See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/49706226

Nanocarriers for pulmonary administration of peptides and therapeutic proteins

Article in Nanomedicine · January 2011 DOI: 10.2217/nnm.10.143 · Source: PubMed

CITATIONS READS 64 413

4 authors:

Fernanda Andrade Mafalda Videira University of Porto University of Lisbon

50 PUBLICATIONS 1,402 CITATIONS 45 PUBLICATIONS 1,399 CITATIONS

SEE PROFILE SEE PROFILE

Domingos Ferreira Bruno Sarmento University of Porto University of Porto

139 PUBLICATIONS 6,631 CITATIONS 504 PUBLICATIONS 15,039 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

COST Action MP1404, “SimInahle”, 2015-2019 View project

Resveratrol Drug Delivery View project

All content following this page was uploaded by Mafalda Videira on 26 December 2013.

The user has requested enhancement of the downloaded file. Review

Nanocarriers for pulmonary administration of peptides and therapeutic proteins

Peptides and therapeutic proteins have been the target of intense research and development in recent years by the pharmaceutical and biotechnology industry. Preferably, they are administered through the parenteral route, which is associated with reduced patient compliance. Formulations for noninvasive administration of peptides and therapeutic proteins are currently being developed. Among them, inhalation appears as a promising alternative for the administration of such products. Several formulations for pulmonary delivery are in various stages of development. Despite positive results, conventional formulations have some limitations such as reduced bioavailability and side effects. Nanocarriers may be an alternative way to overcome the problems of conventional formulations. Some nanocarrier-based formulations of peptides and therapeutic proteins are currently under development. The results obtained are promising, revealing the usefulness of these systems in the delivery of such drugs.

1 keywords: inhalation n nanocarriers n noninvasive delivery n peptides Fernanda Andrade , n therapeutic proteins Mafalda Videira2, Domingos Ferreira1 & Bruno Sarmento†1,3 Recent advances in biotechnology and genetic pulmonary administration are currently under 1Department of Pharmaceutical engineering have resulted in the promotion of development and in clinical trials. Some of them Technology, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha peptides and proteins as an important class of showed positive results. However, conventional 164 4050-04, Portugal therapeutic agents. Despite the emergence of formulations have some limitations, such as 2iMed.UL – Research Institute for Medicines and Pharmaceutical several peptides and proteins with therapeutic reduced bioavailability and side effects [2,7]. Sciences, Faculty of Pharmacy, potential, their administration in the active con­ Nanocarriers may be an alternative way to University of Lisbon, Portugal formation has been shown to be an enormous overcome the problems of conventional formu­ 3Centro de Investigação em Ciências da Saúde (CICS), Department of challenge for the pharmaceutical industry. There lations. They allow the protection of the proteins Pharmaceutical Sciences, Instituto are several limitations that are imposed such as from degradation, enhance transepithelial trans­ Superior de Ciências da Saúde – Norte, Gandra, Portugal low bioavailability, physical or chemical instabil­ port and reduce their immunogenicity [8–10]. †Author for correspondence: ity and side effects [1]. This paper is a review of studies on pulmo­ Tel.: +351 222 078 949 Currently, owing to instability and reduced nary delivery of peptides and therapeutic pro­ Fax: +351 222 003 977 [email protected] permeability of proteins through biomembranes, teins encapsulated into nanocarriers. the parenteral route is the most commonly used route of administration for such drugs. However, Peptides & therapeutic this is an invasive route, which can lead to a proteins: characteristics, stability reduced acceptance by patients and, conse­ & administration quently, increased costs of therapy, especially „„Characteristics when is required a prolongedAuthor or chronic treat­ Peptides and therapeutic Proof proteins, classified as ment [2,3]. biopharmaceuticals, have emerged as useful In order to overcome such drawbacks, non­ and promising drugs in the treatment of vari­ invasive alternative routes have been tested, such ous diseases such as diabetes, cancer or autoim­ as oral, pulmonary, nasal, rectal and transder­ mune diseases [11]. These have gained, in the mal, among others [1]. last decade, an increasing share of the global Although oral administration is considered pharmaceutical market [12]. This is due to the the most attractive route for drug administra­ development of molecular biology that allowed tion, the bioavailability of peptides and proteins the understanding of the role of proteins in after oral administration is greatly reduced [4,5]. pathophysiological processes as well as the grow­ Owing to the physiological characteristics ing development of biotechnology, bioengineer­ of the respiratory system, the pulmonary route ing and recombinant DNA technology, which has received special attention for protein drug allowed large-scale production. The first bio­ delivery [6]. Several peptides and proteins for technologically derived drug product approved

10.2217/NNM.10.143 © 2011 Future Medicine Ltd Nanomedicine (2011) 6(1), xxx–xxx ISSN 1743-5889 1 Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

for market was recombinant human insulin in usual route of administration for such drugs. 1982 [13]. There is a tendency to classify the pro­ However, it does not eliminate the problem of teins as new therapeutic agents; however, insulin instability in the bloodstream [16]. Moreover, was produced at industrial level for the first time this is an invasive route, which can lead to a in 1923 by the company Eli Lilly (Indianapolis, reduced acceptance by patients and, conse­ IN, USA) [14]. quently, increased costs of therapy, especially Owing to their selectivity and ability to foster when it requires prolonged or chronic treatment. a strong and effective action, therapeutic pro­ Besides, there is a need for sterilization and cold teins have a high potential for cure [15]. storage (2–8 °C) of various formulations of pro­ Despite all the therapeutic potential associ­ teins, as well as the need for specialized person­ ated with proteins, they have physicochemical nel for its administration [2,3]. characteristics that limit their therapeutic appli­ In order to overcome the problems associated cations.Because of their high molecular weight with parenteral administration, the pharma­ and general hydrophilicity, proteins have limited ceutical industry has been channeling efforts ability to cross biological membranes and conse­ on developing systems for the administration of quently have reduced bioavailability [16]. biopharmaceuticals without resorting to injec­ tions. Among the different noninvasive routes of „„Stability & formulation challenges administration are the oral, buccal, pulmonary, Owing to their complex structure, peptides and transdermal, ocular, rectal and vaginal routes therapeutic proteins have a limited chemical sta­ [16]. bility in vivo, undergo degradation and proteo­ Oral administration is considered the more lytic cleavage and are removed from the blood­ attractive route for drug administration because stream, thus having reduced systemic half-life. of its convenience of administration and high Besides the above characteristics, which deter­ acceptance by patients. However, the bioavail­ mine their pharmacokinetics and pharmaco­ ability of proteins and peptides after oral admin­ dynamics, they present in vitro barriers to their istration is very low due to their instability in the stability during the pharmaceutical develop­ gastrointestinal tract (acidic pH and proteolytic ment. This is due to the reactivity of some amino degradation) and low permeability through the acids, resulting in degradation reactions such as intestinal mucosa [4,5]. In fact, several studies racemization, oxidation or hydrolysis that are are focused on oral administration of proteins, dependent on conditions during production many of them using nanotechnology to increase or storage as pH, temperature, agitation, ionic their bioavailability. strength, presence of metal ions or surfactants. Parallel to the oral route, inhalation is seen the When unstable, they tend to undergo aggrega­ most effective to deliver proteins and appears as tion with possible precipitation, adsorption and an alternative route to parenteral administration, denaturation, which will limit their concentra­ as demonstrated by the several experimental and tion in vivo and thus will prevent them from clinical assays proposed so far. reaching therapeutic levels after administration [14,16,17]. Pulmonary administration of The problems of in vitro and in vivo instabil­ peptides & therapeutic proteins ity of proteins can be resolved with the addition „„Lungs anatomical & of excipients that act as stabilizers by different physiological characteristics Authormechanisms. Examples are sugars and salts Proof that Through pulmonary administration it is possible increase the thermal stability of proteins, the to access the systemic circulation by an epithe­ nonionic surfactants that reduce their aggrega­ lium of low thickness (0.1–0.5 µm in alveolar tion, metal chelators and enzyme inhibitors that region) [3,18] and high surface area for absorption 2 reduce the ability of various proteolytic enzymes (75–150 m ) [19–21]. In addition, it has intra- and or polymers such as polyethylene glycol (PEG) extracellular enzyme activity and drug efflux to decrease the immunogenicity of proteins and systems below the gastrointestinal tract and an increase their resistance to proteolysis by confor­ extensive blood supply. This is a noninvasive mational restriction in vivo [16]. route of administration, which allows local and systemic therapies with a rapid onset of action „„Administration and avoids the first-pass effect [8,18,22]. Parenteral administration can overcome the Moreover, the bioavailability of proteins and problem of reduced bioavailability of proteins peptides administered via pulmonary adminis­ through biological membranes, thus this is the tration is 10–200-times greater when compared

2 Nanomedicine (2011) 6(1) future science group Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

with other noninvasive routes, owing to a more protein are found in macrophages [28]. Despite permeable epithelium and a reduced volume of the fact that the phagocytic capacity of macro­ fluid that allows high concentrations of drug phages is higher than the endocytic capacity of near the bloodstream (Table 1) [23,24]. pneumocytes, these will not begin to play an All these features make the inhalation tech­ important role in clearance of proteins 48 h after nique a promising alternative to parenteral admin­ exposure [27,28]. In addition, several large pro­ istration of various proteins and peptides [25]. teins reach the circulation in intact form after instillation, suggesting a lower impact of the „„Pulmonary clearance & absorption first three mechanisms in lung protein clearance After administration, peptides and proteins will [26,27]. In the case of proteins that undergo a high undergo lung deposition and be subject to the catabolism, the use of enzyme inhibitors such as existing clearance mechanisms in the respira­ bacitracin, chymostatin, leupeptin or nafmostato tory system. mesylate, reduce proteolysis and thereby increase There are several proposed mechanisms for their bioavailability [17]. the clearance of proteins in the lungs and they As previously mentioned, it appears that the are responsible for reducing the bioavailability alveolar epithelium is considered the main bar­ of these after inhalation. However, these are rier in lung protein clearance [26]. This is com­ complex and not yet fully clarified [26]. Include posed of polarized cells permeable to water, mucociliar escalator, phagocytosis by macro­ gases and lipophilic molecules. However, the phages, intracellular catabolism and permeation permeation of hydrophilic substances of large through the alveolar epithelium [27]. After being molecular size such as proteins, and of ionic spe­ phagocytosed by macrophages, proteins can be cies, is limited [17], with the molecular weight transported by the alveolar surface, undergo cutoff of tight junctions for alveolar type I cells translocation to the lymphatic system or deg­ at 0.6 nm [29]. radation by intracellular enzymatic lysosomal The permeation of molecules through bio­ system [18]. logical membranes is a complex phenomenon. Studies show that the rate of lung protein Simultaneous pathways may be involved in the clearance did not change significantly in the transcellular and paracellular pathway (Figure 1) presence or absence of an endotracheal tube, [30]. suggesting a reduced role of the mucociliar Despite the existing transportation systems, escalator in protein clearance [28]. In addition, in the lung epithelium are poorly character­ they demonstrate that only small amounts of ized and it is known that the absorption rate

