This study demonstrates that a specific combination of PFC liquids (PP2:PP9), that we have previously shown to significantly improve gas exchange, lung function, and attenuate inflammation in animal models of lung injury [13
], also significantly improves the efficiency of rAd gene delivery to the peripheral lung. The generation of PFC liquid/rAd suspensions specifically improved expression in the alveolar epithelial cells.
Recombinant adenovirus can induce high levels of transient transgene expression. Several important improvements have been made in this vector resulting in enhanced gene delivery to the lung. One of earliest improvements was to change from serotype 7 to serotype 5. Since serotype 5 binds the CAR receptor found in airways more efficiently, the viral titer required for gene expression is attenuated, and inflammation significantly reduced. Next, reagents such as EGTA that disrupt tight junctions have been shown to improve expression in the airways by exposing CAR on the basolateral surfaces of airway epithelium. Various PFC liquids have been utilized to improve expression in the distal airways of both healthy and diseased lungs [3
]. Our findings reflect at least two mechanisms thought to be involved with improved expression. First, it has been previously shown that PFC liquid facilitates transient disruption of tight junctions, increasing exposure to CARs, that are distributed throughout the A549 cell membrane [3
]. Second, based on the physicochemical property of this specific PFC liquid combination (with unique kinematic viscosity, vapor pressure, and lipophilicity profiles) [14
], the PFC suspension fosters increased and uniformed distribution of the virus relative to saline, such that more cells are exposed to virus. As such, clinical use of PFC liquids for the delivery of rAd could reduce the amount of virus necessary for the treatment of lung disease. This possibility is an important consideration given the inherent inflammatory response associated with adenovirus. The unique physicochemical properties of the PFC suspension may support further improvement over viscoelastic gels which have been recently shown to improve rAd and AAV expression only in the central airways without increases in alveolar expression [37
]. Our data document significant differences in transgene expression with rAd delivered in PFC versus saline at early time points both in vitro
and in vivo
. In vitro
we demonstrate a higher earlier transduction efficiency and in vivo
a more consistent pattern of temporal expression. We have demonstrated the effect of different PFC liquids on membrane fluidity and packaging using liposomes as model systems [28
]. When the amount of luciferase activity was assessed in the present study, significant differences in expression were only detected at 1
d following administration when PFC liquid suspensions were compared to other methods. We speculate that temporal differences between methods of administration (favoring PFC suspension) may actually be greater than the present analysis; however, we believe that more detailed analyses may be confounded by the high level of focal expression in upper airways with saline instillation as compared to the more peripheral distribution with PFC suspension. As such, this report demonstrates that the major advantages of PFC liquid-suspension delivery of rAd are increased early expression (, ), better distal lung expression, and better retention in the lung ().
While this study was not designed to comprehensively assess ectopic expression, our method of introduction by minitracheal puncture is likely to oppose this possibility since this method bypasses the nasal epithelium as well as minimizes the potential for the virus to move up the mucocililiary tract to transduce the gut. In addition, the alveolar surfaces appeared intact thereby minimizing access for ectopic expression through translocation across the alveolar-capillary membrane. In addition, there was very little ectopic expression detected with the luciferase construct alone.
Previous studies using PFC liquid vehicles for rAd-gene delivery utilized a pulse-chase method of administration, that is, delivery of rAd in saline followed immediately by instillation of PFC liquid [7
]. We reasoned that creating a suspension should increase efficiency by increasing the distribution of the vector compared to the pulse-chase method. We have demonstrated previously that formation of nanocrystal suspensions with PFC liquids improves delivery of biological agents (recombinant proteins and antibiotics) to the lung [33
]. However this technology is dependent on the ability to form powders of the drug/protein of interest. Since rAd is relatively labile and loses viability when lyophilized, we limited our methods to creating liquid suspensions. There have been reports of formulations that stabilize rAd for freeze-dry methods, but our attempts to create nanocrystals using this particular method proved unsuccessful (data not shown). More stable gene delivery vectors or compounds (plasmid DNA, rSV40, siRNA) would be viable candidates for PFC liquid nanocrystal suspension formation. The efficacy of siRNA strategies, delivered as either oligonucleotides or in other gene vector systems, provides the possibility of suppressing gene expression.
No method of gene delivery is ideal, and there are significant problems associated with the use of rAd (e.g., inflammation; cell specificity limited to cells expressing the appropriate receptor for viral entry), especially in the clinical setting. Several strategies have been implemented to try to minimize these problems, including generating “gutted virus” [40
] and the use of chimeric fiber [41
]. In spite of the potential difficulties of rAd, these vectors still hold promise for delivering transient overexpression of genes. As demonstrated in the present study, PFC liquid promotes uniform delivery to the lung and may minimize the inflammatory response to rAd gene products [9
]. This would be particularly important in nonhomogeneous and inflammatory types of lung injury processes where PFC liquids could facilitate distribution of genes or other biologic agents while facilitating gas exchange and improving pulmonary function for management/treatment (i.e., cystic fibrosis, COPD, or ARDS). In contrast, in disease states that affect the central or proximal airways, delivery by saline or the pulse-chase method might be advantageous.
The PFC liquid formulation used in the present studies was selected based on the greatest improvement in gas exchange and lung mechanics and attenuation of lung inflammation in models of acute lung injury [13
]. Within this context, the use of PFC suspensions to enhance gene expression challenges the notion that minor inflammation is required for gene uptake. The major improvement in gene expression in vivo
appears to be in the delivery of the virus to alveolar epithelial cells of the distal lung. Why this increase is observed in these cell types remains unclear. The most likely explanation is that the specific physicochemical properties (e.g., kinematic viscosity, vapor pressure, and lipophilic nature) of PFC liquid enhance delivery to and assist cells in the transduction of the alveoli (i.e., similar to how surfactant is recycled, alterations in membrane dynamics). We suggest that delivery into this compartment prevents clearance by the mucociliary tract.
In summary, PP2:PP9 PFC rAd suspensions increased expression in distal portions of the lung. The suspensions may facilitate receptor-mediated and independent entry of the virus which significantly increases transduction efficiency. This has important therapeutic implications for diseases where expression in the central airways is not sufficient and expression in the alveoli is required. The use of PFC liquid holds promise as a vehicle to deliver gene delivery vectors and other biologic agents in order to more directly and specifically prevent and treat high-risk lung disease processes in critically ill patients. Others have implicated a “surfactant” mediated uptake of PFC into alveolar type II cells, ostensibly associated with an endocytotic process [31
]. If these particles are then secreted out into the lung surface, it would foster recycling of the virus to transduce additional or adjacent alveolar epithelial cells. If this theory holds true, the use of PFC suspensions for gene vector delivery prevents a novel and forward-thinking contribution to the field of gene therapy. Further study of PFC-assisted gene vector delivery methods, including nebulization of PFC liquid suspensions, is warranted to potentially overcome many of obstacles currently associated with gene delivery to the lung epithelium.