In contrast, pDNA/DODAB/MO lipoplexes at ΧDODAB ≤ 0.5 (Fig. 2B) show high-curvature zones where lipid bilayers intercross each other with pDNA monolayers stacked between them. These high-curvature zones have been interpreted as MO-rich domains that alternate with DODAB-rich domains presenting multilamellar organization. The FFT diagrams show that these MO-rich domains possess a distinct structural organization with bi-orientated patterns in angles of 90% between them, consistent with the existence of cubic inverted

The DODAB/MO aggregate organization influences the final structural properties of the resulting pDNA/DODAB/MO lipoplexes, with MO content having a dramatic effect on how

By definition, a *helper* lipid (also termed as co-lipid or simply adjuvant) is any neutral surfactant not directly contributing to NA condensation or to targeting of the cell membrane by the lipoplex. *Helper* lipids enhance transfection efficiency by forming non-lamellar structures that intervene in several steps of the transfection process [73, 74]. These nonlamellar structures influence transfection efficiency in at least two ways: i) lipoplex-cell membrane fusion promoted by the fusogenic character of the *helper*; ii) improved endosomal escape of NA due to the disruption of the endosomal membrane by these structures prior to

Dioleoylphosphatidylethanolamine (DOPE) is the most established *helper* used in non-viral vectors and is known to enhance transfection mediated by different cationic liposomal formulations [76-79]. DOPE stimulates the formation of inverted hexagonal structures (HII –

**2.2. Role of MO as helper lipid in pDNA/DODAB/MO lipoplexes**

not enhance DNA delivery and only increases toxicity in the exposed cells [72].

(Fig. 2A'', 2A''' and 2A'''') [38].

bicontinuous mesophases (Fig. 2B'', 2B''' and 2B'''') [38].

DNA is condensed and protected within the membrane.

endosome/lysosome fusion, which would lead to NA degradation [75].

**Figure 1.** Phase scan imaging of neat DODAB (A, A'); ΧDODAB = 0.7 (B, B'); ΧDODAB = 0.5 (C, C'); ΧDODAB = 0.2 (D, D') and neat MO (E, E') at 25°C. The images on the left side were obtained under polarized light and the images on the right side were obtained with normal light, using DIC lenses. Scale Bar: 200 µm. Abbreviations: Lα, lamellar liquid crystalline phase; Q, cubic isotropic phase; L, isotropic phase. Adapted from [40].

## **2.2. Role of MO as helper lipid in pDNA/DODAB/MO lipoplexes**

248 Cell Interaction

Adapted from [40].

**Figure 1.** Phase scan imaging of neat DODAB (A, A'); ΧDODAB = 0.7 (B, B'); ΧDODAB = 0.5 (C, C'); ΧDODAB = 0.2 (D, D') and neat MO (E, E') at 25°C. The images on the left side were obtained under polarized light and the images on the right side were obtained with normal light, using DIC lenses. Scale Bar: 200 µm. Abbreviations: Lα, lamellar liquid crystalline phase; Q, cubic isotropic phase; L, isotropic phase.

The incubation of nucleic acids with DODAB/MO mixtures or other cationic vesicle formulation leads to the formation of lipoplexes [67, 68]. The electrostatic interaction between opposite charges is the key factor that determines the adsorption of the cationic vesicles to the DNA molecules, a transient state that ends when a critical cationic vesicle concentration is reached. This leads to the disruption of the lipid vesicles which allows the formation of highly organized structures where the DNA molecules are tightly condensed between adjacent bilayers – the so-called lipoplexes [69-71]. The excess of cationic lipid is required for lipoplex binding to the cell surface but any subsequent addition of cationic lipid to the complex does not enhance DNA delivery and only increases toxicity in the exposed cells [72].

Lipoplexes such as the pDNA/DODAB/MO system can be directly visualized by techniques such as cryo-TEM imaging (Fig. 2), which also gives information on the structural properties of the system (size, compactation, organization) [38]. Cryo-TEM imaging reveals that pDNA/DODAB/MO lipoplexes present the same dual phase diagram as obtained for DODAB/MO lipid mixtures [38]. pDNA/DODAB/MO lipoplexes at ΧDODAB > 0.5 (Fig. 2A) exhibit a multilamellar structure consisting of stacked alternating lipid bilayers and pDNA monolayers. The analysis using Fast Fourier Transforms (FFT) corroborates this observation, by denoting a mono-orientated organization pattern at repeating distances of about 5 nm (Fig. 2A'', 2A''' and 2A'''') [38].

In contrast, pDNA/DODAB/MO lipoplexes at ΧDODAB ≤ 0.5 (Fig. 2B) show high-curvature zones where lipid bilayers intercross each other with pDNA monolayers stacked between them. These high-curvature zones have been interpreted as MO-rich domains that alternate with DODAB-rich domains presenting multilamellar organization. The FFT diagrams show that these MO-rich domains possess a distinct structural organization with bi-orientated patterns in angles of 90% between them, consistent with the existence of cubic inverted bicontinuous mesophases (Fig. 2B'', 2B''' and 2B'''') [38].

The DODAB/MO aggregate organization influences the final structural properties of the resulting pDNA/DODAB/MO lipoplexes, with MO content having a dramatic effect on how DNA is condensed and protected within the membrane.

By definition, a *helper* lipid (also termed as co-lipid or simply adjuvant) is any neutral surfactant not directly contributing to NA condensation or to targeting of the cell membrane by the lipoplex. *Helper* lipids enhance transfection efficiency by forming non-lamellar structures that intervene in several steps of the transfection process [73, 74]. These nonlamellar structures influence transfection efficiency in at least two ways: i) lipoplex-cell membrane fusion promoted by the fusogenic character of the *helper*; ii) improved endosomal escape of NA due to the disruption of the endosomal membrane by these structures prior to endosome/lysosome fusion, which would lead to NA degradation [75].

Dioleoylphosphatidylethanolamine (DOPE) is the most established *helper* used in non-viral vectors and is known to enhance transfection mediated by different cationic liposomal formulations [76-79]. DOPE stimulates the formation of inverted hexagonal structures (HII – Fig 3A) which represent a major structural variation from the classic multilamellar sandwich model of lipoplex organization (*Lα*C – Fig. 3B) [80]. However, its application in gene therapy has been strongly limited because of the strong cytotoxicity associated with it [81, 82].

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery 251

This evidence has motivated the search for new *helpers* with higher levels of biocompatibility while maintaining the same efficiency as DOPE. Cholesterol is one of such molecules due to its ability to modify bilayer fluidity [83]. Inclusion of cholesterol results in the formation of complexes that are more stable but less efficient *in vitro* compared to DOPEcontaining lipoplexes. In contrast, addition of cholesterol results in more efficient complexes

MO is another promising alternative to common *helper* lipids, as it seems to combine positive aspects of both DOPE and cholesterol: tendency to promote inverted non-lamellar structures similarly to DOPE (although different from the common inverted hexagonal structures – Fig. 3C) and the fluidizing effect of cholesterol, which increases the fusogenicity

**Figure 3.** Different types of pDNA/cationic lipid structural organizations: A - inverted non-lamellar hexagonal structure characteristic of cationic vesicles containing DOPE at ΧDOPE ≥ 0.5); B - lamellar structural characteristic of cationic vesicles containing ΧHelper ≤ 0.5; and C - inverted bicontinuous cubic structure characteristic of cationic vesicles containing MO at ΧMO ≥ 0.5. Double-tailed surfactant with grey-headgroup represents cationic lipid, double-tailed surfactant with white-headgroup represents DOPE and single-tailed surfactant with white-headgroup represents MO. Grey-coloured regions represent cationic lipid rich-domains and white-coloured regions represent MO or DOPE rich-domains.

The fluidizing effect of MO contributes favourably to the complexation efficiency of DNA, quickening lipoplex formation [41]. At the same time, the formed inverted bicontinuous cubic mesophases improve the resistance of aggregates to extracellular component

for *in vivo* application [84, 85].

of the lipoplexes.

**Figure 2.** Cryo-TEM imaging of pDNA/DODAB:MO lipoplexes at C.R. (+/-) 4.0 (1mM total lipid). Panels A and B represent two different DODAB:MO molar fractions (2:1 and 1:1, respectively) from which have been selected two distinct zones A' and B'. The corresponding FFT diagrams A'' and B'' are shown after the appliance of "Mask" tool in the Digital Micrograph™ (GATAN) software (A''' and B'''). The inverse FFT diagrams of the previous images allow the emergence of distinct structural patterns: mono-orientated organization consistent with the existence of lamellar structures for high DODAB contents (A'''', 2:1) and 90° bi-orientated organization associated with inverted bicontinuous cubic mesophases for high MO contents (B'''', 1:1). Magnification: 50 000x. Adapted from [38].

This evidence has motivated the search for new *helpers* with higher levels of biocompatibility while maintaining the same efficiency as DOPE. Cholesterol is one of such molecules due to its ability to modify bilayer fluidity [83]. Inclusion of cholesterol results in the formation of complexes that are more stable but less efficient *in vitro* compared to DOPEcontaining lipoplexes. In contrast, addition of cholesterol results in more efficient complexes for *in vivo* application [84, 85].

250 Cell Interaction

Fig 3A) which represent a major structural variation from the classic multilamellar sandwich model of lipoplex organization (*Lα*C – Fig. 3B) [80]. However, its application in gene therapy has been strongly limited because of the strong cytotoxicity associated with it [81, 82].

**Figure 2.** Cryo-TEM imaging of pDNA/DODAB:MO lipoplexes at C.R. (+/-) 4.0 (1mM total lipid). Panels A and B represent two different DODAB:MO molar fractions (2:1 and 1:1, respectively) from which have been selected two distinct zones A' and B'. The corresponding FFT diagrams A'' and B'' are shown after the appliance of "Mask" tool in the Digital Micrograph™ (GATAN) software (A''' and B'''). The inverse FFT diagrams of the previous images allow the emergence of distinct structural patterns: mono-orientated organization consistent with the existence of lamellar structures for high DODAB contents (A'''', 2:1) and 90° bi-orientated organization associated with inverted bicontinuous cubic

mesophases for high MO contents (B'''', 1:1). Magnification: 50 000x. Adapted from [38].

MO is another promising alternative to common *helper* lipids, as it seems to combine positive aspects of both DOPE and cholesterol: tendency to promote inverted non-lamellar structures similarly to DOPE (although different from the common inverted hexagonal structures – Fig. 3C) and the fluidizing effect of cholesterol, which increases the fusogenicity of the lipoplexes.

