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Journal of Chinese Integrative Medicine ›› 2010, Vol. 8 ›› Issue (9): 853-862.doi: 10.3736/jcim20100909

• Original Experimental Research • Previous Articles     Next Articles

Polymeric nanoparticle encapsulation of a naturally occurring plant scopoletin and its effects on human melanoma cell A375

Khuda-Bukhsh ARa,Bhattacharyya SSa,Paul Sa,Boujedaini Nb   

  1. a Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India
    b Boiron Laboratory, 20, rue de la Liberation, Sainte-Les-Foy-Lyon, France
  • Received:2010-04-28 Accepted:2010-07-05 Online:2010-09-20 Published:2010-09-15

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Objective

We formulated nano-encapsulation of a naturally occurring coumarin — scopoletin (7-hydroxy-6-methoxy coumarin, HMC, C10H8O4), isolated from plant Gelsemium sempervirens having anticancer potentials, with a bio-adhesive agent — polylactic-co-glycolic acid (PLGA) and tested if its cellular uptake, bioavailability and apoptotic (anticancer) potentials could thus be increased vis-vis unencapsulated HMC.
Methods

A375 melanoma cancer cells were used for testing cellular entry and anticancer potentials of HMC and nano-7-hydroxy-6-methoxy coumarin (NHMC) through several standard protocols. Characterization of NHMC was done by dynamic light scattering for determination of particle size, polydispersity index (PDI), and zeta potential. Surface morphology of nanoparticles was determined by scanning electron microscopy and atomic force microscopy.
Results

HMC was encapsulated with more than 85% entrapment efficiency, the average particle size of NHMC being less than 110 nm and a PDI 0.237, which resulted in enhanced cellular entry and greater bioavailability. NHMC showed a faster cellular uptake (15 min) than its unencapsulated counterpart (30 min). Study of signal molecules through mRNA expressions revealed that NHMC caused down-regulation of cyclin-D1, proliferating cell nuclear antigen (PCNA), survivin and Stat-3, and up-regulation of p53 and caspase-3, that in turn induced a greater number of apoptosis vis-vis unencapsulated HMC.
Conclusion

The formulation yielded small-sized NHMC by biodegradable PLGA that took less time for cellular entry, and caused more apoptosis to cancer cells, but apparently had negligible cytotoxicity against normal skin cells. Nano-encapsulation of bioactive plant ingredients can be a strategy worth trying for designing effective chemopreventive drug products.

Key words: Polylactic-co-glycolic acid, Nanoparticles, Scopoletin, Melanoma, Apoptosis, Antineoplastic agents, Phytogenic, In vitro

Figure 1

Chemical structure of 7-hydroxy-6-methoxy coumarin (HMC)"

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Figure 2

Surface images of NHMC and interaction of NHMC with A375 cells (A) SEM photograph of nanoparticles of HMC prepared from PLGA. (B) Surface topography of NHMC obtained by AFM. (C) Corresponding 3-D image of NHMC obtained by tapping mode AFM and visualized in amplitude mode."

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Figure 3

(A) Average particle size obtained from DLS data for PLGA-encapsulated NHMC. (B) Zeta potential of NHMC"

Figure 4

In vitro release kinetics of NHMC"

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Figure 5

Cell viability of blank nanoparticles, HMC and NHMC with different doses against A375 cell line tested by MTT assay. **P<0.01, vs HMC group. All results were expressed with vehicle control as 100%."

Figure 6

Cellular uptakes of HMC and NHMC in A375 cells"

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Figure 7

Effects of HMC and NHMC on A375 cells Ln1 contains a standard DNA ladder; Ln2 contains DNA from untreated cells; Ln3 contains DNA from HMC (30 μmol/L); Ln4 contains DNA from HMC (60 μmol/L); Ln5 from NHMC (30 μmol/L) and Ln6 from NHMC (60 μmol/L) -treated cells."

Figure 8

Expressions of β-actin, cyclin-D1, survivin, Stat-3, PCNA, caspase-3, and p53 analyzed by RT-PCR Ln1: Control; Ln2: HMC (30 μmol/L); Ln3: HMC (60 μmol/L); Ln4: NHMC (30 μmol/L); Ln5: NHMC (60 μmol/L)."

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