**3. Breaching the physical barriers to gene delivery using ultrasound**

In order to achieve efficient gene expression following ultrasound-mediated gene delivery, multiple barriers need to be overcome to allow pDNA to enter into the nucleus of target cells including penetrating vascular and cellular membranes as well as trafficking through different intracellular compartments.

US contrast agents are intended to be intravascular agents. Whether the gene vector is administered as a simple mixture with the gas body suspension, or is in some way linked to the gas bodies, they are also intravascular agents in vascular UTMD methods. The first barrier encountered is the vascular endothelium. The next are other vascular anatomical features (*e.g.*, the basement membrane, smooth muscle layer, *etc.*) and then the outer cell membrane of the cells one hopes to target. Some intracellular membranous compartments must also be traversed. However, MBs have been used with intramuscular or intraparenchymal injections of vectors, and some successes reported. In UTMD-based gene transfer methods using vascular approaches, to mediate gene therapy *via* acoustic excitation, gas bodies must first exert their influence from within the vascular lumen.

Most of the available evidence from *in vivo* studies indicate that vessel permeabilization effects occur principally in the microcirculation; larger vessels are too robust to be penetrated by cavitation events, even if their vascular endothelium can be effectively destroyed by intraluminal inertial cavitation {Hwang *et al.* 2005}. Extravasation of dyes, nanoparticles or macromolecules through microvessels is almost always accompanied by extravasation of red cells (see below); since these have diameters on the order of 6 µm, breaches in the endothelial wall can be quite large. However, there is also some evidence that more subtle effects, such as partial opening of the tight junctions between endothelial cells, can also contribute. Assuming that the gene vector escapes the intravascular compartment and enters the interstitium, it must then enter the surrounding cells; thus the plasma membrane is the second major barrier encountered by the vectors. Lethal effects of cavitation occurring in the cardiac microcirculation can extend outward into the myocardium {Miller *et al.* 2011}; there is good reason to expect that sub lethal poration of cells located within a few cell diameters of the intravascular cavitation event(s) also occurs.

#### **3.1 Extravasation of dyes, nanoparticles and cells**

Evans blue [EB] is an azo dye which binds serum albumin with high affinity, and is normally unable to pass through the endothelium. Extravasation of EB through reversible or

Ultrasound-Mediated Gene Delivery 221

Two mechanisms by which US and MBs facilitate poration of cell membranes are prominent; these are: (1) cavitational; *e.g*., the opening of transient holes in membranes in consequence to local shear forces exerted on membranes by fluid flow ('micro streaming') around oscillating bubbles, local shock waves (which produce large pressure gradients across a cell), or cavitation microjets, or (2) endocytosis {Doinikov & Bouakaz 2010; Walton & Shohet 2009}. Other mechanisms have been proposed, of course. However, it appears that shear stresses associated with bubble activation is probably the principal mechanism. A third mechanism by which normally 'tight' physiological barriers may be permeabilized is