**Figure 3.** Different types of pDNA/cationic lipid structural organizations: A - inverted non-lamellar hexagonal structure characteristic of cationic vesicles containing DOPE at ΧDOPE ≥ 0.5); B - lamellar structural characteristic of cationic vesicles containing ΧHelper ≤ 0.5; and C - inverted bicontinuous cubic structure characteristic of cationic vesicles containing MO at ΧMO ≥ 0.5. Double-tailed surfactant with grey-headgroup represents cationic lipid, double-tailed surfactant with white-headgroup represents DOPE and single-tailed surfactant with white-headgroup represents MO. Grey-coloured regions represent cationic lipid rich-domains and white-coloured regions represent MO or DOPE rich-domains.

The fluidizing effect of MO contributes favourably to the complexation efficiency of DNA, quickening lipoplex formation [41]. At the same time, the formed inverted bicontinuous cubic mesophases improve the resistance of aggregates to extracellular component

destabilization, thereby potentially enhancing transfection efficiency [38]. MO-based aggregates induce a relatively low cytotoxicity level, which further reinforces its use as a new *helper* lipid in this type of non-viral systems. *In vivo* evaluation of MO-based lipoplexes shall confirm the potential for this neutral surfactant to replace classic *helpers* in lipofection formulations, although some promising results have already been obtained for other cationic lipid formulations that also form inverted bicontinuous cubic structures [86-88]. Ethylphosphatidylcholines are a family of positively charged membrane lipid derivatives that promote the formation of *QIIG* and *QIIP* structures, having been linked to high levels of transfection efficiency with low cytotoxicity in several animal cell lines [89, 90], consubstantiating MO's potential role in gene delivery.

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery 253

dioleoyldimethylammonium propane (DODAP) or cholesteryl hemisuccinate (CHEMS)

Another major breakthrough with impact in gene therapy was the possibility of specific cell targeting by liposomes. Amphiphiles with hydrophilic headgroups could be chemically linked to molecules such as folate, transferrin or the epidermal growth factor that potentiate specific delivery to cancer cells, markedly increasing the therapeutic benefits achieved with

More recently, cationic lipids with amino acid headgroup (serine, alanine) [119, 120] and sugar-based cationic lipids (D-galactose) have appeared as promising families of cationic surfactants [121, 122]. Small molecular weight peptides (glutamate, cysteine) augment the hydrophilicity of the lipoplex surface, as with small surface sugars (galactose, mannose) that

An effective delivery system must confer stability to complexed NA in physiological conditions [123, 124]. Systemic delivery of NA requires a stealth carrier that protects NA from indiscriminate interaction with complement and coagulation pathways that lead to rapid removal from blood circulation of the lipoplexes by opsonization [125-127]. pDNA/DODAB/MO lipoplexes were therefore tested regarding their sensitivity when simulating their interaction with the body (temperature, salt, exposure to serum, nucleases

Fig. 4 shows the variation of free pDNA fraction after incubation of pDNA/DODAB:MO lipoplexes (2:1, 1:1 and 1:2) with different constituents of the plasma. Increasing the temperature from 25°C to physiological temperature (37°C) leads to a reduced but visible release of pDNA from the lipoplex, more evident for lower MO contents. The gel phase of DODAB (ΧDODAB> 0.5) is clearly more disturbed by incubation at higher temperature than the liquid-crystalline phase of DODAB/MO lipid mixtures (ΧDODAB ≤ 0.5). This tendency is maintained upon NaCl addition at physiological concentration (150mM), showing the

DODAB/MO formulations with varying MO content behave very differently when exposed to serum (Fig. 4). Serum may strongly interfere with lipoplexes, both *in vitro* and *in vivo*, causing lipoplex-protein aggregation that lead to degradation of the genetic material and possibly clogging the blood vessels in intravenous application [129]. pDNA/DODAB/MO lipoplexes release up to 30% of the initially complexed pDNA when incubated for 30min with bovine serum albumin, the major constituent of bovine serum, particularly in the case of formulations with low MO content. MO contributes, in fact, to a better resistance of pDNA/DODAB/MO to extracellular components, eventually related to the inverted bicontinuous cubic structures present that reduce the exposure of DNA molecules to the

protective role of MO upon the electrostatic imbalance provoked by salt addition.

[112, 113].

lipoplexes, with little secondary effects [114-118].

additionally allow targetability of the lipoplexes.

**3. Lipoplex interaction with extracellular milieu**

and membrane lipases), to be validated for systemic applications [128].

**3.1. Resistance to components of biological fluids**

## **2.3. Recent progress in gene delivery with cationic lipids**

The quest for the perfect cationic liposome formulation has been based on empirical testing of novel surfactant molecules that had never been previously used for NA delivery [91-93]. The only goals for candidate molecules are the attainance of high transfection efficiency with low cytotoxicity [94, 95].

After the first generation of cationic lipids based on double-chain surfactants with plain ammonium headgroups (DODAB, DOTAP, DOTMA or DMRIE) [96, 97], soon came cationic lipids with poly-ammonium and multivalent functional radicals (DOGS, DOSPA). The latter exhibited higher transfection efficiencies but also higher cytotoxicity due to the immunogenicity of the cationic ammonium headgroups [98, 99]. This negative effect was balanced with the appearance of *helpers* (DOPE) [74] and natural lipid-derivatives such as cholesterol [100] or glycerol [101], although sometimes compromising transfection efficiency. Gemini-dimeric surfactants also presented promising potential but with significant toxicological consequences [102-106].

Polyethylene glycol (PEG)-based lipids emerged as interesting hydrophilic polymer-based surfactants that could provide steric stability to cationic liposomes, increasing lipoplex lifetime in the bloodstream and also decreasing the toxic effects observed *in vitro* and *in vivo*  [107-109]. The polymeric counterpart of the PEG-based surfactants (variable both in chain length and branching) forms a protective surface coating that inhibits the adhesion of plasma components which could promote NA release and particle aggregation [109]. This protective effect is enhanced by including up to 5-10% of PEG in the liposomal formulation, with no visible effects on lipoplex structure [110]. PEG addition reduces net electric charge and increases hydration of the liposome surface, decreasing immunogenicity and cytotoxicity elicited by the particles. Nevertheless, at high concentrations, these polymers are known to be toxic and of difficult clearance from the organism. Therefore, when developing PEGylated particles, one must weigh advantages and disadvantages of including PEG, especially when aiming for long-term therapeutic administration [111].

Inclusion of pH-sensitive molecules in the formulations has been shown to improve transgene expression by favouring DNA release from the endosomal compartment. Examples of pH-sensitive molecules used in non-viral gene delivery include polyhistidine, dioleoyldimethylammonium propane (DODAP) or cholesteryl hemisuccinate (CHEMS) [112, 113].

Another major breakthrough with impact in gene therapy was the possibility of specific cell targeting by liposomes. Amphiphiles with hydrophilic headgroups could be chemically linked to molecules such as folate, transferrin or the epidermal growth factor that potentiate specific delivery to cancer cells, markedly increasing the therapeutic benefits achieved with lipoplexes, with little secondary effects [114-118].

More recently, cationic lipids with amino acid headgroup (serine, alanine) [119, 120] and sugar-based cationic lipids (D-galactose) have appeared as promising families of cationic surfactants [121, 122]. Small molecular weight peptides (glutamate, cysteine) augment the hydrophilicity of the lipoplex surface, as with small surface sugars (galactose, mannose) that additionally allow targetability of the lipoplexes.

## **3. Lipoplex interaction with extracellular milieu**

## **3.1. Resistance to components of biological fluids**

252 Cell Interaction

low cytotoxicity [94, 95].

destabilization, thereby potentially enhancing transfection efficiency [38]. MO-based aggregates induce a relatively low cytotoxicity level, which further reinforces its use as a new *helper* lipid in this type of non-viral systems. *In vivo* evaluation of MO-based lipoplexes shall confirm the potential for this neutral surfactant to replace classic *helpers* in lipofection formulations, although some promising results have already been obtained for other cationic lipid formulations that also form inverted bicontinuous cubic structures [86-88]. Ethylphosphatidylcholines are a family of positively charged membrane lipid derivatives that promote the formation of *QIIG* and *QIIP* structures, having been linked to high levels of transfection efficiency with low cytotoxicity in several animal cell lines [89, 90],

The quest for the perfect cationic liposome formulation has been based on empirical testing of novel surfactant molecules that had never been previously used for NA delivery [91-93]. The only goals for candidate molecules are the attainance of high transfection efficiency with

After the first generation of cationic lipids based on double-chain surfactants with plain ammonium headgroups (DODAB, DOTAP, DOTMA or DMRIE) [96, 97], soon came cationic lipids with poly-ammonium and multivalent functional radicals (DOGS, DOSPA). The latter exhibited higher transfection efficiencies but also higher cytotoxicity due to the immunogenicity of the cationic ammonium headgroups [98, 99]. This negative effect was balanced with the appearance of *helpers* (DOPE) [74] and natural lipid-derivatives such as cholesterol [100] or glycerol [101], although sometimes compromising transfection efficiency. Gemini-dimeric surfactants also presented promising potential but with

Polyethylene glycol (PEG)-based lipids emerged as interesting hydrophilic polymer-based surfactants that could provide steric stability to cationic liposomes, increasing lipoplex lifetime in the bloodstream and also decreasing the toxic effects observed *in vitro* and *in vivo*  [107-109]. The polymeric counterpart of the PEG-based surfactants (variable both in chain length and branching) forms a protective surface coating that inhibits the adhesion of plasma components which could promote NA release and particle aggregation [109]. This protective effect is enhanced by including up to 5-10% of PEG in the liposomal formulation, with no visible effects on lipoplex structure [110]. PEG addition reduces net electric charge and increases hydration of the liposome surface, decreasing immunogenicity and cytotoxicity elicited by the particles. Nevertheless, at high concentrations, these polymers are known to be toxic and of difficult clearance from the organism. Therefore, when developing PEGylated particles, one must weigh advantages and disadvantages of including PEG, especially when aiming for long-term therapeutic administration [111].

Inclusion of pH-sensitive molecules in the formulations has been shown to improve transgene expression by favouring DNA release from the endosomal compartment. Examples of pH-sensitive molecules used in non-viral gene delivery include polyhistidine,

consubstantiating MO's potential role in gene delivery.

significant toxicological consequences [102-106].

**2.3. Recent progress in gene delivery with cationic lipids**

An effective delivery system must confer stability to complexed NA in physiological conditions [123, 124]. Systemic delivery of NA requires a stealth carrier that protects NA from indiscriminate interaction with complement and coagulation pathways that lead to rapid removal from blood circulation of the lipoplexes by opsonization [125-127]. pDNA/DODAB/MO lipoplexes were therefore tested regarding their sensitivity when simulating their interaction with the body (temperature, salt, exposure to serum, nucleases and membrane lipases), to be validated for systemic applications [128].