Table 1. Pulmonary bioavailability of different peptides and therapeutic proteins. Peptide/protein Species Bioavailability Ref. Cetrorelix Rat 48–77% [105] Consensus-interferon Rat ≈70% [106] Cyclosporin A Rat 66–80% [107] Detirelix Dog ≈29% [108] Detirelix Sheep ≈10% [109] Glucagon Rat <1% [110] Human calcitonin Rat ≈17% [110] Human growth factor AuthorRabbit 16–45%Proof[111] Insulin Human ≈50% [112] Insulin Rat ≈12% [113] Insulin Human ≈9% [114] Parathyroid hormone 1–34 Rat ≈40% [110] Parathyroid hormone 1–84 Rat >23% [110] Recombinant-methionyl human granulocyte colony- Hamster ≈46% [115] stimulating factor Salmon calcitonin Rat ≈17% Salmon calcitonin Human ≈28% [50] Somatostatin 1–28 Rat <1% [110] a-interferon Rat >56% [110] Original table using data from various sources.

future science group www.futuremedicine.com 3 Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

Paracellular pathway Transcellular pathway capacity (inspiratory flow rate, breathing frequency, tidal volume) and the inhalation technique used by the patient, as well as fac­ tors inherent to the particles such as the mean diameter, surface and shape, density and their aerodynamic properties [20,36–38]. The delivery efficiency of particles is reduced in patients with lower respiratory capacity, for example, children, elderly persons or adults with certain Figure 1. Schematic representation of the disease conditions that compromises their lung molecule absorption routes. function, resulting in nonreproducible pharma­ cokinetic and pharmcodynamic responses [29]. of various proteins across the epithelium is size- The delivery devices play an important role in dependent [26]. Several authors have demon­ the efficiency of alveolar deposition. The most strated the existence of an inverse relationship common are nebulizers, metered dose inhalers between the molecular weight of macromole­ (MDI) and dry powder inhalers (DPI), which cules and their absorption rates [31–33]. However, were designed for managing small molecules this relationship is particularly true for proteins especially for local delivery and not for biophar­ that undergo absorption by paracellular mecha­ maceuticals. For this reason, in recent years the nism. The temperature also interferes with the development of new and improved devices for absorption of protein and there is a direct rela­ administration of peptides and proteins has tionship between the two [26,27]. been intensified. Examples of such devices are Peptides and proteins of low molecular weight the AERx® from Aradigm, Respimat® from cross the alveolar epithelium mainly by the para­ Boehringer or AeroDose® from Aerogen Inc. cellular pathway. This also prevails when there [17]. Unfortunately, there is no device producing is epithelial injury such as edema or inflamma­ only particles within the size limits appropriate tion. In the permeation of proteins of higher size, to the lung deposition, which results in a very the transcellular route, especially endocytosis, low rate of dose emitted [36]. appears to be more involved [26,34]. Peptides and An issue of relevance arises from the fact that peptidomimetics drugs can be absorbed by active a proportion of patients (over 50%) use the transport using the high-affinity peptide trans­ delivery device improperly, leading to nonre­ porter (PEPT)2 existing in alveolar type II and producibility [37]. Thus, an intensive education in capillary endothelium [21,35]. The presence of of patients by healthcare professionals leads to an caveolin in alveolar type I and endothelial cells increase on the effectiveness of the treatment [36]. of lung and clathrin-coated vesicles in alveolar type I and II suggests the possible involvement of „„State of the art in peptide & protein such pathways in the absorption of proteins [26]. inhalation To increase the absorption of proteins it is pos­ In 1993 the US FDA approved the first protein sible to use permeation enhancers as surfactants, administered via inhalation, the recombinant bile salts and cyclodextrins [17]. human desoxiribonuclease, also known as Dornase a (Pulmozyme®), for the treatment „„Limitations of of cystic fibrosis [39]. Currently there are several Authorpulmonary administration Proofpeptides and proteins with therapeutic potential The main limitation of pulmonary administra­ such as insulin, calcitonin, leuprolide or inter­ tion of drugs relates to the reproducibility of the ferons for which pulmonary administration is dose. Between delivery and absorption in the under development and in clinical trials (Table 2). lungs, the drug undergoes consecutive losses, so the absorbed dose is usually below the dose pres­ Insulin ent in the delivery device (Figure 2). Insulin is the most studied protein and there The deposition of particles at the lower res­ are many studies and formulations developed piratory tract is a complex phenomenon and its for administering insulin by pulmonary route. efficiency depends on several factors including Such formulations are in various stages of devel­ the type of formulation, delivery device used opment, with one approval for marketing by the and their capacity to produce the aerosol. It also FDA and European Medicines Agency (EMA) ® depends on physiological factors such as humid­ (Exubera ) (Table 3). Insulin is a polypeptide hor­ ity and geometry of the airways, respiratory mone with a molecular weight of approximately

4 Nanomedicine (2011) 6(1) future science group Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

6 kDa produced in b cells exist in pancreatic islets of Langerhans [37,38]. Its production has the Dose in the Emitted Deposited Absorbed delivery function to maintain stable glucose levels dur­ dose dose dose ing feeding and fasting, yet regulating lipid and device protein metabolism. Actually, insulin is used in the treatment of diabetes mellitus (2, 5), and its first pulmonary administration in humans was Figure 2. Variation of the drug dose from the delivery device to absorption. reported in 1925 by Gänsslen. In general, patients who received inhaled to inject a long-acting insulin for overnight insulin formulations demonstrate similar post­ glycemic control [44,45]. Moreover, the bioavail­ prandial glycemic control and values of glycated ability of insulin in this formulation was very hemoglobin (HbA1c), faster onset of action, low, 10–20% of subcutaneous, and the complex lower weight gain, lower incidence and sever­ inhalation device increased costs of therapy and ity of hypoglycemia and greater satisfaction decreased the compliance and acceptance by (higher comfort and convenience) compared physicians and patients [2,7]. Also for commercial with patients receiving subcutaneous injection of reasons, clinical trials of the AERx® and AIR® regular insulin [40–43]. However, Pfizer decided were discontinued [46]. With regard to adverse to withdraw Exubera® from the market because effects and their frequency, these are similar it did not achieve the financial expectations. to subcutaneous insulin with the exception of Because it is a short-term insulin, it is necessary coughing, which diminishes with prolonged use,

Table 2. Examples of peptides and proteins under study for pulmonary administration and their development phases. Peptide/protein Therapeutic indication Development Ref. phase a1-antitrypsin a1-antitrypsin deficiency Preclinical [116] Calcitonin Osteoporosis Preclinical [117] Cetrorelix LHRH antagonism Preclinical [118] Ciclosporin A Immunosuppression Preclinical [54,119] Con-IFN Viral infections Preclinical [106] dDAVP Alzheimer’s disease, diabetes insipidus, Preclinical [120] modulation of blood pressure Detirelix LHRH antagonism Preclinical [108,109] DNase Cystic fibrosis Approved [121] Erythropoietin Anemia Preclinical [122] Factor IX, human recombinant Hemophilia B Preclinical [123] GLP-1‡ Diabetes mellitus Phase I [201] Glucagon Hypoglycaemia Preclinical [124] Follicle-stimulating hormone Fertility treatment Preclinical [125] Parathyroid hormone AuthorOsteoporosis ProofPreclinical [126,127] Insulin Diabetes mellitus Different stages [7,41– 43,96,99] INF-a Bronchioalveolar carcinoma Preclinical [128] INF-b Multiple sclerosis Preclinical [129] INF-g Antitumor Phase I [130] Interleukin-2 Renal cell carcinoma, advanced Phase I [59,60,131] melanoma, lung metastasis Leuprolide Prostate cancer, endometriosis Preclinical [85,132,133] Sargramostin Metastatic cancer, sarcoma Phase II [134] Con-IFN: Consensus interferon; Ddavp: 1-Deaminocysteine- 8-d-arginine vasopressin; GLP: Glucagon-like peptide; LHRH: Luteinizing-hormone-releasing hormone. Original table using data from various sources.

future science group www.futuremedicine.com 5 Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

Table 3. Devices and formulations for inhaled insulin, their manufacturers and stages of development. Trademark/ Manufacturer Partner Formulation Development Notes technology type Phase Exubera® Nektar Pfizer DPI Approved (2006) Withdrawn (2007)

AIR® Alkermes Eli Lilly DPI Phase III Discontinued (2008)

Aerodose® Aerogen Liquid inhaler Phase II

Spiros® Dura Pharmaceuticals Eli Lilly DPI Phase I Discontinued

AERx® iDMS Aradigm Novo Nordisk Liquid inhaler Phase III Discontinued (2008)

Afrezza Technosphere® Pharmaceutical Discovery Mannkind DPI Phase III Waiting approval by Corporation the US FDA Microdose® DPI Microdose Technologies Elan Corp. DPI Phase I

Alveair® CoreMed Liquid inhaler Phase I

Bio-Air® BioSante Pharmaceuticals DPI Preclinical

ProMaxx® Baxter Healthcare DPI Phase I Corporation Kos Pharmaceuticals MDI Phase II Qdose Bristol-Myers DPI Phase I Squibb DPI: Dry powder inhaler; Idms: Insulin diabetes management system; MDI: Metered dose inhaler.

and dyspnea in some patients. There is also an calcitonin, it presents a low and variable bioavail­ increase of serum anti-insulin after pulmonary ability (ranging from 0.3–30.6%) and some side administration, which has not yet been associ­ effects such headache, dizziness, nausea or nasal ated with any clinically significant alterations secretions. Pulmonary administration could and stabilizes after 6–12 weeks [41,43]. reduce or avoid these side effects and, generally, Pfizer announced in a statement the occur­ presents higher bioavailability of peptides and rence of a greater number of cases of lung can­ proteins than intrasnasal administration [49]. In cer in diabetic patients also smokers who used one study, a dry power formulation for inhala­ Exubera® compared with other treatments for tion showed 66% of the bioactivity and 28% Diabetes. That it may be due to vasodilatory of the bioavailability of intramuscular calcito­ capacity and promoting the growth of insulin nin [50]. In another study, the bioavailability 1,7 [41]. However, owing to the low number of cases of a (Asu )-eel calcitonin solution containing is not possible to establish a causal relationship sodium glycocholate as absorption enhancer between the emergence of lung cancer and the after intratracheal instillation was 52.9% com­ use of Exubera®. pared with intravenous administration. The formulation did not cause serious damage or Calcitonin local irritation to the pulmonary epithelium AuthorCalcitonin is an endogenous polypeptide Proofhor­ suggesting its possible use in the administration mone with a molecular weight of 3.4 kDa, of calcitonin through the lung [51]. produced by the parafollicular cells of the thy­ roid gland [47]. It plays a crucial role in calcium Cyclosporin A homeostasis and bone remodeling by inhibition Cyclosporin A (CsA) is a hydrophobic cyclic of bone resorption, promoting new bone forma­ peptide with high immunosuppressive activity tion, moving calcium from blood to bone and that could be used in immunological diseases enhancing the rate of calcium deposition [47–49]. such as pulmonary chronic asthma, hyper­ In fact, calcitonin is used intravenously or intra­ sensitivity, bronchiolitis, sarcoidosis [52] or nasally in the clinical treatment of bone meta­ the treatment of lung transplant rejection [53]. bolic disorders such as osteoporosis and Paget’s Pulmonary delivery of CsA seems to be a viable disease and the treatment of hypercalcemia and alternative to parenteral administration [54]. As bone metastasis [47,48]. Although the intranasal a local administration, this will reduce the long- administration is a noninvasive route to deliver term nephrotoxicity of oral and parenteral CsA.

