Recent advances in topical delivery of proteins and peptides mediated by soft matter nanocarriers.

JBA-06892; No of Pages 15 Biotechnology Advances xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Recent advances in topical delivery of proteins and peptides mediated by soft matter nanocarriers Madeleine Witting a, Katja Obst b, Wolfgang Friess a, Sarah Hedtrich b,⁎ a b

Department of Pharmaceutical Sciences, Ludwig-Maximilians-Universität, Munich, Germany Institute for Pharmaceutical Sciences, Freie Universität Berlin, Germany

a r t i c l e

i n f o

Available online xxxx Keywords: Soft matter nanocarrier Topical delivery Biopharmaceuticals Proteins Peptides Targeted drug delivery Dermal Ocular Nasal Pulmonary

a b s t r a c t Proteins and peptides are increasingly important therapeutics for the treatment of severe and complex diseases like cancer or autoimmune diseases due to their high specificity and potency. Their unique structure and labile physicochemical properties, however, require special attention in the production and formulation process as well as during administration. Aside from conventional systemic injections, the topical application of proteins and peptides is an appealing alternative due to its non-invasive nature and thus high acceptance by patients. For this approach, soft matter nanocarriers are interesting delivery systems which offer beneficial properties such as high biocompatibility, easiness of modifications, as well as targeted drug delivery and release. This review aims to highlight and discuss technological developments in the field of soft matter nanocarriers for the delivery of proteins and peptides via the skin, the eye, the nose, and the lung, and to provide insights in advantages, limitations, and practicability of recent advances. © 2015 Elsevier Inc. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ocular protein and peptide delivery . . . . . . . . . . . . . . . . . 2.1. Physiology and diseases in the focus . . . . . . . . . . . . . . 2.2. Nanocarrier driven protein and peptide delivery . . . . . . . . 2.2.1. Liposomes . . . . . . . . . . . . . . . . . . . . . 2.2.2. PLGA/PLA based nanoparticles . . . . . . . . . . . . 2.2.3. Further nanoparticles . . . . . . . . . . . . . . . . 2.3. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . Dermal protein delivery . . . . . . . . . . . . . . . . . . . . . . . 3.1. Skin physiology and challenges . . . . . . . . . . . . . . . . 3.2. Liposomes for dermal protein/peptide delivery . . . . . . . . . 3.3. Biphasic vesicles as topical delivery systems . . . . . . . . . . 3.4. Microemulsions in dermal peptide delivery . . . . . . . . . . 3.5. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . Nasal delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Surface-functionalized nanoparticles and biodegradable polymers 4.3. Further types of nanocarriers . . . . . . . . . . . . . . . . . 4.4. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . Pulmonary delivery of peptides and proteins . . . . . . . . . . . . . 5.1. Pulmonary absorption of peptides and proteins . . . . . . . . . 5.2. Devices and general formulation strategies . . . . . . . . . . .

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⁎ Corresponding author at: Institute for Pharmaceutical Sciences, Pharmacology & Toxicology, Freie Universität Berlin, Königin-Luise-Str. 2-4, 14195 Berlin, Germany. Tel.: +49 30 838 55065. E-mail address: [email protected] (S. Hedtrich).

http://dx.doi.org/10.1016/j.biotechadv.2015.01.010 0734-9750/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Witting M, et al, Recent advances in topical delivery of proteins and peptides mediated by soft matter nanocarriers, Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.01.010

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M. Witting et al. / Biotechnology Advances xxx (2015) xxx–xxx

5.3.

Why nanoparticulate pulmonary delivery of peptides and proteins? . 5.3.1. The fate of nanoparticulate delivery systems in the lung . . 5.3.2. Toxicity of nanoparticulate delivery systems in the lung . . 5.4. Liposomes for pulmonary delivery of peptides and proteins . . . . 5.5. Polymeric nanoparticles for pulmonary delivery . . . . . . . . . . 5.5.1. Chitosan nanoparticles . . . . . . . . . . . . . . . . . 5.5.2. PLGA nanoparticles . . . . . . . . . . . . . . . . . . 5.6. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The constant increase in biotechnologically produced pharmaceuticals reflects the growing impact and demand for biomacromolecules as new therapeutic compounds. The global sales of biologics reached US $163 billion in 2012 and accounted for over 70% of the worldwide revenue (Mitragotri et al., 2014). Especially in the treatment of severe, complex, and highly heterogenic diseases like cancer, autoimmune diseases, or infections, biomacromolecules are more effective than small molecule drugs due to their high specificity and potency (Lu et al., 2014; Mitragotri et al., 2014; Scott et al., 2012). One exemplary success story is trastuzumab (Herceptin®) which today is part of the standard regime for the treatment of HER2-positive breast cancer. Trastuzumab selectively binds to the human epidermal growth factor receptor 2 (HER2) which is overexpressed in about 25% of breast cancer patients. By blocking this receptor, the disease progression rate is significantly reduced and the overall survival prolonged (Scholl et al., 2001). Another blockbuster is the tumor necrosis factor α inhibitor adalimumab which is indicated for the treatment of chronic inflammatory diseases such as rheumatoid arthritis or psoriasis. In 2012, its sales increased to $ 4.6 billion due to increased application in dermatology and the expansion of indications (Aggarwal, 2014). Other successful examples are antibodies for the treatment of colorectal cancer targeting the epidermal growth factor receptor with cetuximab (Erbitux®), or the vascular endothelial growth factor(s) with bevacizumab (Avastin®) or aflibercept (Zaltrap®) (Van der Walle, 2011). The concept of selective biomacromolecules was modified and adapted for several diseases such as multiple sclerosis, macular degeneration, or rheumatoid arthritis and allows dreaming about actual personalized medicine (Marques et al., 2014; Sato et al., 2006). Since the beginning, the delivery of biomacromolecules has challenged pharmaceutical companies and researchers because of their

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specific physicochemical properties. Biomacromolecules exhibit a complex structure with a high molecular weight (between 300 and 1,000,000 Da) and amphiphilic properties (Mitragotri et al., 2014). Moreover, biopharmaceuticals are labile compounds and prone to stability problems like aggregation and denaturation that can occur during manufacturing, storage, and administration (Cromwell et al., 2006; Yadav et al., 2011). If a protein is subjected to agitation or shear stress, changes in pH or ionic strength, light or temperature, its labile secondary structure can easily be disturbed which triggers the unfolding of the amino-acid chain and induces aggregation or denaturation and ultimately the reduction of biological activity (Cromwell et al., 2006; Frokjaer and Otzen, 2005; Mahler et al., 2009; Wang, 2005). Following intravenous, intramuscular or subcutaneous administration, a short in vivo half-life and rapid clearance from the human body are further limitations (Almeida and Souto, 2007; Mitragotri et al., 2014; Sato et al., 2006). Moreover, the administered proteins can undergo degradation due to exposure to proteases and peptidases which are expressed abundantly in the systemic circulation and human organs (Fig. 1). Consequentially, as for today, repeated injections of high doses are inevitable to maintain therapeutic levels. This not only affects the compliance, but also triggers adverse effects such as immunogenicity (Crommelin et al., 2003; Van der Walle, 2011). Based on these considerations, the topical application of proteins/peptides appears an attractive alternative route of administration to reduce systemic side effects, and increase the therapeutic efficacy, and the patients' compliance due to its non-invasive nature. Finally, after application the biomacromolecule has to overcome several biological membranes to reach the systemic circulation or the target site (Fig. 1). This process is again hampered by their physicochemical properties as the passage through biological barriers is limited to rather small, uncharged molecules with a moderate lipophilicity (Mitragotri et al., 2014; Prausnitz and Langer, 2008).

