**3. Results and Discussion**

### **3.1. Generation of carbonate apatite particles**

Addition of only 3 mM Ca2+ to the HCO3 - - buffered cell culture medium (DMEM or Wil‐ lium E, pH 7.5) and incubation at 370 C for 30 min, resulted in microscopically visible parti‐ cles. Generation of these particles only in HCO3 - -, but not in Hepes-buffered media or solution (pH 7.5) containing the same amount of exogenous Ca2+ and phosphate (0.9 mM), indicates the possible involvement of carbonate along with phosphate and Ca2+ in particle formation. Elemental analysis proved the existence of C (3%), P (17%) and Ca2+ (32%) and FT-IR spectra (Fig. 1a) identified carbonate, as evident from the peaks between 1410 and 1540 cm-1 and at approximately 880 cm-1, along with phosphate in the particles, as shown by the peaks at 1000-1100 cm-1 and 550-650 cm-1. X-ray diffraction patterns (Fig. 1b) indicated less crystalline nature, represented by broad diffraction peaks of the particles, compared to that of hydrox‐ yaptite (Fig. 1c) – an intrinsic property of carbonate apatite [12].

**Figure 1.** Infrared spectra of generated carbonate apatite (A) and X-ray diffraction patterns of carbonate apatite (B) and hydroxyapatite (C).

#### **3.2. Influences of pH and temperature on generation of effective particles of carbonate apatite**

We have investigated a long range of pH (7.0 to 7.9) of the HCO3 <sup>−</sup> –buffered medium as well as incubation temperatures (25 °C to 65 °C) in order to make particles by exogenously added Ca2+ and subsequently transfect HeLa cells using the generated particles. Interestingly, the optimal Ca2+ concentrations required for generation of effective number of DNA/carbonate apatite particles leading to the high transfection efficiency, were inversely related to the pHs of the media (Fig. 2-a) and the incubation temperatures (Fig. 2-b). Thus, while 4 mM Ca2+ was sufficient to induce particle formation at pH 7.4 by incubating the Ca2+-supplemented buffered medium for 30 min at 37 °C, only 1 mM2+ was enough to stimulate particle genera‐ tion to the similar level at pH 7.9. Like pH, incubation temperatures have also profound and sensitive effects on particle formulation and subsequent trans-gene delivery. Thus, at the in‐ cubation temperature of 37 °C, 3 mM Ca2+ was able to induce the proper "supersaturation" whereas at 65 °C, only 1 mM Ca2+could stimulate "supersaturation" development to a simi‐ lar extent – a prior need for generation of the particles. The decline below the high efficiency level of transfection was due to the formation of too few particles (microscopically observed) since increase in pH or temperature contributed to the development of "supersaturation" by increasing the ionization of phosphate and carbonate in the solution. The new system of par‐ ticle synthesis is, therefore, very flexible since it allows us to make particles at a wide range of pH and temperatures. The analysis also indicates that induction of "supersaturation" as required for particle formation, can be delicately controlled by manipulating the parameters.

cles. Generation of these particles only in HCO3

270 Advances in Biomaterials Science and Biomedical Applications

and hydroxyapatite (C).

**apatite**

yaptite (Fig. 1c) – an intrinsic property of carbonate apatite [12].


(pH 7.5) containing the same amount of exogenous Ca2+ and phosphate (0.9 mM), indicates the possible involvement of carbonate along with phosphate and Ca2+ in particle formation. Elemental analysis proved the existence of C (3%), P (17%) and Ca2+ (32%) and FT-IR spectra (Fig. 1a) identified carbonate, as evident from the peaks between 1410 and 1540 cm-1 and at approximately 880 cm-1, along with phosphate in the particles, as shown by the peaks at 1000-1100 cm-1 and 550-650 cm-1. X-ray diffraction patterns (Fig. 1b) indicated less crystalline nature, represented by broad diffraction peaks of the particles, compared to that of hydrox‐

**Figure 1.** Infrared spectra of generated carbonate apatite (A) and X-ray diffraction patterns of carbonate apatite (B)

as incubation temperatures (25 °C to 65 °C) in order to make particles by exogenously added Ca2+ and subsequently transfect HeLa cells using the generated particles. Interestingly, the optimal Ca2+ concentrations required for generation of effective number of DNA/carbonate apatite particles leading to the high transfection efficiency, were inversely related to the pHs of the media (Fig. 2-a) and the incubation temperatures (Fig. 2-b). Thus, while 4 mM Ca2+ was sufficient to induce particle formation at pH 7.4 by incubating the Ca2+-supplemented buffered medium for 30 min at 37 °C, only 1 mM2+ was enough to stimulate particle genera‐ tion to the similar level at pH 7.9. Like pH, incubation temperatures have also profound and

**3.2. Influences of pH and temperature on generation of effective particles of carbonate**

We have investigated a long range of pH (7.0 to 7.9) of the HCO3 <sup>−</sup>


–buffered medium as well

**Figure 2.** Regulation of trans-gene delivery and expression facilitated by carbonate apatite particles. Regulation of trans-gene expression by the nanoparticles of carbonate apatite generated at a wide range of pH and temperature. DNA/carbonate apatite particles were generated by addition of 1 to 4 mM Ca2+ and 2 μg plasmid DNA to 1 ml HCO3 <sup>−</sup> (40 mM)-buffered DMEM medium with a pH range from 7.0 to 7.9, followed by incubation for 30 min either at 37 °C (a) or at 25 °C to 65 °C (b). Transfection of HeLa cells, HepG2, NIH3T3 and primary hepatocytes was performed in the same manner as mentioned in 'Materials and methods' section.

#### **3.3. Tranfection efficiency and cell viability assessment**

To evaluate the role of carbonate apatite as a powerful carrier of genetic material, we com‐ pared transfection efficiency of different techniques including two frequently used ones-CaP co-precipitation method and lipofection. In HeLa cell, for example, luciferase expression level for carbonate apatite-mediated transfection was over 25-fold higher than for lipofection and CaP co-precipitation method (Fig. 2c). Nano gram level of DNA was even sufficient for efficient transgene expression (Fig. 2c). Transfection efficiency was also significantly high in HepG2 (Fig. 2d), NIH 3T3 cells (Fig. 2e) and mouse primary hepato‐ cytes (Fig. 2f). We performed MTT assay in HeLa cells (not shown here) to clarify that high transfection efficiency was accompanied by high viability of the cells [12].