Transient pores of a few hundred nanometers in diameter and lifetimes of several seconds can be formed in cell membranes by acoustically-driven, single MB oscillations {Deng *et al.* 2004; Han *et al.* 2007; Zhou *et al.* 2008; Zhou *et al.* 2009}. Voltage clamp studies of *Xenopus* oocytes exposed to 1 MHz US showed that without MBs, there was no change in current at Pr as high as 1.2 MPa. With Optison MBs, opening and resealing of individual pores was observable even at Pr as low as 0.2 MPa. The transmembrane current was carried by influx of Ca2+ ions. The transmembrane current was greater at 0.4 MPa than at 0.2 MPa; in both cases, pore lifetime was ~2 s. More generally, the effect was Pa dependent, increasing slowly with increasing Pa over the range of 0.3 – 0.55 MPa, and then inflecting sharply upward at higher Pa {Deng *et al.* 2004}. Pore opening showed a high level of temporal correlation with inertial cavitation noise {Zhou *et al.* 2008}. Pore size was estimated as 220 ± 80 (mean ± SD) nanometers, and refined estimates of pore resealing times indicated closure in 3 – 5 s {Zhou *et al.* 2009}. *In vitro* studies of cells in contact with SonoVue MBs excited by 1 MHz US at Pa of 0.05 – 2.50 MPa have shown Ca2+ influx associated with poration {Juffermans *et al.* 2008}. Sonoporation to naked DNA *in vitro* is correlated with inertial cavitation activity {Qiu *et al.* 2010}, and depends in part on the fluidity of the membrane at the time of UTMD treatment {Zarnitsyn & Prausnitz 2004}, with higher reporter gene transfection rates at 37 oC than at 21 oC. This is consistent with the need for porated membranes to reseal rapidly in order to maintain viability. Still others have shown that 1 MHz US at Pa as low as 0.1 MPa can permeabilize cells *in vitro* to pDNA {Rahim *et al.* 2006}. In cell suspensions containing Definity MBs, the Pa thresholds for cell permeabilization to 70 kDa FITC-dextran, propidium iodide (indicating cell death) and MB destruction were 75 kPa at 0.5 MHz, 200 kPa at 2.5 MHz, and 600 kPa at 5 MHz {Karshafian *et al.* 2009}. However, the idea that cellular poration results from inertial cavitation has been challenged on the basis of observations of cavitation noise and permeabilization of *in vitro* cell monolayers to 500 kDa FITC dextran; the supporting data indicate that permeabilization precedes the occurrence of broadband noise associated with inertial collapse and rebound cavitation. The authors conclude that the forces associated with microstreaming around activated bubbles is the principal mechanism

of sonoporation {Forbes *et al.* 2008}. More study is clearly needed.

with high cell mortality, this is not always the case {Wang *et al.* 2009}.

US and MB-induced cell lysis and reversible poration in other cell types *in vitro* are strongly correlated with inertial cavitation {Chen *et al.* 2003a; Chen *et al.* 2003b; Lai *et al.* 2006}. Cell surface antigens may be stripped off the surface of viable cells during such events {Brayman *et al.* 1999}. While *in vitro* UTMD-based transfection with naked DNA is often associated

The use of the collapse jets of MBs generated by laser pulses to selectively and directionally sonoporate individual cells has reached a high level of sophistication, producing pore sizes of ~ 200 nm {Sankin *et al.* 2010}, which may someday prove useful for cell therapies. This is very

**3.2 Transient poration of the cell membrane** 

by sonochemical stimulation.

irreversible capillary modification has been observed in small animal (rat) hearts exposed to low-MHz frequency US with the use of various contrast agent MBs, accompanied by premature ventricular contractions {Li *et al.* 2004}. The effect was sensitive to Pa (apparent threshold somewhat less than 1.6 MPa at 1.5 MHz) and to the concentration of the injected MBs {Miller et al. 2004}. The effect appears to be of mechanical origin {Miller *et al.* 2004} With injection of dilute Definity suspensions, extravasation of EB from canine hearts, and cell killing of cardiomycetes within the ultrasonically-interrogated area also occurs under diagnostically realistic exposure conditions {Miller *et al.* 2006}.