Fig. 1. Major hurdles hampering efficient biomacromolecule delivery to target sites occurring during protein formulation, storage and after administration in the human body.



In general, the advantages of biopharmaceuticals far exceed their limitations. Hence, many research efforts are conducted to overcome their drawbacks and to enable sufficient and effective biomacromolecule delivery. In this regard, smart drug delivery systems are of increasing interest but they need to ensure both the maintenance of the biomacromolecule structure and activity as well as efficient and targeted drug delivery. Enabling these visions, soft matter nanocarriers are considered as potential delivery systems (Bao et al., 2013; Lu et al., 2014; Mitragotri et al., 2014; Soussan et al., 2009). Soft matter nanocarriers like liposomes, micelles, nanoparticles based on biological (gelatin, chitosan) or synthetic materials (polyglycerol, poly-lactic-coglycolide) can deliver a broad variety of molecules, show good biocompatibility, can easily be prepared and surface modified, and allow stimuli-responsive payload releases (Fleige et al., 2012; Soussan et al., 2009; Tan et al., 2010; Zhang et al., 2007). The aim of this review is to highlight and discuss technological advances and approaches in the field of soft matter nanocarriers for the topical delivery of therapeutic proteins and peptides, and to provide insights into the advantages, limitations, and practicability of recent developments. We focus on ocular, dermal/transdermal, nasal, and pulmonary routes of biomacromolecule administration which offer several advantages over classic injections. 2. Ocular protein and peptide delivery 2.1. Physiology and diseases in the focus Topical drug application is the safest and easiest way for the therapy of eye diseases. Anatomical structures like the conjunctiva, sclera, retinal pigment epithelium, and the cornea, however, limit the delivery efficiency particularly for the back of the eye (Fig. 2) (El Sanharawi et al., 2010). Additionally, internal structures of the eye are protected by tight junctions of the corneal epithelium and a mucosa which is an important barrier for drug delivery systems (Fröhlich and Roblegg, 2014). Various drugs are used for the therapy of ocular diseases such as anti-inflammatory, antiviral, and antibacterial agents, as well as antiangiogenic antibodies like bevacizumab or pegaptinib. The most common formulations are eye drops, hydrogels, or ointments, but their efficiency is limited by effective clearance mechanism like lacrimation

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and tear flow preventing a prolonged stay and sufficient penetration of applied drugs which ultimately leads to bioavailabilities ≤ 5%. 95% of the substance is absorbed into the systemic circulation through transnasal and conjunctival pathways (Hughes et al., 2005; Liu et al., 2012). Even more challenging is the drug delivery to the posterior part of the eye due to the vitreous body that reduces the diffusivity of molecules. This is a major problem since particularly diseases of the posterior part are the underlying cause for blindness (Del Amo and Urtti, 2008). Age-related vision disorders like diabetic retinopathy and agerelated macular degeneration (AMD) are the major causes for blindness in the world (Rowe-Rendleman et al., 2014; Schwartz et al., 2014). For their treatment, local drug administration still is the application route of choice, but its treatment efficacy and patient compliance are limited by repeated applications, systemic exposure, and poor availability in the posterior segments of the eye (Ding, 1998). Hence, there is an unmet demand for novel pharmaceutical formulations enabling the maintenance of high drug levels at the site of action for a prolonged time. In this course, it is also noteworthy that particularly the delivery of therapeutic proteins and peptides is challenging because of their physiochemical properties and instabilities. Today, the angiogenesis inhibitors pegaptinib (molecular weight: 50 kDa), ranibizumab (molecular weight 48 kDa) and aflibercept (molecular weight 97 kDa) are the first line treatment for AMD. These macromolecules target the vascular endothelial growth factor (VEGF), which is involved in choroidal neovascularization during AMD and, hence, is responsible for severe vision loss (Schwartz et al., 2014). To allow an efficient biomacromolecule delivery to the posterior segment, most frequently intravitreal injections are performed which are related to disadvantages such as patient discomfort, infections of the eye, elevated intraocular pressure, retinal vascular occlusion, or retinal detachment (Rowe-Rendleman et al., 2014). Hence, alternative delivery strategies are required, but as for today most of the studies focus on the ocular delivery of small drugs and the delivery of biomacromolecules is still at the beginning. 2.2. Nanocarrier driven protein and peptide delivery The therapeutic potential of proteins and peptides for the treatment of AMD and retinal angiogenesis is increasingly realized in

Fig. 2. Anatomy and diseases of the human eye. AMD, age-related macular degeneration; DME, diabetic macular edema; DR, diabetic retinopathy; RD, retinal degeneration. Adapted and reprinted with permission from Zhang et al. (2012).


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ophthalmology (Gariano and Gardner, 2005). Their physiochemical properties, instabilities, and fast vascular drainage, however, impede the development of protein/peptide-loaded nanocarriers. An ideal ocular delivery system should enable drug targeting and controlled and sustained release, prolonged retention time, as well as a patient-friendly frequency of administration (Liu et al., 2012). Nanocarriers might have the potential to fulfill these requirements due to different reasons: The small size which allows a high diffusivity across the corneal epithelium and the high surface area facilitating interactions with the mucosal layer which ultimately prolongs the retention time (Nagarwal et al., 2009). Although major advances were made in nanocarrier-mediated drug delivery (Juliano, 2007; Tan et al., 2010), comparatively few publications are available on ocular delivery of proteins and peptides. The carrier systems that have been studied most are liposomes (Abrishami et al., 2011; Bochot and Fattal, 2012), micelles, and poly(lactic-co-glycolic) acid (PLGA) or polylactic acid (PLA) based carrier systems (Yandrapu et al., 2013). 2.2.1. Liposomes The use of liposomes in biomedical applications constantly increased during the last decades. Liposomal drug encapsulation provides several advantages for drug delivery to the anterior and posterior parts of the eye because the corneal penetration is increased and, for the posterior segment, longer residence time at the site of action, less side effects, and targeted delivery to the retina were achieved (Ebrahim et al., 2005). Additionally, by slight surface modifications the extent of drug absorption can be further enhanced: The corneal surface is negatively charged and, hence, positively charged liposomes increase the drug delivery efficiency (Law et al., 2000). For the therapy of ocular inflammation, one interesting study described the liposomal encapsulation of the vasoactive intestinal peptide (VIP), a potent immunosuppressive neuropeptide (Camelo et al., 2007). They found significant accumulation of VIP loaded liposomes in the posterior segment of the eye, 24 h post-injection. The liposomes were internalized by immune cells such as macrophages suggesting that intravitreal injections can increase the ocular immune response. The intravitreous injection of VIP-loaded liposomes resulted in 15-times higher VIP concentrations compared to the control solution. However, one drawback of liposomes is their fast clearance when being injected in an inflamed eye due to an impaired blood–retinal barrier. To prevent rapid elimination, VIP-loaded liposomes were embedded into a hyaluronic acid gel resulting in a long lasting protective effect in vivo (Lajavardi et al., 2009). Another study reported the encapsulation of the anti-VEGF antibody bevacizumab into liposomes and the subsequent injection into a rabbit eye; bevacizumab solution served for control. 1.5-fold higher areas under the curve were achieved and the residence time was slightly prolonged using the encapsulated antibody compared with unloaded bevacizumab (5.27 and 3.88 μg × d/mL) (Abrishami et al., 2011). Overall, the data showed a slight enhancement of drug concentration and retention time, but lacked striking effects. For drug delivery to the posterior segment, an early study by Alghadyan et al., showed that an intravitreal injection of cyclosporineloaded liposomes yielded slightly increased drug concentrations and prolonged drug half-lives (Alghadyan et al., 1988a, 1988b). Back then, this was a promising result since the topical or even systemic application of cyclosporine showed poor intravitreal penetration. Cyclosporine is a potent immunosuppressant peptide, which is frequently used to prevent corneal graft rejection, treat autoimmune uveitis, and the dry eye syndrome. Aside from few promising results, liposomes can suffer from the general limitations such as long-term stability and low drug loading capacity. Additionally, the commercial production of sterile liposomes is expensive and technically challenging which reduces their applicability for ocular delivery (Lallemand et al., 2003).