#### **3.4. Estimation of particle sizes and cellular uptake of particle-associated plasmid DNA**

To explore why carbonate apatite is so efficient as a vector for gene delivery, we investigat‐ ed two basic properties of carbonate apatite [12]. Carbonate, when present in the apatite structure, limits the size of the growing apatite crystals and increases the dissolution rate [12]. We carried out scanning electron microscopic observation of generated carbonate apa‐ tite (Fig. 3A) which revealed reduced growth of the crystals, most of which had diameters of 50 to 300 nm. We verified this size limiting effect of carbonate by observing cellular uptake of the PI (propidium iodide)-labeled plasmid DNA adsorbed to the apatites, since large par‐ ticles are phagocytosed less efficiently than small ones [12]. DNA was carried into the cells by carbonate apatite (Fig. 3B-c) at least 10 times more efficiently than hydroxyapatite, gener‐ ated by 1 min incubation (Fig. 3B-d). Longer period (30 min) incubation resulted in large hy‐ droxyapatite particles [12], showing significantly reduced transfection efficiency [12] (Fig. 2A) due to extremely low cellular uptake of DNA [12]. Our findings, therefore, clearly sug‐ gest that carbonate apatite is superior over hydroxyapatite for its intrinsic property of pre‐ venting crystal growth, leading to high efficiency cellular uptake of DNA.

**Figure 3.** A, scanning electron microscopy, showing limited growth of generated carbonate apatite cryatals. Scale bar, 600 nm. B, cellular uptake of PI-labeled plasmid DNA associated with carbonate apatite and hydroxyapatite. a, no up‐ take of DNA (control), since endocytosis was blocked by energy depletion (50 mM 2-deoxy glucose and 1 mM Naazide). DNA/carbonate apatite particles were prepared in 1 ml serum-free media (described in legend to Fig. 3) using 6 mM Ca2+ and 2 μg DNA. 40 ng (b) and 200 ng (c) of DNA in 20 μl and 100 μl of 1ml suspension respectively, were allowed for cellular uptake for 4 hr. d, 2 μg of DNA adsorbed to hydroxyapatite (described in experimental protocol) was allowed for uptake for the same period of time. Bar indicates 50 μM.

#### **3.5. Endosomal escape of plasmid DNA carried by nanoparticles**

expression level for carbonate apatite-mediated transfection was over 25-fold higher than for lipofection and CaP co-precipitation method (Fig. 2c). Nano gram level of DNA was even sufficient for efficient transgene expression (Fig. 2c). Transfection efficiency was also significantly high in HepG2 (Fig. 2d), NIH 3T3 cells (Fig. 2e) and mouse primary hepato‐ cytes (Fig. 2f). We performed MTT assay in HeLa cells (not shown here) to clarify that high

**3.4. Estimation of particle sizes and cellular uptake of particle-associated plasmid DNA**

To explore why carbonate apatite is so efficient as a vector for gene delivery, we investigat‐ ed two basic properties of carbonate apatite [12]. Carbonate, when present in the apatite structure, limits the size of the growing apatite crystals and increases the dissolution rate [12]. We carried out scanning electron microscopic observation of generated carbonate apa‐ tite (Fig. 3A) which revealed reduced growth of the crystals, most of which had diameters of 50 to 300 nm. We verified this size limiting effect of carbonate by observing cellular uptake of the PI (propidium iodide)-labeled plasmid DNA adsorbed to the apatites, since large par‐ ticles are phagocytosed less efficiently than small ones [12]. DNA was carried into the cells by carbonate apatite (Fig. 3B-c) at least 10 times more efficiently than hydroxyapatite, gener‐ ated by 1 min incubation (Fig. 3B-d). Longer period (30 min) incubation resulted in large hy‐ droxyapatite particles [12], showing significantly reduced transfection efficiency [12] (Fig. 2A) due to extremely low cellular uptake of DNA [12]. Our findings, therefore, clearly sug‐ gest that carbonate apatite is superior over hydroxyapatite for its intrinsic property of pre‐

**Figure 3.** A, scanning electron microscopy, showing limited growth of generated carbonate apatite cryatals. Scale bar, 600 nm. B, cellular uptake of PI-labeled plasmid DNA associated with carbonate apatite and hydroxyapatite. a, no up‐ take of DNA (control), since endocytosis was blocked by energy depletion (50 mM 2-deoxy glucose and 1 mM Naazide). DNA/carbonate apatite particles were prepared in 1 ml serum-free media (described in legend to Fig. 3) using 6 mM Ca2+ and 2 μg DNA. 40 ng (b) and 200 ng (c) of DNA in 20 μl and 100 μl of 1ml suspension respectively, were allowed for cellular uptake for 4 hr. d, 2 μg of DNA adsorbed to hydroxyapatite (described in experimental protocol)

was allowed for uptake for the same period of time. Bar indicates 50 μM.

transfection efficiency was accompanied by high viability of the cells [12].

272 Advances in Biomaterials Science and Biomedical Applications

venting crystal growth, leading to high efficiency cellular uptake of DNA.

To evaluate the role of endosomal escape of DNA in transgene expression, following endo‐ cytosis of PI-labeled plasmid DNA, we labeled endosomes with LysoSensor (a fluorescence probe for endosomes). Following 6 hr of DNA uptake by cells, a significant portion of DNA (red colour) appeared to be released from endosomes (green colour) after colocalization of plasmid DNA with endosomes (Fig. 4).

**Figure 4.** Endosomal escape of endocytosed PI–labeled DNA, as evident after colocalization with a fluorescence probe (Lyso-Sensor) for endosomes.