Erythrocyte extravasation has been observed in rat kidney glomerular capillaries exposed to diagnostic US with contrast agent MBs in the circulation {Miller et al. 2010a, Miller et al., 2010b}; such damage was associated with tissue inflammation at 1 week and fibrosis at 4 weeks {Miller *et al.* 2009}. A threshold Pa of ~0.8 MPa was indicated {Miller *et al.* 2007}. Glomerular capillary hemorrhage with contrast-enhanced diagnostic US also occurs in large animals (pigs) exposed to 1.5 MHz, 1.7 MPa US {Miller *et al.* 2010}. Petechial hemorrhage on intestinal blood vessels in an *in vivo* murine model was observed with the contrast agent Albunex, with apparent Pa thresholds of 0.4 MPa at 0.4 MHz, 0.8 MPa at 1.1 MHz, or 2.3 MPa at 2.3 MHz; the thresholds were lower with the agent Levovist than with Albunex {Miller & Gies 1998a, Miller & Gies 1998b}. At 2.25 MHz, a threshold Pa of between 0.85 and 1.0 MPa was indicated for vessel damage in exteriorized rat cremaster muscle containing Definity MBs {Samuel *et al.* 2009}. Similarly, intravital observation of red cell extravasation from rat cremaster muscle capillaries containing MB contrast agent was observed more than a decade ago {Price *et al.* 1998; Skyba *et al.* 1998}. Microscopic observations of US and MB-induced red cell extravasation have been reported in glass catfish {Maruvada & Hynynen 2004}. In exteriorized rat spinotrapezius muscle, 1 MHz US at a Pa of 0.75 MPa was shown to result in extravasation of 100 nm diameter microspheres co-injected with contrast agent MBs {Song *et al.* 2002}. In rats, *in vivo* exposure of the kidneys following injection of MBs resulted in red cell extravasation from the glomerular capillaries into Bowman's space with an apparent threshold Pa of ~ 0.73 MPa (at ~ 1 MHz), with nearly 40% of histological sections taken from the focal plane showing extravasation at a Pa of 1.8 MPa {Miller *et al.* 2007}. Intravital microscopy and concurrent cavitation detection was used to study the relationship between bubble dynamics and extravasation of red cells from rat cremaster muscle using 2.25 MHz US, and Definity contrast agent infused *via* the tail vein. Vascular damage and acoustic emissions from the MBs were correlated. The greatest amount of red cell extravasation and the greatest cumulated bubble acoustic emissions occurred at 10 Hz pulse repetition frequency **[PRF]**, indicating that the time for tissue refill with bubbles following each pulse was ~100 ms. In experiments in which pulses were applied at 100 Hz PRF and the Pa varied from 0 – 2.0 MPa, there was no vascular damage at Pr ≤ 0.85 MPa, but unambiguous damage occurred at Pr ≥ 1.0 MPa {Samuel *et al.* 2009}. Finally, there is evidence that low amplitude US induced MB oscillations (1 MHz, Pr = 0.l MPa, SonoVue bubbles) can increase the permeability of primary endothelial monolayer cultures *in vitro*. Insonation produced immediate influx of Ca2+ ions into the cells, indicating poration of the endothelial plasma membranes. The effect was essentially abolished by application of catalase, strongly suggesting a role for extracellularly-produced H2O2 associated with nonlinear bubble oscillations. Moreover, histochemical staining for a protein associated with gap junctions showed an approximately 50% increase immediately after insonation, but returned to control levels within 30 minutes of insonation {Juffermans *et al.* 2009}.

irreversible capillary modification has been observed in small animal (rat) hearts exposed to low-MHz frequency US with the use of various contrast agent MBs, accompanied by premature ventricular contractions {Li *et al.* 2004}. The effect was sensitive to Pa (apparent threshold somewhat less than 1.6 MPa at 1.5 MHz) and to the concentration of the injected MBs {Miller et al. 2004}. The effect appears to be of mechanical origin {Miller *et al.* 2004} With injection of dilute Definity suspensions, extravasation of EB from canine hearts, and cell killing of cardiomycetes within the ultrasonically-interrogated area also occurs under

