Journal of Controlled Release 161 (2012) 214–224

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Journal of Controlled Release

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Review

Controlled pulmonary drug and gene delivery using polymeric nano-carriers

Moritz Beck-Broichsitter, Olivia M. Merkel 1, Thomas Kissel ⁎Department of Pharmaceutics and Biopharmacy, Philipps-Universität, Ketzerbach 63, D-35037 Marburg, Germany

⁎ Corresponding author at: Department of PharPhilipps-Universität Marburg, Ketzerbach 63, D-350326421 28 25881; fax: +49 6421 28 27016.

E-mail address: kissel@staff.uni-marburg.de (T. Kiss1 Current address: Department of Pharmaceutical Sci

259 Mack Ave, Detroit, MI 48201, USA.

0168-3659/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.jconrel.2011.12.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 August 2011Accepted 6 December 2011Available online 13 December 2011

Keywords:Pulmonary drug deliveryGene therapyInhalationNanoparticlesIsolated lung model

Pulmonary drug and gene delivery to the lung represents a non-invasive avenue for local and systemic ther-apies. However, the respiratory tract provides substantial barriers that need to be overcome for successfulpulmonary application. In this regard, micro- and nano-sized particles offer novel concepts for the develop-ment of optimized therapeutic tools in pulmonary research. Polymeric nano-carriers are generally preferredas controlled pulmonary delivery systems due to prolonged retention in the lung. Specific manipulation ofnano-carrier characteristics enables the design of “intelligent” carriers specific for modulation of the durationand intensity of pharmacological effects. New formulations should be tested for pulmonary absorption anddistribution using more advanced ex vivo and in vivo models. The delivery of nano-carriers to the air-spaceenables a detailed characterization of the interaction between the carrier vehicle and the natural pulmonaryenvironment. In summary, polymeric nanoparticles seem to be especially promising as controlled deliverysystems and represent a solid basis for future advancement for pulmonary delivery applications.

© 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2142. Structure and function of the respiratory tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2153. Controlled pulmonary drug and gene delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

3.1. Current strategies to prolong the retention of therapeutic agents within the lung. . . . . . . . . . . . . . . . . . . . . . . . . . . 2163.1.1. Encapsulation of the therapeutic agent in polymeric nano-carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2163.1.2. Avoiding the clearance mechanisms of the lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

3.2. Models to evaluate the potential of polymeric nano-carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2183.3. Pulmonary gene delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

3.3.1. Polymeric vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2203.3.2. Models to determine successful in vivo gene delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

4. Conclusion & perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

1. Introduction

The lung is a directly accessible organ from the outside. Thus, in-halation therapy represents an attractive application route for thetargeted delivery of medications to their desired site of action. Thelocal application of therapeutic agents to the respiratory system has

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several advantages over other routes of administration, like increasedselectivity, high local concentration, and lower systemic exposure andhence emerged as a prevalent approach in the treatment of respirato-ry diseases [1,2]. Site-specific delivery also facilitates a reduction ofthe necessary dose to be administered. In addition, inhalation repre-sents a non-invasive alternative for systemic delivery of pharmaceu-ticals (e.g. peptides and proteins). The lower enzymatic activity(compared to the oral route), large alveolar surface area and thinepithelial air-blood barrier allow rapid absorption of macromoleculesfrom the alveolar airspace [3].

In general, the advantage of inhalation therapy depends on thefate of the delivered medication (mechanism and rate of elimination)in the respiratory tract. Once the therapeutic agent has been

Fig. 1. Schematic of the microstructure of the human respiratory tract [reproducedfrom [3] with permission of the copyright holder].

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deposited in the lung, elimination is immediately initiated, decreasingthe initial high local concentrations in lung tissue [3–6]. Therapid decay of drug concentrations often requires multiple daily inha-lations (up to 9 times), conflicting with the patients' convenience andcompliance [7]. Moreover, “conventional” inhalation therapy doesnot permit targeted delivery to specific lung cells (e.g. alveolar typeII cells) and a deposition of the therapeutic compound in differentlung areas is often poorly controlled.

To overcome these short-comings of “conventional” inhalationtherapy, more sophisticated, “intelligent” pulmonary delivery sys-tems are desirable. Among the large number of potential carriersystems, micro- and nano-sized vehicles have attracted growingattention due to their controlled release and targeting properties[8]. Moreover, the design of vehicles with specific physicochemicalproperties (e.g. size, shape, surface chemistry, and bioadhesive prop-erties) that bypass the clearance mechanisms of the lung couldprovide prolonged residence times of the therapeutic agent withinthe respiratory tract [9].

Nano-carriers composed of polymers with particular physico-chemical and biological properties have been identified as attractivecandidates [10–15]. These delivery systems fulfill the stringentrequirements placed on pulmonary delivery devices, such as suffi-cient association of the therapeutic agent with the carrier particles,targeting of specific sites or cell populations in the lung, protectionof the therapeutic agent against degradation, release of the therapeu-tic agent at a therapeutically optimal rate, ability to be transferredinto an aerosol, stability against forces generated during aerosoliza-tion as well as low toxicity.

The biological environment strongly influences the performanceof delivery vehicles at the target site. Consequently, conventionalin vitro studies may not reflect and predict the delivery situationunder in vivo conditions impeding the development of more ad-vanced pulmonary delivery systems. Accordingly, ex vivo and in vivomodels were commonly employed to investigate lung-specificpharmacokinetics and pharmacodynamics of inhaled therapeutics[16–21].