#### **3.6. Relationship of endosomal pH and crystalline properties of particles affecting transfection**

Treatment with bafilomycin A1, a specific inhibitor of v-ATPase (a proton pump for acidifi‐ cation of endocytic vesicles) resulted in drastic reduction of transfection efficiency in HeLa cells (Fig. 5A), which indicated that acidic environment might be necessary for solubilization of carbonate apatite to release DNA from the apatite. To establish this notion, we generated fluoridated carbonate apatite to see the effect of solubility of the particles on transfection ef‐ ficiency, since incorporation of fluoride reduces the solubility of the apatite [12]. Surprising‐ ly, transfection efficiency was reduced gradually to a significant extent (100 fold) with increasing fluoride level in carbonate apatite (Fig. 5B).

**Figure 5.** A, Effect of bafilomycin A1 (an inhibitor of v-ATPase) on transfection. HeLa cells were incubated with DNA/ carbonate apatite particles and 200 nM bafilomycin A1 for 6 hr. After washing with 5 mM EDTA in PBS, cells were grown for 1 day and luciferase expression was detected. B, Changes in luciferase expression for increasing concentra‐ tions of F- (0.01 to 3 mM) and strontium (0.01 to 3 mM) added during generation of DNA/carbonate apatite particles.

To establish a relationship between transfection efficiency and dissolution rates of the apa‐ tites, turbidity (320 nm) measurement was done as an indicator of their solubilization, fol‐ lowing an acid load in solution of generated apatites. Carbonate apatite generated in presence of increasing concentrations of NaF, showed gradual decrease in dissolution rates, as evident from changes in turbidity, following adjustment of pH from 7.5 to 7.0 with 1 N HCl (Fig. 6 A, B), which is consistent with gradually reduced transfection efficiency of fluo‐ ridated carbonate apatites (Fig. 5 B). With decreasing pH from 7.0 to 6.8, carbonate apatite was completely solubilized within 1 min, whereas fluoridated carbonate apatite was partial‐ ly dissolved (Fig. 6 B).

To examine whether dissolution rates of apatites are correlated with their degree of crystalli‐ zation, we studied x-ray diffraction of the apatites (Fig. 7), which clearly indicates that apa‐ tite with higher degree of crystallization, had lower solubility (Fig. 6A). In other words, apatites with higher crystallinity (Fig. 7) showed lower transfection efficiency (Fig. 5 B). The gradual increase in crystallinity owing to increased level of incorporated fluoride in carbo‐ nate apatite (Fig. 7) resulted in gradual decrease in transfection efficiency (Fig. 5B).

**Figure 6.** A, Dissolution rates (at pH 7.0) of fluoridated carbonate apatites prepared by addition of 0-3 mM F- during generation of carbonate apatite at pH 7.5 (described in experimental protocol), were studied by turbidity measure‐ ment at 320 nm of apatite suspensions just after being adjusted to the pH 7.0 with 1 N HCl. B, Dissolution rates of carbonate apatite, fluoridated carbonate apatite and strontium-containing carbonate apatite at pHs of 7.0 and 6.8.

pH-Sensitive Nanocrystals of Carbonate Apatite- a Powerful and Versatile Tool for Efficient Delivery of Genetic... http://dx.doi.org/10.5772/ 53107 275

To establish a relationship between transfection efficiency and dissolution rates of the apa‐ tites, turbidity (320 nm) measurement was done as an indicator of their solubilization, fol‐ lowing an acid load in solution of generated apatites. Carbonate apatite generated in presence of increasing concentrations of NaF, showed gradual decrease in dissolution rates, as evident from changes in turbidity, following adjustment of pH from 7.5 to 7.0 with 1 N HCl (Fig. 6 A, B), which is consistent with gradually reduced transfection efficiency of fluo‐ ridated carbonate apatites (Fig. 5 B). With decreasing pH from 7.0 to 6.8, carbonate apatite was completely solubilized within 1 min, whereas fluoridated carbonate apatite was partial‐

To examine whether dissolution rates of apatites are correlated with their degree of crystalli‐ zation, we studied x-ray diffraction of the apatites (Fig. 7), which clearly indicates that apa‐ tite with higher degree of crystallization, had lower solubility (Fig. 6A). In other words, apatites with higher crystallinity (Fig. 7) showed lower transfection efficiency (Fig. 5 B). The gradual increase in crystallinity owing to increased level of incorporated fluoride in carbo‐

**Figure 6.** A, Dissolution rates (at pH 7.0) of fluoridated carbonate apatites prepared by addition of 0-3 mM F- during generation of carbonate apatite at pH 7.5 (described in experimental protocol), were studied by turbidity measure‐ ment at 320 nm of apatite suspensions just after being adjusted to the pH 7.0 with 1 N HCl. B, Dissolution rates of carbonate apatite, fluoridated carbonate apatite and strontium-containing carbonate apatite at pHs of 7.0 and 6.8.

nate apatite (Fig. 7) resulted in gradual decrease in transfection efficiency (Fig. 5B).

ly dissolved (Fig. 6 B).

274 Advances in Biomaterials Science and Biomedical Applications

**Figure 7.** X-ray diffraction patterns of carbonate apatite (A) and fluoridated carbonate apatites, containing 0.65% (B), 1.43% (C) and 2.5% (D). Carbonate apatite was generated by addition of 6 mM Ca2+ and fluoridated carbonate apa‐ tites by addition of 1 mM (B), 2 mM (C) and 3 mM (D) NaF along with 6 mM Ca2+ to HCO3 - -buffered medium (pH7.5), followed by incubation at 370C

To establish that decreased transfection efficiency was only due to decreased solubility of fluoridated carbonate apatite, but not by any other fluoride-mediated effects, we examined the effects of strontium which, when incorporated into carbonate apatite, is known to im‐ prove the crystallinity and reduce the solubility of the apatite, but to a lesser extent than flu‐ oride [12]. As expected, addition of strontium chloride during preparation of carbonate apatite reduced its dissolution rate but to a level less than that observed for fluoride (Fig 6B). Moreover, transfection efficiency was gradually decreased with increasing concentrations of strontium chloride during generation of DNA/carbonate apatite particles (Fig. 5B). Taken to‐ gether, our findings suggest that intracellular release of DNA through dissolution of apatite should play a major role in carbonate apatite-mediated transfection.