6 Nanomedicine (2011) 6(1) future science group Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

Furthermore, it allows a higher concentration of by macrophages) and thus increase systemic CsA at the site of action [55]. However, the use circulation time [6,8,20,22]. They also allow the of solvents such as ethanol and propylene glycol reduction of the immunogenicity of proteins, in conventional formulations for pulmonary thus decreasing the toxicity of the formulation administration can cause adverse effects [55]. [45]. These advantages are common to different administration routes, including pulmonary IL-2 administration. IL‑2, also known as aldesleukin, a cytokine Nanoparticle delivery to the lungs is an attrac­ of 15.5 kDa, is an immunomodulatory drug tive concept because it can cause retention of approved for the treatment of renal cell carci­ the particles in the lungs accompanied with a noma and advanced melanoma, especially when prolonged drug release, drug protection and they are metastatic in the lungs. It is also used improved bioavailability over the conventional in other metastatic cancers and diseases char­ pulmonary drug delivery systems. acterized by states of immunodeficiency [56,57]. Today there are already some approved and Intravenous administration of IL‑2 is associated marketed nanotechnology-based drug delivery with severe and dose-limiting side effects in the systems (nanoDDS) for the administration of kidneys, cardiovascular system and liver, among different type of drugs, namely AmBisome® ® others [58]. In recent years, alternative methods (amphotericin B), Macugen (pegaptanib), of administration, including inhalation, have Abraxane® (paclitaxel), Doxil® (doxorubicin), been studied and are the subject of clinical trials, DaunoXome® (daunorubicin) and Rapamune® obtaining positive results, especially in reducing (sirolimus). Examples of nanoDDS for admin­ associated side effects and increasing the median istration of proteins (PEGylated proteins) with survival of patients [59,60]. Since 2006, the EMA market authorization are summarized in Table 4. has granted orphan designation to an inhaled human IL‑2 produced by Immunservice GmbH, Pulmonary biodistribution for the treatment of renal cell carcinoma. of nanocarriers „„Deposition Nanomedicine & drug As referred above, the deposition of particles in delivery systems the lungs depends on several factors; however, The application of nanotechnology in medicine the mass median aerodynamic diameter of par­ has been the target of growing interest over recent ticles, which is a function of particle size, shape years, with the emergence of the concept of nano­ and density, plays a major role in their deposi­ medicine. This is because the cellular and subcel­ tion [29,63]. lular structures are at the nano and micrometer Alveolar deposition of particles is bimodal. scale [61]. The goal of nanomedicine is to allow a The highest rates occur with aerodynamic more accurate and timely diagnosis and provide diameters in the range of 1 to 5 µm and 1 to the most effective treatment without side effects 100 nm. Particles larger than 5 µm and less [62]. Currently, the main application areas of than 1 nm are deposited in the nasopharynx nanomedicine are imaging and cancer therapy, and then swallowed. Particles below 1 µm and however, studies have been carried out in various above 100 nm, deposit in tracheobronchial area areas, such as peptide and protein delivery, vac­ or are exhaled. The higher alveolar deposition cination, gene therapy, tissue engineering or pro­ efficiency occurs in particles with 20 nm diam­ duction of devices for administrationAuthor of drugs [61]. eter (~50%) [6,20,64–66] Proof. However, these limits Nanocarriers, including nanoparticles, lipo­ are not consensual (Figure 3). somes and micelles, may be used for the incor­ poration of drugs allowing their protection from Table 4. Protein-based nanotechnology-based drug delivery systems degradation, targeting to desired organs or tis­ with market authorization. sues and the reduction of side effects [8–10]. In the case of protein formulations, the use of nanocar­ Therapeutic protein Trademark riers have several advantages such as improved Pegademase bovine Adagen® stability and transepithelial transport, obtaining Peginterferon a-2a Pegasys® modified-release formulations, deep penetration Peginterferon a-2b PEG-Intron/ViraferonPeg® in tissues, increased internalization by cells, high Pegaspargase Oncaspar® strength, conductivity and durability, and their ® ability to escape the in vivo defensive system Pegfilgrastim Neulasta (e.g., mucociliary clearance and phagocytosis Pegvisomant Somavert®

future science group www.futuremedicine.com 7 Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

The main mechanism underlying the pulmo­ nary deposition of nanocarriers is Brownian dif­ >5 µm fusion. As the diameter decreases, the mobility increases, which will promote the deposition of particles in all areas of the lungs [20,64]. However, as referred to above, the deposition of particles 100 nm–1 µm also depends on the nature of the particles. Studies show that polymeric nanoparticles with 400 nm [67] and lipid nanoparticles with 1–100 nm and 1–5 µm 300 nm [7] present an alveolar deposition approx­ imately 75% and 45% w/w of the dose delivered, respectively. Figure 3. Particle deposition profile in the It is possible to produce aggregates of nano­ respiratory tract. carriers (nanocomposites) with aerodynamic diameters between 1 and 5 µm in order to receptor-mediated endocytosis [26,61]. Caveolin-1 increase alveolar deposition, however, they protein is abundant in alveolar type I and in should be easily dispersed when they come into the pulmonary capillary endothelium, and the contact with the alveolar fluid [20,68]. caveolae-mediated endocytosis is a possible important pathway in epithelial translocation „„Clearance & permeation of nanoparticles [64,72,73]. The type and surface of After lung deposition, nanocarriers are sub­ nanoparticles modulate their endocytotic uptake merged in the airway fluid (mucus and surfac­ pathways in lung epithelium. For example, the tant) and exposed to the mechanisms of perme­ inhibition of caveolin- and clathrin-mediated ation and clearance [20]. Nanocarriers that are endocytosis by methyl-b-cyclodextrin results in liposoluble or soluble intra- and extracellular a significant reduction in the uptake of golden fluids undergoin situ dissolution. Hydrophobic nanoparticles by human alveolar epithelial cells, encapsulated molecules of low molecular weight with the inhibition more pronounced for PEG- are absorbed by passive diffusion across the lung coated nanoparticles [74]. epithelial membrane and the hydrophilic mol­ The level and duration of nanoparticle reten­ ecules can be absorbed through tight junctions tion and accumulation in lungs varies for the or by active transport [20,64]. Insoluble or poorly different nanoparticles. For example, significant soluble particles in the mucus and surfactant levels of ferric oxide nanoparticles with 22 nm are not able to be readily absorbed. Owing to diameter pass through the alveolar-capillary their proximity to the epithelial and defen­ barrier into systemic circulation within 10 min, sive system cells, these may suffer cell-particle after intratracheally instillation into rats. Only a interaction. Consequently, defense mechanisms small percentage of the particles suffer phagocy­ such as mucociliary clearance, phagocytosis by tosis, because the nanoparticles escaped phago­ macrophages and endocytosis will remove the cytosis by alveolar macrophages and entered into inhaled particles [20]. Although phagocytosis is alveolar epithelium, where they accumulate. the predominant mechanism in the clearance of 50 days after instillation is still possible to detect solid particles, the percentage of nanoparticles ferric oxide nanoparticles in the lungs [75]. In present in macrophages after inhalation is, in another study conducted in humans, only 25% Authormany cases, reduced. There appears to someProof of the deposited 100 nm diameter carbon par­ inefficiency of macrophages in the depletion ticles were cleared within 1 day by mucociliary of particles in the order of nanometers [23,64,69]. action, and 75% of the particles were persistent Particles below 260 nm suffer reduced phagocy­ in the airways. Clearance in the lung periphery tosis [70,71] and below 70 nm are not recognized is much slower than from the airways, being by macrophages [20]. This may be explained by only approximately 3% of the deposited particles the absence or reduced promotion of chemo­ within 24 h, while the remaining particles were tactic signal from the nanoparticles [64]. Thus, retained for more than 48 h [76]. The pulmonary endocytosis appears to play an important role pharmacokinetics of a CsA-loaded liposome for­ in epithelial translocation of nanoparticles to mulation was determined, and it was found that cellular level. There are several existing endo­ the liposome carrier is retained up to 16.9-times cytic pathways involved with both types of longer than the CsA half-life in normal lung and pneumocytes: pinocytosis, adsorptive endo­ 7.5-times longer in inflamed lungs in mice [77]. cytosis (nonspecific binding to receptors) and In the case of cationic liposomes, they exhibited

8 Nanomedicine (2011) 6(1) future science group Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

an accumulation up to twofold in the inflamed lung tissue as compared with healthy lungs in Lipid bilayer rats [78]. After being translocated within the lung epithelium, the particles can enter the blood­ Hydrophilic stream or lymphatic system. The latter can still drug be accessed by the phagocytozed particles [64,79]. Hydrophobic They can also reach the CNS via sensory nerve drug endings that exist in the pulmonary epithelium [66], thus overcoming the blood–brain barrier and targeting the drugs to the CNS. Figure 4. Schematic representation of a liposome. Pulmonary administration of peptides & therapeutic proteins- proved to be stable after 3 months of storage, loaded nanocarriers (state of the art promoting a decrease in systemic levels of glu­ & clinical trials) cose for 12 h when compared with solutions „„Liposomes for pulmonary administration of insulin and Liposomes are small spherical vesicles com­ subcutaneous injection [83]. Chono et al. (2009) posed of one or more bilayers of phospholipids, produced liposomes containing different deriva­ cholesterol, and/or other lipids [62]. Owing to tives of phosphatidylcholine by hydration of lipid their structure, they allow the incorporation of film technique [84]. In vivo studies using rats as hydrophilic drugs in the aqueous core, and lipo­ an animal model showed a greater reduction of philic drugs within the lipid bilayer (Figure 4) [10]. serum glucose after intratracheal administration Depending on the number and composition of of insulin encapsulated in liposomes compared the bilayers and the coating when present, it is with insulin solution. However, this reduction possible to obtain systems with modified-release was only significant for liposomes containing characteristics [80,81]. The use of liposomes for the derivative dipalmitoylphosphatidylcholine. pulmonary administration of several drugs This study also assessed the influence of the size including peptides and therapeutic proteins of liposomes in the absorption of insulin. There and DNA for gene therapy has been suggested was a greater reduction in levels of plasma glu­ [17]. Owing to their interaction with endogenous cose and increased serum insulin after admin­ phospholipids, liposomes promote an increased istration of liposomes with an average diameter retention time in the lungs. Furthermore, the use of 100 nm. In the case of liposomes of 1000 nm, of phospholipids similar to the surfactant pro­ the authors observed an increased retention by motes the absorption of the incorporated drugs, macrophages. In vitro tests performed in cell although the mechanism underlying the promo­ cultures of Calu-3 showed the existence of an tion of absorption is not yet clear [80]. absorption-promoting effect of liposomes, pos­ As discussed above, insulin is the most stud­ sibly by opening of tight junctions [84]. ied protein for pulmonary delivery. Several for­ Leuprolide, also known as leuprorrelin, is a mulations containing nanocarriers have been highly hydrophilic luteinizing-hormone-releas­ developed for pulmonary delivery of insulin in ing hormone agonist peptide with a molecu­ order to overcome the problems associated with lar weight of 1.2 kDa, used in the treatment conventional formulationsAuthor [41,43]. Huang et al. of prostate cancer, Proofendometriosis, and preco­ (2006) produced liposomes using the mem­ cious puberty [85]. In order to develop a DPI brane destabilization/dialysis method with an for pulmonary administration of leuprolide, average diameter of 200 nm and encapsulation Shahiwala et al. (2005) produced liposomes by efficiency of 52% [82]. In vivo studies demon­ reverse-phase evaporation technique followed by strate a homogeneous deposition of liposomes lyophilization [85]. The diameter of the aggre­ in alveoli with reduction of systemic levels of gates of liposomes obtained lies within the limits glucose and absence of immunoreaction of lung compatible with alveolar deposition of particles tissue [82]. The liposomes obtained by Bi et al. of 3.5–4.3 µm, possessing high encapsulation (2008) using the reverse-phase evaporation tech­ efficiency (66–72%). After intratracheal instil­ nique possessed an average diameter of 295 nm lation in rats, liposomes promoted an increase and an encapsulation efficiency of 43%. They of serum luteinizing hormone (LH) than the were then were subjected to spray congealing solution of leuprolide and an increased half-life before solvent evaporation [83]. The formulation when compared with solutions for pulmonary