This review will give an overview of the application of polymericnano-carriers for pulmonary drug and gene delivery. Anatomy andphysiology of the respiratory tract, methods for nanoparticle prepara-tion, attributes of the employed polymeric materials, toxicologicalconsiderations, as well as strategies to bypass lung clearance mecha-nisms are discussed. Recent results from ex vivo and in vivo studiesare included to underline the unique potential of nanoparticulatedelivery systems for the treatment of respiratory diseases.

2. Structure and function of the respiratory tract

A detailed knowledge of lung physiology is an important prerequi-site for the development of new pulmonary delivery systems[1–3,22]. The respiratory tract is divided into two distinct zones: theconducting airways and the respiratory zone. The conducting airwaysact as an air transport system and include the mouth/nasal cavity,pharynx, larynx, trachea, bronchi and bronchioles. In the respiratoryzone, i.e. respiratory bronchioles and alveoli, the gas exchange takesplace. The conducting airways exhibit 16 bifurcations, followed byanother 6 bifurcations of the respiratory bronchioles representingthe passage to the respiratory zone where the alveolar ducts withalveolar sacs finally branch off.

The cell layer thickness of the air-blood barrier gradually de-creases from ≥10 μm in the tracheo-bronchial region to ≤0.3–1 μmin the alveolar region (Fig. 1). The walls of the conducting airwaysare coated by an adhesive, viscoelastic mucus layer (thickness:5–55 μm) secreted by goblet and submucosal gland cells.

The major components of respiratory mucus are glycoproteins andwater [23,24]. Clearance of mucus from the lung is driven by the mo-tion of ciliated cells (broncho-tracheal escalator), which generate a

mucus flow rate of ~5 mm/min. Thus, the respiratory mucus blanketis replaced every 20 min in healthy subjects [5,24]. The composition,thickness, physicochemical properties (e.g. viscosity) and clearanceof respiratory mucus is often altered in patients suffering from airwaydiseases such as asthma, chronic obstructive pulmonary disease andcystic fibrosis [5]. Pathological conditions may affect the efficacy ofpulmonary drug and gene delivery systems especially if the payloadhas to be delivered to the tissue of the conducting airways. The alve-olar space is coated by a complex surfactant lining that reducessurface tension to minimize the work of breathing and preventscollapse of the alveoli during expiration. Pulmonary surfactant issecreted by cuboidal type II pneumocytes and contains 90% lipidsand 10% proteins [25]. Type I pneumocytes are thin cells (≤200 nm)with a large extension (~200 μm), covering over ~95% of the alveolarepithelial surface [26].

The airways also fulfill additional physiological functions, such aswarming, humidifying and cleaning of the inhaled air. Effective clear-ance mechanisms are needed to eliminate foreign materials fromthe airways. The majority of insoluble particles deposited in theupper airways are eliminated by mucociliary clearance [5]. The mostprominent defense mechanism of the respiratory region is macro-phage clearance. Particles deposited in the deeper lung will betaken up by alveolar macrophages, which slowly migrate out of thelung, either following the broncho-tracheal escalator or the lymphaticsystem [6,27]. Particle clearance by macrophages appears most effi-cient for particles having a geometric size between 1 and 3 μm [6].

3. Controlled pulmonary drug and gene delivery

Although the lung possesses effective barrier systems and clear-ance mechanisms, much attention has been paid to this organ fordrug and gene delivery applications. The large alveolar surface area(~150 m2) is enveloped by a capillary network less than 1 μm be-neath the epithelial surface, from which many agents can be readilyabsorbed to the bloodstream [1,22]. Moreover, therapeutic aerosolsare effective treatment modalities for local respiratory diseases likebronchial asthma, chronic obstructive pulmonary disease or life-threatening pulmonary hypertension, providing superior pulmonaryselectivity compared to the oral or intravenous route of administra-tion [1,28]. However, “conventional” pulmonary dosage forms lackcontrolled release properties. Thus, local and systemic inhalationtherapies would benefit from pulmonary formulations which regulaterelease rates over extended periods of time [7,29]. Among the diversecarriers described for pulmonary applications, polymeric nanoparti-cles show potential as controlled delivery devices due to prolongedretention in the lung [10–12,14,30].

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3.1. Current strategies to prolong the retention of therapeutic agentswithin the lung

3.1.1. Encapsulation of the therapeutic agent in polymeric nano-carriersRelease of therapeutic agents from polymeric nanoparticles is

thought to be controlled by a combination of diffusion through thepolymer matrix and polymer degradation [31]. Consequently, the re-lease rates from polymeric nanoparticles in vitro are normally fast dueto the short distance the drugs have to cover to diffuse out of the par-ticles. However, the evidence falls short of proving the usefulness ofpolymeric nanoparticles for drug and gene delivery to the lung byemploying simple in vitro release tests. The evaluation of nanoparticlebehavior in physiologically relevant contexts (e.g. ex vivo and in vivo)must be done to support the claim that polymeric nanoparticles mayimprove pulmonary drug and gene delivery. Encapsulation of thera-peutic agents in polymeric nano-sized controlled delivery systemshas been shown to modulate the pharmacokinetic and -dynamic pro-file in the lung [12,14,32–36]. Moreover, the medication is protectedfrom metabolic degradation.