#### **3.7. Immobilization of cell-adhesive molecules on nano-particle surface**

Since embryonic stem cells produce substantial amount of fibronectin-specific integrins as well as E-cadherin as transmembrane proteins [17], we hypothesized that if the nano-parti‐ cles of carbonate apatite could be complexed with fibronectin and E-cadherin, either indi‐ vidually or together, they might recognize in the immobilized state the corresponding receptors on cell surface in order to facilitate quick internalization of the composite particles across the plasma membrane through endocytosis. These nano-apatite particles possess anion- and cation-binding domains which enable them to bind to both acidic and basic ami‐ no acids of protein molecules [18, 19]. On the other hand, fibronectin as well as E-cadherin are rich in acidic amino acid residues [19, 20] which make them excellent candidates for pos‐ sible binding with the apatite particles. We have examined whether these "cell adhesive molecules" could, in deed, bind to the particles, by SDS-PAGE and Western blot analysis, following generation of apatite-protein composites and decomplexation through EDTAmediated particle dissolution. Whereas binding affinity of the particles for fibronectin was relatively lower requiring higher amount of initially added fibronectin as observed by SDS-PAGE, almost all E-cad-Fc was found to be associated with the particles as verified by West‐ ern blot analysis (Fig 8). Very high affinity for E-cadherin could be interpreted by the previous report that E-cadhein has many exposed acidic residues in several loop structures responsible for binding divalent cation Ca2+ [20].

**Figure 8.** Analysis of the binding of cell-adhesive proteins to nano-particles. Particles were prepared by addition of 3 μl of 1 M CaCl2 and 5 to 20 μg of fibronectin or 1 to 5 μg of E-cad-Fc to 1 ml bicarbonate-buffered DMEM and incuba‐ tion for 30 min at 370C. Generated particles were centrifuged at 15000 rpm for 5 min and washed 2 times with H2O to remove the unbound proteins, followed by EDTA treatment to dissolve the particles. SDS-PAGE and Western blot analysis were performed in order to see, respectively, particle-associated fibronectin (A) or E-cad-Fc (B).

#### **3.8. Enhanced cellular uptake of DNA by immobilized cell-adhesive molecules**

In order to explore whether apatite particles functionalized with fibronectin and E-cad-Fc can facilitate enhanced delivery of apatite-associated plasmid DNA across the plasma mem‐ brane, we examined cellular uptake of the DNA labeled with propidium iodide (PI) [19], fol‐ lowing 4 hr incubation of F9 cells with various particle formulations. As shown in Fig. 9, while with only apatite particles, delivery of PI-labeled DNA into the cells was extremely low, complexation of the particles with either fibronectin or E-cad-Fc resulted in significant‐ ly improved DNA delivery, suggesting that immobilized fibonectin or E-cad-Fc retained their functionalities in order to recognize specific cell surface integrin or E-cadherin, respec‐ tively for enabling subsequent internalization of the whole particle composite through endo‐ cytosis [19, 21]. Moreover, the apatite particles when complexed with both fibronectin and E-cad-Fc, demonstrated more pronounced DNA delivering activity compared to the parti‐ cles embedded with either fibronectin or E-cad-Fc, indicating a synergistic effect of the mul‐ tifunctional particles on endocytosis through simultaneous recognition of extracellular domains of specific integrin as well as E-cadherin molecules.

sible binding with the apatite particles. We have examined whether these "cell adhesive molecules" could, in deed, bind to the particles, by SDS-PAGE and Western blot analysis, following generation of apatite-protein composites and decomplexation through EDTAmediated particle dissolution. Whereas binding affinity of the particles for fibronectin was relatively lower requiring higher amount of initially added fibronectin as observed by SDS-PAGE, almost all E-cad-Fc was found to be associated with the particles as verified by West‐ ern blot analysis (Fig 8). Very high affinity for E-cadherin could be interpreted by the previous report that E-cadhein has many exposed acidic residues in several loop structures

**Figure 8.** Analysis of the binding of cell-adhesive proteins to nano-particles. Particles were prepared by addition of 3 μl of 1 M CaCl2 and 5 to 20 μg of fibronectin or 1 to 5 μg of E-cad-Fc to 1 ml bicarbonate-buffered DMEM and incuba‐ tion for 30 min at 370C. Generated particles were centrifuged at 15000 rpm for 5 min and washed 2 times with H2O to remove the unbound proteins, followed by EDTA treatment to dissolve the particles. SDS-PAGE and Western blot

In order to explore whether apatite particles functionalized with fibronectin and E-cad-Fc can facilitate enhanced delivery of apatite-associated plasmid DNA across the plasma mem‐ brane, we examined cellular uptake of the DNA labeled with propidium iodide (PI) [19], fol‐ lowing 4 hr incubation of F9 cells with various particle formulations. As shown in Fig. 9,

analysis were performed in order to see, respectively, particle-associated fibronectin (A) or E-cad-Fc (B).

**3.8. Enhanced cellular uptake of DNA by immobilized cell-adhesive molecules**

responsible for binding divalent cation Ca2+ [20].

276 Advances in Biomaterials Science and Biomedical Applications

**Figure 9.** Effects of particle-immobilized proteins on cellular internalization of plasmid DNA. Particles were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of PI-labelled plasmid DNA and 5 μg of fibronectin and/or 5 μg of E-cad-Fc to 1 ml bicarbonate-buffered DMEM and incubation for 30 min at 370C. F9 cells were incubated with the generated parti‐ cles for 4 hr, washed with 5 mM EDTA in PBS and visualized by a fluorescence microscope (scale bar, 50 μm).