2.2.2. PLGA/PLA based nanoparticles Polymers such as PLA and PLGA are biodegradable and biocompatible, are approved by regulatory authorities, and were repeatedly investigated for the delivery of biomacromolecules (Geroski and Edelhauser, 2000; Hermans et al., 2014; Lu et al., 2014). The method of particle production, however, should be chosen carefully because commonly used organic solvents during preparation affect protein stability and activity (Jain, 2000). Most of the studies focused on the encapsulation of anti-VEGF agents for the treatment of neovascularization. For example, Yandrupa et al. developed bevacizumab-loaded PLA nanoparticles (mean size: 251 ± 15 nm) which were loaded onto PLGA microparticles (mean size: 1.6 ± 1.03 μm) using a supercritical pressure quench technology (Yandrapu et al., 2013). A significantly prolonged in vitro release of bevacizumab for up to 4 months was achieved without any loss in protein activity after intravitreal injection. Unfortunately, the efficacy of these particles was not assessed in an AMD model. An alternative approach targeting the neovascularization of AMD was the delivery of a serpin-derived peptide that exhibits strong antiangiogenic properties by inducing endothelial cell apoptosis and decreasing their migration (Karagiannis and Popel, 2008; Koskimaki et al., 2012). Therefore, biodegradable nanoparticles were prepared by self-assembly of the anionic peptides using a cationic polymer and subsequent particle coating with PLGA. The final particles exhibited zero-order kinetics and drug release for up to 200 days. Studies in a laser-induced neovascularization mouse model showed efficient inhibition of angiogenesis following a single application (Shmueli et al., 2013). However, the superiority of the encapsulation still needs to be verified since the effects of encapsulated and free peptide were similar, and an advantage of the particulate formulation was only observed after 14 weeks. New molecules targeting neovascularization are of interest since anti-VEGF antibodies like bevacizumab are not effective for all patients. For the treatment of retinal ischemia, the peptide pigment epithelium-derived factor (PEDF), a protective factor produced by the retinal pigment epithelium, could be a therapeutic option (Li et al., 2006). Encapsulation of PEDF into PLGA particles resulted in superior protection of the retina compared to the free peptide, and delayed retinal response to ischemic injury, most likely by protection of the peptide from degradation and clearance. As discussed previously, encapsulating proteins into nanocarriers can impair their bioactivity asking for additives to protect proteins during particle preparation (Tamber et al., 2005). Among others, proteinic excipients such as albumin exhibit protective effects in different steps of protein encapsulation. Hence, Varshochian et al. tested different stabilizers for the fabrication of PLGA nanoparticles loaded with bevacizumab using the water/oil emulsion method which can lead to aggregation and inactivation of the biomacromolecule triggered by interfacial adsorption (Varshochian et al., 2013). Albumin efficiently protected bevacizumab from entrapment stress and resulted in significantly enhanced stability due to its surface active properties. It is assumed that albumin has a higher affinity to the interface and is sacrificed by aggregation which ultimately avoids the interfacialadsorption of the antibody. Interestingly, the typically observed burst release of PLGA nanoparticles was missing which might be also attributed to the co-formulation with albumin. Nevertheless, ex vivo studies in rabbit vitreous showed a prolonged release of the antibody resulting in therapeutically relevant bevacizumab levels. To improve the bioavailability of cyclosporine, Liu et al. developed poly(D,L-lactide)-b-dextran nanoparticles and functionalized them with phenylboronic acid (Liu et al., 2012). The particles' surface consisted of a linear block copolymer, and PLA–dextran was conjugated with phenylboric acid to generate mucoadhesive properties. Encapsulation of cyclosporine resulted in a sustained release and facilitated interactions with the corneal mucus layer. Unfortunately, no continuative studies in animals were conducted so that no conclusion about the performance in vivo was possible.