Fig. 4 shows the variation of free pDNA fraction after incubation of pDNA/DODAB:MO lipoplexes (2:1, 1:1 and 1:2) with different constituents of the plasma. Increasing the temperature from 25°C to physiological temperature (37°C) leads to a reduced but visible release of pDNA from the lipoplex, more evident for lower MO contents. The gel phase of DODAB (ΧDODAB> 0.5) is clearly more disturbed by incubation at higher temperature than the liquid-crystalline phase of DODAB/MO lipid mixtures (ΧDODAB ≤ 0.5). This tendency is maintained upon NaCl addition at physiological concentration (150mM), showing the protective role of MO upon the electrostatic imbalance provoked by salt addition.

DODAB/MO formulations with varying MO content behave very differently when exposed to serum (Fig. 4). Serum may strongly interfere with lipoplexes, both *in vitro* and *in vivo*, causing lipoplex-protein aggregation that lead to degradation of the genetic material and possibly clogging the blood vessels in intravenous application [129]. pDNA/DODAB/MO lipoplexes release up to 30% of the initially complexed pDNA when incubated for 30min with bovine serum albumin, the major constituent of bovine serum, particularly in the case of formulations with low MO content. MO contributes, in fact, to a better resistance of pDNA/DODAB/MO to extracellular components, eventually related to the inverted bicontinuous cubic structures present that reduce the exposure of DNA molecules to the plasma constituents. In fact, the results suggest a direct correlation between lipoplex stability and MO content.

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery 255

The adsorption and uptake of lipoplexes may be affected by the presence of proteoglycans at the plasma cell membrane surface. It is therefore important to study how lipoplexes interact with these extracellular matrix components during cell transfection. Association of lipoplexes with negative polyelectrolytes free in solution might also be useful to evaluate

Proteoglycans (membrane receptors consisting of a protein core and one or more anionic glycosaminoglycan chains including heparin, dermatan and chondroitin sulphates) were identified as the mediating agents for cationic liposome/DNA cellular uptake both *in vitro* and *in vivo* [135]. Lipoplex/proteoglycan interaction is suggested to depend upon three major aspects: the ionic strength, the effect of *helper* lipids and of the glycosaminoglycan

When the lipoplexes interact with heparin and heparin sulphate, the negative charge of the polyelectrolytes determines NA release from the lipoplex through the same type of

On subjecting pDNA/DODAB:MO (2:1 and 1:1) lipoplexes to increasing amounts of heparin (HEP), the improved resistance and stability of the lipoplexes obtained with increasing amounts of MO could be confirmed (Fig. 5). The fact that the system with higher MO content (ΧDODAB= 0.5) shows enhanced resistance to heparin relatively to pDNA/DODAB:MO (2:1) lipoplexes suggests that pDNA dissociation is mainly dependent on structural

In addition to the efficiency of MO based lipoplexes, patient tolerability is determinant for therapeutic application of these systems. *In vitro* toxicity tests are a useful, time and costeffective first approach in the validation process of a therapeutic agent. The adverse effects of liposomes on cells can be identified through different assays that look at particular aspects of cell behavior, such as metabolism, proliferation or cell membrane integrity. To determine if liposomal formulations will be well tolerated by all cells it will contact with, it is important to test cytotoxicity using different cell types. The cell lines should be selected: i) to evaluate how target cells will react; ii) to predict eventual toxicity for the heart and liver, by using cardiomyocytes and hepatocytes, respectively; iii) to screen if the liposomes can be applied to all types of cells. In the case of DODAB:MO liposomes, four different mammalian cell lines (HEK 293, BJ5ta, L929 and C2C12) were exposed for two days to increasing concentrations of these systems, after which different analytical methods were applied (Figs. 6

The cell lines presented here are routinely used for toxicity studies and are commercially available. The human Embryonic Kidney (HEK) 293 cell line was originally derived from

cooperative process that is responsible for lipoplex formation [136-138].

properties (Fig. 2) rather than physicochemical properties of the lipoplexes.

**4. Modulation of cell behaviour by lipoplexes**

**3.2. Lipoplex adhesion to the cell surface**

eventual loss of pDNA at the cell surface [134].

structure [134, 135].

**4.1. Cytotoxicity**

to 8).

Some authors have managed to transiently overcome this inhibitory effect of serum on lipofection by increasing the charge ratio (+/-) of cationic liposome to DNA [130, 131]. Significantly enhanced gene transfer has also been achieved by pre-incubating the delivery system with serum proteins prior to NA complexation [132, 133].

**Figure 4.** Resistance of pDNA/DODAB:Monoolein lipoplexes to components of biological fluids. Variation on the percentage of free pDNA upon incubation with DODAB:Monoolein liposomes (2:1, 1:1, and 1:2) at CR (+/-) 2.0, and subsequently exposed to a temperature increase from 25°C to 37°C in the presence of NaCl salt (150mM) and BSA (0.5g/L) at incubation times of 30 min. The values were calculated through spectral decomposition of ethidium bromide steady-state fluorescence, as described elsewhere [38].

## **3.2. Lipoplex adhesion to the cell surface**

254 Cell Interaction

elsewhere [38].

stability and MO content.

plasma constituents. In fact, the results suggest a direct correlation between lipoplex

Some authors have managed to transiently overcome this inhibitory effect of serum on lipofection by increasing the charge ratio (+/-) of cationic liposome to DNA [130, 131]. Significantly enhanced gene transfer has also been achieved by pre-incubating the delivery

**Figure 4.** Resistance of pDNA/DODAB:Monoolein lipoplexes to components of biological fluids. Variation on the percentage of free pDNA upon incubation with DODAB:Monoolein liposomes (2:1, 1:1, and 1:2) at CR (+/-) 2.0, and subsequently exposed to a temperature increase from 25°C to 37°C in the presence of NaCl salt (150mM) and BSA (0.5g/L) at incubation times of 30 min. The values were calculated through spectral decomposition of ethidium bromide steady-state fluorescence, as described

system with serum proteins prior to NA complexation [132, 133].

The adsorption and uptake of lipoplexes may be affected by the presence of proteoglycans at the plasma cell membrane surface. It is therefore important to study how lipoplexes interact with these extracellular matrix components during cell transfection. Association of lipoplexes with negative polyelectrolytes free in solution might also be useful to evaluate eventual loss of pDNA at the cell surface [134].

Proteoglycans (membrane receptors consisting of a protein core and one or more anionic glycosaminoglycan chains including heparin, dermatan and chondroitin sulphates) were identified as the mediating agents for cationic liposome/DNA cellular uptake both *in vitro* and *in vivo* [135]. Lipoplex/proteoglycan interaction is suggested to depend upon three major aspects: the ionic strength, the effect of *helper* lipids and of the glycosaminoglycan structure [134, 135].

When the lipoplexes interact with heparin and heparin sulphate, the negative charge of the polyelectrolytes determines NA release from the lipoplex through the same type of cooperative process that is responsible for lipoplex formation [136-138].

On subjecting pDNA/DODAB:MO (2:1 and 1:1) lipoplexes to increasing amounts of heparin (HEP), the improved resistance and stability of the lipoplexes obtained with increasing amounts of MO could be confirmed (Fig. 5). The fact that the system with higher MO content (ΧDODAB= 0.5) shows enhanced resistance to heparin relatively to pDNA/DODAB:MO (2:1) lipoplexes suggests that pDNA dissociation is mainly dependent on structural properties (Fig. 2) rather than physicochemical properties of the lipoplexes.

## **4. Modulation of cell behaviour by lipoplexes**

## **4.1. Cytotoxicity**

In addition to the efficiency of MO based lipoplexes, patient tolerability is determinant for therapeutic application of these systems. *In vitro* toxicity tests are a useful, time and costeffective first approach in the validation process of a therapeutic agent. The adverse effects of liposomes on cells can be identified through different assays that look at particular aspects of cell behavior, such as metabolism, proliferation or cell membrane integrity. To determine if liposomal formulations will be well tolerated by all cells it will contact with, it is important to test cytotoxicity using different cell types. The cell lines should be selected: i) to evaluate how target cells will react; ii) to predict eventual toxicity for the heart and liver, by using cardiomyocytes and hepatocytes, respectively; iii) to screen if the liposomes can be applied to all types of cells. In the case of DODAB:MO liposomes, four different mammalian cell lines (HEK 293, BJ5ta, L929 and C2C12) were exposed for two days to increasing concentrations of these systems, after which different analytical methods were applied (Figs. 6 to 8).

The cell lines presented here are routinely used for toxicity studies and are commercially available. The human Embryonic Kidney (HEK) 293 cell line was originally derived from human embryonic kidney cells grown in tissue culture, from which 293T cell line is derived. BJ5ta cells are normal human foreskin fibroblasts immortalized with telomerase. Murine cell lines L929 and C2C12 are fibroblasts and myoblasts, respectively.

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery 257

cell lines, especially with a lipid concentration ≥ 20 µg/ml (Fig. 6). At these higher concentrations, DODAB:MO (1:1) induced lower levels of cytotoxicity in all the cell types, which probably reflects the higher content of MO and concomitant lower content of cationic lipid. Interestingly, the cell membrane integrity assay did not reveal such obvious, concentration-dependent variations in cytotoxicity (Fig. 7). The results obtained with the proliferation test (Fig. 8) were quite concordant with those from the metabolic assay (Fig. 6), indicating again the L929 and C2C12 cells as more sensitive, while BJ5ta proliferation was

Lipoplexes prepared from these liposomal formulations, at concentrations typically used in transfection experiments, constantly leading to slightly lower viability rates compared to the

The fact that MO-based aggregates cause reduced levels of cytotoxicity for concentrations typically used on transfection assays, reinforcing the use of MO as a new *helper* lipid in this type of non-viral systems. Even if there is general agreement in the reduced toxicity of liposomes as non-viral vectors, these results emphasize the need for accurate liposome/lipoplex evaluation to better assess human risk prior to using them as lipofection

**Figure 6.** Evaluation of the cytotoxicity (metabolic assay) in four different mammalian cell lines (BJ5-ta, L929, 293 and C2C12) induced by varying concentrations of DODAB:MO-based liposomes after 48 h of incubation. C\_DMSO: cells incubated with 30 % DMSO; C\_Cells: cells alone. The mean (+/−) SD was obtained from two independent experiments. MTT assay can be used to estimate cell viability, specifically as marker of the cell metabolic capacity. The soluble tetrazolium MTT is reduced by metabolically active cells, thus the developed purple color proportional to the number of viable cells.

clearly increased when incubated with up to 20 µg/ml lipid (Fig. 8).

base DODAB:MO liposomes (data not shown).

vectors.

**Figure 5.** Resistance of pDNA/DODAB:Monoolein lipoplexes to model proteoglycans. Variation on the percentage of free pDNA upon incubation of pDNA/DODAB:Monoolein lipoplexes (2:1, 1:1, and 1:2) at CRs (+/-) 2.0/4.0 with increasing amounts of heparin (HEP) at incubation times of 30 min. The values were calculated through spectral decomposition of ethidium bromide steady-state fluorescence, as described elsewhere [38]. Adapted from [38].