#### **3.9. Quantitation and validation of trans-gene expression facilitated by cell-adhesive molecules**

Since expression of a trans-gene is the result of overcoming a number of barriers including entry into the cells, release from the particle and endosomes and finally nuclear transloca‐ tion [17, 18], we have investigated whether improved DNA delivery as a result of integrinand E-cadherin-mediated endocytosis of composite particles (Fig. 9) contributed to the similar extent to final protein expression. Quantitative luciferase expression analysis indicat‐ ed that particles complexed with fibronectin or E-cad-Fc promoted trans-gene expression

with a value which was almost 20 times higher than that achieved for the particles only (Fig 10). A prior optimization study demonstrated that 1 to 5 μg/ml of fibronectin as well as Ecad-Fc conferred the best transfection efficiency and was, therefore, maintained for all sub‐ sequent experiments. Finally, synergistic activity of fibronectin and E-cad-Fc which caused huge cellular uptake of DNA (Fig. 9), further accelerated gene expression efficiency with a value almost 3 times higher than that observed for commercially available lipofectamine (Fig. 10). With increasing the total amount of initially added DNA up to 4 μg, a further in‐ crease in trasfection efficiency was observed (data not shown here) possibly due to the high‐ er loading of DNA into the crystals with the consequence of more DNA getting inside the cells. The high level of expression could directly be observed by fluorescence microscopy which demonstrated many GFP-expressing F9 cells (Fig. 11). Fluorescence Activated Cell Sorting (FACS) analysis demonstrated that almost 60% cells were GFP-positive following transfection with the particles carrying, in addition to pEGFP plasmid DNA, both fibronec‐ tin and E-cad-Fc (Fig 11). MTT assay was performed in F9 cells to clarify that high transfec‐ tion efficiency was not accompanied by significant toxicity of the cells (data not shown here). In order to establish that such organic-inorganic hybrid particles promote trans-gene delivery and expression through specific interactions with cell-surface molecules (integrin or E-cadherin), we added increasingly high amounts of free fibronectin to the preformed parti‐ cle suspension carrying both fibronectin and E-cadherin and incubated with the cells for the same period of time (4 hr) as followed in usual transfection procedure. Transfection efficien‐ cy decreased as the concentration of free fibronectin increased from 5 to 100μg/ml, indicat‐ ing the involvement of specific interactions between immobilized fibronectin and the corresponding specific integrin receptors (Fig 12). At a sufficiently high concentration (300 μg/ml), free fibronectin drastically reduced luciferase expression suggesting that high amount of fibronectin molecules not only saturate their specific integrins and block binding of immobilized fibronectin needed for particle internalization, but also shield cell-surface Ecadherin and prevent specific binding of particle surface-embedded E-cad-Fc chimera lead‐ ing to very low cellular uptake of particle-associated DNA and diminished luciferase expression. Since embryonic stem cells are the final target for genetic modification in regen‐ erative medicine, we applied the new transfection approach to mouse embryonic stem cells. As shown in Fig. 13, only apatite particles were extremely inefficient in transfecting the cells, whereas fibronectin-bound particles to some extent promoted GFP expression and fibronec‐ tin and E-cad-Fc-bound particles to a significant extent accelerated trans-gene expression, thus proposing that the synergistic effect is a universal way of accelerating trans-gene deliv‐ ery and expression using inorganic nano-particle-associated cell recognizable proteins. Quantitative luciferase expression in embryonic stem cells indicated that particles com‐ plexed with fibronectin and E-cad-Fc individually, promoted trans-gene expression with ef‐ ficiency approximately 9 and 7 times higher, respectively, than that achieved with the particles only (Fig. 14). However, when the particles were associated with both fibronectin and E-cadherin-Fc, a synergistic effect resulted in remarkable level of transgene expression leading to almost 40 and 28 times higher efficiency than that obtained by apatite particles and widely used lipofectamine 2000 system [14].

pH-Sensitive Nanocrystals of Carbonate Apatite- a Powerful and Versatile Tool for Efficient Delivery of Genetic... http://dx.doi.org/10.5772/ 53107 279

with a value which was almost 20 times higher than that achieved for the particles only (Fig 10). A prior optimization study demonstrated that 1 to 5 μg/ml of fibronectin as well as Ecad-Fc conferred the best transfection efficiency and was, therefore, maintained for all sub‐ sequent experiments. Finally, synergistic activity of fibronectin and E-cad-Fc which caused huge cellular uptake of DNA (Fig. 9), further accelerated gene expression efficiency with a value almost 3 times higher than that observed for commercially available lipofectamine (Fig. 10). With increasing the total amount of initially added DNA up to 4 μg, a further in‐ crease in trasfection efficiency was observed (data not shown here) possibly due to the high‐ er loading of DNA into the crystals with the consequence of more DNA getting inside the cells. The high level of expression could directly be observed by fluorescence microscopy which demonstrated many GFP-expressing F9 cells (Fig. 11). Fluorescence Activated Cell Sorting (FACS) analysis demonstrated that almost 60% cells were GFP-positive following transfection with the particles carrying, in addition to pEGFP plasmid DNA, both fibronec‐ tin and E-cad-Fc (Fig 11). MTT assay was performed in F9 cells to clarify that high transfec‐ tion efficiency was not accompanied by significant toxicity of the cells (data not shown here). In order to establish that such organic-inorganic hybrid particles promote trans-gene delivery and expression through specific interactions with cell-surface molecules (integrin or E-cadherin), we added increasingly high amounts of free fibronectin to the preformed parti‐ cle suspension carrying both fibronectin and E-cadherin and incubated with the cells for the same period of time (4 hr) as followed in usual transfection procedure. Transfection efficien‐ cy decreased as the concentration of free fibronectin increased from 5 to 100μg/ml, indicat‐ ing the involvement of specific interactions between immobilized fibronectin and the corresponding specific integrin receptors (Fig 12). At a sufficiently high concentration (300 μg/ml), free fibronectin drastically reduced luciferase expression suggesting that high amount of fibronectin molecules not only saturate their specific integrins and block binding of immobilized fibronectin needed for particle internalization, but also shield cell-surface Ecadherin and prevent specific binding of particle surface-embedded E-cad-Fc chimera lead‐ ing to very low cellular uptake of particle-associated DNA and diminished luciferase expression. Since embryonic stem cells are the final target for genetic modification in regen‐ erative medicine, we applied the new transfection approach to mouse embryonic stem cells. As shown in Fig. 13, only apatite particles were extremely inefficient in transfecting the cells, whereas fibronectin-bound particles to some extent promoted GFP expression and fibronec‐ tin and E-cad-Fc-bound particles to a significant extent accelerated trans-gene expression, thus proposing that the synergistic effect is a universal way of accelerating trans-gene deliv‐ ery and expression using inorganic nano-particle-associated cell recognizable proteins. Quantitative luciferase expression in embryonic stem cells indicated that particles com‐ plexed with fibronectin and E-cad-Fc individually, promoted trans-gene expression with ef‐ ficiency approximately 9 and 7 times higher, respectively, than that achieved with the particles only (Fig. 14). However, when the particles were associated with both fibronectin and E-cadherin-Fc, a synergistic effect resulted in remarkable level of transgene expression leading to almost 40 and 28 times higher efficiency than that obtained by apatite particles

278 Advances in Biomaterials Science and Biomedical Applications

and widely used lipofectamine 2000 system [14].