Among various techniques known for the preparation of polymer-ic nanoparticles [37], the nanoprecipitation method has the advan-tage that no additional surfactant is required during nanoparticleformation [38]. Since the surface properties of nanoparticles will notbe masked by adsorbed surfactant molecules, their absence duringnanoparticle preparation will be an advantage in drug loading by ad-sorption [39]. In addition to nanoparticle preparation in aqueousmedia, additional methods have been conceived to prepare submi-cron polymeric particles by spray-drying techniques [40]. Spray-drying allows the continuous conversion of fluids into powdersusing a one-step process [41]. Moreover, the adjustment of spray-drying process parameters enables the manipulation of various parti-cle properties (e.g. size, density and shape) [9,42]. Recent develop-ments in this field rely on improving the fluid breakdown by“electro-spraying” or “vibrating-mesh nozzles”; while electrostaticcollectors accomplish high process yields [43–47].

Fig. 2. Schematic of the structural organization of nanocomposites prepared from apositively-charged branched polyester (P(68)-10) and pDNA in the presence of differ-ent excipients [reproduced from [65] with permission of the copyright holder].

3.1.1.1. Polymeric materials with potential for pulmonary drug deliveryapplications. A number of polymers have been utilized in formulatingdelivery systems for pulmonary applications [12,14]. The selection ofan appropriate polymer is mainly based on criteria such as its bio-compatibility and degradability [48]. Aliphatic polyesters like poly(-lactide-co-glycolide) (PLGA) have been most extensively used dueto their low toxicity [49–51]. The slow degradation rate (weeks tomonths), however, would lead to an unwanted accumulation in therespiratory tract, especially when frequent dosing is required[52,53]. Moreover, for an effective nanoparticulate drug delivery sys-tem, sufficient drug loading and controlled drug release over a prede-termined period of time must be ensured [12,14]. The low affinity ofdrugs to the polymeric matrix often leads to relatively fragile interac-tions between drug and polymer accompanied by fast release rates[54,55].

One way to overcome this problem is to synthesize polymers withparticular characteristics, such as faster degradation rates and definedfunctionality to promote interactions with therapeutic compounds[52,53,56]. Fast-degrading branched polyesters have been synthe-sized for drug delivery applications by grafting short PLGA chainsonto charge-modified poly(vinyl alcohol) (PVA) backbones [57–59].

Amphiphilic properties of branched polyesters make them highlysuitable for pulmonary formulations in several ways. On the onehand, the flexibility of this type of polymer was shown with regardto tuning degradation rates [60,61]. Degradation can be tailoredthrough modifications in the degree of charge substitution andPLGA chain length from a few hours up to several weeks, which isan important aspect for pulmonary application. On the other hand,the introduction of charged functional groups within the polymer

backbone enhances the drug loading and release profile ofoppositely-charged therapeutic agents [12,14,32,34,35,39,58,59].

Nanoparticles can be fabricated from branched polyesters withoutthe use of additional surfactants or stabilizers [38,39,62]. It has beenreported that the addition of different excipients during the nanopar-ticle preparation process, such as lung surfactant (Alveofact®), car-boxymethyl cellulose or poloxamer (Fig. 2), can generate compositenanoparticles with modified surface charges, degradation rates, sta-bility, toxicity and biological activity [60–65]. Other types of biode-gradable polymers suitable for pulmonary application are based onbiodegradable poly(anhydride)s and poly(ketal)s [66,67].

3.1.1.2. Pulmonary toxicity of polymeric nano-carriers. The same prop-erties that make polymeric nanoparticles attractive as drug deliverysystems (i.e. size, surface characteristics, chemical composition),also evoke toxicity concerns, particular following pulmonary appli-cation [68–70]. Safety evaluations of polymeric nanoparticles forpulmonary application are currently a subject of intense research.So far, evidence has accumulated that the toxicological potential ofnanoparticles depends on their degradability, chemical composition,particle size, and concentration at the target site [68,69].

For instance, positively-charged poly(styrene) nanoparticlesshowed a higher incidence of pulmonary reactions (cell recruitment,total protein, and lactate dehydrogenase release) than theirnegatively-charged counterparts [71]. Cationic biodegradable nano-particles also caused increased toxic effects after pulmonary instilla-tion. By contrast, anionic biodegradable nanoparticles were muchbetter tolerated [72]. These observations point to the fact that notonly size but also material properties play an important role in induc-ing inflammatory responses in the lung. Dailey et al. compared thetoxicological potential of non-biodegradable and biodegradablenanoparticles in vivo [64]. To characterize the inflammatory potential,various response parameters were quantified in bronchoalveolarlavage fluid. Biodegradable nanoparticles, made from a linear poly-ester (PLGA) and an amine-modified branched polyester revealedsignificantly lower inflammatory potential compared to non-biodegradable poly(styrene) nanoparticles, especially in recruitmentof inflammatory cells (Fig. 3).

These experimental findings are in general agreement with therecently proposed “nanotoxicological classification system” (NCS)

Fig. 3. Inflammatory cell recruitment after pulmonary nanoparticle application to mice(PMN: polymorphonucleocyte; PS-NP: poly(styrene) nanoparticles; DEAPA-NP: amine-modified branched polyester nanoparticles; PLGA-NP: poly(lactide-co-glycolide) nano-particles) [reproduced from [64] with permission of the copyright holder].

Fig. 4. Influence of polymeric nanoparticles on the minimum surface tension of pulmo-nary surfactant examined in a pulsating bubble surfactometer [reproduced from [74]with permission of the copyright holder].

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[73], which allows a precise differentiation of toxicological risksfor polymeric nanoparticles by a combination of size related effectswith risks derived from polymer degradability.

The slow degradation rate of PLGA is a disadvantage for pulmo-nary drug delivery, especially when repeated administrations are re-quired. The faster degradation rates of branched polyesters makethem more suitable for pulmonary formulations [52,53].