2.2.3. Further nanoparticles Sakai et al. prepared fibroblast growth factor (FGF)-loaded gelatin nanoparticles to protect photoreceptors from degeneration by improved targeting and sustained drug release (Sakai et al., 2007). In rats, FGF exhibited protective activities, but a single intravitreal injection was insufficient to ensure long-term effects because of its rapid elimination. Interestingly, intravitreal injection of the FGF-nanoparticles resulted in a prolonged residence time for about 8 weeks in the eye and a significant reduction of photoreceptor cell death in the FGF-nanoparticle treated group was observed. Gurny and his coworkers followed a different strategy and developed methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) (MPEG-hexPLA) micelle carriers for the delivery of cyclosporine to the eye. MPEG-hexPLA micelles showed good biocompatibility in rabbits, as well as high drug loading capacity, and stability in aqueous formulations (Mondon et al., 2011). Aside from this preliminary work, however, the efficiency of these micelles was not assessed. More advanced work was published by Calvo et al. (1996) who loaded polyestercaprolactone nanocapsules with cyclosporine. Following corneal application, cyclosporine levels were 5 times higher compared to the control solution (319.98 versus 74.34), but failed to prevent corneal graft rejection in animal studies (Juberias et al., 1998). Overall, no major improvements in terms of cyclosporine delivery were made to date and currently available formulations using oils are still associated with poor tolerability and low bioavailability (Di Tommaso et al., 2011; Lallemand et al., 2003). 2.3. Outlook To date, only few studies on ocular delivery of proteins and peptides using soft matter nanocarriers were published, all of them at an early stage of development. In this regard, a very general aspect was hardly addressed, too: The fate of the nanoparticles after ocular application. Do they accumulate, are they systemically absorbed, how and how fast are they eliminated? Most eye diseases discussed in this section require continuous treatment. Most compounds used for ocular applications are biodegradable and, thus, presumably biocompatible. Nevertheless, the fate of the nanocarriers needs to be investigated comprehensively not only for protein/peptide delivery. Overall, the impact of biopharmaceuticals for the treatment of eye diseases, however, will continuously increase requesting efficient delivery strategies and further developments in this field. Methods aiming to bioengineer peptides and proteins might also be an appropriate option. Here, biomacromolecules are fused to protein polymers to provide a platform for controlled release and nanoparticle assembly (Fleige et al., 2012; Lu et al., 2014). For example, Wang and coworkers formulated a 20 amino acid mini-peptide that required a drug release system due to its small size. Hence, protein polymer nanoparticles of the mini-peptide were produced to increase the molecular weight, to enhance the potency, and to extend the residence time at the site of action. This was achieved by conjugating the peptide with protein polymers resulting in high molecular weight (~ 40 kDa), drastically increased the cellular uptake, nuclear localization, and high efficacy (Wang et al., 2014a). Another promising approach is the incorporation of biomacromolecules into or onto contact lenses enabling a more sustained release. This approach was already successfully applied for the delivery of wound healing and epidermal growth factors (Chow and Di Girolamo, 2014; Holland et al., 2012). 3. Dermal protein delivery 3.1. Skin physiology and challenges Dermal and transdermal applications are important drug delivery pathways. A large amount of topical formulations are on the market and approximately about 33% of drugs in clinical trials aim for topical

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applications (Paudel et al., 2010). Dermal application allows painless drug administration with the possibility to control its release and to avoid the first-pass effect (Jepps et al., 2013; Mitragotri et al., 2014). The skin is a complex structure that protects the human body from environmental factors such as UV radiation, microorganisms, or mechanical damage and controls essential processes like thermal regulation (Menon et al., 2012; Natarajan et al., 2014; Pasparakis et al., 2014). When applying a drug topically, its dermal absorption is hampered by the stratum corneum (SC), the main skin barrier, which needs to be overcome to reach its target site, which can either be intradermal or the systemic circulation. The SC consists of flattened and tightly packed corneocytes that are embedded in a highly lipophilic lipid matrix consisting of ceramides, cholesterol and fatty acids (Fig. 3) (Menon et al., 2012; Wertz, 2013). Underneath the SC, the viable epidermis is composed of viable keratinocytes which are arranged in three sub-structures: the stratum granulosum, stratum spinosum, and stratum basale. The dermis, which contains lymphatic and blood vessels as well as nerve fibers, consists of a dense network formed by proteins, collagen, and elastic fibers and gives structural support for the viable epidermis (Fig. 3) (McGrath et al., 2008). Due to the unique properties of the SC, efficient skin absorption is only obtained with rather small drugs (molecular weight ≤ 500 Da, total cutoff 800 Da) which also exhibit a moderate lipophilicity (log P = 1–3) (Brown et al., 2006, 2008). Hence, biomacromolecules are almost completely restricted from dermal penetration due to their high molecular weight (between 300 and 1,000,000 Da) and amphoteric properties (Kalluri and Banga, 2011; Uzor et al., 2011). Nevertheless, topical application of biomacromolecules seems promising for specific indications. When administered systemically, proteins and peptides show a short half-life which makes frequent injections inevitable. Topical applications could prolong their halflife and allow for a controlled release from the formulations which helps to maintain steady blood concentrations (Amsden and Goosen, 1995). Based on these considerations, the dermaland transdermal delivery of biomacromolecules such as calcitonin (Chang et al., 2000; Manosroi et al., 2013; Tas et al., 2012) and insulin (Hadebe et al., 2014; Ling and Chen, 2013; Sintov and Wormser, 2007) have repeatedly been pursued. Moreover, using topically applied biomacromolecules for the treatment of severe skin diseases is a rather new but highly interesting approach. This is of particular interest for a substitution therapy of proteins that are deficient in the skin due to genetic mutations and, thus, cause severe skin diseases like ichthyosis vulgaris (Smith et al., 2006) or autosomal recessive congenital ichthyosis. In order to achieve efficient protein/peptide delivery into the skin, several attempts have been employed that often involve invasive or skin barrier altering methods like iontophoresis, sonophoresis, or microporation. However, these methods mostly lack broad applicability (Antosova et al., 2009; Degim and Celebi, 2007; Jain et al., 2013). Since plain biomacromolecules are restricted from dermal penetration, suitable formulations and carrier system are needed and soft matter nanocarriers might have the potential to meet the demands. In particular, various liposomal formulations, micellar systems, or biphasic vesicles were employed yielding interesting results which will be discussed hereinafter.

3.2. Liposomes for dermal protein/peptide delivery Topical vaccination is one potential field of application and became of increasing interest during the last years. Administered via intramuscular or subcutaneous injections, classic vaccination is accompanied by infection risks, needle anxiety, or pain at the injection site. Hence, the development of alternative and non-invasive routes is explored, and dermal vaccination is of particular interest as the skin contains a complex network of immune cells (Langerhans and dendritic cells) and is highly responsive to antigens (Li et al., 2011).


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Fig. 3. Schematic depiction of the different skin layers, appendages and vessels.

Li et al. developed two types of flexible liposomes composed of phospholipids S 100 and cholesterol to allow for efficient transcutaneous delivery of the model antigen ovalbumin (molecular weight approx. 45 kDa). 30 min after topical application, particle accumulation in the hair follicles and sweat ducts was detected, but a further distribution of the liposomes in the skin was not observed. No significant differences in the immune response of normal and barrier disrupted skin were detected, and in combination with the adjuvant imiquimod a strong and equivalent immunization was achieved (Li et al., 2011). For topical vaccination, hair follicles are of particular interest since they are shunt pathways across the SC which can facilitate the absorption of large molecules such as antigens. Here, the nanocarrier size is a determining factor since especially larger particles (about 600 nm) penetrate beyond the infundibulum towards the abundant perifollicular Langerhans cells (Hansen and Lehr, 2014). Hence, several groups studied the administration of antigens for transcutaneous immunization (Baleeiro et al., 2013; Mattheolabakis et al., 2010; Mittal et al., 2013; Raber et al., 2014). An adjuvant, however, seemed to be essential for efficient immune responses in non-invasive hair follicle mediated immunization (Mittal et al., 2014). The local application of biomacromolecules can also serve other purposes, for example the treatment of severe skin conditions. Aufenvenne et al. (2013) developed sterically stabilized liposomes, equipped with a cationic lipopeptide, and encapsulated recombinant human transglutaminase 1 (molecular mass 92 kDa) in order to address the underlying cause of autosomal recessive congenital ichthyosis. The unilamellar vesicles were composed of phosphatidylcholine and poly(ethylene glycol)-2000-dipalmitoyl-phosphatidylethanolamine and were about 200 nm in size. To demonstrate the feasibility of the concept in vivo, a skin-humanized mouse model was used. Following the application of the liposomal formulations, a substantial improvement of the phenotype was seen already after two applications. Accordingly, a study of Stout et al. demonstrated the successful engineering of a cell penetrating peptide — filaggrin complex and its efficient penetration into epidermal tissue after topical application as well as the restoration of the pathological phenotype in filaggrin deficient flaky-tail mice (Stout et al., 2014). These two studies provided the proof of concept for a topical protein substitution therapy and described promising results with potential applicability in the future.