**Figure 10.** Comparison of luciferase expression for differentially formulated particles. Particles were prepared by addi‐ tion of 3 μl of 1 M CaCl2, 2 μg of luciferase plasmid DNA and 5 μg of either fibronectin, E-cad-Fc or both to 1 ml bicar‐ bonate-buffered DMEM and incubation for 30 min at 370C. F9 cells were incubated with the generated particles for 4 hr and after replacement of particle-containing media with fresh media, further incubated for 1 day in order to quan‐ titate luciferase expression. Transfection efficiency was normalized after estimation of total proteins in cell lysate.

**Figure 11.** Comparison of GFP expression between only particles and fibronectin/E-cad-Fc-embedded-particles. Parti‐ cles were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of pEGFP plasmid DNA and 5 μg of fibronectin and 5 μg of Ecad-Fc to 1 ml bicarbonate-buffered DMEM and incubation for 30 min at 370C. F9 cells were incubated with the generated particles for 4 hr and after replacement of particle-containing media with fresh media, further incubated for 1 day in order to both observe and quantitate GFP expression by fluorescence microscopy and flow cytometry, re‐ spectively (scale bar, 50 μm).

**Figure 12.** Blocking of integrin-mediated trans-gene delivery by excess free fibronectin. Particles were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of luciferase plasmid DNA and 5 μg of fibronectin and 5 μg of E-cad-Fc to 1 ml bicarbonate-buffered DMEM and incubation for 30 min at 370C. F9 cells were incubated with the generated particles in presence or absence of increasingly high concentrations of free fibronectin for 4 hr and after replacement of particlecontaining media with fresh media, further incubated for 1 day in order to quantitate luciferase expression. Transfec‐ tion efficiency was normalized after estimation of total proteins in cell lysate.

**Figure 13.** Enhancement of GFP expression in mouse embryonic stem cells with fibronectin/E-cad-Fc-embedded-parti‐ cles. Particles were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of pEGFP plasmid DNA and 5 μg of fibronectin and 5 μg of E-cad-Fc to 1 ml bicarbonate-buffered DMEM and incubation for 30 min at 370C. Embryonic stem cells were incu‐ bated with the generated particles for 4 hr and after replacement of particle-containing media with fresh media, fur‐ ther incubated for 1 day in order to see GFP expression by a fluorescence microscope (scale bar, 50 μm).

**Figure 12.** Blocking of integrin-mediated trans-gene delivery by excess free fibronectin. Particles were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of luciferase plasmid DNA and 5 μg of fibronectin and 5 μg of E-cad-Fc to 1 ml bicarbonate-buffered DMEM and incubation for 30 min at 370C. F9 cells were incubated with the generated particles in presence or absence of increasingly high concentrations of free fibronectin for 4 hr and after replacement of particlecontaining media with fresh media, further incubated for 1 day in order to quantitate luciferase expression. Transfec‐

**Figure 13.** Enhancement of GFP expression in mouse embryonic stem cells with fibronectin/E-cad-Fc-embedded-parti‐ cles. Particles were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of pEGFP plasmid DNA and 5 μg of fibronectin and 5 μg of E-cad-Fc to 1 ml bicarbonate-buffered DMEM and incubation for 30 min at 370C. Embryonic stem cells were incu‐ bated with the generated particles for 4 hr and after replacement of particle-containing media with fresh media, fur‐

ther incubated for 1 day in order to see GFP expression by a fluorescence microscope (scale bar, 50 μm).

tion efficiency was normalized after estimation of total proteins in cell lysate.

280 Advances in Biomaterials Science and Biomedical Applications

**Figure 14.** Comparison of luciferase expression for differentially formulated apatite particles and liposomes. 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. Embryonic stem cells were incubated with the generated particles for 4 hr and after replacement of particle-containing media with fresh media, further in‐ cubated for 1 day in order to quantitate luciferase expression. Transfection efficiency was normalized after estimation of total proteins in cell lysate. Transfection by lipofectamine was performed according to the instructions provided by Invitrogen.

#### **3.10. DNA binding with differentially formulated cell adhesive protein-embedded particles**

Since direct mixing of DNA and cell-adhesive proteins in Ca2+ and PO4 3--containing medi‐ um prior to induction of particle formation by incubation at 370 C for 30 min, could interfere with maximum DNA loading due to the competitive binding of the proteins to the growing crystals, we investigated DNA binding efficiency by first adding DNA to the particle-prepa‐ ration medium prior to time-dependent addition of the proteins [22]. As shown in Fig. 15, in the direct mixing process (control), DNA binding is much higher for E-cadherin-Fc com‐ pared to fibronectin, indicating that E-cadherin-Fc facilitates DNA loading probably by accel‐ erating particle growth because turbidity of particle suspension was higher for E-cadherin-Fc than for fibronectin (not shown). It is worth mentioning that only particles have also higher affinity towards DNA (almost 40%) that the particles associated with fibronectin which showed lower turbidity than the particles (mentioned before), suggesting again that particle growth has a significant role in the observed DNA binding efficiency. When cell adhesive proteins were added after 5, 10 and 20 min from the start of incubation of DNA-containing particlepreparation medium, followed by incubation for an additional 25, 20 and 10 min respective‐ ly, DNA binding to the particles was enhanced to a significant extent for fibronectin, Ecadherin and fibronectin/E-cadherin-Fc compared to the control, suggesting than a competitive inhibition of DNA binding happens in the direct mixing procedure while delaying addition of the proteins to the growing crystals and DNA favors optimal DNA binding to the parti‐ cles. Decreased DNA binding to the particles with which E-cadherin and fibronectin/E-cadherin-Fc were incubated for only 1 min, could be due to the reduced growth of the particles for too long time absence of E-cadherin-Fc in particle-preparation medium.