Another aspect to be taken into account is the possibility that the liposomes and lipoplexes may differently affect parameters such as metabolism, cell membrane structure and chemistry, cell proliferation and mobility. For a comprehensive study, a minimum of three different methodologies, monitoring at least two of these parameters, should be used. From our own results, it was observable that the metabolism of L929 and C2C12 cells was more pronouncedly affected by the contact with DODAB:MO liposomes compared to the other cell lines, especially with a lipid concentration ≥ 20 µg/ml (Fig. 6). At these higher concentrations, DODAB:MO (1:1) induced lower levels of cytotoxicity in all the cell types, which probably reflects the higher content of MO and concomitant lower content of cationic lipid. Interestingly, the cell membrane integrity assay did not reveal such obvious, concentration-dependent variations in cytotoxicity (Fig. 7). The results obtained with the proliferation test (Fig. 8) were quite concordant with those from the metabolic assay (Fig. 6), indicating again the L929 and C2C12 cells as more sensitive, while BJ5ta proliferation was clearly increased when incubated with up to 20 µg/ml lipid (Fig. 8).

256 Cell Interaction

human embryonic kidney cells grown in tissue culture, from which 293T cell line is derived. BJ5ta cells are normal human foreskin fibroblasts immortalized with telomerase. Murine cell

**Figure 5.** Resistance of pDNA/DODAB:Monoolein lipoplexes to model proteoglycans. Variation on the percentage of free pDNA upon incubation of pDNA/DODAB:Monoolein lipoplexes (2:1, 1:1, and 1:2) at CRs (+/-) 2.0/4.0 with increasing amounts of heparin (HEP) at incubation times of 30 min. The values were calculated through spectral decomposition of ethidium bromide steady-state fluorescence, as

Another aspect to be taken into account is the possibility that the liposomes and lipoplexes may differently affect parameters such as metabolism, cell membrane structure and chemistry, cell proliferation and mobility. For a comprehensive study, a minimum of three different methodologies, monitoring at least two of these parameters, should be used. From our own results, it was observable that the metabolism of L929 and C2C12 cells was more pronouncedly affected by the contact with DODAB:MO liposomes compared to the other

described elsewhere [38]. Adapted from [38].

lines L929 and C2C12 are fibroblasts and myoblasts, respectively.

Lipoplexes prepared from these liposomal formulations, at concentrations typically used in transfection experiments, constantly leading to slightly lower viability rates compared to the base DODAB:MO liposomes (data not shown).

The fact that MO-based aggregates cause reduced levels of cytotoxicity for concentrations typically used on transfection assays, reinforcing the use of MO as a new *helper* lipid in this type of non-viral systems. Even if there is general agreement in the reduced toxicity of liposomes as non-viral vectors, these results emphasize the need for accurate liposome/lipoplex evaluation to better assess human risk prior to using them as lipofection vectors.

**Figure 6.** Evaluation of the cytotoxicity (metabolic assay) in four different mammalian cell lines (BJ5-ta, L929, 293 and C2C12) induced by varying concentrations of DODAB:MO-based liposomes after 48 h of incubation. C\_DMSO: cells incubated with 30 % DMSO; C\_Cells: cells alone. The mean (+/−) SD was obtained from two independent experiments. MTT assay can be used to estimate cell viability, specifically as marker of the cell metabolic capacity. The soluble tetrazolium MTT is reduced by metabolically active cells, thus the developed purple color proportional to the number of viable cells.

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery 259

In spite of extensive efforts to unravel the *in vitro/in vivo* mechanisms of internalization of lipoplexes, doubts remain as to whether the topology of lipoplexes facilitates the entry of DNA by fusion with the plasma membrane or with endosomal vesicles. Other studies have indicated that endocytosis is possibly the preferred mechanism of lipoplex internalization by cells [139, 140]. After the formation of the endocytic vesicle containing the lipoplexes, the internal pH of the endosomes decreases to about 5.5 [141].The endosomes then fuse with the lysosomes, in which the condensed NA component may be hydrolysed by lysosomal enzymes [142, 143]. Endosomal release of the NA should occur, avoiding the lysosomal

Different mechanisms for complex internalization have been proposed, in particular for lipoplexes and polyplexes. Endocytosis at the plasma membrane may be clathrin-dependent or -independent. Clathrin-independent mechanisms include fusion of lipoplexes with the plasma membrane, phagocytosis, macropinocytosis and caveolae-mediated uptake [144]. *In vitro* cell culture systems provide the opportunity to experimentally address how lipoplexes interact with the plasma membrane. Although it is widely accepted that endocytosis is the most important route for lipoplex entry, different endocytic pathways may be used in parallel. The most likely explanation is that different cell types prefer a particular mechanism but use

Intracellular trafficking of lipoplexes can be followed by co-localization studies of labeled particle components and dyes, or antibodies that recognize cell organelles or molecules playing a role in the process (e.g. clathrin coating endocytic pits in the plasma membrane) [139, 145] (Fig. 9). Cell lines harboring mutations in some of these molecules may also be used to evaluate their importance for the internalization process of specific formulations. The use of inhibitors of endocytosis has also been widely used but has two major limitations: the significant toxicity induced by the inhibitors themselves and the evidence corroborating that internalization can be simultaneously mediated by different pathways.

The endosomal escape is thought to be the major limitation for efficient gene transfection [146]. A number of strategies have been explored to enhance NA endosomal release. For example, the incorporation of a non-lamellar forming lipid such as DOPE that disrupts the endosome membrane or inclusion of a pH-dependent molecule that senses the acidification

Modulation of the endosomal escape during lipoplex intracellular trafficking was replicated by exposing pDNA/DODAB:MO (2:1, 1:1 and 1:2) lipoplexes to acidic conditions in the presence of increasing amounts of hydrochloric acid (pH ranging from 7.5 to 2.5) (Fig. 10). The percentage of released DNA steadily increased upon milieu acidification from pH 7.4 to 4.5, which is the pH range typical in the endosome. This trend correlates negatively with the MO content in the formulation, suggesting that MO's inverted bicontinuous cubic structures may protect more efficiently the lipoplex structure in this environment. More stringent acidification of the environment (pH 4.5 to pH 2.5) inverts the release tendency, which can

in the endosome compartment leading to disruption of its membrane [147].

be related to degradation of naked pDNA in solution.

more than one. Therefore, optimization remains largely dependent of trial and error.

**4.2. Cellular uptake and intracellular trafficking**

lythic pathway, leading to successful transfection.

**Figure 7.** Evaluation of the cytotoxicity (cell membrane integrity) in four different mammalian cell lines (BJ5-ta, L929, 293 and C2C12) induced by varying concentrations of DODAB:MO-based liposomes after 48 h of incubation. C\_DMSO: cells incubated with 30 % DMSO; C\_Cells: cells alone. The mean (+/−) SD was obtained from two independent experiments. The LDH assay is used to estimate cell viability, as the intracellular enzyme LDH is released into the extracellular medium when cell membranes are damaged.

**Figure 8.** Evaluation of the cytotoxicity (proliferation) in four different mammalian cell lines (BJ5-ta, L929, 293 and C2C12) of varying concentrations of DODAB:MO-based liposomes after 48 h of incubation. C\_DMSO: cells incubated with 30 % DMSO; C\_Cells: cells alone. The mean (+/−) SD was obtained from two independent experiments. Sulforhodamine B (SRB) is considered a proliferation assay, used for cell density determination, based on the determination of the cellular protein content.

## **4.2. Cellular uptake and intracellular trafficking**

258 Cell Interaction

**Figure 7.** Evaluation of the cytotoxicity (cell membrane integrity) in four different mammalian cell lines (BJ5-ta, L929, 293 and C2C12) induced by varying concentrations of DODAB:MO-based liposomes after 48 h of incubation. C\_DMSO: cells incubated with 30 % DMSO; C\_Cells: cells alone. The mean (+/−) SD was obtained from two independent experiments. The LDH assay is used to estimate cell viability, as the intracellular enzyme LDH is released into the extracellular medium when cell membranes are damaged.

**Figure 8.** Evaluation of the cytotoxicity (proliferation) in four different mammalian cell lines (BJ5-ta, L929, 293 and C2C12) of varying concentrations of DODAB:MO-based liposomes after 48 h of incubation. C\_DMSO: cells incubated with 30 % DMSO; C\_Cells: cells alone. The mean (+/−) SD was obtained from two independent experiments. Sulforhodamine B (SRB) is considered a proliferation assay, used for cell density determination, based on the determination of the cellular protein content. In spite of extensive efforts to unravel the *in vitro/in vivo* mechanisms of internalization of lipoplexes, doubts remain as to whether the topology of lipoplexes facilitates the entry of DNA by fusion with the plasma membrane or with endosomal vesicles. Other studies have indicated that endocytosis is possibly the preferred mechanism of lipoplex internalization by cells [139, 140]. After the formation of the endocytic vesicle containing the lipoplexes, the internal pH of the endosomes decreases to about 5.5 [141].The endosomes then fuse with the lysosomes, in which the condensed NA component may be hydrolysed by lysosomal enzymes [142, 143]. Endosomal release of the NA should occur, avoiding the lysosomal lythic pathway, leading to successful transfection.

Different mechanisms for complex internalization have been proposed, in particular for lipoplexes and polyplexes. Endocytosis at the plasma membrane may be clathrin-dependent or -independent. Clathrin-independent mechanisms include fusion of lipoplexes with the plasma membrane, phagocytosis, macropinocytosis and caveolae-mediated uptake [144]. *In vitro* cell culture systems provide the opportunity to experimentally address how lipoplexes interact with the plasma membrane. Although it is widely accepted that endocytosis is the most important route for lipoplex entry, different endocytic pathways may be used in parallel. The most likely explanation is that different cell types prefer a particular mechanism but use more than one. Therefore, optimization remains largely dependent of trial and error.

Intracellular trafficking of lipoplexes can be followed by co-localization studies of labeled particle components and dyes, or antibodies that recognize cell organelles or molecules playing a role in the process (e.g. clathrin coating endocytic pits in the plasma membrane) [139, 145] (Fig. 9). Cell lines harboring mutations in some of these molecules may also be used to evaluate their importance for the internalization process of specific formulations. The use of inhibitors of endocytosis has also been widely used but has two major limitations: the significant toxicity induced by the inhibitors themselves and the evidence corroborating that internalization can be simultaneously mediated by different pathways.

The endosomal escape is thought to be the major limitation for efficient gene transfection [146]. A number of strategies have been explored to enhance NA endosomal release. For example, the incorporation of a non-lamellar forming lipid such as DOPE that disrupts the endosome membrane or inclusion of a pH-dependent molecule that senses the acidification in the endosome compartment leading to disruption of its membrane [147].