future science group www.futuremedicine.com 9 Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

and sub­cutaneous administration. However, primary lung carcinoma in another animal. The the bioavailability of liposomes is only 50% most significant results were observed in treating compared with subcutaneous injection and it pulmonary metastasis from osteosarcoma [88]. In is necessary to change the formulation in order a Phase I clinical trial, the pulmonary delivery to increase the bioavailability of leuprolide [85]. of liposomes containing IL‑2 to patients with Letsou et al. produced liposomes containing various carcinomas affecting the lungs (pulmo­ CsA that were administered by inhalation to nary sarcoma, renal cell carcinoma, melanoma dogs [55]. There was a higher concentration of and metastatic osteosarcoma) was well tolerated CsA in the lungs instead of the liver, kidneys, and there was no significant toxicity observed spleen, heart and blood compartment. However, at doses that may possess therapeutic effect 3 h after administration, there was some accu­ [57]. However, this study did not determine the mulation of CsA in the kidney and spleen, so the clinical efficacy of the formulation. In another effects on the kidney should be studied [55]. In Phase I clinical trial, which studied the utility another study, pulmonary delivery of liposome- of inhaled lipossomal IL‑2 in patients with com­ encapsulated CsA promoted an accumulation mon variable immunodeficiency (CVID) [89], of CsA in the lungs. The formulation was stable IL‑2 retained its biological activity after encap­ after nebulization and maintains the immuno­ sulation. No changes were observed in pulmo­ suppressive activity of CsA after encapsulation nary function or significant side effects during [52]. Gilbert et al. administered liposomal CsA treatment. Although the patients treated with by inhalation to healthy humans and found a IL‑2 liposome declared a sense of improvment in preferential deposition of the particles in the their condition, the investigators did not detect alveolar region (70% of inhaled dose) [86]. When alterations in the immune response within the the formulation was administered via a mouth- blood compartment. Such lack of response evi­ only face mask for 45 min, no changes were dence may be due to low specificity of the mark­ observed in pulmonary function. By contrast, ers used in the study [89]. the administration using a nebulizer mouthpiece Superoxide dismutase (SOD) is an anti­oxidant has proved troublesome, leading to coughing enzyme, kidnapper of free radicals, ubiquitous and throat irritation and a slight decrease in lung in mammalian cells. It causes a decrease of function. This may be due to the fact that par­ reactive oxygen species responsible for oxidative ticles, when administered by a nebulizer mouth­ stress, involved in phenomena such as carcino­ piece, suffer greater impaction in the throat. The genesis, inflammation and neurodegeneration observed differences do not appear to be due [90]. Studies demonstrate the success of the use to the formulation, because both cases used the of SOD in the treatment of rheumatoid arthri­ same formula, with the same particle size at the tis and ischemia-reperfusion injury. However, same dose [86]. after intravenous and oral administration, the In an in vivo study conducted by Khanna et al. SOD has a reduced circulation half-life and a in healthy dogs, the pulmonary administration high-level gastrointestinal degradation, respec­ of IL‑2-loaded liposomes led to an activation of tively. With the aim of developing a noninvasive the immune response by a significant increase formulation that promotes an increased half-life in leukocyte levels in the lung and serum mono­ of SOD, Kaipel et al. produced liposomes for nuclear cells [87]. This response was statistically pulmonary administration [90]. In vivo studies significant when compared with inhaled free conducted in pigs showed a prolonged release AuthorIL‑2. The observed differences may be dueProof to of SOD into the systemic circulation and an increased cellular uptake or decreased clearance increase in its half-life. There were no side effects of liposomal IL‑2. In vitro studies showed that such as irritation or inflammation in the lung,

activation of pulmonary leukocytes leads to inhi­ as well as changes in pH and plasmatic O2 and

bition of proliferation of tumor cells [87]. Another CO2 pressure [90]. study of the same researchers conducted in dogs Lange et al. developed different liposomal with primary lung carcinoma or lung metastasis formulations for aerosol delivery of a cationic of other carcinomas, has shown that there is no a-helical peptide (CM3) with antimicrobial significant toxicity associated with pulmonary and antiendotoxin activity [91]. The pulmonary administration of liposomal IL‑2 [88]. It was delivery of CM3 allows the treatment of local also found that liposomal IL‑2 stimulates the infections and reduction of systemic effects. immune system after inhalation. Moreover, in Of the several formulations developed, the best some animals was observed the complete regres­ results in terms of the encapsulation and nebuli­ sion of metastasis and the nonprogression of zation efficiencies and maintenance of liposomal

10 Nanomedicine (2011) 6(1) future science group Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

integrity during nebulization were achieved with A B the combination of dimyristoyl phosphatidyl­ Lipidic matrix choline and dimyristoyl phosphatidylglycerol Matrix imperfections with a 3:1 molar ratio. With this formulation it was possible to obtain liposomes with diameters of 262 nm and an encapsulation efficiency of 73%. Using a mathematical model it was pos­ Drug sible to predict the deposition profile and distri­ bution in the lungs of liposomes in adults and children of different ages. The model results Figure 5. Schematic representation of (A) solid lipid nanoparticle and showed a pulmonary deposition in all lungs, (B) nanostructured lipid carrier. particularly in the tracheobronchial region. In this region, the minimum inhibitory levels of liposomes and lipid particles, such as low encap­ CM3 can be reached in the adult model, and sulation efficiency and stability during storage can be exceeded in pediatric model subjects [91]. and rapid release of encapsulated compounds. They can be obtained through polymerization „„Lipid nanoparticles of monomers or polymer dispersion (Figure 6) Lipid nanoparticles generally comprise two types [9,81]. Among polymeric nanocarriers, those of structures, the solid lipid nanoparticles (SLNs) containing natural polysaccharides, including and nanostructured lipid carriers (NLCs) [10,92]. chitosan, alginate or hyaluronic acid, enjoy high They consist of a solid lipid matrix at both ambi­ popularity in the production of drug delivery ent and body temperatures, dispersed in aqueous systems because they are natural, biocompatible, solution and stabilized with an layer of emulsifier bio­degradable, nontoxic and hydrophilic com­ agent, usually phospholipids [81]. They emerged pounds, possessing in general low-cost produc­ as an alternative to liposomes and pharmaceuti­ tion and many sources in nature [94]. Among the cal emulsions, because they are more stable in synthetic polymers, poly(lactic-co-glycolic acid) biological fluids. They are also less toxic than (PLGA) is a well studied polymer, accepted by the inorganic and polymeric particles owing to regulatory authorities and widely used in bio­ their biocompatibility and biodegradability [81]. medical applications owing to its biocompatibil­ Unlike the SLNs, the NLCs are composed not ity, biodegradation and usefulness in production only of solid lipids, but also by a mixture of solid of modified-release formulations [45,95]. and liquid lipids, resulting in a solid matrix less Polymeric nanocarriers have been highly rigid and ordered. This difference will allow explored for the pulmonary administration of NLCs to increase the loading capacity of drugs insulin. Huang et al. developed nanoparticles of and the stability during storage (Figure 5) [10,92]. low molecular weight chitosan using the emulsi­ The lipid nanoparticles have been proposed for fication/solvent evaporation technique[96] . The pulmonary administration of insulin. Examples resulting particles have a spherical shape with include the nanoparticles of lecithin obtained by an average diameter of approximately 400 nm, emulsification method followed by lyophilization zeta potential of approximately +42 mV and an with an average diameter of 300 nm, and alveolar encapsulation efficiency of 95.5%. The release deposition of 45% (w/w) of the dose delivered. profile of insulin was characterized by a burst The retention of the primary, secondary and effect followed by prolonged release for 24 h. tertiary structure of insulinAuthor after processing was When administered Proof to diabetic rats, the for­ confirmed [7]. In another study, Liu et al. pro­ mulation demonstrated a hypoglycemic effect duced SLN-containing micelles inside by double emulsion with an average diameter of 115 nm and an encapsulation efficiency of 98% [93]. In vitro studies showed prolonged release of insulin. It was Polymeric also demonstrated to retain the integrity of insulin matrix Drug after encapsulation and the stability of the formu­ lation after 6 months of preparation at 4 °C [93].