3.3. Biphasic vesicles as topical delivery systems Aside from liposomes, biphasic vesicles, built up from different nanoscale components, might be beneficial for the topical delivery of drugs and antigens. Biphasic vesicles are lipid-based delivery systems that combine the structures of liposomes and emulsions. In general, these vesicles have aqueous, oily, and micellar compartments which are surrounded by concentric phospholipid bilayers and allow a flexible composition for optimized encapsulation efficiencies and tailored viscosity (Foldvari, 2010). Biphasic vesicles were studied for the topical delivery of, e.g., insulin (6 kDa) (King et al., 2002, 2003), interferon alpha (19 kDa) (Foldvari et al., 2010; King et al., 2013), albumin (60 kDa) (Foldvari, 2010), or antigens like hen egg lysozyme (14 kDa), leukotoxin (100 kDa), or staphylococcal enterotoxin (28 kDa) (Baca-Estrada et al., 2000). One interesting application was described by King et al. who investigated the effectiveness of lipid-based biphasic vesicles (Biphasix™) for dermal insulin delivery (King et al., 2002). The biphasic vesicles consisted of a complex mixture of soya phosphatidylcholine, cholesterol, propylene glycol, and linoleamidopropyl-PG-dimonium chloride phosphate. Insulin-loaded vesicles were eventually incorporated into a patch which was then applied onto diabetic Sprague Dawley rats. A pharmacologically relevant decrease in blood glucose was observed for about 52 h, and the serum insulin levels did not differ from the effects achieved by subcutaneous insulin injection. Further studies substantiated these findings (Foldvari, 2010; King et al., 2003). However, dermally applied insulin is still less effective in glucose lowering compared to oral, pulmonary, or buccal application (oral: 20–50%, pulmonary: 45–80%, buccal: 15–75% versus 25–38% in dermal) (King et al., 2002). 3.4. Microemulsions in dermal peptide delivery In addition to proteins, peptide-based therapeutics (chains of 50 amino acids or less) are interesting biopharmaceutical products due to their higher target affinity and specificity, the easy production process, and the possibility of synthesis rather than expression. In general, due to their size, peptides exhibit better tissue penetrating properties and are less immunogenic than proteins (Sato et al., 2006). In this course,



Goebel et al. aimed for enhanced skin penetration of the antioxidative dipeptides carnosine (molecular weight 226.23 Da) and N-acetyl-Lcarnosine (molecular weight 268.27 Da) (Goebel et al., 2012). The peptides were formulated in a microemulsion, containing phospholipon 90 G, plantacare 2000 UP, polyglycerol, cetiol B and purified water. A distinct skin penetration enhancement was achieved, but a rapid and substantial loss of L-carnosine due to instabilities and biodegradation was also observed, which is a well-known problem for biomacromolecules. This study underlines the importance of an adequate and protective drug carrier system for labile compounds. 3.5. Outlook As for today, the dermal delivery of biomacromolecules using soft matter nanocarriers is still scarcely addressed. In contrast, skin barrier modifying methods like sonophoresis, iontophoresis, or microporation were investigated more intensively. The nanoparticles' properties mainly influence the biodistribution and elimination of loaded biomacromolecules. The nanocarriers should be rather small since the size is determining the penetration depth. Furthermore, moderate ionization and amphiphilic properties facilitate interactions between the carrier system and the skin surface. At this point, it is also noteworthy to mention potential risks since particularly dermal protein application can trigger immunologic responses. The likeliness of these events further increases in diseased skin which is characterized by an impaired barrier function (Berard et al., 2003). Another subject of controversial debate is the fate of the topically applied nanoparticles: Do the drug carrier systems themselves overcome the stratum corneum and penetrate into or through the skin? What happens in diseased skin which is characterized by an impaired barrier function? As for today, some publications describe skin absorption of nanoparticles while others did not substantiate these findings even in diseased skin. One problem is the huge diversity of fabricated nanoparticles mainly differing in particle size, surface properties, components, etc. Hence, the nanoparticles' fate needs to be assessed individually; a final and general prediction is not possible. Taken together, dermal proteinand peptide delivery are currently mainly investigated in the course of topical vaccination. Indeed, at least two clinical trials aiming for transcutaneous vaccine delivery via the hair follicles were initiated or conducted just recently. Moreover, recent studies provided the

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proof of concept for a local protein substitution therapy which may open a new treatment option for severe skin diseases (Aufenvenne et al., 2013; Stout et al., 2014). These studies, however, are at a very early stage so that further investigations are crucial to allow for conclusions about the practicability of this approach. 4. Nasal delivery The human nasal cavity is composed of the respiratory region, the nasal vestibule, and the olfactory region and covers a surface area of 180 cm2. Nasally applied drugs can enter the systemic circulation, penetrate into the brain, or follow both pathways (Fig. 4). Located in the upper section of the nasal cavity, the olfactory region offers the best entry way for proteins or peptides into the brain. Transcellular or paracellular pathways are possible for drugs crossing the olfactory epithelium; intranasally applied proteins/peptides most likely reach the brain through perineural channels of the paracellular pathway (Malerba et al., 2011; Vyas et al., 2005). Another pathway is an intracellular axonal carriage directly to the olfactory bulb. When the delivery via the olfactory nerve pathway results in endocytotic uptake, transport into the brain occurs (Fig. 4) (Graff and Pollack, 2005). A mucosal layer covers the nasal epithelium, entraps foreign particles and thus contributes to their clearance from the nasal cavity by cilia movement. These barriers need to be overcome when aiming for nasal drug delivery. Lipophilic drugs are well absorbed in the nasal epithelium, whereas the penetration of polar and high molecular weight drugs such as proteins or peptides through the nasal epithelium is limited (Illum, 2003; Luppi et al., 2010). Overall, nasal drug delivery is painless, the application is easy, the hepatic and gastrointestinal first-pass effects are avoided, and no sterility is required (Graff and Pollack, 2005; Teijeiro-Osorio et al., 2009). Moreover, the nasal mucosa might be a suitable application site for non-invasive vaccination as mucosal and systemic immune reactions are initiated (Luppi et al., 2010; Wang et al., 2014b). As for today, several nasal sprays or drops are commercially available for instance for the treatment of respiratory tract diseases, headache, or allergic reactions and the intranasal application of peptides such as buserelin (Suprefact nasal®), calcitonin (Miacalcin®), and desmopressin (Minirin®) is implemented in the market. The delivery of proteins, however, is less explored but could be a convenient way to deliver

Fig. 4. Distribution of drugs from the nasal cavity and possible routes for brain delivery. BBB = blood–brain barrier, CSF = cerebrospinal fluid.