Modulation of the endosomal escape during lipoplex intracellular trafficking was replicated by exposing pDNA/DODAB:MO (2:1, 1:1 and 1:2) lipoplexes to acidic conditions in the presence of increasing amounts of hydrochloric acid (pH ranging from 7.5 to 2.5) (Fig. 10). The percentage of released DNA steadily increased upon milieu acidification from pH 7.4 to 4.5, which is the pH range typical in the endosome. This trend correlates negatively with the MO content in the formulation, suggesting that MO's inverted bicontinuous cubic structures may protect more efficiently the lipoplex structure in this environment. More stringent acidification of the environment (pH 4.5 to pH 2.5) inverts the release tendency, which can be related to degradation of naked pDNA in solution.

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery 261

Interestingly, DODAB:MO based liposomes and lipoplexes were found to induce production of low levels of TNF-α by macrophages, comparable or lower than

DOTMA/DOPE and DOTMA/cholesterol lipoplexes (data not shown) [150].

**Figure 10.** Resistance of pDNA/DODAB:Monoolein lipoplexes to pH decrease (modulation of

hydrochloric acid at incubation times of 30 min. The values were calculated through spectral decomposition of ethidium bromide steady-state fluorescence, as described elsewhere [38].

pDNA/DODAB:Monoolein lipoplexes (2:1, 1:1, and 1:2) at CRs (+/-) 2.0/4.0 with increasing amounts of

Transfection efficiency of plasmid DNA can be directly evaluated by detecting the protein encoded by the reporter gene. Examples of reporter genes are: green fluorescent protein (GFP) and similar, detectable by techniques as microscopy or flow cytometry; βgalactosidase, whose activity can be evaluated by a colorimetric assay; luciferase, whose

endosomal escape). Variation on the percentage of free pDNA upon incubation of

**4.3. Transfection efficiency**

**Figure 9.** Visualization of cellular uptake of DODAB:MO lipoplexes by HEK 293T cells. Liposomes are labelled with Bodipy-PE (green) and endo-lisosomes with dextran (red). Co-localization (yellow) indicates sites of active endocytosis of lipoplexes. DODAB:MO (2:1) (+/-) 4.0, 1 µg pDNA/well, amplification 200x.

Lipoplex charge ratio (+/-) also affects the intensity of pDNA release. Using the same DODAB:MO base formulation, increasing charge ratio (+/-) seems to prevent pDNA release from the lipoplex. This effect was already visible in the destabilization of pDNA/DODAB/MO lipoplexes by plasma constituents such as serum and salt, and probably reflects more efficient pDNA condensation in presence of excess cationic lipid.

Increasing ammonium/phosphate ratio carries the risk of increased cytotoxicity. One possible solution may be using increasing amounts of MO in lipoplex formulation for better protection of pDNA integrity without imposing major toxic effects to the target cell.

Non-viral vectors, although less toxic than viral vectors, may still elicit a strong, nonspecific immune response. Toxicity frequently results from characteristics of the encapsulating polymer or lipid such as the length, saturation, or branching of the polymer. Efforts to reduce the toxicity of nonviral vectors have largely resulted in attempts to make the vectors more biodegradable and biocompatible. Many of the aforementioned systems (i.e. triggered release with disulfides, PEG copolymers) incorporated more biologically active components, thereby reducing the elicited immune response. For example, the incorporation in liposomes of molecules known to suppress the production of the cytokine tumor necrosis factor (TNFα), as compared to lipoplex alone, succeeded in maintain its levels low while achieving comparable levels of transgene expression [148]. Another method explored by Tan [149] significantly reduced toxicity through the sequential injection of liposome and later of DNA, as opposed to using formed lipoplexes. With this approach, cytokine levels (IL-12, TNF-α) were reduced by greater than 80% compared to lipoplex delivery [149]. Thus, significant advances have been made towards decreasing the toxicity of these non-viral vectors. Interestingly, DODAB:MO based liposomes and lipoplexes were found to induce production of low levels of TNF-α by macrophages, comparable or lower than DOTMA/DOPE and DOTMA/cholesterol lipoplexes (data not shown) [150].

**Figure 10.** Resistance of pDNA/DODAB:Monoolein lipoplexes to pH decrease (modulation of endosomal escape). Variation on the percentage of free pDNA upon incubation of pDNA/DODAB:Monoolein lipoplexes (2:1, 1:1, and 1:2) at CRs (+/-) 2.0/4.0 with increasing amounts of hydrochloric acid at incubation times of 30 min. The values were calculated through spectral decomposition of ethidium bromide steady-state fluorescence, as described elsewhere [38].

#### **4.3. Transfection efficiency**

260 Cell Interaction

amplification 200x.

**Figure 9.** Visualization of cellular uptake of DODAB:MO lipoplexes by HEK 293T cells. Liposomes are labelled with Bodipy-PE (green) and endo-lisosomes with dextran (red). Co-localization (yellow) indicates sites of active endocytosis of lipoplexes. DODAB:MO (2:1) (+/-) 4.0, 1 µg pDNA/well,

Lipoplex charge ratio (+/-) also affects the intensity of pDNA release. Using the same DODAB:MO base formulation, increasing charge ratio (+/-) seems to prevent pDNA release from the lipoplex. This effect was already visible in the destabilization of pDNA/DODAB/MO lipoplexes by plasma constituents such as serum and salt, and probably

Increasing ammonium/phosphate ratio carries the risk of increased cytotoxicity. One possible solution may be using increasing amounts of MO in lipoplex formulation for better

Non-viral vectors, although less toxic than viral vectors, may still elicit a strong, nonspecific immune response. Toxicity frequently results from characteristics of the encapsulating polymer or lipid such as the length, saturation, or branching of the polymer. Efforts to reduce the toxicity of nonviral vectors have largely resulted in attempts to make the vectors more biodegradable and biocompatible. Many of the aforementioned systems (i.e. triggered release with disulfides, PEG copolymers) incorporated more biologically active components, thereby reducing the elicited immune response. For example, the incorporation in liposomes of molecules known to suppress the production of the cytokine tumor necrosis factor (TNFα), as compared to lipoplex alone, succeeded in maintain its levels low while achieving comparable levels of transgene expression [148]. Another method explored by Tan [149] significantly reduced toxicity through the sequential injection of liposome and later of DNA, as opposed to using formed lipoplexes. With this approach, cytokine levels (IL-12, TNF-α) were reduced by greater than 80% compared to lipoplex delivery [149]. Thus, significant advances have been made towards decreasing the toxicity of these non-viral vectors.

reflects more efficient pDNA condensation in presence of excess cationic lipid.

protection of pDNA integrity without imposing major toxic effects to the target cell.

Transfection efficiency of plasmid DNA can be directly evaluated by detecting the protein encoded by the reporter gene. Examples of reporter genes are: green fluorescent protein (GFP) and similar, detectable by techniques as microscopy or flow cytometry; βgalactosidase, whose activity can be evaluated by a colorimetric assay; luciferase, whose activity can be measured with a luminometer, after a substrate is converted into a luminescent form by luciferase. In Figure 11 is depicted an experiment that allows to identify the effect on transfection efficiency of varying the content of MO in the liposomal formulations, lipid:DNA charge ratio in the lipoplexes and also the quantity of pDNA added to the cells, as pDNA dosage is known to affect transfection efficiency. It can be observed that the incorporation of MO in the liposomes resulted in a transfection efficiency improvement when compared to the cationic lipid DODAB alone. When using 1 µg DNA/well, the transfection levels of pDNA/DODAB:MO systems are of the same order of magnitude as Lipofectamine™ LTX. For a lower MO content (pDNA/DODAB:MO (2:1) formulation), a dose effect response (0.5 µg and 1 µg of pDNA) was observed. For higher MO content (pDNA/DODAB:MO (1:1) formulation), the transfection efficiencies remained constant at both CRs. This result strengthens the role of MO as *helper* lipid in the transfection agent.

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery 263

The identification of the most important formulation parameters and how they influence macromolecule delivery and bioactivity will give direction towards the development of novel therapeutic solutions. The morphology and structure of the lipoplex is influenced by the surrounding environment and the chemical nature of its constituents. Physicochemical properties of the systems define the course of most events when lipoplex interact with the body, tissues and cells. The effectiveness of vector internalization, its intracellular trafficking and successful transgene expression in target cells, is directly dependent on the *helper* lipid features, net charge of the lipoplex and the degree of NA compactation within the complex. Different target cells may impose specific challenges to transfection and many inherent factors are unknown. The advent of controlled cell targeting for improved specificity holds

A good lipofection system must protect NA from deleterious interaction with biological fluids and cell components, while remaining biocompatible and efficient as delivery agent. In summary, with this work we intend to demonstrate that MO can be used safely and efficiently as *helper* lipid in the preparation of non-viral vectors for transfection. The presence of this natural lipid in the formulations reduces the net positive charge necessary

for successful NA complexation, thus decreasing transfection associated cytoxicity.

*Centre of Molecular & Environmental Biology - CBMA, University of Minho, Campus of Gualtar,*

This work was supported by FCT research project PTDC/QUI/69795/2006, which is cofunded by the program COMPETE from QREN with co-participation from the European Community fund FEDER; CFUM [PEst-C/FIS/UI0607/2011]; CBMA [Pest C/BIA/UI4050/2011]; J.P.N. Silva holds a PhD Grant (SFRH/BD/46968/2008); A. C.N. Oliveira

[1] Patil, S.D., D.G. Rhodes, and D.J. Burgess, *DNA-Based Therapeutics and DNA Delivery Systems - A Comprehensive Review.* American Association of Pharmaceutical Scientists

great promise for application of these formulations in nanomedicine.

*Centre of Physics, University of Minho, Campus of Gualtar, Braga, Portugal*

**5. Conclusions**

**Author details**

*Braga, Portugal*

**6. References**

Corresponding Author

 \*

**Acknowledgement**

J. P. Neves Silva and M. E. C. D. Real Oliveira\*

holds a PhD grant (SFRH/BD/68588/2010).

Journal, 2005. 7: p. E61-E77.

A. C. N. Oliveira and A. C. Gomes

**Figure 11.** Transfection efficiency of HEK 293T cells by MO-based lipoplexes. Transfected pDNA encoded the β-galactosidase gene whose activity was evaluated by a colorimetric assay after 48 h of incubation. Lipoplexes prepared at charge ratio (+/-) 4.0 or 2.0, 0.5 or 1.0 µg pDNA/well. Controls: cells incubated with free pDNA; cells transfected using Lipofectamine® as lipofection agent. The mean (+/−) SD was obtained from three independent experiments. Adapted from [38].