Erythrocyte extravasation has been observed in rat kidney glomerular capillaries exposed to diagnostic US with contrast agent MBs in the circulation {Miller et al. 2010a, Miller et al., 2010b}; such damage was associated with tissue inflammation at 1 week and fibrosis at 4 weeks {Miller *et al.* 2009}. A threshold Pa of ~0.8 MPa was indicated {Miller *et al.* 2007}. Glomerular capillary hemorrhage with contrast-enhanced diagnostic US also occurs in large animals (pigs) exposed to 1.5 MHz, 1.7 MPa US {Miller *et al.* 2010}. Petechial hemorrhage on intestinal blood vessels in an *in vivo* murine model was observed with the contrast agent Albunex, with apparent Pa thresholds of 0.4 MPa at 0.4 MHz, 0.8 MPa at 1.1 MHz, or 2.3 MPa at 2.3 MHz; the thresholds were lower with the agent Levovist than with Albunex {Miller & Gies 1998a, Miller & Gies 1998b}. At 2.25 MHz, a threshold Pa of between 0.85 and 1.0 MPa was indicated for vessel damage in exteriorized rat cremaster muscle containing Definity MBs {Samuel *et al.* 2009}. Similarly, intravital observation of red cell extravasation from rat cremaster muscle capillaries containing MB contrast agent was observed more than a decade ago {Price *et al.* 1998; Skyba *et al.* 1998}. Microscopic observations of US and MB-induced red cell extravasation have been reported in glass catfish {Maruvada & Hynynen 2004}. In exteriorized rat spinotrapezius muscle, 1 MHz US at a Pa of 0.75 MPa was shown to result in extravasation of 100 nm diameter microspheres co-injected with contrast agent MBs {Song *et al.* 2002}. In rats, *in vivo* exposure of the kidneys following injection of MBs resulted in red cell extravasation from the glomerular capillaries into Bowman's space with an apparent threshold Pa of ~ 0.73 MPa (at ~ 1 MHz), with nearly 40% of histological sections taken from the focal plane showing extravasation at a Pa of 1.8 MPa {Miller *et al.* 2007}. Intravital microscopy and concurrent cavitation detection was used to study the relationship between bubble dynamics and extravasation of red cells from rat cremaster muscle using 2.25 MHz US, and Definity contrast agent infused *via* the tail vein. Vascular damage and acoustic emissions from the MBs were correlated. The greatest amount of red cell extravasation and the greatest cumulated bubble acoustic emissions occurred at 10 Hz pulse repetition frequency **[PRF]**, indicating that the time for tissue refill with bubbles following each pulse was ~100 ms. In experiments in which pulses were applied at 100 Hz PRF and the Pa varied from 0 – 2.0 MPa, there was no vascular damage at Pr ≤ 0.85 MPa, but unambiguous damage occurred at Pr ≥ 1.0 MPa {Samuel *et al.* 2009}. Finally, there is evidence that low amplitude US induced MB oscillations (1 MHz, Pr = 0.l MPa, SonoVue bubbles) can increase the permeability of primary endothelial monolayer cultures *in vitro*. Insonation produced immediate influx of Ca2+ ions into the cells, indicating poration of the endothelial plasma membranes. The effect was essentially abolished by application of catalase, strongly suggesting a role for extracellularly-produced H2O2 associated with nonlinear bubble oscillations. Moreover, histochemical staining for a protein associated with gap junctions showed an approximately 50% increase immediately after insonation, but returned to control levels within 30 minutes of insonation {Juffermans *et al.* 2009}.

diagnostically realistic exposure conditions {Miller *et al.* 2006}.

#### **3.2 Transient poration of the cell membrane**

Two mechanisms by which US and MBs facilitate poration of cell membranes are prominent; these are: (1) cavitational; *e.g*., the opening of transient holes in membranes in consequence to local shear forces exerted on membranes by fluid flow ('micro streaming') around oscillating bubbles, local shock waves (which produce large pressure gradients across a cell), or cavitation microjets, or (2) endocytosis {Doinikov & Bouakaz 2010; Walton & Shohet 2009}. Other mechanisms have been proposed, of course. However, it appears that shear stresses associated with bubble activation is probably the principal mechanism. A third mechanism by which normally 'tight' physiological barriers may be permeabilized is by sonochemical stimulation.