Despite many studies focusing on the toxicological responses ofpolymeric nanoparticles administered to the lung, their influenceon the biophysical properties of pulmonary surfactant is largely un-known [68]. Therapeutic doses of polymeric nanoparticles with dis-tinct physicochemical properties impeded the ability of pulmonarysurfactant to decrease surface tension rapidly during compression/expansion cycles (Fig. 4). The ability of the different nanoparticleformulations to interact with surfactant proteins was regarded askey factor for their inhibition of surfactant function in vitro [74].

The translocation of polymeric nanoparticles across the alveolarbarrier has been tested ex vivo and in vivo after nanoparticle instilla-tion [75,76]. The applied particles were mainly located in the alveolarspace and in macrophages [75]. Particles with geometric sizesb100 nm were detected in other organs outside the lung tissue [76].However, the relevance of these findings still remains to be estab-lished, as polymeric nanoparticles are currently under investigationas potential drug delivery systems to the lung.

3.1.2. Avoiding the clearance mechanisms of the lungDue to the effective clearance mechanisms of the lung, the ability

to control kinetics of the therapeutic compound remains a consider-able challenge. Current strategies to overcome mucociliary clearanceare accomplished by an aerodynamic targeting of the alveolar air-space via manipulation of the aerodynamic particle size (b5 μm), sur-face modified particles that rapidly penetrate the mucus barrier ormucoadhesive formulations [77]. Following inspiration, a particlecan collide with the walls of the airways, where it will be depositedupon contact with the airway surfaces. It is evident that the extentand efficiency of particle deposition in the respiratory tract dependon particle characteristics, lung morphology and breathing pattern[78,79]. Three physical mechanisms govern the pulmonary depositionof inhaled particles, namely inertial impaction, gravitational sedimen-tation and Brownian diffusion. The aerodynamic diameter (dae) of anaerosol particle is defined in terms of its aerodynamic behavior withconsideration of size, density and shape. In the range above dae of1 μm, particle deposition mainly occurs by inertial and gravitationalforces. For particles with dae smaller than 0.2 μm, diffusion is thedominating deposition mechanism. Diffusional and gravitationalforces govern particle transport for the intermediate range. To targetthe smaller airways and the alveolar region, dae below 0.1 μm andbetween 1 and 5 μm are preferable (Fig. 5A). Particle deposition inthe upper airways predominantly occurs due to inertial impaction ifparticles possess dae of >5 μm or have a high velocity [78,79].

Understanding the release kinetics from polymeric nanoparticlestogether with the physiological aspects of the mucus barrier is an im-portant prerequisite for the development of particles designed to tar-get the bronchial tissue. Particles need to overcome the mucus barrierbefore the therapeutic agent is completely released from the carriersystem and at rates markedly faster than mucus replacement.Particles with sizes below 1 μm are able to penetrate the capillarymucus network to reach the periciliary fluid layer and eventuallyenter bronchial epithelial cells [23,24]. However, bare nanoparticlesfail to penetrate the mucus barrier due to strong interactions withmucus. One strategy to minimize interactions with mucus andenhance particle diffusion in mucosal fluids is done by the chemicalmodification of nanoparticle surfaces, e.g. by grafting with poly(eth-ylene glycol) (PEGylation) [24]. Application of mucolytic agents likePulmozyme® (rhDNase, Dornase alfa) and Mucinex® (N-acetyl-L-cysteine) may be of importance as an adjuvant to particle transport,when respiratory mucus is abnormally viscoelastic. Although themacro-viscoelastic properties of mucus were significantly reducedby a treatment with mucolytic agents, several authors found only amoderate improvement of particle diffusion in medicated mucosalfluids, which was attributed to the increased micro-viscosity within

Fig. 5. Extent of regional (A) and total (B) particle deposition in the human respiratorytract following particle inhalation [reproduced from [80,81] with permission of thecopyright holder].

Fig. 6. Scanning electron microscopy pictures of composite particles prepared by spray-drying from aqueous nanosuspensions containing poly(styrene) (A) and poly(lactide-co-glycolide) nanoparticles (B), respectively. Scale bars: 1 μm.

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mucus pores [24]. Another approach to circumvent mucociliary clear-ance can be accomplished by means of adhesion of the pulmonaryformulation to the mucosal tissue (mucoadhesion). The increasedcontact time between the delivery formulation and the mucus layerresults in a prolonged residence time of the therapeutic agent in thelung [82,83].

Different strategies have been proposed to overcome phagocytosisof inhaled particles by alveolar macrophages present in the deeperlung [77]. The size discriminating particle uptake properties of alveo-lar macrophages represent a basis for the design of pulmonary con-trolled release formulations with prolonged residence time [6]. Inthis regard, large porous particles were developed as controlled pul-monary drug delivery systems [84]. Due to their unique geometry,these particles are not taken up by alveolar macrophages. In a recentapproach, swellable microparticles were introduced to bypass macro-phage clearance [85]. Dry microparticles display inhalable particlesizes, but once in contact with the moisture of the lung a significantincrease in particle size is observed. Another approach to delay lungclearance can be achieved by inhalation of submicron particles[10–12,14]. Although their small size limits pulmonary deposition,as nanoparticles alone are expected to be exhaled after inhalation(dae: 0.1–1 μm (Fig. 5B)) [78,79], researchers are encouraged to de-velop suitable application forms for inhalation. Aerosol particles suit-able for deposition in the deeper lung can be generated bynebulization of polymeric nanosuspensions [32,62,86] or aerosoliza-tion of nanoparticle-containing microparticles (composite particles)

[87]. Vibrating-mesh nebulizers (e.g. Aeroneb® Pro) are preferredfor the delivery of polymeric nanoparticles from suspension [32,86].As an alternative to nebulization, polymeric nanoparticles can be de-livered to the lung by means of dry powder aerosolization [87,88].Therefore, polymeric nanoparticles are encapsulated into compositeparticles using standard techniques like spray-drying (Fig. 6). Thedelivery of nanoparticles as micro-particulate composites has beenintensively investigated as these “Trojan” particles combine the ben-efits of both microparticles (aerodynamic behavior) and nanoparti-cles (avoidance of macrophage clearance) [87].