„„Polymeric nanoparticles The polymeric nanocarriers have been adopted as the preferred drug delivery system because Figure 6. Schematic representation of a they overcome some disadvantages presented by polymeric nanoparticle.

future science group www.futuremedicine.com 11 Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

similar to subcutaneous administration of eliminated more slowly from the lung compared insulin solution but prolonged in time [96]. In with those not coated. Moreover, the opening another study, Yamamoto et al. produced PLGA of tight junctions also promoted by chitosan led nanoparticles using the emulsification/solvent to an increased absorption of calcitonin and a evaporation technique followed by granulation decrease in in vivo systemic levels of calcium [97]. In vitro and in vivo showed an alveolar depo­ [101]. The pharmacological effect was prolonged sition of approximately 45% (w/w) of emitted for 24 h after inhalation. The particles with a dose and pharmacological effect of prolonged diameter of 650 nm and were nebulized with over 12 h when compared with solutions of success and the release profile of calcitonin is insulin administered intratracheally and intra­ characterized by a burst effect followed by pro­ venously [97]. Kawashima et al. obtained PLGA longed release over time [101]. nanoparticles with average diameters of 400 nm, The different nanocarriers presented in sec­ and an alveolar deposition of 75% (w/w) using tion six were produced by different research the modified emulsification/solvent evaporation groups and were characterized differently mak­ method [67]. In vitro dissolution tests showed an ing it difficult to compare. However, the poly­ insulin release profile characterized by an initial meric nanocarriers appear in general to have a burst effect followed by extended release. In vivo larger diameter, which can leave to a reduced studies show a significant reduction in systemic alveolar deposition, and promote a more pro­ levels of glucose, which lasts for a period exceed­ longed drug release compared with the other ing 48 h, compared with an aqueous solution of nanocarriers. Table 5 summarizes the different insulin for inhalation [67]. This biphasic release formulations proposed for pulmonary admin­ of insulin can mimic insulin mixtures of short istration of peptides and therapeutic proteins and long duration of action in the market for described above. injection. In a study, Grenha et al. produced chitosan nanoparticles with and without lipid Pulmonary toxicity of nanocarriers coating obtained by ionic gelation and then Despite the many advantages presented by spray-dried to obtain nanocomposites [68,80]. The nanocarriers, they may have a high reactivity nanoparticles obtained had an average diameter with cellular components due to small size, between 380 and 450 nm and encapsulation which raises some safety concerns. In vivo and efficiency between 65 and 81%.In vitro studies in vitro data showed induction of inflammatory demonstrated a rapid release of insulin in the responses and epithelial damage in the lungs, as case of lipid nanoparticles without coating and well as extrapulmonary effects, such as oxida­ a prolonged release in formulations containing tive stress or increased blood clotting [64,76,102]. lipid coating [68,80]. In vivo studies in rats show Such studies rely on very high doses of nanopar­ that after intratracheal administration, uncoated ticles, particularly ultrafine inorganic particles, lipid nanoparticles reach the alveolar region and that do not correspond to the actual exposure promote a greater reduction of systemic levels doses [64]. Moreover, because of the more homo­ of glucose, compared with insulin solution [98]. geneous nature of the nanoparticles produced Poly(n-butyl cyanoacrylate) (PBCA)/dextran for drug delivery, the risk associated with their nanoparticles obtained by Zhang et al. using the exposure may not be similar to that observed for in situ polymerization method have a diameter of the ultrafine particles[103] , however, this should 255 nm and an encapsulation efficiency of 79% not be overlooked, requiring further studies on Author[99]. In vitro assays demonstrate an insulin releaseProof risk assessment. profile characterized by an initial burst effect In the case of nanoparticles as drug deliv­ followed by prolonged release. In vivo studies ery systems, those who raise more safety issues are characterized by a prolonged therapeutic are the polymeric and the inorganic particles. effect over time when compared with insulin Despite their known biocompatibility for cer­ solution administered intratracheally and a bio­ tain routes of administration such as intrave­ availability of 57% compared with subcutaneous nous, the toxicity of polymeric nanoparticles administration [99]. to the lung epithelium should be investigated. PLGA nanoparticles coated with chitosan Thus, the use of cellular models, for example, obtained by the emulsion and solvent diffusion A549 (alveolar epithelial), BEAS-2B, Calu-3 technique have already been proposed to admin­ or 16HBE14o-(bronchial epithelium) will be ister calcitonin via the oral [100] and pulmonary useful in toxicology studies because they allow [101] routes. Owing to the mucoadhesion pro­ the study of metabolic activity, membrane moted by chitosan, coated nanoparticles were integrity and the release of proinflammatory

12 Nanomedicine (2011) 6(1) future science group Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

Table 5. Summary of published studies relating to peptide- and protein-loaded nanocarriers for inhalation. Type of Formulation components Preparation technique Peptide/ Ref. nanocarrier therapeutic protein Liposomes DLPC Hydration of lipid film Cyclosporin A [52,55,86] HPC, Chol, PEG-DPPE (70:30:1 molar ratio) Membrane destabilization/dialysis Insulin [82] Soya lecithin, Chol Reverse phase evaporation Insulin [83] Derivates of PC, Chol, DCP (7:2:1 molar Hydration of lipid film Insulin [84] ratio) DMPC Freezing and thawing IL-2 [57,87–89] HSPC, Chol (4:1 molar ratio) Reverse phase evaporation Leuprolide [85] DPPC, Chol:E-PG Crossflow injection Superoxide [90] dismutase DMPC, DMPG (3:1 molar ratio) Hydration of lipid film a-helical cationic [91] peptide Lipid nanoparticles Lecithin Emulsification Insulin [7] SC, SPC, stearic acid, palmitic acid Reverse micelle-double emulsion Insulin [93] Polymeric PLGA/chitosan Emulsion and solvent diffusion Calcitonin [101] nanoparticles Low molecular weight chitosan Emulsification/solvent evaporation Insulin [96] PLGA Emulsification/solvent evaporation Insulin [97] PLGA Modified emulsification/solvent Insulin [67] evaporation chitosan/TPP Ionic gelation Insulin [68] chitosan/TPP, DPPC/DMPG Ionic gelation-hydration of lipid film Insulin [80] PBCA/dextran In situ polymerization Insulin [99] Chol: Cholesterol; DCP: Dicetylphosphate; DLPC: Dilauroylphosphatidylcholine; DMPC: Dimyristoylphosphatidylcholine; DMPG: Dimyristoylphosphatidylglycerol; DPPC: Dipalmitoylphosphatidylcholin; E-PG: Egg-phosphatidylglycerol; HPC: Hydrogenated egg yolk phosphatidylcholine; HSPC: Hydrogenated soya phosphatidylcholine; PBCA: Poly(n-butyl cyanoacrylate); PC: Phosphatidylcholine; PEG-DPPE: N-methoxypolyethyleneglycol succinoyl-2-N-dipalmitoylphosphatidyl ethanolamine; PLGA: Poly(lactic-co-glycolic acid); SC: Sodium cholate; SPC: Soybeann phosphatidylcholine; TPP: Pentasodium tripolyphosphate. Original table using data from various sources.

and inflammatory mediators after internaliza­ such as lactate dehydrogenase (LDH) release, tion of the nanoparticles. Besides the interac­ cell count and protein concentration in the tion of particles with lung epithelial cells and bronchial alveolar lavaged fluid, recruitment of macrophages, is also necessary to study their polymorphonuclear cells and chemotactic factor effect on mucus and surfactant. Surfactants MIP-2 induction were measured. The results playing important roles in gas exchange and were compared with the intratracheal instilla­ surface tension in the alveolar space and their tion of nonbiodegradable nanospheres polysty­ destabilization by inhaled particles can have rene containing 75 and 220 nm. It was found considerable consequences for health [71]. An that particles of biodegradable DEAP-PVAL- in vitro study performedAuthor with gelatin nanopar­ g-PLGA and PLGA Proof when administered by ticles in contact with surfactant film showed pulmonary route have an lower inflammatory that the interaction between them does not effect than the nonbiodegradable particles of cause destabilization of the film. In another similar size [70]. Studies with chitosan particles in vitro study, nanoparticles of inorganic polio­ in Calu-3 and 16HBE14o- cell cultures demon­ rganosiloxano with 22 nm interfered with the strated that it is nontoxic or of a low toxicity to components of the surfactant film and provoked cells of the pulmonary route [80]. their destabilization. Dailey et al. studied the In the case of lipid carriers, they are in most proinflammatory potential of diethylamino­ cases composed of physiological components, propylamine polyvinyl alcohol grafted-PLGA so the body has metabolic pathways that reduce (DEAP-PVAL-g-PLGA) and PLGA nanopar­ the toxic effects derivative from short and long- ticles with approximately 215 and 82 nm in term exposure. However, it is necessary to take diameter, respectively, administered to mice by into account the emulsifiers and preservatives intratracheal instillation [70]. Several parameters agents used in formulations [10]. Chono et al.

future science group www.futuremedicine.com 13 Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

studied the cytotoxicity of dipalmitoylphos­ bioavailability, and physical or chemical insta­ phatidylcholine liposomes and found that, after bility, which impose their administration by pulmonary administration, the LDH levels were injection. similar to those obtained with administration In order to overcome the drawbacks of par­ of phosphate buffered saline [84]. However, enteral administration, noninvasive alternative administration of ethylenediaminetetraacetic routes, such as oral, pulmonary, nasal, rectal and acid and sodium taurocholate, two possible transdermal, have been investigated. permeability enhancers in pharmaceutical for­ Although the oral route is considered more mulations, has led to increased levels of LDH attractive and convenient for the administration [84]. In another study, Thomas et al. investigated of drugs, peptides and proteins show reduced the effects of soy phosphatidylcholine liposomes bioavailability after administration via this route. on pulmonary function in healthy adults. The Owing to the physiology of the lungs, inha­ results showed that there are no adverse effects lation is presented as a promising noninvasive on lung function, as well as oxygen saturation, alternative to parenteral administration of sev­ and the liposomes used in the study were well eral drugs. tolerated [104]. Currently there are different formulations under study for pulmonary delivery of thera­ Conclusion peutic peptides and proteins, including insulin, Recent advances in biotechnology and genetic interferons, sargramostin, parathyroid hormone engineering have resulted in the promotion of and erythropoietin. pharmaceutical peptides and proteins as impor­ Of all the proteins under development, insu­ tant classes of therapeutic agents. Despite the lin has been the target of most studies. This is emergence of several peptides and proteins with because diabetes is a chronic disease that affects potential therapeutic effects, their administra­ a large number of people around the world tion in the active conformation has been shown which requires, in specific cases, the subcutane­ to be a huge challenge to the pharmaceutical ous injection of insulin. The noninvasive admin­ industry. There are several limitations as low istration of insulin can increase the acceptance

Executive summary Peptides & therapeutic proteins: characteristics, stability & administration ƒƒ Owing to instability and reduced permeability of peptides and proteins through biomembranes, the parenteral route is the most commonly used for the administration of such drugs. However, this is an invasive and sometimes painful route, requiring the production of sterile formulations and has a low acceptance among patients. Pulmonary administration of peptides & therapeutic proteins ƒƒ Despite the difficulties associated with nonparenteral administration of peptides and proteins with regards to bioavailability and stability, pulmonary administration of such drugs is possible and a treatment of cystic fibrosis is currently on the market dornase a (Pulmozyme®) . ƒƒ The low acceptance by patients to treatment that involves injections can be minimized by pulmonary administration, which increases the control of disease and reduces the costs associated with complications arising from the failure of the treatment regimen. ƒƒ Although pulmonary administration appears as a promising noninvasive alternative to injection, inhalation of peptides and therapeutic proteins in conventional formulations presents some disadvantages, such reduced bioavailability and immunogenicity. Nanomedicine & drug delivery systems ƒƒ The use of nanocarriers allows, among other things, protection of proteins from degradation, reduction in their immunogenicity and side effects, increases in the transepithelial transport and cellular internalization and enables controlled release formulations. Pulmonary biodistribution ofAuthor nanocarriers Proof ƒƒ The deposition of particles at the lower respiratory tract is a complex phenomenon that depends on several factors, especially the aerodynamic diameter of particles. ƒƒ There are several possible mechanisms involved in clearance and permeation of nanoparticles through the lung epithelium, however, endocytosis appears to play the major role in epithelial translocation of nanoparticles to cellular level. Pulmonary administration of peptide- & therapeutic protein-loaded nanocarriers ƒƒ Several formulations containing nanocarriers are being developed for inhalation of peptides and therapeutic proteins, such as insulin, calcitonin and IL‑2. Some of these obtained promising results, pending studies on humans to evaluate their therapeutic potential. Pulmonary toxicity of nanocarriers ƒƒ Despite the advantages of nanoparticulate drug delivery systems, the toxicological aspects of these inhaled drug delivery systems have to be considered. Currently, most of the existing information on the pulmonary toxicity of nanoparticles is based on environmental or occupational exposure to nanomaterials, especially ultrafine particles. Several in vitro and in vivo studies suggest the appearance of side effects to health derived from such exposure. The lack of data on engineered particles must be balanced with studies to determine the toxicological potential of such particles in the lung, before their commercialization.