**Figure 15.** Binding affinities of DNA to differentially formulated cell adhesive protein-embedded particles. Particles in the control samples 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 at 370C for 30 min. Formation of the particles in experimental samples was done by addition of fibronectin, E-cadherin-Fc or both after 5, 10, 20 and 29 min from the start of incubation of DNA-containing particle preparation medium, followed by incubation for an addi‐ tional 25, 20, 10 and 1 min respectively. F9 cells were incubated with the generated particles for 4 hr and after re‐ placement of particle-containing media with fresh media, further incubated up to 1 day for quantitation of luciferase expression. Transfection efficiency was normalized after estimation of total proteins in cell lysate.

#### **3.11. Size determination for differentially formulated cell adhesive protein-embedded particles**

Particle growth kinetics is correlated to the size of the finally formed particles and exces‐ sive growth lead to big size particles being inefficient for intracellular DNA delivery [11]. Since E-cadherin-Fc favors particle growth by making bridges among the neighboring Ecadherin-anchored crystals [14], prolonged incubation together with DNA for generation of functional particles might lead to large complex particles. As shown in Fig. 16, fibronectin association maintained the average particle diameter close to 300 nm whereas E-cadherin-Fc or fibronectin/E-cadherin-Fc induced the particle growth with an average diameter of approximately 900 nm. However, addition of E-cadherin-Fc or fibronectin/E-cadherin-F af‐ ter 5, 10, 20 and 29 min from the start of incubation of DNA-containing particle-prepara‐ tion medium, followed by incubation for an additional 25, 20, 10 and 1 min respectively, resulted in the particles of decreasing sizes with a minimum average value of approximate‐ ly 300 nm. On the other hand, time-dependent association of fibronectin having no role in particle growth induction, demonstrated no significant change in overall particle diame‐ ter, suggesting that particle growth is the size-determining factor for cell-adhesive proteinembedded particles.

inhibition of DNA binding happens in the direct mixing procedure while delaying addition of the proteins to the growing crystals and DNA favors optimal DNA binding to the parti‐ cles. Decreased DNA binding to the particles with which E-cadherin and fibronectin/E-cadherin-Fc were incubated for only 1 min, could be due to the reduced growth of the particles for too

**Figure 15.** Binding affinities of DNA to differentially formulated cell adhesive protein-embedded particles. Particles in the control samples 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 at 370C for 30 min. Formation of the particles in experimental samples was done by addition of fibronectin, E-cadherin-Fc or both after 5, 10, 20 and 29 min from the start of incubation of DNA-containing particle preparation medium, followed by incubation for an addi‐ tional 25, 20, 10 and 1 min respectively. F9 cells were incubated with the generated particles for 4 hr and after re‐ placement of particle-containing media with fresh media, further incubated up to 1 day for quantitation of luciferase

**3.11. Size determination for differentially formulated cell adhesive protein-embedded**

Particle growth kinetics is correlated to the size of the finally formed particles and exces‐ sive growth lead to big size particles being inefficient for intracellular DNA delivery [11]. Since E-cadherin-Fc favors particle growth by making bridges among the neighboring Ecadherin-anchored crystals [14], prolonged incubation together with DNA for generation of functional particles might lead to large complex particles. As shown in Fig. 16, fibronectin association maintained the average particle diameter close to 300 nm whereas E-cadherin-Fc or fibronectin/E-cadherin-Fc induced the particle growth with an average diameter of approximately 900 nm. However, addition of E-cadherin-Fc or fibronectin/E-cadherin-F af‐ ter 5, 10, 20 and 29 min from the start of incubation of DNA-containing particle-prepara‐ tion medium, followed by incubation for an additional 25, 20, 10 and 1 min respectively, resulted in the particles of decreasing sizes with a minimum average value of approximate‐ ly 300 nm. On the other hand, time-dependent association of fibronectin having no role in

expression. Transfection efficiency was normalized after estimation of total proteins in cell lysate.

**particles**

long time absence of E-cadherin-Fc in particle-preparation medium.

282 Advances in Biomaterials Science and Biomedical Applications

**Figure 16.** Estimation of sizes for differentially formulated cell adhesive protein-embedded particles. Following prepa‐ ration of different particles as mentioned in the legend to Figure 4, dynamic light scattering (DLS) measurement was performed with a Super-dynamic Light Scattering Spectrophotometer.

#### **3.12. Cellular delivery of DNA in association with cell adhesive protein-embedded particles**

Both DNA binding to the particles and particle size contribute to the overall uptake of DNA by cells. As shown in Fig. 17, only particles were very inefficient in delivering pro‐ pidium (PI)-labeled plasmid DNA into F9 cells whereas particles being associated with fibronectin or E-cadherin-Fc significantly increased cellular delivery of labeled DNA in a 4 hr uptake study. Moreover, particles when complexed with both fibronectin and E-cad‐ herin-Fc in direct mixing with DNA, synergistically accelerated delivery of PI-labeled DNA into the cells. Particles prepared by addition of fibronectin or fibronectin/E-cadherin-Fc after 5, 10 and 20 min from the start of incubation of labeled DNA-containing particle preparation medium and incubation for an additional 25, 20 and 10 min respectively, medi‐ ated increased cellular delivery of labeled DNA, indicating that transgene delivery is wellcontrolled by the sizes as well as the DNA-loading efficiency of cell adhesive proteinembedded particles. Reduced DNA uptake level for the small size particles with which cell-adhesive proteins were incubated for a very short time (1 min) could be accounted for their inefficient binding with the cell-recognition molecules. The reason for low DNA up‐ take for the particles to which only E-cadherin-Fc was adsorbed in a time-dependent man‐ ner, is still not clear and might be related to the serum instability of the complex particles at the time of transgene delivery.