3.2. Models to evaluate the potential of polymeric nano-carriers

Different preclinical models can be used to examine the potentialof polymeric nanoparticles after pulmonary administration, whichwill be described briefly hereafter. Interested readers are referred toexcellent reviews on this topic [17,20,21,89].

Nanoparticle behavior should not only be monitored in vitro, butin a physiologically relevant context to obtain reliable drug releaseand distribution data. In general, cell culture models of the respiratorytract are employed to explore cellular mechanisms of drug transportprocesses using both continuous and primary cells [89,90]. Ex vivoisolated, perfused, and ventilated lung (IPL) models display the ad-vantage of structural and functional integrity of the organ. The impactof lung-specific factors enables a realistic extrapolation of absorptionand distribution profiles to the in vivo situation [17–20]. Informationabout the fate of inhaled therapeutics gained from in vivo analysis isrestricted by limited screening capacity and the potential for non-linear dose–response relationships in animal models [21,91]. Overall,the application and comparison of different models to elucidate thebehavior of pulmonary formulations at the target site could be attrac-tive to establish in vitro-in vivo correlations.

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The IPL technique was utilized to study the pulmonary absorptionand distribution characteristics of a hydrophilic model drug afteraerosolization as solution or entrapped into nanoparticles composedof charge-modified branched polyesters [32]. Pulmonary retentionof the encapsulated model drug was observed for the nanoparticleformulation as reflected by a reduced perfusate concentration.

The performance of salbutamol-loaded nanoparticles preparedfrom charge-modified branched polyesters has been characterizedin an IPL model (Fig. 7A and B). After inhalative administration of dif-ferent salbutamol formulations (drug solution and drug-loaded nano-particles) to an ex vivo rabbit lung, salbutamol showed a reproducible

Fig. 7. Ex vivo (A, B) and in vivo (C) performance of salbutamol-loaded biodegradablenanoparticles after pulmonary administration. The pulmonary retention profile clearlydemonstrates a significantly slower absorption of salbutamol from the isolated, per-fused, and ventilated lung model for nanoparticles compared to salbutamol solution(A). The final distribution of salbutamol in the different compartments of the lungmodel is illustrated in (B). The bronchoprotection of salbutamol applied as solution(grey squares) or entrapped in nanoparticles (black triangles) presents a prolongeddrug action for the nanoparticle formulation (C) [reproduced from [34,35] with per-mission of the copyright holder].

pulmonary absorption profile. The initial drug absorption phasefrom both formulations exhibited superimposed curves, whereasthe second absorption phase disclosed slower drug absorption forsalbutamol-loaded nanoparticles (kβ=−0.0005 min−1) comparedto salbutamol solution (kβ=−0.0026 min−1) (Fig. 7A). The signifi-cantly decreased total recovery of salbutamol from nanoparticles(~49%) compared to that from solution (~75%) (Fig. 7B) has been at-tributed to internalization of salbutamol-loaded nanoparticles intolung cells [34].

Additionally, a number of in vivo studies underline the potential ofnanoparticles as controlled pulmonary drug delivery vehicles. Ryttinget al. prepared salbutamol-loaded nanoparticles from a new type ofbranched polyester [35]. Drug transport studies across pulmonarycell monolayers revealed a delayed transport of salbutamol fromnanoparticles. Finally, a prolonged bronchoprotection was observedin vivo for salbutamol-loaded nanoparticles (Fig. 7C). Sung et al.fabricated “porous nanoparticle-aggregate particles” composed ofrifampicin-loaded nanoparticles for drug delivery to the lung [33]. Al-though the in vitro drug release exhibited an initial burst of rifampi-cin, pharmacokinetic studies performed in vivo demonstrated anincreased retention of rifampicin in the lung tissue. Roa et al. encap-sulated doxorubicin-loaded nanoparticles into inhalable effervescentpowder formulations and tested the prepared formulations in atumor-bearing mouse model [36]. Animals treated with inhalable ef-fervescent nanoparticle powders showed an improved survival com-pared to control groups. Moreover, nanoencapsulation of doxorubicinreduced toxic side effects.

Overall, these results demonstrate that polymeric nanoparticlesare a promising tool for the treatment of severe pulmonary diseases.Furthermore, a good agreement between ex vivo and in vivo resultswas obtained (Fig. 7), which serves as an example for the potentialof the IPL technique to predict drug absorption and distributionfrom the intact animal. The choice of an appropriate test animal to as-sess the pulmonary pharmacology of inhaled therapeutics is influ-enced by several factors, e.g. size and airway geometry of the lung.The most popular species include the mouse, rat and guinea pig[21]. The lung geometry of small animals, however, impedes highlung deposition of therapeutic aerosols. Consequently, test formula-tions are intratracheally instilled, which in turn causes an inhomoge-neous lung distribution. Additionally, the volume and the number ofblood samples required for analytical tests need to be considered.The IPL approach may overcome some of the limitations to experi-mentation with animals and may assist the currently used in vivostudies. Experimental parameters remain controllable in IPL prepara-tions, while in the test animal these are likely to change in responseto the application of a drug formulation. Formulations can be admin-istered directly to the airspace via inhalation in a quantitative and re-producible manner. The absorptive profile can be monitored bysequential analysis of the perfusate medium [17,20].