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biomacromolecules. In this course, different soft matter nanocarrier based systems were developed and investigated. The delivery of proteins/peptides into the brain is of particular interest. Several proteins including growth factors, cytokines, neuropeptides, or recombinant antibodies have been identified as potential therapeutics for the treatment of neurological diseases like Alzheimer's, Parkinson, or Huntington's disease, but a safe and effective delivery to the brain is mainly restricted by the BBB and still not satisfying. A non-invasive alternative could be protein/peptide-loaded nanocarriers which were shown to deliver peptides/proteins efficiently into the brain when ranging from 20 to 200 nm in size (Malerba et al., 2011; Vyas et al., 2005).

Neuropeptides are also potential compounds for direct nose to brain transport (Kubek et al., 2009). Here, PLA nanoparticles (110 nm) were loaded with the endogenous neuropeptide protirelin which is also known as thyrotropin-releasing hormone. Protirelin is discussed as an anticonvulsant agent for epileptic patients but due to rapid degradation and the BBB systemic protirelin administration does not achieve therapeutic levels in the CNS. These obstacles could be overcome following the application of protirelin-loaded PLA nanoparticles which significantly increased the neuropeptide concentrations in the central nervous system. 4.3. Further types of nanocarriers

4.1. Liposomes Liposomes are potential candidates for nasal protein/peptide delivery due to their amphiphilic properties, the possibility to encapsulate hydrophilic and lipophilic compounds, and their high biocompatibility (Kammona and Kiparissides, 2012; Mainardes et al., 2006). Some groups investigated liposomes for the delivery of insulin (Jain et al., 2007) and calcitonin aiming for a maximization of intranasal and systemic delivery. The intranasal application of positively and negatively charged liposomes loaded with calcitonin resulted in significantly enhanced bioavailability of calcitonin; the positively charged liposomes being superior due to electrostatic interactions with the negatively charged mucosa. Moreover, the residence time of positively charged liposomes was prolonged which further facilitated the transmucosal calcitonin transport (Law et al., 2001). Another approach was the encapsulation of anionic liposomes in mucoadhesive chitosan/DNA complexes for the intranasal delivery of DNA vaccines. In vivo application demonstrated significantly enhanced cellular uptake, high loading efficiencies, DNA stabilizing effects and significantly increased secretory immunoglobulin (Ig) levels, as well as prolonged mucosal immunity (Chen et al., 2013). Moreover, galactosemodified liposomes, specifically targeting macrophages, have been studied (Wang et al., 2013). Intranasal application of ovalbuminloaded liposomes resulted in a higher uptake rate, higher secretory IgA and IgG levels, as well as enhanced release of tumor necrosis factor-α and interleukin-6 than plain liposomes. Migliore et al. aimed for efficient protein delivery into the brain and formulated ovalbumin-loaded cationic liposomes which indeed induced higher ovalbumin levels in the central nervous system (CNS) and lower protein concentrations in the periphery being beneficial in terms of systemic side effects (Migliore et al., 2010). 4.2. Surface-functionalized nanoparticles and biodegradable polymers To deliver antigens for vaccination against hepatitis B, PLGA nanoparticles coated with chitosan and glycol chitosan were developed (Pawar et al., 2013). Both types of surface-functionalized nanoparticles were about 200 nm in size, positively charged, and exhibited a smooth surface. Coated and uncoated PLGA nanoparticles were tested in vivo whereby the glycol chitosan coated nanoparticles exhibited the best absorption properties due to high mucoadhesiveness. Concordantly with other studies, intranasal application of the nanoparticles significantly enhanced the immune response, whereas again the glycol chitosan coated nanoparticles were most efficient. Surface modified PLGA nanoparticles were also employed to enhance the delivery of the basic fibroblast growth factor (bFGF) into the brain since bFGF does not overcome the BBB when administered intravenously (Zhang et al., 2014). Physiologically, bFGF contributes to the development of neurons and is tested as a potential agent for the treatment of Alzheimer's disease. To further improve the delivery efficacy of PEG–PLGA nanoparticles, the particle surface was modified with lectins which facilitate mucoadhesion and endocytosis. As intended, significantly higher brain concentrations and prolonged retention time were achieved following the application of lectin modified nanoparticles.

To enable the nasal delivery of insulin, hybrid chitosan/cyclodextrin (CS/CD) nanoparticles were developed (Teijeiro-Osorio et al., 2009). CS/ CD nanoparticles (200 nm–400 nm in size) reversibly reduced the transepithelial resistance in vitro, suggesting an increase of membrane permeability. In vivo studies in rats verified that CS/CD nanoparticles overcome the nasal mucosa and reduced the plasma glucose levels more efficiently compared to nasally administrated insulin solutions. These positive effects were also observed for mucoadhesive and enzymatic inhibitory nanoparticles consisting of polyacrylic acid and polygalactose elements (Wang et al., 2014b). Insulin-loaded nanoparticles (100 nm) were developed and exhibited strong enzymatic inhibitory activity towards leucine aminopeptidase which triggered enzymatic protein degradation. The nanoparticles maintained the insulin stability for up to 140 h, resulted in higher bioavailability and lowered the blood glucose concentration for 9 h. Similar results were obtained using amphiphilic glycopolymer nanoparticles (Zheng et al., 2013). For intranasal vaccination, IgA-loaded chitosan–dextran nanoparticles were employed which exhibited a particle size range from 150 nm to 520 nm depending on the chitosan/dextran ratio. The particles allowed high loading efficiencies and efficient uptake in the nasal epithelia was achieved in vivo. The induction of immunological responses, however, still needs to be assessed (Sharma et al., 2013). 4.4. Outlook Intranasal application could be an attractive alternative to oral or parenteral application of proteins and peptides when aiming for local, brain or systemic delivery. Moreover, current preclinical and clinical investigations mainly focus on mucosal immunization, protein delivery into the brain and systemic delivery of insulin; all of them current medical needs. There are several clinical studies aiming for intranasal insulin delivery and, as discussed above, intranasal application of peptides is established and various products can be found on the market. The application of soft matter nanocarriers, however, does not play a critical role to date. Same holds true for intranasal vaccination, whereas at least one clinical trial was conducted using liposomes for intranasal antigen delivery. These facts indicate that intranasal protein delivery can be realized in the future; the impact of nanocarriers in this course still needs to be elucidated. In this regard, systematic studies of biocompatibility, systemic availability, distribution and excretion of intranasally administered nanocarriers are crucial since systemic absorption is a very likely event. Thus, for nanocarrier formulation biocompatible and biodegradable compounds should preferentially be used (Kammona and Kiparissides, 2012). As for today, however, the fate and distribution of intranasally applied nanocarriers were hardly investigated. 5. Pulmonary delivery of peptides and proteins Pulmonary delivery of proteins and peptides is fueled by two aspects. Firstly, for both peptides like LH-RH-antagonists or calcitonin and proteins like insulin delivery via a non-invasive alternative to parenteral application is highly desirable. The huge absorptive area of about 140 m2 closely interacting with the blood circulation, the low