Transient pores of a few hundred nanometers in diameter and lifetimes of several seconds can be formed in cell membranes by acoustically-driven, single MB oscillations {Deng *et al.* 2004; Han *et al.* 2007; Zhou *et al.* 2008; Zhou *et al.* 2009}. Voltage clamp studies of *Xenopus* oocytes exposed to 1 MHz US showed that without MBs, there was no change in current at Pr as high as 1.2 MPa. With Optison MBs, opening and resealing of individual pores was observable even at Pr as low as 0.2 MPa. The transmembrane current was carried by influx of Ca2+ ions. The transmembrane current was greater at 0.4 MPa than at 0.2 MPa; in both cases, pore lifetime was ~2 s. More generally, the effect was Pa dependent, increasing slowly with increasing Pa over the range of 0.3 – 0.55 MPa, and then inflecting sharply upward at higher Pa {Deng *et al.* 2004}. Pore opening showed a high level of temporal correlation with inertial cavitation noise {Zhou *et al.* 2008}. Pore size was estimated as 220 ± 80 (mean ± SD) nanometers, and refined estimates of pore resealing times indicated closure in 3 – 5 s {Zhou *et al.* 2009}. *In vitro* studies of cells in contact with SonoVue MBs excited by 1 MHz US at Pa of 0.05 – 2.50 MPa have shown Ca2+ influx associated with poration {Juffermans *et al.* 2008}. Sonoporation to naked DNA *in vitro* is correlated with inertial cavitation activity {Qiu *et al.* 2010}, and depends in part on the fluidity of the membrane at the time of UTMD treatment {Zarnitsyn & Prausnitz 2004}, with higher reporter gene transfection rates at 37 oC than at 21 oC. This is consistent with the need for porated membranes to reseal rapidly in order to maintain viability. Still others have shown that 1 MHz US at Pa as low as 0.1 MPa can permeabilize cells *in vitro* to pDNA {Rahim *et al.* 2006}. In cell suspensions containing Definity MBs, the Pa thresholds for cell permeabilization to 70 kDa FITC-dextran, propidium iodide (indicating cell death) and MB destruction were 75 kPa at 0.5 MHz, 200 kPa at 2.5 MHz, and 600 kPa at 5 MHz {Karshafian *et al.* 2009}. However, the idea that cellular poration results from inertial cavitation has been challenged on the basis of observations of cavitation noise and permeabilization of *in vitro* cell monolayers to 500 kDa FITC dextran; the supporting data indicate that permeabilization precedes the occurrence of broadband noise associated with inertial collapse and rebound cavitation. The authors conclude that the forces associated with microstreaming around activated bubbles is the principal mechanism of sonoporation {Forbes *et al.* 2008}. More study is clearly needed.

US and MB-induced cell lysis and reversible poration in other cell types *in vitro* are strongly correlated with inertial cavitation {Chen *et al.* 2003a; Chen *et al.* 2003b; Lai *et al.* 2006}. Cell surface antigens may be stripped off the surface of viable cells during such events {Brayman *et al.* 1999}. While *in vitro* UTMD-based transfection with naked DNA is often associated with high cell mortality, this is not always the case {Wang *et al.* 2009}.

The use of the collapse jets of MBs generated by laser pulses to selectively and directionally sonoporate individual cells has reached a high level of sophistication, producing pore sizes of ~ 200 nm {Sankin *et al.* 2010}, which may someday prove useful for cell therapies. This is very

Ultrasound-Mediated Gene Delivery 223

allowing DOX penetration into the cell nuclei. Whether MBs and/or nanodroplet emulsions