3.3. Pulmonary gene delivery

One specialty within pulmonary drug delivery that is gaining moreand more interest is the delivery of nucleic acids via the lung [92].Therefore, pulmonary delivery of nucleic acids plays a major role inthe development of new therapeutics against genetic and chroniclung disorders [93–95]. Although pulmonary delivery of nucleicacids bypasses the first pass effect of the liver, all barriers that applyto pulmonary drug delivery must be still overcome [96]. Additionalbarriers come into play as therapeutic DNA has to be delivered intothe cell nucleus, whereas siRNA needs to be delivered to the cytosol.Therefore, DNA and RNA delivery present different delivery require-ments, which we have recently separately reviewed in detail[97,98]. Since nucleic acids are prone to degradation by nucleasesand are not readily endocytosed, delivery vectors that mediate

Fig. 8. Knock-down in actin-EGFP mice. A: Bronchiolus of a control EGFP-mouse dis-playing high fluorescence intensity. B: Bronchiolus of a PEI(25 kDa)-PEG(2 kDa)10/siRNA treated EGFP-mouse displaying lower fluorescence intensity. C: Alveolar epithe-lium of a control EGFP-mouse displaying high fluorescence intensity. D: Alveolar epi-thelium of a PEI(25 kDa)-PEG(2 kDa)10/siRNA treated EGFP-mouse displaying almostcomplete loss of fluorescence signal. E: Knock down in actin-EGFP mice compared tonon-EGFP-expressing mice (blank). EGFP-expression in lungs of siEGFP/PEI(25 kDa)-PEG(2 kDa)10 treated mice was down regulated by 42% and is significantly (pb0.05)lower than in mice treated with siFLuc/PEI(25 k)-PEG(2 k)10 [reproduced from [112]with permission of the copyright holder].

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protection against degradation but foster cellular uptake are neces-sary for successful pulmonary delivery of nucleic acids [96].

3.3.1. Polymeric vectorsIn contrast to viral delivery vectors, which are highly efficient,

non-viral counterparts cause less immunostimulatory, mutagenic,and oncogenic complications, and some can approach viruses con-cerning their transfection efficiencies [99]. Whereas viral vectorswere designed by nature, non-viral vectors have to be synthesizedin a smart way to maintain biologic activity of their loads. According-ly, non-viral vectors must exhibit the ability to package genetic mate-rial into nano-sized particles, deliver it intracellularly, and to mediateendosomal escape for incorporation of siRNA into the RNA-inducedsilencing complex (RISC) and for translocation of DNA into thenucleus.

A number of lipid-based and polymer-based non-viral vectorshave been developed to formulate nucleic acids into nano-sized par-ticles for pulmonary delivery [93]. Whereas PLGA plays a major rolefor pulmonary delivery of small molecules as described above, itmust be modified for efficient gene delivery. One of the most promi-nent polymeric gene delivery vectors is poly(ethylene imine) (PEI)[100]. Additionally, the polysaccharide chitosan [101], SuperFect®, ageneration four fractured PAMAM dendrimer [102], and PLGA-basedpolymers [62,103] have also been described for pulmonary deliveryof nucleic acids. Due to its high cationic charge density, PEI is ableto efficiently condense negatively-charged DNA into nano-sized com-plexes, and thereby protects it from degradation by nucleases [104].However, this positive charge causes toxicity, which can be reduced,e.g. by decreasing the molecular weight [104] or by grafting withPEG [105,106]. Accordingly, Kleemann et al. compared the efficiencyof low molecular weight (LMW) PEI (5 kDa), high molecular weightPEI (25 kDa), and high molecular weight PEI (25 kDa) grafted withPEG (5 kDa) for pulmonary gene delivery. The results showed thatLMW PEI most efficiently mediated luciferase expression in both thebronchial and alveolar cells, whereas polyplexes of 25 kDa PEI weremainly found in bronchial cells. PEGylated PEI, which caused en-hanced luciferase expression in vitro, failed to deliver DNA in vivo[107]. This can be explained by the reduced interaction of the PEG-grafted polyplexes with the cell surface, which in turn causes reducedcellular uptake [108]. To overcome this lack of interaction of PEI-g-PEG with the cell surface, TAT, a cell-penetrating peptide derivedfrom the HI-virus [109], was coupled to PEG (3.4 kDa)-grafted PEI(25 kDa). The authors showed that TAT–PEG–PEI formed polyplexeswith higher stability against polyanions, Alveofact®, bronchial alveo-lar lining fluid and DNase. Although TAT–PEG–PEI polyplexesperformed poorly in vitro, they were more efficient in pulmonaryin vivo gene delivery than PEI alone [110].

In recent years, we also explored the suitability of PEG-PEI forsiRNA delivery in vitro [111] and in vivo [112]. The expression of en-hanced green fluorescent protein (EGFP) in the lung tissue of EGFPmice was significantly and sequence-specifically knocked downwith no histological indications of inflammation (Fig. 8). In a relatedstudy not only PEGylated PEI (25 kDa) but also fatty acid-modifiedPEI (8.3 kDa) increased cytokine levels in the lung lavage fluid,although the cytotoxicity of PEGylated polymers was found to bereduced compared to unmodified PEI as measured by in vitro WST-and LDH assays and in vivo LDH release [113].