14 Nanomedicine (2011) 6(1) future science group Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

by patients who start therapy early, thereby Future perspective optimizing glycemic control and reducing the Evolution has happened in recent years, but is macro- and microvascular effects derived from expected in the near future an increase in the high levels of plasma glucose. development of formulations containing nano­ Although results from clinical trials demon­ carriers to improve the properties of various strate the usefulness of inhaled insulin in relation drugs, such as higher bioavailability, controlled to blood glucose control and patient satisfaction, release and targeting to specific organs and tis­ the conventional formulations as Exubera, have sues. This approach also allows the administra­ reduced bioavailability and side effects such as tion of stable drugs, as well as insoluble hydro­ increased anti-insulin antibodies. phobic drugs that otherwise would be excluded With the aim of overcoming the problems during the pharmaceutical development despite associated with conventional formulations for their high therapeutic potential. pulmonary delivery of peptides and proteins, Owing to the advantages presented by nano­ several research groups have studied the poten­ carriers, we will not be surprised by an increase tial of using nanotechnology systems, including in marketing authorization and commercializa­ the use of nanocarriers. tion of nanotechnology products, with particu­ In recent years, there have been few published lar emphasis on the administration of peptides studies concerning to the pulmonary adminis­ and therapeutic proteins by alternative routes to tration of peptide- and protein-loaded nanocar­ parenteral administration. riers. These studies vary in the peptide or protein It is also expected that the development of used, as well as the nature of nanocarrier and the new and improved delivery devices will allow technique used in their preparation. us to overcome the limitations of pulmonary Among the various peptides and proteins, administration of drugs, with regards to the insulin is the most studied and the results are nonreproducibility of the dose. promising, especially due to the increase of bio­ availability and the potential of obtaining con­ Financial & competing interests disclosure trolled release formulations. Disclose any financial interests. The authors have no Similar results were obtained with promising other relevant affiliations or financial involvement with formulations containing IL‑2, calcitonin, leup­ any organization or entity with a financial interest in or rolide and others. Despite some Phase I studies financial conflict with the subject matter or materials dis- already being carried out, clinical trials to deter­ cussed in the manuscript apart from those disclosed. mine the therapeutic efficacy of such formula­ No writing assistance was utilized in the production of tions in humans is still awaited. this manuscript.

Pharm. 356(1–2), 259–266 (2008). 12 Evaluatepharma: World preview 2016 Bibliography . 6 Bailey M, Berkland C: Nanoparticle (2010). 1 Umashankar M, Sachdeva R, Gulati M: formulations in pulmonary drug delivery. 13 Tang L, Meibohm B: Pharmacokinetics of Aquasomes: a promising carrier for peptides Med. Res. Rev. 29(1), 196–212 (2009). peptides and proteins. In: Pharmacokinetics and protein delivery. Nanomedicine 6, and Pharmacodynamics of Biotech Drugs - 419–426 (2010). 7 Nyambura B, Kellaway I, Taylor K: Insulin nanoparticles: stability and aerosolization Principles and Case Studies in Drug 2 Klingler C, Müller B, Steckel H: Insulin- from pressurized metered dose inhalers. Int. J. Development. Meibohm B (Ed.). Wiley-VCH micro- and nanoparticles for pulmonary Pharm. 375(1–2), 114–122 (2009). Verlag GmbH & Co. KGaA, Weinheim, delivery. Int. J. Pharm. 377(1–2), 173–179 Germany (2006). (2009). 8 Sung J, Pulliam B, Edwards D: Nanoparticles Author Proof14 for drug delivery to the lungs. Trends Hillery A: Drug delivery: The basic concepts. 3 Amidi M, Mastrobattista E, Jiskoot W, Biotechnol. 25(12), 563–570 (2007). In: Drug Delivery and Targeting for Hennink W: Chitosan-based delivery systems Pharmacists and Pharmaceutical Scientists. 9 Soppimath K, Aminabhavi T, Kulkarni A, for protein therapeutics and antigens. Adv. Hillery A, Lloyd A, Swarbrick J (Eds). Taylor Rudzinski W: Biodegradable polymeric Drug Deliv. Rev. 62(1), 59–82 (2010). & Francis, London, UK (2001). nanoparticles as drug delivery devices. 4 Sarmento B, Ribeiro A, Veiga F, Ferreira D: J. Control. Release 70(1–2), 1–20 (2001). 15 Morishita M, Peppas N: Is the oral route Development and characterization of new possible for peptide and protein drug delivery? 10 Martins S, Sarmento B, Ferreira D, Souto E: insulin containing polysaccharide Drug Discov. Today 11(19–20), 905–910 Lipid-based colloidal carriers for peptide and nanoparticles. Colloids Surf. B Biointerfaces (2006). 53(2), 193–202 (2006). protein delivery-liposomes versus lipid nanoparticles. Int. J. Nanomedicine 2(4), 16 Antosova Z, Mackova M, Kral V, Macek T: 5 Bayat A, Dorkoosh F, Dehpour A et al.: 595–607 (2007). Therapeutic application of peptides and Nanoparticles of quaternized chitosan proteins: parenteral forever? Trends Biotechnol. 11 Tauzin B: Biotechnology medicines in derivatives as a carrier for colon delivery of 27(11), 628–635 (2009). insulin: ex vivo and in vivo studies. Int. J. development. (2008). 17 Cryan S: Carrier-based strategies for targeting

future science group www.futuremedicine.com 15 Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

protein and peptide drugs to the lungs. 31 Folkesson H, Weström B, Karlsson B: inhaled human insulin (Exubera) in adult AAPS J. 7(1), E20–E41 (2005). Permeability of the respiratory tract to patients with Type 2 diabetes. Diabetes Care 18 Cryan S, Sivadas N, Garcia-Contreras L: different-sized macromolecules after 31(9), 1723–1728 (2008). In vivo animal models for drug delivery across intratracheal instillation in young and adult 44 Karathanasis E, Bhavane R, Annapragada A: the lung mucosal barrier. Adv. Drug Deliv. rats. Acta Physiol. Scand. 139(2), 347–354 Glucose-sensing pulmonary delivery of Rev. 59(11), 1133–1151 (2007). (1990). human insulin to the systemic circulation of 19 Iazzetti G, Rigutti E (Eds): Atlas of Anatomy. 32 Conhaim R, Watson K, Lai-Fook S, rats. Int. J. Nanomedicine 2(3), 501–513 Taj Books, Surrey, UK (2005). Harms B: Transport properties of alveolar (2007). epithelium measured by molecular hetastarch 20 Yang W, Peters J, Williams RR: Inhaled 45 Ungaro F, D’emmanuele Di Villa Bianca R, absorption in isolated rat lungs. J. Appl. nanoparticles – a current review. Int. J. Giovino C et al.: Insulin-loaded plga/ Physiol. 91(4), 1730–1740 (2001). Pharm. 356(1–2), 239–247 (2008). cyclodextrin large porous particles with 33 Holter J, Weiland J, Pacht E, Gadek J, improved aerosolization properties: in vivo 21 Groneberg D, Fischer A, Chung K, Daniel H: Davis W: Protein permeability in the adult deposition and hypoglycaemic activity after Molecular mechanisms of pulmonary respiratory distress syndrome. Loss of size delivery to rat lungs. J. Control. Release peptidomimetic drug and peptide transport. selectivity of the alveolar epithelium. J. Clin. 135(1), 25–34 (2009). Am. J. Respir. Cell Mol. Biol. 30(3), 251–260 Invest. 78(6), 1513–1522 (1986). (2004). 46 Garg S, Kelly W: Insulin delivery via lungs – 34 Matsukawa Y, Yamahara H, Yamashita F, is it still possible? Diabetes Technol. Ther. 22 Grenha A, Carrión-Recio D, Teijeiro-Osorio Lee V, Crandall E, Kim K: Rates of protein 11(2 Suppl. 2), S1–S3 (2009). D, Seijo B, Remuñán-López C (Eds): Nano transport across rat alveolar epithelial cell and Microparticulate Carriers for Pulmonary 47 Peppas N, Carr D: Impact of absorption and monolayers. J. Drug Target 7(5), 335–342 Drug Delivery. American Scientific Publishers, transport on intelligent therapeutics and (2000). Valencia, CA, USA (2008). nano-scale delivery of protein therapeutic 35 Bahadduri P, D’souza V, Pinsonneault J et al.: agents. Chem. Eng. Sci. 64(22), 4553–4565 23 Tomoda K, Ohkoshi T, Nakajima T, Functional characterization of the peptide (2009). Makino K: Preparation and properties of transporter pept2 in primary cultures of inhalable nanocomposite particles: effects of 48 Lee Y, Sinko P: Oral delivery of salmon human upper airway epithelium. Am. J. Respir the size, weight ratio of the primary calcitonin. Adv. Drug Deliv. Rev. 42(3), Cell Mol. Biol. 32(4), 319–325 (2005). nanoparticles in nanocomposite particles and 225–238 (2000). temperature at a spray-dryer inlet upon 36 Islam N, Gladki E: Dry powder inhalers 49 Patton J: Pulmonary delivery of drugs for properties of nanocomposite particles. (DPIs) – a review of device reliability and bone disorders. Adv. Drug Deliv. Rev. 42(3), Colloids Surf. B Biointerfaces 64(1), 70–76 innovation. Int. J. Pharm. 360(1–2), 1–11 239–248 (2000). (2008). (2008). 50 Deftos L, Nolan J, Seely B et al.: 24 Smola M, Vandamme T, Sokolowski A: 37 Brunton L, Lazo J, Parker K (Eds): Goodman Intrapulmonary drug delivery of salmon Nanocarriers as pulmonary drug delivery & gilman’s The Pharmacological Basis of calcitonin. Calcif. Tissue Int. 61(4), 345–347 systems to treat and to diagnose respiratory therapeutics. McGraw-Hill Professional, (1997). Berkshire, UK (2006). and non respiratory diseases. Int. J. 51 Yamamoto A, Okumura S, Fukuda Y, Nanomedicine 3(1), 1–19 (2008). 38 Katzung B (Ed.): Basic & Clinical Fukui M, Takahashi K, Muranishi S: 25 Cefalu W: Concept, strategies, and feasibility Pharmacology. Lange Medical Books/ Improvement of the pulmonary absorption of of noninvasive insulin delivery. Diabetes Care McGraw-Hill, New York, NY, USA (2001). (asu1,7)-eel calcitonin by various 27(1), 239–246 (2004). 39 Cheng K, Mahato R: Biopharmaceutical absorption enhancers and their pulmonary 26 Kim K, Malik A: Protein transport across the challenges: pulmonary delivery of proteins toxicity in rats. J. Pharm. Sci. 86(10), lung epithelial barrier. Am. J. Physiol Lung and peptides. In: Pharmacokinetics and 1144–1147 (1997). Cell. Mol. Physiol. 284(2), L247–L259 Pharmacodynamics of Biotech Drugs – 52 Waldrep J, Arppe J, Jansa K, Vidgren M: (2003). Principles and Case Studies in Drug Experimental pulmonary delivery of Development, Meibohm B (Ed.): Wiley-VCH 27 Hastings R, Folkesson H, Matthay M: cyclosporin a by liposome aerosol. Int. J. Verlag GmbH & Co. KGaA, Weinheim, Mechanisms of alveolar protein clearance in Pharm. 160, 239–249 (1998). Germany (2006). the intact lung. Am. J. Physiol. Lung Cell Mol. 53 Mitruka S, Pham S, Zeevi A et al.: Aerosol Physiol. 286(4), L679–L689 (2004). 40 Wolzt M, De La Peña A, Berclaz P, Tibaldi F, cyclosporine prevents acute allograft rejection Gates J, Muchmore D: Air inhaled insulin 28 Berthiaume Y, Albertine K, GradyAuthor M, Fick G, Proofin experimental lung transplantation. versus subcutaneous insulin: Matthay M: Protein clearance from the air J. Thorac. Cardiovasc. Surg. 115(1), 28–36, pharmacokinetics, glucodynamics, and spaces and lungs of unanesthetized sheep over Discussion 36–27 (1998). pulmonary function in asthma. Diabetes Care 144 h. J. Appl. Physiol. 67(5), 1887–1897 54 Keenan R, Iacono A, Dauber J et al.: 31(4), 735–740 (2008). (1989). Treatment of refractory acute allograft 41 De Galan B, Simsek S, Tack C, Heine R: 29 Agu R, Ugwoke M, Armand M, Kinget R, rejection with aerosolized cyclosporine in lung Efficacy and safety of inhaled insulin in the Verbeke N: The lung as a route for systemic transplant recipients. J. Thorac. Cardiovasc. treatment of diabetes mellitus. Neth. J. Med. delivery of therapeutic proteins and peptides. Surg. 113(2), 335–340, Discussion 340–331 64(9), 319–325 (2006). Respir. Res. 2(4), 198–209 (2001). (1997). 42 Skyler J, Cefalu W, Kourides I et al.: Efficacy 30 Pezron I, Mitra R, Pal D, Mitra A: Insulin 55 Letsou G, Safi H, Reardon M et al.: of inhaled human insulin in Type 1 diabetes aggregation and asymmetric transport across Pharmacokinetics of liposomal aerosolized mellitus: a randomised proof-of-concept human bronchial epithelial cell monolayers cyclosporine a for pulmonary study. Lancet 357(9253), 331–335 (2001). (calu-3). J. Pharm. Sci. 91(4), 1135–1146 immunosuppression. Ann. Thorac. Surg. (2002). 43 Rosenstock J, Cefalu W, Hollander P et al.: 68(6), 2044–2048 (1999). Two-year pulmonary safety and efficacy of

