Stability of the conjugate in PBS and growth media and its in vitro activity on HCT-116 were studied. Cell cycle arrest and nuclear fragmentation upon PAMAM-CPT treatment were investigated.

Results

The conjugate was stable under physiological pH (7.4) in PBS and in growth media (with 10% FBS) with minimal release of 4% and 6% drug, respectively, at 48 h. PAMAM-CPT inhibited proliferation of HCT-116 cells with an IC50 value of 1.6 ± 0.3 µM. The conjugate induced signs of cell cycle arrest with up to 68% of cells blocked in the G2 phase. Confocal images of cells treated with PAMAM-CPT suggest nuclear fragmentation and formation of apoptotic bodies.

Conclusions

Results show that the PAMAM-CPT conjugate was active against colorectal cancer cells in vitro, inhibiting their growth and inducing nuclear fragmentation. Coupled with the ability to target macromolecular therapeutics to tumors, this conjugate shows promise for cancer chemotherapy.

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Abbreviations

ATCC:

American type culture collection

BOC:

tert-Butyloxycarbonyl

CCK:

cell counting kit

CPT:

camptothecin

DAPI:

4′, 6-diamidino-2-phenylindole

DCM:

dichloromethane

DI:

deionized

DIPC–N:

N’–diisopropyl carbodiimide

DIPEA–N:

N–diisopropyl ethylamine

DLS:

dynamic light scattering

DMAP:

4-dimethyl amino pyridine

DMSO:

dimethyl sulfoxide

DNA:

deoxyribonucleic acid

EDC (m):

1-[3-(dimethyl amino) propyl]-3 ethyl carbodiimide methiodide

EPR:

enhanced permeability and retention

FBS:

fetal bovine serum

FPLC:

fast protein liquid chromatography

G4.0:

Generation 4.0

HPLC:

high performance liquid chromatography

IC50 :

half maximal inhibitory concentration

MW:

molecular weight

MWCO:

molecular weight cutoff

NHS-s:

N-hydroxy sulfosuccinimide sodium salt

P-gp:

permeability glycoprotein

PAMAM:

Poly (amido amine)

PBS:

phosphate buffered saline

PEG:

Poly (ethylene glycol)

RNAse:

ribonuclease

SEC:

size exclusion chromatography

TFA:

trifluoroacetic acid

THF:

tetrahydrofuran

TLC:

thin layer chromatography

UV:

ultraviolet

WST:

water-soluble tetrazolium salt

Wt:

weight

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ACKNOWLEDGEMENTS

This work was supported by a National Institutes of Health grant (R01-EB007470) and the Utah Science Technology and Research (USTAR) Initiative.

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Authors and Affiliations

  1. Department of Bioengineering,, University of Utah, Salt Lake City, Utah, 84108, USA

    Giridhar Thiagarajan & Hamidreza Ghandehari

  2. Utah Center for Nanomedicine, Nano Institute of Utah, University of Utah, Salt Lake City, Utah, 84108, USA

    Giridhar Thiagarajan, Abhijit Ray, Alexander Malugin & Hamidreza Ghandehari

  3. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah, 84108, USA

    Abhijit Ray, Alexander Malugin & Hamidreza Ghandehari

Authors
  1. Giridhar Thiagarajan
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  2. Abhijit Ray
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  3. Alexander Malugin
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  4. Hamidreza Ghandehari
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Corresponding author

Correspondence to Hamidreza Ghandehari.

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Supplementary Fig. 1

Mass spectrometry (ESI, m/z, M+1) images of intermediate compounds. A) glycine-camptothecin (m/z, M+1, 406.0); B) succinic acid-glycine-camptothecin (m/z, M+1, 506.1) (TIFF 890 kb)

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(GIF 138 kb)

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Thiagarajan, G., Ray, A., Malugin, A. et al. PAMAM-Camptothecin Conjugate Inhibits Proliferation and Induces Nuclear Fragmentation in Colorectal Carcinoma Cells. Pharm Res 27, 2307–2316 (2010). https://doi.org/10.1007/s11095-010-0179-6

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  • DOI: https://doi.org/10.1007/s11095-010-0179-6

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