Additionally, we reported that PEGylated PEI can activate both thecomplement system [114] and induce the expression of apoptosis-related genes [115]. Therefore, the need for biodegradable vectorsseems obvious. As mentioned above, PLGA-based, biodegradable vec-tors need to be modified with cationic side chains to enable the con-densation of nucleic acids. While others have investigated PLGA–PEInanoparticles which exhibited cytotoxicity at PEI/DNA ratios above1 [116], Wittmar et al. have grafted diethylaminopropylamine poly(-vinyl alcohol) with PLGA (DEAPA-PVA-g-PLGA) to obtain polyesters

that degrade faster than PLGA [58,117]. This PLGA derivative waslater used to prepare nanoparticles for pulmonary application [62].Additionally, Dailey et al. found that this biodegradable pulmonarydelivery system had a lower proinflammatory potential in vivo thannon-biodegradable nanoparticles of polystyrene [64]. DEAPA-PVA-g-PLGA was later investigated concerning siRNA delivery and showedgreat efficiency in vitro even after nebulization [60]. When ternarynanocomposites made of pDNA, modified PLGA and lung surfactantwere generated, the transfection efficiency of this biodegradablegene delivery system was further increased [65].

As far as pulmonary siRNA delivery is concerned, mucoadhesivechitosan enjoys great popularity. So called “nanochitosan” wasreported to deliver siRNA against RSV in mice [118] and rats [119].siRNA- or locked nucleic acid (LNA)-loaded complexes of 114 kDachitosan with a deacetylation degree of 84% were able to reduceEGFP expression in EGFP mice when administered daily [120,121].Interestingly, the biological efficiency could be maintained upondecreased dosing when the chitosan complexes were administeredby intratracheal aerosolization [122].

However, the systems described above rely on adsorptive endocy-tosis for intracellular delivery. In contrast, targeted vectors exploit theexpression of an internalizing cell surface receptor which is engulfed

Fig. 9. Biodistribution of siRNA-loaded polyplexes 2 h after intranasal (left) or intratra-cheal (right) administration. While intratracheal administration leads to quantitativesiRNA delivery to the lung lobes with subsequent excretion via the kidneys, intranasaldelivery causes strong deposition in the oropharynx [reproduced from [98,112] withpermission of the copyright holder].

221M. Beck-Broichsitter et al. / Journal of Controlled Release 161 (2012) 214–224

upon activation and thus shuttles extracellular material into the lungtissue [95]. In the past, various targeting moieties, such as galactose[123], lactose [124], lactoferrin [125], antibodies against the endothe-lial cell adhesion molecule-1 (PECAM-1 or CD31) [126], and Fabfragments of polyclonal antibodies directed against the polymeric im-munoglobulin receptor [127] were successfully employed to improvethe transfection efficiency in the lung. Except for one report in the lit-erature where biotinylated TAT-RGD peptide – which in addition toTAT contains the arginine-glycine-aspartate (RGD) sequence – wasused as targeting moiety [128], the expression of various integrinson airway epithelium [129] has so far not been extensively exploited.Specific targeting to the correct cell population in gene delivery iscrucial to avoid side effects; targeting approaches will most certainlygain importance in non-viral delivery of nucleic acids to the lung.

3.3.2. Models to determine successful in vivo gene deliverySince delivery studies of nucleic acids involving polymeric vectors

display strong differences concerning the biologic activity of the loadin vitro vs. in vivo, the need for valid in vivo models becomes veryclear. Much research is performed to optimize models of lung disor-ders, infectious diseases, allergies or delivery of insulin and vaccines[21]. Before biologic efficiency can be tested, the biodistribution andlung retention must first be examined to determine if the deliverysystem will be available at the site of action. In gene delivery, biodis-tribution is often measured by quantification of reporter gene expres-sion in several organs ex vivo [127], or fluorescence or confocal laserscanning microscopy is performed in dissected lungs [107]. However,ex vivo techniques only allow the investigation of distribution andtransfection efficiency at one distinct time point. Information aboutbiodistribution and gene expression over a certain time period andkinetics of gene delivery vectors can be non-invasively obtainedby live bioluminescence imaging [130], fluorescence imaging [131]or gamma camera imaging [112]. The downside of fluorescence imag-ing is that it is rather semi-quantitative while radioactive signals canbe normalized to the injected dose. The attractiveness of molecularimaging approaches is certainly reflected in the ability to measure bi-ological processes in living animals, in the same animal at certaintime points or even dynamically [132]. Additionally, molecular imag-ing approaches can be used to determine which administration routeor device leads to the most extensive deposition in the deep lung.Intratracheal administration, for example, was described to lead topreferential deposition in the left lung lobes [112], and intranasalvs. intratracheal application was shown to result in strong depositionin the oropharynx (Fig. 9).

As imaging ligands are coupled to nanomaterials, a merely thera-peutic nanoparticle can become what is called a “theragnostic” parti-cle. Thus, therapy and diagnosis may be combined by deliveringnucleic acids and imaging the therapeutic effects [133]. However,many theragnostics are currently based on paramagnetic iron oxideparticles, which are traced by magnetic resonance imaging (MRI).However, the lung can only be imaged by MRI if it is vented withgases such as hyperpolarized Helium-3 [134]. Although developmentof lung-specific theragnostics based on superparamagnetic iron oxidewill be challenging, the latter can be used to prepare “magnetic aero-sols” that can be directed towards a magnet in a defined manner totarget a specific region in the lung [135].