Once pDNA enters the nucleus, some DNA can be condensed by histones and form persistent nucleosome-like structures. Persistent gene expression from these stable episomal pDNA genomes requires the introduction of specific cis-acting elements in the gene transfer constructs. We have demonstrated that incorporation of locus control region and intron elements into specific gene transfer constructs can achieve persistent expression of therapeutic-levels of coagulation proteins in the liver following nonviral gene therapy {Miao *et al.* 2001; Miao *et al.* 2003; Ye *et al.* 2003}. The locus control region {Yant *et al.* 2003} can contribute to the open chromatin structure of the pDNA genome and avoid silencing of the transgene expression cassette {Miao *et al.* 2005}. Furthermore, addition of an intron element significantly enhanced the transcription efficiency of stable mRNA {Miao *et al.* 2005}. Thus, gene transfer of episomal pDNA into slow-dividing or terminally differentiated cells facilitated by UTMD has high potential to achieve a therapeutic effect to treat specific diseases. For gene transfer into dividing cells where integration of target genes are required, incorporation of other systems such as sleeping beauty transposons {Aronovich *et al.* 2011; Yant *et al.* 2007} or PhiC31 integrase {Keravala *et al.* 2011} with UTMD-mediated

can significantly augment DNA delivery to the nucleus needs further investigation.

nonviral gene transfer methodology can lead to long-term transgene expression.

**4.1 Naked DNA: Plasmids** 

Song *et al.* 2011}.

**4. The vectors: Packaging desired genes for ultrasound-mediated delivery** 

Naked plasmid DNA provides many advantages as a nonviral gene transfer vector, including: (1) ease of preparation, (2) cost-effectiveness, (3) minimum toxicity, and (4) it is least immunogenic of the vectors. Indeed, the immunogenic CpG moiety can be modified easily if needed. Most importantly, quality control is quite easy compared to other pDNA complexes containing synthetic vehicles. However, it has been a very challenging problem to deliver naked pDNA into specific cells due to its large size and negative charge. *In vivo* delivery of naked pDNA is especially difficult with additional impediments of instability of pDNA in blood serum as well as in cellular sub-compartments such as cytosol, endosome, and nucleus after cell entry. Recently a hydrodynamic approach has been developed to drive efficient gene delivery into liver {Liu *et al.* 1999} and muscle {Danko *et al.* 1997} resulting in therapeutic levels of transgene expression in animal disease models, including hemophilia B {Miao *et al.* 2003} and others {Zhang *et al.* 2000}. This method in its current form is not suitable for clinical use; however, notable recent advances have been made in large animal models (*vide, e.g*., {Fabre *et al.* 2008; Kamimura *et al.* 2009; Suda *et al.* 2008}. Alternatively, innovative US and MB technology to facilitate delivery of naked pDNA is a potential clinically feasible nonviral gene therapy approach {Miao *et al.* 2005; Shen *et al.* 2008;

The potential for sonoporation to increase pDNA loading of cells was recognized years ago {Fechheimer *et al.* 1987}. Sonoporation-enhanced transport of nanoparticles into cells is dependent on molecular size; uptake of particles ≤ 37 nm diameter was enhanced by sonoporation without gross damage, but particle uptake generally declined as particle size increased {Mehier-Humbert *et al.* 2005a}. pDNA gene expression is faster with sonoporation than with liposome-based methods which depend on endocytosis {Mehier-Humbert *et al.* 2005}, indicating that pDNA enters cells through transient pores. Moreover, due to the risk of pDNA degradation by serum nucleases and removal by phagocytes {Niidome & Huang

similar to pore size determined by sonoporation of *Xenopus* oocytes using 1.07 MHz US of 0.3 MPa Pa with Definity MBs; *viz*., a mean pore size of 220 ± 80 nm diameter {Zhou Y *et al.* 2009}. Lipoplexes, which are normally taken up by cells *via* endocytosis, are taken up poorly when 'PEGylated' (coated with polyethylene glycol). However, when PEGylated pDNA/lipolexes were attached to acoustically activated MBs, large increases in PEGylated lipoplex transfection were achieved in an *in vitro* model system, relative to ether 'free' PEGylated lipolexes, or lipolexes + MB simple (un-linked) mixtures {Lentacker *et al.* 2009}. The investigators also used a number of endocytosis inhibitors; their results indicate that endocytosis was not the primary mechanism involved. They postulate that lipoplex-loaded MBs collapsing near cell monolayers in culture plates released the lipolexes, resulting in high local lipoplex concentrations, that some of the released lipolexes became entrained in MB collapse jets and were 'injected' into the cells with the fluid jet (see also {Miller 2000}). However, Meijering and colleagues, using fluorescently labeled dextrans of ~4 – 500 kDa, present data which indicate that the principal mechanism by which UTMD treatment (1 MHz US, 0.2 MPa Pr, SonoVue MBs) facilitates macromolecule delivery across the plasma membrane is *via* induction of endocytosis. Sonoporation was also observed, but did not seem to be the mechanism for macromolecular uptake {Meijering *et al.* 2009}. It is clear that the our understanding of the mechanism(s) of UTMD-enhanced uptake of macromolecules remains incomplete.