## **5. Conclusions**

262 Cell Interaction

agent.

activity can be measured with a luminometer, after a substrate is converted into a luminescent form by luciferase. In Figure 11 is depicted an experiment that allows to identify the effect on transfection efficiency of varying the content of MO in the liposomal formulations, lipid:DNA charge ratio in the lipoplexes and also the quantity of pDNA added to the cells, as pDNA dosage is known to affect transfection efficiency. It can be observed that the incorporation of MO in the liposomes resulted in a transfection efficiency improvement when compared to the cationic lipid DODAB alone. When using 1 µg DNA/well, the transfection levels of pDNA/DODAB:MO systems are of the same order of magnitude as Lipofectamine™ LTX. For a lower MO content (pDNA/DODAB:MO (2:1) formulation), a dose effect response (0.5 µg and 1 µg of pDNA) was observed. For higher MO content (pDNA/DODAB:MO (1:1) formulation), the transfection efficiencies remained constant at both CRs. This result strengthens the role of MO as *helper* lipid in the transfection

**Figure 11.** Transfection efficiency of HEK 293T cells by MO-based lipoplexes. Transfected pDNA encoded the β-galactosidase gene whose activity was evaluated by a colorimetric assay after 48 h of incubation. Lipoplexes prepared at charge ratio (+/-) 4.0 or 2.0, 0.5 or 1.0 µg pDNA/well. Controls: cells incubated with free pDNA; cells transfected using Lipofectamine® as lipofection agent. The mean (+/−)

SD was obtained from three independent experiments. Adapted from [38].

The identification of the most important formulation parameters and how they influence macromolecule delivery and bioactivity will give direction towards the development of novel therapeutic solutions. The morphology and structure of the lipoplex is influenced by the surrounding environment and the chemical nature of its constituents. Physicochemical properties of the systems define the course of most events when lipoplex interact with the body, tissues and cells. The effectiveness of vector internalization, its intracellular trafficking and successful transgene expression in target cells, is directly dependent on the *helper* lipid features, net charge of the lipoplex and the degree of NA compactation within the complex. Different target cells may impose specific challenges to transfection and many inherent factors are unknown. The advent of controlled cell targeting for improved specificity holds great promise for application of these formulations in nanomedicine.

A good lipofection system must protect NA from deleterious interaction with biological fluids and cell components, while remaining biocompatible and efficient as delivery agent. In summary, with this work we intend to demonstrate that MO can be used safely and efficiently as *helper* lipid in the preparation of non-viral vectors for transfection. The presence of this natural lipid in the formulations reduces the net positive charge necessary for successful NA complexation, thus decreasing transfection associated cytoxicity.

## **Author details**

J. P. Neves Silva and M. E. C. D. Real Oliveira\* *Centre of Physics, University of Minho, Campus of Gualtar, Braga, Portugal*

A. C. N. Oliveira and A. C. Gomes *Centre of Molecular & Environmental Biology - CBMA, University of Minho, Campus of Gualtar, Braga, Portugal*

## **Acknowledgement**

This work was supported by FCT research project PTDC/QUI/69795/2006, which is cofunded by the program COMPETE from QREN with co-participation from the European Community fund FEDER; CFUM [PEst-C/FIS/UI0607/2011]; CBMA [Pest C/BIA/UI4050/2011]; J.P.N. Silva holds a PhD Grant (SFRH/BD/46968/2008); A. C.N. Oliveira holds a PhD grant (SFRH/BD/68588/2010).

## **6. References**

[1] Patil, S.D., D.G. Rhodes, and D.J. Burgess, *DNA-Based Therapeutics and DNA Delivery Systems - A Comprehensive Review.* American Association of Pharmaceutical Scientists Journal, 2005. 7: p. E61-E77.

<sup>\*</sup> Corresponding Author

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**Chapter 11**

© 2012 Pelaez-Vargas et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Micropatterned Coatings for Guided Tissue**

A. Pelaez-Vargas, D. Gallego-Perez, N. Higuita-Castro, A. Carvalho, L. Grenho, J.A. Arismendi, M.H. Fernandes, M.P. Ferraz, D.J. Hansford and F.J. Monteiro

Dento-alveolar trauma and congenital absences are the most important causes of edentulism that are not associated with bacteria. However, the World Health Organization reports show that dental caries and periodontitis, two conditions of bacterial origin, are the most frequent oral diseases in humans [1]. These conditions might be avoided if an adequate oral preventive health policy is implemented, including preventive and educational measures that, regardless of the population´s socioeconomic factors, have shown their effectiveness. Despite these facts, tooth extraction1, defined as the surgical removal of a tooth, is currently

Tooth loss or edentulism affects the aesthetics and function of the stomatognathic system. The missing interproximal contact produces an intra-arch imbalance that is visible as dental misalignment and the formation of anterior/posterior diastema. Additionally, the tooth distal to the extraction site will drift mesially into the space, thus creating an oclusal collapse. Inter-arch disharmonies are observed as occlusal collapse, supereruption of antagonist teeth, and alteration of the vertical dimension of occlusion. The synergy of inter-/ intra- arch disequilibrium is associated with ATM dysfunction, muscle hyperactivity, nutritional imbalances, tooth wear, mobility, and potential harmful contact areas during

Recovering aesthetics and function is only possible using some oral rehabilitation procedures such as fixed or removable prostheses. In the fixed restorations, titanium implant-based therapy appears as the "gold standard', considering that successful rates of

1 Tooth extraction was indexed (1965) in the MESH of the National Library of Medicine (NLM). Some synonyms are

and reproduction in any medium, provided the original work is properly cited.

**Regeneration in Dental Implantology**

Additional information is available at the end of the chapter

the most frequent surgical procedure in the world [1].

mandibular eccentric movements or otologic symptoms [2].

~95% after 5 years have been reported.

dental extraction, exodontia or pulling teeth.

http://dx.doi.org/10.5772/50838

**1. Introduction**

**Chapter 11**

## **Micropatterned Coatings for Guided Tissue Regeneration in Dental Implantology**

A. Pelaez-Vargas, D. Gallego-Perez, N. Higuita-Castro, A. Carvalho, L. Grenho, J.A. Arismendi, M.H. Fernandes, M.P. Ferraz, D.J. Hansford and F.J. Monteiro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50838

## **1. Introduction**

272 Cell Interaction

1968.

673-682.

542.

[145] Elouahabi, A. and J. Ruysschaert, *Formation and Intracellular Trafficking of Lipoplexes and*

[146] Bhushan, B., *Handbook of Nanotechnology*. 2nd ed, ed. S. Verlag. 2010, Berlin (Germany).

[147] Caracciolo, G., et al., *Efficient Escape from Endosomes Determines the Superior Efficiency of Multicomponent Lipoplexes.* Journal of Physical Chemistry B, 2009. 113: p. 4995-4997. [148] Liu, F., et al., *Effect of Non-Ionic Surfactants on the Formation of DNA-Emulsion Complexes and Emulsion-Mediated Gene Transfer.* Pharmaceutical Research, 1996. 13: p. 1642-1646. [149] Tan, Y., et al., *Sequential Injection of Cationic Liposome and Pasmid DNA Effectively Transfects the Lung with Minimal Inflammatory Toxicity.* Molecular Therapy, 2001. 3: p.

[150] Yasuda, S., et al., *Comparison of the Type of Liposome Involving Cytokine Production Induced by Non-CpG Lipoplex in Macrophages.* Molecular Pharmaceutics, 2010. 7: p. 533-

*Polyplexes.* Molecular Therapy, 2005. 11: p. 336-347.

Dento-alveolar trauma and congenital absences are the most important causes of edentulism that are not associated with bacteria. However, the World Health Organization reports show that dental caries and periodontitis, two conditions of bacterial origin, are the most frequent oral diseases in humans [1]. These conditions might be avoided if an adequate oral preventive health policy is implemented, including preventive and educational measures that, regardless of the population´s socioeconomic factors, have shown their effectiveness. Despite these facts, tooth extraction1, defined as the surgical removal of a tooth, is currently the most frequent surgical procedure in the world [1].

Tooth loss or edentulism affects the aesthetics and function of the stomatognathic system. The missing interproximal contact produces an intra-arch imbalance that is visible as dental misalignment and the formation of anterior/posterior diastema. Additionally, the tooth distal to the extraction site will drift mesially into the space, thus creating an oclusal collapse. Inter-arch disharmonies are observed as occlusal collapse, supereruption of antagonist teeth, and alteration of the vertical dimension of occlusion. The synergy of inter-/ intra- arch disequilibrium is associated with ATM dysfunction, muscle hyperactivity, nutritional imbalances, tooth wear, mobility, and potential harmful contact areas during mandibular eccentric movements or otologic symptoms [2].

Recovering aesthetics and function is only possible using some oral rehabilitation procedures such as fixed or removable prostheses. In the fixed restorations, titanium implant-based therapy appears as the "gold standard', considering that successful rates of ~95% after 5 years have been reported.

<sup>1</sup> Tooth extraction was indexed (1965) in the MESH of the National Library of Medicine (NLM). Some synonyms are dental extraction, exodontia or pulling teeth.

<sup>© 2012</sup> Pelaez-Vargas et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The history of implantology has been divided in two different phases, namely the preosseointegration and post-osseointegration eras. Many audacious designs were developed to be used in sub- or endosteal areas in the former. The most commonly used implants were blades and plaques based on metallic alloys such as cobalt-chromium-molybdenum and stainless steel (Figure 1). These types of devices have been associated to doubtful long-term clinical success. Initial clinical research reported by Branemark was criticized, but the basis for a new philosophy in dental therapy had been initiated. After completing training in Sweden, Zarb and colleagues from Toronto started a longitudinal study to verify the Micropatterned Coatings for Guided Tissue Regeneration in Dental Implantology 275

**Figure 3.** Ten-year follow-up of a successful implant-based rehabilitation after trauma. (a) Initial panoramic radiography, where radiopaque zones compatible with metallic residues are observed. (b) Initial clinical view. Note the extensive loss of mandibular teeth and their alveolar process. (c, d) Surgical phase: ten implants were placed in two surgical treatment phases after a procedure of distraction-based osteogenesis for vertical bone augmentation. (e,f) Final restorations exhibited an increased length to obtain a compensation of the vertically lost bone dimension. (g,h) Panoramic radiography and intraoral view after ten years of function. Note that the restoration is maintained in a

possibilities of osseointegrated implants [3]. In 1982, the concept of osseointegration was discussed in a meeting in Toronto attended by people from the most important dental schools in North America. For more detailed information about the first steps of osseointegrated implants, the reader is referred to more specific literature [3, 4]. Branemark's work described osseointegration as a biological phenomenon involving direct

contact between bone and Ti surfaces, allowing for a new philosophy of therapy.

good aesthetic condition. (i) Clinical view in labial resting position.