16 Nanomedicine (2011) 6(1) future science group Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

56 Thipphawong J: Inhaled cytokines and et al.: Efficient elimination of inhaled (2008). cytokine antagonists. Adv. Drug Deliv. Rev. nanoparticles from the alveolar region: 81 Faraji A, Wipf P: Nanoparticles in cellular 58(9–10), 1089–1105 (2006). Evidence for interstitial uptake and drug delivery. Bioorg. Med. Chem. 17(8), 57 Skubitz K, Anderson P: Inhalational subsequent reentrainment onto airways 2950–2962 (2009). epithelium. Environ. Health Perspect. 115(5), interleukin-2 liposomes for pulmonary 82 Huang Y, Wang C: Pulmonary delivery of 728–733 (2007). metastases: a Phase I clinical trial. Anticancer insulin by liposomal carriers. J. Control. Drugs 11(7), 555–563 (2000). 70 Dailey L, Jekel N, Fink L et al.: Investigation Release 113(1), 9–14 (2006). of the proinflammatory potential of 58 Huland E, Heinzer H: Renal cell carcinoma 83 Bi R, Shao W, Wang Q, Zhang N: Spray- biodegradable nanoparticle drug delivery – innovative medical treatments. Curr. Opin. freeze-dried dry powder inhalation of systems in the lung. Toxicol. Appl. Pharmacol. Urol. 14(4), 239–244 (2004). insulin-loaded liposomes for enhanced 215(1), 100–108 (2006). 59 Huland E, Burger A, Fleischer J et al.: pulmonary delivery. J. Drug Target 16(9), Efficacy and safety of inhaled recombinant 71 Azarmi S, Roa W, Löbenberg R: Targeted 639–648 (2008). delivery of nanoparticles for the treatment of interleukin-2 in high-risk renal cell cancer 84 Chono S, Fukuchi R, Seki T, Morimoto K: lung diseases. Adv. Drug Deliv. Rev. 60(8), patients compared with systemic Aerosolized liposomes with dipalmitoyl 863–875 (2008). interleukin-2: an outcome study. Folia Biol. phosphatidylcholine enhance pulmonary (Praha) 49(5), 183–190 (2003). 72 Kathuria H, Cao Y, Ramirez M, Williams M: insulin delivery. J. Control. Release 137(2), 60 Heinzer H, Mir T, Huland E, Huland H: Transcription of the caveolin-1 gene is 104–109 (2009). differentially regulated in lung type I Subjective and objective prospective, 85 Shahiwala A, Misra A: A preliminary epithelial and endothelial cell lines. A role for long-term ana­lysis of quality of life during pharmacokinetic study of liposomal ets proteins in epithelial cell expression. inhaled interleukin-2 immunotherapy. J. Clin. leuprolide dry powder inhaler: a technical J. Biol. Chem. 279(29), 30028–30036 Oncol. 17(11), 3612–3620 (1999). note. AAPS PharmSciTech 6(3), E482–E486 (2004). 61 Hartig S, Greene R, Dasgupta J et al.: (2005). 73 Roy I, Vij N: Nanodelivery in airway Multifunctional nanoparticulate 86 Gilbert B, Knight C, Alvarez F et al.: diseases: challenges and therapeutic polyelectrolyte complexes. Pharm. Res. Tolerance of volunteers to cyclosporine applications. Nanomedicine 6(2), 237–244 24(12), 2353–2369 (2007). a-dilauroylphosphatidylcholine liposome (2010). 62 Mishra B, Patel B, Tiwari S: Colloidal aerosol. Am. J. Respir. Crit. Care Med. 156(6), nanocarriers: a review on formulation 74 Brandenberger C, Mühlfeld C, Ali Z et al.: 1789–1793 (1997). Quantitative evaluation of cellular uptake and technology, types and applications toward 87 Khanna C, Hasz D, Klausner J, Anderson P: trafficking of plain and polyethylene targeted drug delivery. Nanomedicine 6(1), Aerosol delivery of interleukin 2 liposomes is glycol-coated gold nanoparticles. Small 6(15), 9–24 (2010). nontoxic and biologically effective: Canine 1669–1678 (2010). 63 Crowder T, Rosati J, Schroeter J, Hickey A, studies. Clin. Cancer Res. 2(4), 721–734 Martonen T: Fundamental effects of particle 75 Zhu M, Feng W, Wang Y et al.: (1996). Particokinetics and extrapulmonary morphology on lung delivery: predictions of 88 Khanna C, Anderson P, Hasz D, Katsanis E, translocation of intratracheally instilled ferric stokes’ law and the particular relevance to dry Neville M, Klausner J: Interleukin-2 liposome oxide nanoparticles in rats and the potential powder inhaler formulation and development. inhalation therapy is safe and effective for health risk assessment. Toxicol. Sci. 107(2), Pharm. Res. 19(3), 239–245 (2002). dogs with spontaneous pulmonary metastases. 342–351 (2009). 64 Oberdörster G, Oberdörster E, Oberdörster J: Cancer 79(7), 1409–1421 (1997). 76 Möller W, Felten K, Sommerer K et al.: Nanotoxicology: an emerging discipline 89 Ten R, Anderson P, Zein N, Temesgen Z, Deposition, retention, and translocation of evolving from studies of ultrafine particles. Clawson M, Weiss W: Interleukin-2 ultrafine particles from the central airways Environ. Health Perspect. 113(7), 823–839 liposomes for primary immune deficiency and lung periphery. Am. J. Respir. Crit. Care (2005). using the aerosol route. Int. Med. 177(4), 426–432 (2008). 65 Azarmi S, Tao X, Chen H et al.: Formulation Immunopharmacol. 2(2–3), 333–344 (2002). 77 Arppe J, Vidgren M, Waldrep JC: Pulmonary and cytotoxicity of doxorubicin nanoparticles 90 Kaipel M, Wagner A, Wassermann E et al.: pharmacokinetics of cyclosporin a liposomes. carried by dry powder aerosol particles. Int. J. Increased biological half-life of aerosolized Int. J. Pharm. 161, 205–214 (1998). Pharm. 319(1–2), 155–161 (2006). liposomal recombinant human Cu/Zn 66 Vega-Villa K, Takemoto J, Yáñez J, 78 Herber-Jonat S, Mittal R, Gsinn S, superoxide dismutase in pigs. J. Aerosol Med. Remsberg C, Forrest M,Author Davies N: Clinical Bohnenkamp H, Guenzi E,Proof Schulze A: Pulm. Drug Deliv. 21(3), 281–290 (2008). Comparison of lung accumulation of cationic toxicities of nanocarrier systems. Adv. Drug 91 Lange C, Hancock R, Samuel J, Finlay W: liposomes in normal rats and lps-treated rats. Deliv. Rev. 60(8), 929–938 (2008). In vitro aerosol delivery and regional airway Inflamm. Res. DOI 10.1007/s00011-010- 67 Kawashima Y, Yamamoto H, Takeuchi H, surface liquid concentration of a liposomal 0260-y (2010) (Epub ahead of print). Fujioka S, Hino T: Pulmonary delivery of cationic peptide. J. Pharm. Sci. 90(10), insulin with nebulized dl-lactide/glycolide 79 Videira M, Botelho M, Santos A, Gouveia L, 1647–1657 (2001). De Lima J, Almeida A: Lymphatic uptake of copolymer (PLGA) nanospheres to prolong 92 Müller R, Petersen R, Hommoss A, pulmonary delivered radiolabelled solid lipid hypoglycemic effect. J. Control. Release Pardeike J: Nanostructured lipid carriers nanoparticles. J. Drug Target 10(8), 607–613 62(1–2), 279–287 (1999). (NLC) in cosmetic dermal products. Adv. (2002). 68 Grenha A, Seijo B, Remuñán-López C: Drug Deliv. Rev. 59(6), 522–530 (2007). 80 Grenha A, Remuñán-López C, Carvalho E, Microencapsulated chitosan nanoparticles for 93 Liu J, Gong T, Fu H et al.: Solid lipid Seijo B: Microspheres containing lipid/ lung protein delivery. Eur. J. Pharm. Sci. nanoparticles for pulmonary delivery of chitosan nanoparticles complexes for 25(4–5), 427–437 (2005). insulin. Int. J. Pharm. 356(1–2), 333–344 pulmonary delivery of therapeutic proteins. 69 Semmler-Behnke M, Takenaka S, Fertsch S (2008). Eur. J. Pharm. Biopharm. 69(1), 83–93

future science group www.futuremedicine.com 17 Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