#### **3.3 Entry of pDNA into the nucleus**

Following delivery of pDNA across the plasma membrane of cells into cytoplasm, pDNA may travel through multiple cellular compartments and finally enter the nucleus *via*  diffusion {Liang *et al.* 2004} or other assisted mechanism to produce efficient transgene expression. UTMD may facilitate overcoming some of these barriers most likely *via* cavitation bioeffects. It was reported that long-term exposure to therapeutic ultrasound (1 MHz, 2 W/cm2, 30% duty cycle for 30 mins) can overcome the rate-limiting step of driving DNA into the cell nucleus {Duvshani-Eshet & Machluf 2005}. One thousand fold higher gene expression levels of luciferase was achieved with minimal loss in cell viability (<20%) in three different cell types (BHK, LNCaP, and BCE). These data implied that therapeutic US is the main driving force delivering pDNA not only to the cell cytoplasm but also to the nucleus. However, in this case, no MB was used. The same group {Duvshani-Eshet *et al.* 2006} showed that adding Optison further increased transfection levels. Confocal and atomic force microscopy studies indicated that long-term therapeutic US application localizes the pDNA in cell and nucleus regardless of Optison addition. In addition, the use Optison did not affect the kinetics of protein expression, indicating Optison did not affect DNA trafficking to the nucleus. They hypothesized that US application by itself plays a major role in delivering DNA to the nucleus.

An interesting recent report used Doxorubicin (DOX) as a molecular nanotheranostic agent to study UTMD-mediated intracellular delivery and nuclear trafficking {Mohan & Rapoport 2010}. DOX is a popular research tool due to its inherent fluorescence and was encapsulated in poly(ethylene glycol)-co-polycaprolactone (PEG-PCL) micelles or PEG-PCL stabilized perfluorocarbon nanodroplets in this study. US triggered DOX trafficking into cell nuclei; the trafficking was further enhanced in the presence of phase-transition nanodroplets which become gas MBs upon US exposure of sufficient Pr. This was believed to be due to cavitation induced transient permeabilization of both plasma and nuclear membranes, thus

similar to pore size determined by sonoporation of *Xenopus* oocytes using 1.07 MHz US of 0.3 MPa Pa with Definity MBs; *viz*., a mean pore size of 220 ± 80 nm diameter {Zhou Y *et al.* 2009}. Lipoplexes, which are normally taken up by cells *via* endocytosis, are taken up poorly when 'PEGylated' (coated with polyethylene glycol). However, when PEGylated pDNA/lipolexes were attached to acoustically activated MBs, large increases in PEGylated lipoplex transfection were achieved in an *in vitro* model system, relative to ether 'free' PEGylated lipolexes, or lipolexes + MB simple (un-linked) mixtures {Lentacker *et al.* 2009}. The investigators also used a number of endocytosis inhibitors; their results indicate that endocytosis was not the primary mechanism involved. They postulate that lipoplex-loaded MBs collapsing near cell monolayers in culture plates released the lipolexes, resulting in high local lipoplex concentrations, that some of the released lipolexes became entrained in MB collapse jets and were 'injected' into the cells with the fluid jet (see also {Miller 2000}). However, Meijering and colleagues, using fluorescently labeled dextrans of ~4 – 500 kDa, present data which indicate that the principal mechanism by which UTMD treatment (1 MHz US, 0.2 MPa Pr, SonoVue MBs) facilitates macromolecule delivery across the plasma membrane is *via* induction of endocytosis. Sonoporation was also observed, but did not seem to be the mechanism for macromolecular uptake {Meijering *et al.* 2009}. It is clear that the our understanding of the mechanism(s) of UTMD-enhanced uptake of macromolecules