**Figure 17.** Differentially formulated cell adhesive protein-embedded particles for cellular delivery of DNA. Particles in the control samples were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of PI-labelled plasmid DNA and 2 μg of either fibronectin, E-cad-Fc or both to 1 ml bicarbonate-buffered DMEM and incubation at 37<sup>0</sup>C for 30 min. Formation of the particles in experimental samples was done by addition of fibronectin, E-cadherin-Fc or both after 5, 10, 20 and 29 min from the start of incubation of DNA-containing particle preparation medium, followed by incubation for an addi‐ tional 25, 20, 10 and 1 min respectively. F9 cells were incubated with the generated particles for 4 hr, washed with 5 mM EDTA in PBS and visualized by a fluorescence microscope (scale bar, 100 μm).

#### **3.13. Transfection efficiency achieved with cell adhesive protein-embedded particles**

Since transgene expression is the result of overcoming a number of barriers including entry into the cells, release from the particles and endosomes, and finally nuclear translocation [11], we checked whether accelerated DNA delivery owing to the improved DNA loading capacity and smaller sizes of fibronectin and E-cadherin-Fc-anchored carbonate apatite par‐ ticles, contributed to the similar extent to final protein expression (Fig. 18). Quantitative luci‐ ferase expression demonstrated that particles generated by addition of fibronectin and fibronectin/E-cadherin-Fc after 5 min from the start of incubation of DNA-containing medi‐ um and incubation for an additional 25 min, enhanced 2 and 3-fold higher transgene expres‐ sion than the control samples prepared by direct mixing with DNA. This is a significant achievement considering the high expression level already achieved with control samples [15]. The decline in luciferase expression for other samples is consistent with the low effi‐ ciency of DNA delivery as described before.

**Figure 18.** Intracellular expression of luciferase gene delivered by differentially formulated cell adhesive protein-em‐ bedded particles. Particles in the control samples were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of PI-labelled plasmid DNA and 2 μg of either fibronectin, E-cad-Fc or both to 1 ml bicarbonate-buffered DMEM and incubation at 370C for 30 min. Formation of the particles in experimental samples was done by addition of fibronectin, E-cadherin-Fc or both after 5, 10, 20 and 29 min from the start of incubation of DNA-containing particle preparation medium, followed by incubation for an additional 25, 20, 10 and 1 min respectively. F9 cells were incubated with the generated particles for 4 hr and after replacement of particle-containing media with fresh media, further incubated up to 1 day in order to quantitate luciferase expression. Transfection efficiency was normalized after estimation of total proteins in cell lysate.

#### **3.14. Role of protein kinase C on immobilized fibronectin and E-cad-Fc-mediated gene delivery**

**Figure 17.** Differentially formulated cell adhesive protein-embedded particles for cellular delivery of DNA. Particles in the control samples were prepared by addition of 3 μl of 1 M CaCl2, 2 μg of PI-labelled plasmid DNA and 2 μg of either fibronectin, E-cad-Fc or both to 1 ml bicarbonate-buffered DMEM and incubation at 37<sup>0</sup>C for 30 min. Formation of the particles in experimental samples was done by addition of fibronectin, E-cadherin-Fc or both after 5, 10, 20 and 29 min from the start of incubation of DNA-containing particle preparation medium, followed by incubation for an addi‐ tional 25, 20, 10 and 1 min respectively. F9 cells were incubated with the generated particles for 4 hr, washed with 5

**3.13. Transfection efficiency achieved with cell adhesive protein-embedded particles**

Since transgene expression is the result of overcoming a number of barriers including entry into the cells, release from the particles and endosomes, and finally nuclear translocation [11], we checked whether accelerated DNA delivery owing to the improved DNA loading capacity and smaller sizes of fibronectin and E-cadherin-Fc-anchored carbonate apatite par‐ ticles, contributed to the similar extent to final protein expression (Fig. 18). Quantitative luci‐ ferase expression demonstrated that particles generated by addition of fibronectin and

mM EDTA in PBS and visualized by a fluorescence microscope (scale bar, 100 μm).

284 Advances in Biomaterials Science and Biomedical Applications

Since protein kinase C (PKC) in "inside-out" signaling cascade enhances integrin affinity towards ECM proteins promoting cell adhesion and spreading [23, 24] and up regulates endocytosis and recycling of E-cadherin [21], we have investigated the effect of Phorbol 12-myristate 13-acetate (PMA), a specific activator of PKC on trans-gene delivery mediat‐ ed by particle-immobilized fibronectin and E-cadherin-Fc. As shown in Fig. 19, while on‐ ly carbonate apatite particles are very inefficient in transfecting F9 cells even in presence of increasing doses of PMA (0 to 100 nM), fibronectin- or E-cad-Fc-embedded particles showed significant increment in luciferase gene expression ( 2 to 10 times) depending on PMA concentrations. Surprisingly, particles when associated with both of the "cell adhe‐ sive molecules" remarkably enhanced trans-gene expression resulting in almost 8, 14, 20 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‐ ed DNA in cytoplasm [27] (Fig 19).

**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 expression.

#### **3.15. Transfection efficiency achieved in leukemia cells with cell adhesive proteinembedded particles**

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 through integrin-mediated endocytosis [22].

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) being able to bind fibronectin [22], particles with electrostatically associated fibronectin could recognize any of the two receptors for efficient endocytosis in Jurkat cells leading to high transgene expression. However, particles with adsorbed E-cadherin-Fc reduced trans‐ fection efficiency below the level obtained with particles only, indicating that binding of Ecadherin-Fc probably neutralizes the positive charges of the particles as required for their subsequent interaction with anionic cell surface and additionally, E-cadherin-Fc on the par‐ ticle surface might have low affinity interaction with the cell membrane integrin (αEβ7). On the other hand, the highest gene expression obtained with the particles complexed with both fibronectin and E-cadherin-Fc could be interpreted by the strong affinity of the composite particles towards the cell membrane due to the specific and synchronized recognition of the two different ligands on particle surface to their corresponding integrin receptors on plasma membrane, resulting in fast endocytosis of the particles along with DNA.

**Figure 20.** Comparison of luciferase expression among differentially formulated apatite particles. Particles were pre‐ pared 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. Jurkat cells were incubated with the generated particles for 1 day followed by quantitation of luciferase expression. Transfection efficiency was normalized after esti‐ mation of total proteins in cell lysate. Transfection by lipofectamine was performed according to the instructions pro‐ vided by Invitrogen. Reproducibility of the result was established by performing same the experiment in another day.