4. Conclusion & perspective

Pulmonary drug and gene delivery via inhalation facilitates a site-specific treatment of lung diseases. However, the rapid decline oftherapeutic agent concentration in the lung is considered as a signif-icant disadvantage of “conventional” inhalation therapy. Hence, thedevelopment of pulmonary controlled release formulations wouldbe highly beneficial for patients suffering from airway diseases. Alarge number of carrier systems have been investigated as potential

controlled delivery formulations to the lung, including polymerbased particles. Encapsulation of the therapeutic agent in polymericparticles has been shown to protect the medication from metabolicdegradation and to sustain the release rate.

Chemical modifications to PLGA add several benefits in the selec-tion of a suitable polymeric material for pulmonary administration.Moreover, the adjustment of the polymer structure can alter the en-capsulation efficiency and release profile of therapeutic agents [136].

Different strategies have been proposed to avoid rapid particleclearance from the lung, in order to control the pharmacokineticsand pharmacodynamics of the applied therapeutic agents at thetarget site. In this context, polymeric nanoparticles are generallypreferred as pulmonary delivery devices owing to a prolonged reten-tion in the lung. Pulmonary delivery limitations have been resolvedby generating micro-sized aerosol particles.

The shortcomings of conventional nebulizers have led to the de-velopment of efficient breath-synchronized devices (AKITA® technol-ogy and AAD® system) that offer the possibility to deliver precisedoses of drug to the lung [137,138]. Adaptation of these novel aerosoldelivery systems to relevant organ and animal models seems to bereasonable, as high lung deposition with minimal environmental ex-posures will be required for the evaluation of potent drugs, e.g. vaso-dilators, and their inhalative controlled release formulations [7,139].

Unfortunately, the appropriate technology to generate nano-sizedaerosols from polymeric nanoparticle formulations is not yet suffi-ciently developed. The aerosol delivery of individual polymericnanoparticles (dae: b0.1 μm), however, would be very promising toachieve high peripheral lung deposition (Fig. 5), potentially allowingaccess to alveolar tissue. Indeed, nanoparticles with geometric sizes ofb100 nm are taken up to a greater extent by alveolar type II cells thanalveolar macrophages [140]. Hence, deposition of polymeric nanopar-ticles to the alveolar space may be of relevance for targeting drugs toalveolar type II cells.

The shape (aspect ratio) of polymeric particles may also be a de-terminant for regional lung deposition, since elongated particleshave the additional deposition mechanism of interception, contrary

222 M. Beck-Broichsitter et al. / Journal of Controlled Release 161 (2012) 214–224

to spherical particles [141]. Depending on their aspect ratio, fiberscould be preferentially deposited in the alveolar or tracheo-bronchial region. With adequate geometric dimensions of fibers,engulfment by alveolar macrophages would be ruled out, leading toprolonged retention in the respiratory system [77].

The isolated perfused lung is a powerful tool to analyze the phar-macokinetic profiles of formulations administered to the lung underex vivo conditions. Areas in which IPL models have not yet beenused include the evaluation of the pharmacodynamic action of con-trolled release systems. With suitable modifications, the applicationof IPL preparations for these investigations will become technicallyfeasible. Clearly, the IPL technique is able to assist the currently andwidely used in vivo studies and will therefore find a more widespreadutilization in pulmonary research.

In terms of pulmonary delivery of nucleic acids, efficient vectorshave been identified which are less toxic than 25 kDa PEI [107]. How-ever, later investigations showed that both LMW PEI and PEG-PEI arepotent activators of the complement system [114], and various modi-fications can be highly immunogenic [113]. Therefore, alternatives forPEI must be found that are more promising in vivo and suitable forpotential clinical translation. These alternatives may either be basedon partially biodegradable cross-linked very short PEIs, oligo ethyleneimines, or on completely different structures. Since chitosan enjoyspopularity in pulmonary siRNA delivery [118–121], the biocompatibil-ity of this biopolymer needs to be tested in the future. If chitosanproves safe, modifications to enhance its efficacy may potentially beintroduced. Alternatively, PLGA-based vectors currently seem to bevery promising. However, for in vivo administration, the preparationtechnique described by Nguyen et al. [60] needs to be further opti-mized to yield higher siRNA concentrations in the nanoparticles.

In terms of targeted delivery of nucleic acids, which is needed toobtain specificity, one approach that may prove useful and has notyet been exploited extensively is integrin targeting in the lung [128].

While currently most of the reports describing novel pulmonarygene delivery vectors focus on the delivery of reporter genes, thera-peutic effects in vivo must be achieved to move to the next step.Various in vivo models are available [21]. The same applies forsiRNA delivery. Certainly, a novel vector must first of all be character-ized concerning suitability for pulmonary delivery and later concern-ing its in vivo behavior. Molecular imaging techniques will proveuseful in the assessment of pharmacokinetics and biodistribution ina non-invasive manner [132].

However, one concern that is neglected in most of the studies onpulmonary delivery of nucleic acids is the suitability of the formula-tion for aerosolization, lyophilization and packaging into metereddose inhalers or dry powder inhalers. For clinical translation, thesechallenges in the development of pulmonary drug delivery systemsneed to be overcome.

Acknowledgements

The German Research Foundation (DFG) is acknowledged for thefinancial support. We thank Leigh Marsh for language editing.

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