Following delivery of pDNA across the plasma membrane of cells into cytoplasm, pDNA may travel through multiple cellular compartments and finally enter the nucleus *via*  diffusion {Liang *et al.* 2004} or other assisted mechanism to produce efficient transgene expression. UTMD may facilitate overcoming some of these barriers most likely *via* cavitation bioeffects. It was reported that long-term exposure to therapeutic ultrasound (1 MHz, 2 W/cm2, 30% duty cycle for 30 mins) can overcome the rate-limiting step of driving DNA into the cell nucleus {Duvshani-Eshet & Machluf 2005}. One thousand fold higher gene expression levels of luciferase was achieved with minimal loss in cell viability (<20%) in three different cell types (BHK, LNCaP, and BCE). These data implied that therapeutic US is the main driving force delivering pDNA not only to the cell cytoplasm but also to the nucleus. However, in this case, no MB was used. The same group {Duvshani-Eshet *et al.* 2006} showed that adding Optison further increased transfection levels. Confocal and atomic force microscopy studies indicated that long-term therapeutic US application localizes the pDNA in cell and nucleus regardless of Optison addition. In addition, the use Optison did not affect the kinetics of protein expression, indicating Optison did not affect DNA trafficking to the nucleus. They hypothesized that US application by itself plays a

An interesting recent report used Doxorubicin (DOX) as a molecular nanotheranostic agent to study UTMD-mediated intracellular delivery and nuclear trafficking {Mohan & Rapoport 2010}. DOX is a popular research tool due to its inherent fluorescence and was encapsulated in poly(ethylene glycol)-co-polycaprolactone (PEG-PCL) micelles or PEG-PCL stabilized perfluorocarbon nanodroplets in this study. US triggered DOX trafficking into cell nuclei; the trafficking was further enhanced in the presence of phase-transition nanodroplets which become gas MBs upon US exposure of sufficient Pr. This was believed to be due to cavitation induced transient permeabilization of both plasma and nuclear membranes, thus

remains incomplete.

**3.3 Entry of pDNA into the nucleus** 

major role in delivering DNA to the nucleus.

allowing DOX penetration into the cell nuclei. Whether MBs and/or nanodroplet emulsions can significantly augment DNA delivery to the nucleus needs further investigation.

Once pDNA enters the nucleus, some DNA can be condensed by histones and form persistent nucleosome-like structures. Persistent gene expression from these stable episomal pDNA genomes requires the introduction of specific cis-acting elements in the gene transfer constructs. We have demonstrated that incorporation of locus control region and intron elements into specific gene transfer constructs can achieve persistent expression of therapeutic-levels of coagulation proteins in the liver following nonviral gene therapy {Miao *et al.* 2001; Miao *et al.* 2003; Ye *et al.* 2003}. The locus control region {Yant *et al.* 2003} can contribute to the open chromatin structure of the pDNA genome and avoid silencing of the transgene expression cassette {Miao *et al.* 2005}. Furthermore, addition of an intron element significantly enhanced the transcription efficiency of stable mRNA {Miao *et al.* 2005}. Thus, gene transfer of episomal pDNA into slow-dividing or terminally differentiated cells facilitated by UTMD has high potential to achieve a therapeutic effect to treat specific diseases. For gene transfer into dividing cells where integration of target genes are required, incorporation of other systems such as sleeping beauty transposons {Aronovich *et al.* 2011; Yant *et al.* 2007} or PhiC31 integrase {Keravala *et al.* 2011} with UTMD-mediated nonviral gene transfer methodology can lead to long-term transgene expression.