**Figure 1.** History of dental implants. (a,b) Panoramic radiography and general view of a failed total rehabilitation supported on blade/plate-shaped implants. (c) Parallel pins implant, (d) Endosteal endodontic stabilizers and (e) different designs and surfaces of screw dental implants.

**Figure 2.** Double implants to replace a mandibular molar. (a,b) Periapical radiography and occlusal view of the final rehabilitation. (c,d) Periapical radiography and detailed occlusal view after 10 years of function.

Micropatterned Coatings for Guided Tissue Regeneration in Dental Implantology 275

274 Cell Interaction

The history of implantology has been divided in two different phases, namely the preosseointegration and post-osseointegration eras. Many audacious designs were developed to be used in sub- or endosteal areas in the former. The most commonly used implants were blades and plaques based on metallic alloys such as cobalt-chromium-molybdenum and stainless steel (Figure 1). These types of devices have been associated to doubtful long-term clinical success. Initial clinical research reported by Branemark was criticized, but the basis for a new philosophy in dental therapy had been initiated. After completing training in Sweden, Zarb and colleagues from Toronto started a longitudinal study to verify the

**Figure 1.** History of dental implants. (a,b) Panoramic radiography and general view of a failed total rehabilitation supported on blade/plate-shaped implants. (c) Parallel pins implant, (d) Endosteal

**Figure 2.** Double implants to replace a mandibular molar. (a,b) Periapical radiography and occlusal view of the final rehabilitation. (c,d) Periapical radiography and detailed occlusal view after 10 years of function.

endodontic stabilizers and (e) different designs and surfaces of screw dental implants.

**Figure 3.** Ten-year follow-up of a successful implant-based rehabilitation after trauma. (a) Initial panoramic radiography, where radiopaque zones compatible with metallic residues are observed. (b) Initial clinical view. Note the extensive loss of mandibular teeth and their alveolar process. (c, d) Surgical phase: ten implants were placed in two surgical treatment phases after a procedure of distraction-based osteogenesis for vertical bone augmentation. (e,f) Final restorations exhibited an increased length to obtain a compensation of the vertically lost bone dimension. (g,h) Panoramic radiography and intraoral view after ten years of function. Note that the restoration is maintained in a good aesthetic condition. (i) Clinical view in labial resting position.

possibilities of osseointegrated implants [3]. In 1982, the concept of osseointegration was discussed in a meeting in Toronto attended by people from the most important dental schools in North America. For more detailed information about the first steps of osseointegrated implants, the reader is referred to more specific literature [3, 4]. Branemark's work described osseointegration as a biological phenomenon involving direct contact between bone and Ti surfaces, allowing for a new philosophy of therapy.

In general, it is accepted that implant therapy is a predictable treatment without any contraindications for partially and fully edentulous patients. However, several factors, such as implant design, surgical procedure, anatomic and osseous conditions, systemic diseases, prosthetic design (Figure 2), and two-stage or immediate loading may affect the prognosis and long-term success (Figure 3). A poor prognosis was observed in patients with insufficient quality and/or quantity of bone receptor. Patients exhibiting poor quality of bone (type IV) in the posterior area of the maxilla had a 35% implant failure. This retrospective study indicated that patients with type I, II and III showed only 3% failure [5].

Micropatterned Coatings for Guided Tissue Regeneration in Dental Implantology 277

under gingival tissue and maintained unloaded during a 3 to 6-month period. However, new approaches have been introduced that include immediate loading based on the primary

**Figure 4.** Fourteen-year follow-up of a successful implant-based rehabilitation. (a) Full-thickness flap reflection reveals the extension of missing bone in the buccal aspect of the maxilla. (b) The use of monocortical block graft to reconstruct the horizontal deficiency of the maxilla. (c) After 6 months tissue exhibits good healing. (d) Three implants were placed in this site and submerged for a six-month period. (e,f) Periapical radiography and clinical photography of complete restoration after treatment

The long-term success of an implant largely depends on the equilibrium between osseointegration and epithelial/connective tissue attachment. A complete soft tissue seal protects the newly formed bone from bacterial products originated in the oral environment. Several animal and *in vitro* studies showed similar epithelial and connective structures between the gingiva and the peri-implant mucosa. The outer surface of the peri-implant mucosa is lined by a stratified keratinized oral epithelium that is continuous, with a junctional epithelium attached to the Ti surface by a basal lamina and hemidesmosomes.

and (g,h) Periapical radiography and clinical view after 14 years of function.

stability during surgical procedure and surface bioactivity [13].

Systemic diseases are potential factors that affect the prognosis. The available information is derived from empirical observational studies where multifactorial risks are considered. Moy et al. showed a 10-year retrospective cohort study where 1140 patients were evaluated. Their results presented evidence that smoking, history of diabetes, head and neck radiation and postmenopausal estrogen therapy were correlated with a significantly increased failure rate, while gender, hypertension, coronary arterial disease, pulmonary disease, steroid therapy, chemotherapy and absence of hormone replacement therapy for post-menopausal women were not associated with increased risk in implant failure [6]. This study concluded that there are no absolute medical contraindications to dental implant rehabilitation, although they must be individually considered for each new patient.

The decision to put implants in patients treated for aggressive periodontitis is a challenge since controlled studies with large sample sizes are not available. Some case reports showed approximately 8% failure in patients previously treated for aggressive periodontitis. However, all the evaluated implants were placed in patients that had been previously treated for several years before the implants were put in place [7]. Until more evidence becomes available, factors such as time before therapy, the presence of natural treated teeth or immune compromise might render these clinical situations unpredictable.

Reduced alveolar bone height appears as an important consideration when evaluating the prognosis of dental implants. Two different approaches are used to achieve clinical success. In situations of an extremely reduced amount of bone, the surgeon may employ bone augmentation procedures (Figure 3 and 4), which result in higher costs, greater morbidity, and longer treatment times. Another possibility includes the use of short implants, which are defined as devices shorter than 6 to 10 mm [8]. Conflicting information is available about the success of this type of implants, with some authors reporting that short implants are unpredictable in cases of poor bone quality. However, alternative treatments are viable if other favourable factors are considered. A survival rate of approximately 94% for a fiveyear observation period was observed [8-10]. However, short implants have mechanical disadvantages as a consequence of implant-crown ratios and the amount of osseointegrated area around the implant [11].

In the last decade, there has been an important discussion related to loading implants immediately. Branemark's group postulated that early loading affects the prognosis due to a fibrous capsule that may develop due to micro-movements, thus affecting osseointegration [12]. They introduced the two-phase surgical procedure, in which the implant is submerged under gingival tissue and maintained unloaded during a 3 to 6-month period. However, new approaches have been introduced that include immediate loading based on the primary stability during surgical procedure and surface bioactivity [13].

276 Cell Interaction

In general, it is accepted that implant therapy is a predictable treatment without any contraindications for partially and fully edentulous patients. However, several factors, such as implant design, surgical procedure, anatomic and osseous conditions, systemic diseases, prosthetic design (Figure 2), and two-stage or immediate loading may affect the prognosis and long-term success (Figure 3). A poor prognosis was observed in patients with insufficient quality and/or quantity of bone receptor. Patients exhibiting poor quality of bone (type IV) in the posterior area of the maxilla had a 35% implant failure. This retrospective study indicated that patients with type I, II and III showed only 3% failure [5]. Systemic diseases are potential factors that affect the prognosis. The available information is derived from empirical observational studies where multifactorial risks are considered. Moy et al. showed a 10-year retrospective cohort study where 1140 patients were evaluated. Their results presented evidence that smoking, history of diabetes, head and neck radiation and postmenopausal estrogen therapy were correlated with a significantly increased failure rate, while gender, hypertension, coronary arterial disease, pulmonary disease, steroid therapy, chemotherapy and absence of hormone replacement therapy for post-menopausal women were not associated with increased risk in implant failure [6]. This study concluded that there are no absolute medical contraindications to dental implant rehabilitation, although

The decision to put implants in patients treated for aggressive periodontitis is a challenge since controlled studies with large sample sizes are not available. Some case reports showed approximately 8% failure in patients previously treated for aggressive periodontitis. However, all the evaluated implants were placed in patients that had been previously treated for several years before the implants were put in place [7]. Until more evidence becomes available, factors such as time before therapy, the presence of natural treated teeth

Reduced alveolar bone height appears as an important consideration when evaluating the prognosis of dental implants. Two different approaches are used to achieve clinical success. In situations of an extremely reduced amount of bone, the surgeon may employ bone augmentation procedures (Figure 3 and 4), which result in higher costs, greater morbidity, and longer treatment times. Another possibility includes the use of short implants, which are defined as devices shorter than 6 to 10 mm [8]. Conflicting information is available about the success of this type of implants, with some authors reporting that short implants are unpredictable in cases of poor bone quality. However, alternative treatments are viable if other favourable factors are considered. A survival rate of approximately 94% for a fiveyear observation period was observed [8-10]. However, short implants have mechanical disadvantages as a consequence of implant-crown ratios and the amount of osseointegrated

In the last decade, there has been an important discussion related to loading implants immediately. Branemark's group postulated that early loading affects the prognosis due to a fibrous capsule that may develop due to micro-movements, thus affecting osseointegration [12]. They introduced the two-phase surgical procedure, in which the implant is submerged

or immune compromise might render these clinical situations unpredictable.

they must be individually considered for each new patient.

area around the implant [11].

**Figure 4.** Fourteen-year follow-up of a successful implant-based rehabilitation. (a) Full-thickness flap reflection reveals the extension of missing bone in the buccal aspect of the maxilla. (b) The use of monocortical block graft to reconstruct the horizontal deficiency of the maxilla. (c) After 6 months tissue exhibits good healing. (d) Three implants were placed in this site and submerged for a six-month period. (e,f) Periapical radiography and clinical photography of complete restoration after treatment and (g,h) Periapical radiography and clinical view after 14 years of function.

The long-term success of an implant largely depends on the equilibrium between osseointegration and epithelial/connective tissue attachment. A complete soft tissue seal protects the newly formed bone from bacterial products originated in the oral environment.

Several animal and *in vitro* studies showed similar epithelial and connective structures between the gingiva and the peri-implant mucosa. The outer surface of the peri-implant mucosa is lined by a stratified keratinized oral epithelium that is continuous, with a junctional epithelium attached to the Ti surface by a basal lamina and hemidesmosomes. The non-keratinized junctional epithelium is only a few cell layers thick in the apical portion and is separated from the alveolar bone by collagen-rich connective tissue. This 3 to 4 mm biological barrier, formed irrespective of the original mucosal thickness, protects the osseointegration zone from factors released by the plaque and the oral cavity [14]. The main differences between the soft tissues around natural teeth and those around implants are the collagen fibres orientation, which run parallel from the implant surface to the crest bone, the low number of fibroblasts, and the reduced vascularization of the scar tissue.

