**4. Conclusions**

and 92-fold higher efficiency due to the presence of PMA at 1, 10, 50 and 100 nM concen‐ trations, respectively. Immobilization of either fibronectin or E-cad-Fc on the particles al‐ so showed a dramatic increment in transgene expression, indicating clearly that both of the transmembrane proteins integrin and E-cadherin are up-regulated in response to PKC activation to promote efficient internalization of the bio-functional particles across the plas‐ ma membrane (data not shown here) and subsequent expression of the particle-associat‐

**Figure 19.** Effects of PMA on trans-gene expression mediated by fibronectin/E-cad-Fc-embedded-particles. Particles were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of luciferase plasmid DNA and 2 μg of either fibronectin, E-cad-Fc or both to 1 ml bicarbonate-buffered DMEM and incubation for 30 min at 370C. F9 cells were incubated with the gen‐ erated particles in presence of increasingly high concentrations of PMA (o to 100 nM) for 1 hr and after replacement of particle- and PMA-containing media with fresh media, further incubated for 1 day in order to quantitate luciferase

**3.15. Transfection efficiency achieved in leukemia cells with cell adhesive protein-**

T cell expresses on its membrane α4β1 and α5β1 integrins which can bind fibronectin dur‐ ing lymphocyte adhesion and migration from vascular compartment to the injured tissues [22]. Moreover, αEβ7 integrin on some T cells can interact with epithelial E-cadherin for tis‐ sue-specific retention of lymphocytes [22]. We, therefore, aimed to functionalize the surface of DNA-associated nanocrystals with fibronectin and E-cadherin-Fc for transgene delivery

As shown in Fig. 20, luciferase expression in Jurkat cells was significantly low after delivery of luciferase gene-containing plasmid DNA with the help of carbonate apatite particles. A 3 fold enhancement in transgene expression was observed following delivery with fibronec‐ tin-embedded particles. Transgene expression could be further increased to the level (up to 6 times) equivalent to that of lipofection with the particles complexed with both fibronectin and E-cadherin-Fc. Since lymphocytes posses 2 different types of integrins (α4β1 and α5β1)

ed DNA in cytoplasm [27] (Fig 19).

286 Advances in Biomaterials Science and Biomedical Applications

expression.

**embedded particles**

through integrin-mediated endocytosis [22].

Stem cells possessing the inherent capability of transforming into many cell types, have been shown tremendous potential for cell-based therapies in regenerative medicine for neurologi‐ cal disease or injury [28], diabetes [29] and myocardial infarct [30]. The *in vitro* differentiated derivatives of stem cells are thought to be able to repair or replace damaged cells, tissues or organs. However, compared to embryonic stem cells, adult stem cells are likely more diffi‐ cult to be implemented into useful therapies considering their limited pluripotency. Trans‐ gene delivery could be a powerful strategy for specific differentiation of embryonic stem cells since several transcription factors have been demonstrated to regulate stem cell differ‐ entiation to specific cell types of heart, pancreas, liver and neurons [31-36]. On the other hand, tumor cells such as leukemia and lymphoma cells are obvious and attractive targets for gene therapy. Gene transfer and expression for cytokine and immunomodulatory mole‐ cules in various kinds of tumor cells have been shown to mediate tumor regression and anti‐ metastatic effects [37-40]. Moreover, genetically modified leukemia cells expressing costimulatory molecules or cytokines are likely to have significant therapeutic roles for patients with leukemia [41, 42].

Among the existing approaches for transgene delivery, viral systems suffer from their po‐ tential life-threatening effects of immunogenicity and carcinogenicity whereas non-viral ones, although safe, possess significant limitation in terms of efficacy [43]. Development of a safe as well as an efficient carrier is, therefore, an urgent requirement for effective imple‐ mentation of the stem cells in regenerative medicine and the leukemia (or lymphocytes) in cancer treatment.

We have established a novel type of non-viral gene delivery systems based on pH-sensitive inorganic nanoparticles and revealed an innovative strategy for surface-functionalization of these biodegradable nanoparticles through their ionic interactions with "cell-adhesive mole‐ cules". Moreover, the new approach has directly been applied for highly efficient delivery and expression of a trans-gene into "hard-to-transfect" embryonic stem cells- a success with tremendous future for stem-cell based therapeutic development. The involvement of E-cad‐ herin and fibronectin in intercellular and extracellular interactions of cultured undifferenti‐ ated embryonic stem cells may exclude the possibility of stem cell differentiation following transfection with the new nano-apatite carriers associated with E-cad-Fc and fibronectin. More the same approach has successfully used to transfect the leukemia cells having poten‐ tial application in cancer therapy.

### **Acknowledgements**

This work has financially been supported by a research grant (Project ID 02-02-09-SF0013) of the Ministry of Science, Technology and Innovation (MOSTI), Malaysia.