level of metabolizing enzymes as compared to the GI tract, as well as the avoidance of the first pass effect in general render the highest bioavailability of proteins for any non-invasive route. The strive for inhalative insulin treatment stimulated industrial and academic research, but the withdrawal of Exubera® was a major setback which demonstrated some of the difficulties of this approach. Nevertheless, Afrezza™, a fast-acting insulin for inhalation therapy of type 1 and 2 diabetes, has just recently received approval by the FDA. Secondly, local treatment of respiratory diseases like asthma or chronic obstructive pulmonary disease is state of the art. The fascinating direct access for peptides and proteins to the site of action with maximized local and minimized systemic drug concentration also creates high potential for treatment of other diseases like lung malignancies or lung transplant rejection. 5.1. Pulmonary absorption of peptides and proteins The respiratory system presents a unique compartment for drug delivery (Kohlhäufl, 2007). The trachea divides into the bronchi, the bronchioles, and finally the alveoli. The proximal conducting airways are characterized by a columnar epithelium whereas the alveoli are covered by a very thin epithelium of approx. 0.1 μm thickness. This thin cell layer in the alveoli facilitates the uptake of peptides and proteins. Furthermore, drug absorption benefits from the high blood flow through the lung, which is required for gas exchange and nutrient supply, as well as from the lymphatic vessels in close proximity. But proteins and peptides deposited in the lung also face unfavorable conditions limiting their effect or absorption. For example, mucociliary clearance is an important defense mechanism which eliminates not only dust and microorganisms but also drugs. Furthermore, alveolar macrophages are responsible for chemotaxis, phagocytosis, and microbial killing which also affect the efficacy of delivered drugs, specifically when incorporated in particulate systems. In addition, various peptidases distributed on the lung surface degrade protein and peptide drugs. In the tracheobronchial tree, macromolecules dissolve or nanoparticles disperse in the mucus. Either they become cleared by the cilia or dissolved molecules can diffuse through the mucus and across the airway epithelium. Inhaled particles deposit in the alveolar region, dissolve, or disperse in the thin layer of lining fluid which coats the alveolar epithelium, have to navigate through the viscous mucus, and are subjected to clearance by alveolar macrophages. Eventually, the drug molecules can transfer across the alveolar epithelium into the bloodstream. Additionally, peptides and proteins may get digested by extraor intracellular enzymes. If the rate of adsorption is accelerated, the potential for enzymes degrading the drug molecule is strongly reduced. Overall, the bioavailability of proteins following pulmonary delivery ranges from 3.5 to almost 50% (Patton et al., 2004). Whether absorption takes places paracellularly, transcellularly, or via transcytosis depends on the macromolecule's molecular weight (Koussoroplis and Vanbever, 2013). Up to approximately 40 kDa, peptides and proteins are mainly transported paracellularly and show Cmax after 5 to 90 min. Above this molecular size, more time consuming transcytosis takes place (Gumbleton et al., 2007). The bioavailability, however, does not necessarily decrease with increasing molecular weight. It is also very much affected by the protein stability in the lung. If degradation is limited, systemic absorption can be high despite a long residence time of several hours in case of slow transport by transcytosis (Gumbleton et al., 2007; Patton et al., 2004). Absorption enhancers or protease inhibitors can further enhance protein absorption (Yamamoto et al., 1994) but the routine use of these additives in patients is limited by safety concerns. 5.2. Devices and general formulation strategies Aerosol generation has only a minor impact on the integrity of small molecular drugs including peptides. But physicochemical stability is of utmost importance for successful inhalation of biopharmaceuticals.

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General protein formulation is extensively covered in the literature with excellent books available (Jameel and Hershenson, 2010; Mahler et al., 2010). Mainly three different device strategies are used: metered dose inhalers (MDIs), nebulizers, and dry powder inhalers (DPIs). In MDIs, the drug is dissolved or suspended in a propellant such as HFA 134a. The feasibility of MDIs for proteins and protein containing nanoparticles was demonstrated previously (Bechtold-Peters, 2007; Nyambura et al., 2009), but the approach still gives rise to major concerns regarding chemical and physical stability of proteins including oxidation, unfolding, and aggregation. Consequently, DPIs and nebulizers are preferred which are less stressful for protein molecules. Additionally, they typically provide a more efficient delivery into the lung. But both DPIs and nebulizers also face the challenge that the protein integrity needs to be preserved. Lyophilization is the standard method to obtain dry formulations of biopharmaceuticals. These may either be inhaled directly (Claus et al., 2013) or be milled to the appropriate size and filled into the device of choice (Maa and Prestrekski, 2000). The standard method for preparation and design of dry protein, peptide or nanoparticle containing particles to be used in DPIs is spray-drying. Besides the requirements for inhalative delivery with respect to size, aerodynamic behavior, or flow properties, the powders have to provide a stabilizing effect on the biopharmaceuticals and nanocarriers which can be provided by carefully adjusting pH, ionic strength, addition of surfactants, and stabilizers like sucrose forming glassy matrices. Among inert excipients available for the production of nanoparticle-based dry powders, lactose and mannitol are the first choice as they are also used in marketed DPIs. Nebulizers are typically less convenient in handling as compared to DPIs. During nebulization, the drug molecules and nanoparticulate systems may be exposed to heat, depending on the type of nebulizer used, as well as an enormously increased air–liquid interface (Hertel, 2014). Therefore, liquid protein or nanoparticulate based inhalation formulations typically include general protein protectants like sucrose and protectants against surface induced stress created upon nebulization like surfactants. Additionally, formulation development requires careful selection of pH and ionic strength conditions for the liquid to be nebulized to provide thermal stability and optimize colloidal stability of protein molecules and nanoparticles. For pulmonary applications, limitations with respect to pH (3.5–8.0, preferably N 5.0) and osmolarity (150–550 mOsm, preferably isotonic) apply. 5.3. Why nanoparticulate pulmonary delivery of peptides and proteins? Nanocarriers, specifically nanoparticles and liposomes, may be used for the incorporation of drugs to protect it from degradation, to target specific tissues or cells, and to modify the release as compared to conventional pulmonary delivery systems. For protein drugs, nanocarriers may specifically improve the stability and, consequently, the bioavailability by reducing the contact time to proteases and by avoiding the sites of enzymatic degradation such as macrophages or neutrophils (Loira-Pastoriza et al., 2014). Furthermore, nanocarriers can facilitate the transepithelial transport of the large, hydrophilic peptides and proteins and enable the escape from macrophages which attack the nanometer sized drug molecules. Technically, they allow co-localized deposition of different drugs or excipients like protease inhibitors, too. 5.3.1. The fate of nanoparticulate delivery systems in the lung In the lung, nanocarriers that are lipo-soluble or water soluble undergo in situ dissolution. Particles, which are insoluble or poorly soluble cannot be readily absorbed. They face the challenge of movement in the mucus before reaching cell surfaces and become subject of cell–particle interaction. The viscoelastic mucus is particularly obstructive in certain lung diseases, and the thickness of the mucus layer can increase by a factor of 100 in the case of cystic fibrosis (CF) (Lopez-Vidriero and Reid, 1978). Small nanoparticles are less hindered and can therefore more easily overcome the mucosal barrier (Rytting et al., 2008). Moreover,