Micropatterned Coatings for Guided Tissue Regeneration in Dental Implantology 279

Mobility is the most evident sign of implant failure and can be presented as rotational, lateral or horizontal, and axial or vertical mobility [15]. There are different terms in the literature associated with biological implant failure or complications like peri-implant diseases, peri-mucositis and peri-implantitis, where the first two are reversible inflammatory reactions around a functioning implant. Peri-implantitis is a chronic inflammation with loss of the supporting tissues around the implant induced by bacterial colonization, facilitated by the implant/abutment gap and by the chemistry and surface

Bacteria colonize and develop biofilms on the transmucosal abutment of osseointegrated dental implants. Like the gingival crevice around the natural tooth, the peri-implant mucosa covering the alveolar bone is closely adapted to the implant. In partially edentulous subjects, the developing microbiota around implants closely resembles the microflora of natural teeth [14]. In addition to the dark-pigmented, gram-negative anaerobic rods, other bacteria are associated with peri-implant infections (*Bacteroides forsythus, Fusobact. nucleatum, Campylobacter, Peptostreptcc. micros and Prevotella intermedia*) [17], and eventually *Staphylocc.*

Metals, including Ti, may induce non-specific immunomodulation and self-immunity. In immunologic *in vitro* tests, sensitization to Ti was observed [19]. Such problems with Ti and the ever-growing expectations on aesthetics lead to increasing interest in all-ceramic

The surface texture of dental implants affects the rate of osseointegration [20] and biomechanical fixation. Surface roughness may be classified as "macro", "micro" and "nano" sized topologies. The "macro" range, from millimetres to 10 μm, is directly related to implant geometry, with threaded screws and macroporous coatings helping the primary stability of the implants during the early phases of implantation. However, high surface roughness may increase peri-implantitis risk compared with moderate roughness (1-2 μm) within the "micron" range (1-10 μm), maximizing bone/implant interlocking. Surface profiles in the "nano" range play an important role in protein adsorption and osteoblast adhesion and thus, in osseointegration [21]. No reproducible surface roughness is currently

Dental implant failure is an active clinical research area. A number of strategies have been studied to modulate cell/material interactions, which play an important role in determining the short- and long-term implant success rate. This chapter will be mainly focused on the basic aspects to study cell/material interactions in dental implants using progenitor cells and *in vitro* biofilm formation approaches, as well as basic information related with micro-

Mesenchymal Stem Cells (MSCs) are commonly isolated from perinatal tissues (i.e, placenta, umbilical cord and blood from the umbilical cord) and postnatal tissues (bone marrow,

engineering technologies to modify dental implant surfaces.

**2. Strategies to study cell/material interactions**

roughness of the restorative components [14, 16].

*spp, enterics,* and *Candida spp* [18].

implants.

clinically available.

An osseointegrated implant is a good alternative to replace missing teeth, but they are not exempt from failure and complications (Figure 5). Oral implant failures have been classified as: 1) biological failures, which can be observed before loading and are associated with reduced osseointegration. If they take place after loading, they are associated with failing to maintain the achieved osseointegration; 2) mechanical failures, which can be observed as implant or prosthetic structural failures; 3) iatrogenic failures, mainly associated with procedures that affect anatomical structures or the misalignment of implants, which render them impossible to restore; and 4) failures by inadequate patient adaptation that include phonetical, aesthetical or psychological problems [15].

**Figure 5.** Different failure types in dental implantology. (a,b) Periapical radiography shows a radiolucent lesion on the mesial surface associated with bone resorption and sign of mobility. Photography shows the implant after removal. (c,d) Periapical radiographies show two posterior fractured implants. Fracture was apparently caused by bruxism. Note in the radiography (d) that the more anterior implant exhibits a large radiolucent zone as bone losses and the adjacent tooth shows an apical lesion. (e,f) Failure of full restoration on implants and natural teeth was caused presumably by poor design and support.

Mobility is the most evident sign of implant failure and can be presented as rotational, lateral or horizontal, and axial or vertical mobility [15]. There are different terms in the literature associated with biological implant failure or complications like peri-implant diseases, peri-mucositis and peri-implantitis, where the first two are reversible inflammatory reactions around a functioning implant. Peri-implantitis is a chronic inflammation with loss of the supporting tissues around the implant induced by bacterial colonization, facilitated by the implant/abutment gap and by the chemistry and surface roughness of the restorative components [14, 16].

278 Cell Interaction

The non-keratinized junctional epithelium is only a few cell layers thick in the apical portion and is separated from the alveolar bone by collagen-rich connective tissue. This 3 to 4 mm biological barrier, formed irrespective of the original mucosal thickness, protects the osseointegration zone from factors released by the plaque and the oral cavity [14]. The main differences between the soft tissues around natural teeth and those around implants are the collagen fibres orientation, which run parallel from the implant surface to the crest bone, the

An osseointegrated implant is a good alternative to replace missing teeth, but they are not exempt from failure and complications (Figure 5). Oral implant failures have been classified as: 1) biological failures, which can be observed before loading and are associated with reduced osseointegration. If they take place after loading, they are associated with failing to maintain the achieved osseointegration; 2) mechanical failures, which can be observed as implant or prosthetic structural failures; 3) iatrogenic failures, mainly associated with procedures that affect anatomical structures or the misalignment of implants, which render them impossible to restore; and 4) failures by inadequate patient adaptation that include

low number of fibroblasts, and the reduced vascularization of the scar tissue.

**Figure 5.** Different failure types in dental implantology. (a,b) Periapical radiography shows a radiolucent lesion on the mesial surface associated with bone resorption and sign of mobility. Photography shows the implant after removal. (c,d) Periapical radiographies show two posterior fractured implants. Fracture was apparently caused by bruxism. Note in the radiography (d) that the more anterior implant exhibits a large radiolucent zone as bone losses and the adjacent tooth shows an apical lesion. (e,f) Failure of full restoration on implants and natural teeth was caused presumably by

phonetical, aesthetical or psychological problems [15].

poor design and support.

Bacteria colonize and develop biofilms on the transmucosal abutment of osseointegrated dental implants. Like the gingival crevice around the natural tooth, the peri-implant mucosa covering the alveolar bone is closely adapted to the implant. In partially edentulous subjects, the developing microbiota around implants closely resembles the microflora of natural teeth [14]. In addition to the dark-pigmented, gram-negative anaerobic rods, other bacteria are associated with peri-implant infections (*Bacteroides forsythus, Fusobact. nucleatum, Campylobacter, Peptostreptcc. micros and Prevotella intermedia*) [17], and eventually *Staphylocc. spp, enterics,* and *Candida spp* [18].

Metals, including Ti, may induce non-specific immunomodulation and self-immunity. In immunologic *in vitro* tests, sensitization to Ti was observed [19]. Such problems with Ti and the ever-growing expectations on aesthetics lead to increasing interest in all-ceramic implants.

The surface texture of dental implants affects the rate of osseointegration [20] and biomechanical fixation. Surface roughness may be classified as "macro", "micro" and "nano" sized topologies. The "macro" range, from millimetres to 10 μm, is directly related to implant geometry, with threaded screws and macroporous coatings helping the primary stability of the implants during the early phases of implantation. However, high surface roughness may increase peri-implantitis risk compared with moderate roughness (1-2 μm) within the "micron" range (1-10 μm), maximizing bone/implant interlocking. Surface profiles in the "nano" range play an important role in protein adsorption and osteoblast adhesion and thus, in osseointegration [21]. No reproducible surface roughness is currently clinically available.

Dental implant failure is an active clinical research area. A number of strategies have been studied to modulate cell/material interactions, which play an important role in determining the short- and long-term implant success rate. This chapter will be mainly focused on the basic aspects to study cell/material interactions in dental implants using progenitor cells and *in vitro* biofilm formation approaches, as well as basic information related with microengineering technologies to modify dental implant surfaces.

## **2. Strategies to study cell/material interactions**

Mesenchymal Stem Cells (MSCs) are commonly isolated from perinatal tissues (i.e, placenta, umbilical cord and blood from the umbilical cord) and postnatal tissues (bone marrow, trabecular bone, alveolar bone, cartilage, hair follicles, fat, skin and dental pulp) [22]. Bone marrow and pulp derived cells are frequently used in oral biology research to evaluate the biocompatibility of different dental materials. However, the frequency of MSCs in both tissues is not well known (0.001 to 0.01%). Bernardo *et al.* reported that MSCs frequency in bone marrow exhibits an age-related behaviour from 1:10,000 in a new-born to 1:1,000,000 in an 80-year-old subject [23]. Another important aspect is associated with anatomic skeletal site-specific differences [24], where MSCs obtained from calvaria have proven more successful for grafting in craniofacial application than cells obtained from others donor sites.

Micropatterned Coatings for Guided Tissue Regeneration in Dental Implantology 281

promoting or inhibiting cell proliferation and differentiation since it is a complex mixture of

Fetal Bovine Serum (FBS) is an inexpensive choice to conduct *in vitro* bone regeneration studies, but Human Serum is another option that can help to obtain more realistic results, and although a direct extrapolation from *in vitro* studies to clinical situations would be desirable, this has not been possible [29]. Recently, Deorosan and Nauman (2011) evaluated the effects of the concentrations of serum and glucose on the metabolic activity of murine MSCs, and concluded that the effects of the serum percentage (e.g. 2%, 5% and 10%) were negligible. However, a high correlation was found between cell viability and glucose

Antibacterial and antimycotic agents are other types of supplements that can be controversial. Both should be avoided because they may affect cell physiology and mask improper aseptic conditions. However, from a realistic point of view, considering the risk of losing irreplaceable MSCs, the standard use of antibacterials (e.g., Penicilin/Streptomycin, 100 IU/ml and 10mg/ml, respectively; Gentamicin, 10μg/ml) and antimycotics (e.g., Amphotericin B, 0.25μg/ml) is required. Also, tetracyclines are often used to take advantage of their Ca-binding and fluorescence properties under UV light, allowing for the quantification of matrix mineralization. Other tetracyclines, like doxycycline and minocycline, when in low concentrations, may stimulate the proliferation of human

The main goal of long-term *in vitro* MSCs cell cultures is their differentiation. To promote osteoblastic differentiation, the most frequently used supplements are ascorbic acid (AA, 50μg/ml), dexamethasone (Dex, 10nM), and β-glycerolphosphate (βGP, 10mM) [32]. When tested *in vitro*, these compounds are able to promote growth and accelerate the differentiation process of osteoprogenitor cells, thus reducing the proliferation period of the developmental sequence in the expression of the osteoblastic phenotype [33]. Coelho and Fernandes studied the proliferation/differentiation of hBMSCs cultured under different conditions of mineralization supplements. Their results show high proliferation in all the tested conditions, but mineralization was only achieved in the presence of βGP and this