94 Liu Z, Jiao Y, Wang Y, Zhou C, Zhang Z: Jornacion C: Systemic absorption and activity containing absorption enhancers in rats. Polysaccharides-based nanoparticles as drug of recombinant consensus interferons after Pharm. Res. 13(1), 80–83 (1996). delivery systems. Adv. Drug Deliv. Rev. intratracheal instillation and aerosol 118 Lizio R, Klenner T, Borchard G et al.: 60(15), 1650–1662 (2008). administration. Pharm. Res. 12(12), Systemic delivery of the gnrh antagonist 95 Yang Y, Bajaj N, Xu P, Ohn K, Tsifansky M, 1889–1895 (1995). cetrorelix by intratracheal instillation in Yeo Y: Development of highly porous large 107 Taljanski W, Pierzynowski S, Lundin P et al.: anesthetized rats. Eur. J. Pharm. Sci. 9(3), plga microparticles for pulmonary drug Pulmonary delivery of intratracheally instilled 253–258 (2000). delivery. Biomaterials 30(10), 1947–1953 and aerosolized cyclosporine a to young and 119 Iacono A, Johnson B, Grgurich W et al.: A (2009). adult rats. Drug Metab. Dispos. 25(8), randomized trial of inhaled cyclosporine in 96 Huang X, Du Y, Yuan H, Hu F: Preparation 917–920 (1997). lung-transplant recipients. N. Engl. J. Med. and pharmacodynamics of low-molecular- 108 Bennett D, Tyson E, Nerenberg C, Mah S, 354(2), 141–150 (2006). weight chitosan nanoparticles containing De Groot J, Teitelbaum Z: Pulmonary 120 Folkesson H, Weström B, Dahlbäck M, insulin. Carbohydr. Poly. 76, 368–373 (2009). delivery of detirelix by intratracheal Lundin S, Karlsson B: Passage of aerosolized 97 Yamamoto H, Hoshina W, Kurashima H instillation and aerosol inhalation in the bsa and the nona-peptide dDAVP via the et al.: Engineering of poly(dl-lactic-co- briefly anesthetized dog.Pharm. Res. 11(7), respiratory tract in young and adult rats. Exp. glycolic acid) nanocomposite particles for dry 1048–1055 (1994). Lung Res. 18(5), 595–614 (1992). powder inhalation dosage forms of insulin 109 Schreier H, Mcnicol K, Bennett D, 121 Christopher F, Chase D, Stein K, Milne R: with the spray-fluidized bed granulating Teitelbaum Z, Derendorf H: Rhdnase therapy for the treatment of cystic system. Adv. Powder Technol.18(2), 215–228 Pharmacokinetics of detirelix following fibrosis patients with mild to moderate lung (2007). intratracheal instillation and aerosol disease. J. Clin. Pharm. Ther. 24(6), 415–426 98 Grenha A: Microencapsulación de inhalation in the unanesthetized awake sheep. (1999). Pharm. Res. 11(7), 1056–1059 (1994). nanopartículas de quitosano para la 122 Bitonti A, Dumont J, Low S et al.: Pulmonary administración pulmonar de macromoléculas 110 Patton JS, Trinchero P, Platz RM: delivery of an erythropoietin fc fusion protein terapéuticas. Departamento de Farmacia y Bioavailability of pulmonary delivered in non-human primates through an Tecnología Farmacéutica PhD Thesis, 306 peptides and proteins – a-interferon, immunoglobulin transport pathway. Proc. (2006). calcitonins and parathyroid hormones. Natl Acad. Sci. USA 101(26), 9763–9768 99 Zhang Q, Shen Z, Nagai T: Prolonged J. Control. Release 28(1–3), 79–85 (1994). (2004). hypoglycemic effect of insulin-loaded 111 Colthorpe P, Farr S, Smith I, Wyatt D, 123 Russell K, Read M, Bellinger D et al.: polybutylcyanoacrylate nanoparticles after Taylor G: The influence of regional Intratracheal administration of recombinant pulmonary administration to normal rats. Int. deposition on the pharmacokinetics of human factor ix (benefix) achieves J. Pharm. 218(1–2), 75–80 (2001). pulmonary-delivered human growth hormone therapeutic levels in hemophilia b dogs. 100 Kawashima Y, Yamamoto H, Takeuchi H, in rabbits. Pharm. Res. 12(3), 356–359 Thromb. Haemost. 85(3), 445–449 (2001). (1995). Kuno Y: Mucoadhesive dl-lactide/glycolide 124 Onoue S, Yamamoto K, Kawabata Y, copolymer nanospheres coated with chitosan 112 Pfützner A, Mann A, Steiner S: Hirose M, Mizumoto T, Yamada S: Novel dry to improve oral delivery of elcatonin. Pharm. Technosphere/insulin – a new approach for powder inhaler formulation of glucagon with Dev. Technol. 5(1), 77–85 (2000). effective delivery of human insulin via the addition of citric acid for enhanced 101 Yamamoto H, Kuno Y, Sugimoto S, pulmonary route. Diabetes Technol. Ther. pulmonary delivery. Int. J. Pharm. 382(1–2), Takeuchi H, Kawashima Y: Surface-modified 4(5), 589–594 (2002). 144–150 (2009). plga nanosphere with chitosan improved 113 Pang Y, Sakagami M, Byron P: The 125 Low S, Nunes S, Bitonti A, Dumont J: Oral pulmonary delivery of calcitonin by pharmacokinetics of pulmonary insulin in the and pulmonary delivery of fsh-fc fusion mucoadhesion and opening of the in vitro isolated perfused rat lung: proteins via neonatal fc receptor-mediated intercellular tight junctions. J. Control. Release implications of metabolism and regional transcytosis. Hum. Reprod. 20(7), 1805–1813 102(2), 373–381 (2005). deposition. Eur. J. Pharm. Sci. 25(4–5), (2005). 369–378 (2005). 102 Renwick L, Brown D, Clouter A, 126 Codrons V, Vanderbist F, Ucakar B, Préat V, Donaldson K: Increased inflammation and 114 RAVE K, Bott S, Heinemann L et al.: Vanbever R: Impact of formulation and altered macrophage chemotactic responses Time-action profile of inhaled insulin in methods of pulmonary delivery on absorption caused by two ultrafine particle types.Occup. comparison with subcutaneously injected of parathyroid hormone (1–34) from rat Environ. Med. 61(5), 442–447 Author(2004). insulin lispro and regular human Proofinsulin. lungs. J. Pharm. Sci. 93(5), 1241–1252 103 Chang C: The immune effects of naturally Diabetes Care 28(5), 1077–1082 (2005) (2004). occurring and synthetic nanoparticles. 115 Niven R, Lott F, Cribbs J: Pulmonary 127 Codrons V, Vanderbist F, Verbeeck R et al.: J. Autoimmun. 34(3), J234–J246 (2010). absorption of recombinant methionyl human Systemic delivery of parathyroid hormone 104 Thomas D, Myers M, Wichert B, Schreier H, granulocyte colony stimulating factor (1–34) using inhalation dry powders in rats. Gonzalez-Rothi R: Acute effects of liposome (r-hug-csf) after intratracheal instillation J. Pharm. Sci. 92(5), 938–950 (2003). to the hamster. Pharm. Res. 10(11), aerosol inhalation on pulmonary function in 128 Van Zandwijk N, Jassem E, Dubbelmann R, 1604–1610 (1993). healthy human volunteers. Chest 99(5), Braat M, Rumke P: Aerosol application of 1268–1270 (1991). 116 Hubbard R, Crystal R: Strategies for aerosol interferon-a in the treatment of 105 Lizio R, Klenner T, Sarlikiotis A et al.: therapy of a1-antitrypsin deficiency by the bronchioloalveolar carcinoma. Eur. J. Cancer Systemic delivery of cetrorelix to rats by a new aerosol route. Lung 168(Suppl.), 565–578 26(6), 738–740 (1990). (1990). aerosol delivery system. Pharm. Res. 18(6), 129 Martin P, Vaidyanathan S, Lane J et al.: 771–779 (2001). 117 Kobayashi S, Kondo S, Juni K: Pulmonary Safety and systemic absorption of pulmonary 106 Niven R, Whitcomb K, Woodward M, Liu J, delivery of salmon calcitonin dry powders delivered human ifn-b1a in the nonhuman

18 Nanomedicine (2011) 6(1) future science group Review Andrade, Videira, Ferreira & Sarmento Nanocarriers for pulmonary administration of peptides & therapeutic proteins Review

primate: comparison with subcutaneous therapy in cardiac high risk patients with 134 Tazawa R, Nakata K, Inoue Y, Nukiwa T: dosing. J. Interferon Cytokine Res. 22(6), metastatic renal carcinoma. J. Urol. 179(4), Granulocyte-macrophage colony-stimulating 709–717 (2002). 167–167 (2008). factor inhalation therapy for patients with 130 Halme M, Maasilta P, Repo H et al.: Inhaled 132 Adjei A, Sundberg D, Miller J, Chun A: idiopathic pulmonary alveolar proteinosis: a recombinant interferon‑g in patients with Bioavailability of leuprolide acetate following pilot study; and long-term treatment with lung cancer: pharmacokinetics and effects on nasal and inhalation delivery to rats and aerosolized granulocyte-macrophage chemiluminescence responses of alveolar healthy humans. Pharm. Res. 9(2), 244–249 colony-stimulating factor: a case report. macrophages and peripheral blood (1992). Respirology 11(Suppl.) S61–S64 (2006). neutrophils and monocytes. Int. J. Radiat. 133 Adjei A, Garren J: Pulmonary delivery of Oncol. Biol. Phys. 31(1), 93–101 (1995). peptide drugs: effect of particle size on „„Patent 131 Eichelberg C, Andreas A, Heuer R, bioavailability of leuprolide acetate in healthy 201 Nilsson T, Calander S, Niemi A, Friberg C, Huland H, Heinzer H, Huland E: Long-term male volunteers. Pharm. Res. 7(6), 565–569 Kax L, Myrman M tumor control with inhalational interleukin 2 (1990).

Author Proof

future science group www.futuremedicine.com 19

View publication stats