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the covalent attachment of polyethylene glycol (PEG) facilitates the movement of nanoparticles in the mucus (Lai et al., 2009) and masks them against phagocytosis. Another strategy to shuttle nanoparticles across the mucosal barrier is doping with magnetic properties by embedding super-paramagnetic iron oxide structures in combination with an externally applied magnetic force (Rubin and Williams, 2014; Upadhyay et al., 2012). Furthermore, mucolytic agents could be coadministered. Recombinant human DNAse (rhDNAse) is a mucolytic used in CF treatment. But hardly any to only moderate improvement in nanoparticles transport through rhDNAse-treated CF sputum was observed although the macroviscoelastic properties were significantly improved (Dawson et al., 2004; Sanders et al., 2000). N-acetyl-L-cysteine, a mucolytic drug which cleaves disulfide bonds and thus reduces mucin fibers' cross-linking, enabled non-viral gene vectors to at least partially overcome the mucus barrier (Ferrari et al., 2001). Although passing the mucus layer can present a major hurdle, there is at the same time a need for mucoadhesive systems. Mucoadhesive nanoparticles, typically based on hydrophilic polymers such as chitosan, sodium alginate, or hyaluronic acid, can benefit from a prolonged residence time and higher drug concentration gradient at a given target or adsorption site. After inhalation, the particle size mainly determines the phagocytosis rate by macrophages. Particles below approx. 100 nm may not be recognized by macrophages, whereas larger nanoparticles become phagocytized (Andrade et al., 2011; Yang et al., 2008). But already structures of the size of protein drugs are subjected to macrophage uptake. Whereas bigger proteins like IgG and human chorionic gonadotropin are affected, the smaller peptide insulin and the protein human growth hormone are not phagocytized (Ducreux and Vanbever, 2007). Due to their unique geometry, large porous particles in the micrometer range carrying nanoparticulate payload escape alveolar macrophages (Cryan, 2005). Coating of the nanoparticles with phospholipids or PEG further reduces phagocytosis, whereas coating with gammaglobulin, fibronectin, surfactant-associated protein, and gelatin enhances the uptake (Andrade et al., 2011; Loira-Pastoriza et al., 2014; Ruge et al., 2012). Whereas evading the reticuloendothelial system is a problem for systemically applied nanoparticles, the uptake of nanoparticulate therapeutics by alveolar macrophages can be beneficial for lung cancer treatment or vaccination. Pulmonary vaccination could be a promising needle-free alternative to vaccination by injection. Besides a systemic immune response, pulmonary vaccination can lead to an additional mucosal immune response against airborne pathogens. Nanoparticles exhibit an adjuvant-like effect which relies on the stimulation of specialized antigen processing dendritic cells that affect T-cell responses, induce B-cell differentiation, and antibody production (Banchereau and Ralph, 1998). Nanoparticle size and surface properties are important for the potency of particle based vaccines and both nanometer scale considerations for vaccination and micrometer scale effects on pulmonary delivery and aerolization have to be combined (Garcia-Contreras et al., 2008). For review, see Blank et al. who nicely summarized opportunities and obstacles of pulmonary vaccination (Blank et al., 2011). 5.3.2. Toxicity of nanoparticulate delivery systems in the lung Besides the beneficial effects, nanoparticle delivery to the lung gives also rise to toxicological concerns due to (i) a correlation between particulate air pollution and increased morbidity and mortality, (ii) since inhalation of rigid nanoparticles can lead to pneumoconiosis and bronchitis, and (iii) that nanoparticles prompt oxidative stress and cellular toxicity (Marianecci et al., 2011). Moreover, protein and lipid absorption by the nanoparticles may affect gas exchange and phosphatidylcholine, phosphatidylinositol and phosphatidylinositol phosphates which are important regulators of cell functions (Schleh et al., 2011). Hence, the biocompatibility of nanoparticles in the alveolar environment has to be considered. In this context, it is noteworthy that biodegradable polymeric nanoparticles do not induce the same inflammatory response as non-biodegradable nanoparticles (Mohamud et al., 2014).

5.4. Liposomes for pulmonary delivery of peptides and proteins Using liposomes for parenteral delivery of drugs including peptides and proteins has a long history and many key properties and various targeting strategies have been subjected to recent reviews (Allen and Cullis, 2013; Arias, 2013; Deshpande et al., 2013; Paliwal et al., 2013; Piroyan et al., 2014). Intensive research focused on inhalation of antibiotic-loaded liposomes to improve drug pharmacokinetics and biodistribution and decrease toxicity (Cipolla et al., 2014). Furthermore, lipid cationic nanocarriers are widely used to condense and intracellularly deliver negatively charged nucleic acid based macromolecules to the lung. Liposomes may be transferred into the lung in liquid or dry powder form. Using ultrasonic nebulization, narrowly distributed droplets in the desired low μm range can be created and insulin loaded liposomes were stable upon nebulization (Huang and Wang, 2006). Typically liposomal protein formulations for inhalation are DPIs to ensure storage stability. They can be prepared by spray-drying optimized to achieve the optimal particle size for inhalation and to preserve the protein activity (Bosquillon et al., 2004; Codrons et al., 2004). Liposomal dry powder aerosols for β-glucuronidase based on dimyristoylphosphatyl choline:cholesterol (7:3) were successfully prepared by lyophilization and jet-milling (Lu and Hickey, 2005). The concepts of liposomal encapsulation and design of DPI particle design can also be combined. Yang et al. prepared porous nanoparticle-aggregate particles by spray freeze-drying and increased the entrapment efficiency of octreotide acetate by forming a hydrophobic ion pair complex with sodium deoxycholate. In a hepatic ischemia–reperfusion injury model, inhalation of the particles rendered significantly increased bioavailability and improved plasma aspartate aminotransferase levels when compared to subcutaneous injection of octreotide acetate (Yang et al., 2013). Finally, the PulmoSphere® technology has gained FDA approval as part of the Novartis TOBI podhaler in 2013. These large porous particles are based on long chain phospholipids and besides for insulin, they have also been employed for the pulmonary delivery of other protein and peptides like PTH, human growth hormone, sCT, glucagon-like peptide-1 and immunoglobulins (Hertel, 2014). The pharmacokinetics of protein and peptide drugs can be modified by changing the liposome composition. Chono et al. generated liposomes containing different phosphatidyl choline variants. A significant reduction in plasma glucose levels was only reached with dipalmitoylphosphatidyl choline and the effect was most pronounced for 100 nm liposomes most likely by opening tight junctions (Chono et al., 2009). Consistently, Huang et al. and Bi et al. achieved a reduction of the systemic glucose levels for 12 h after pulmonary administration (Bi et al., 2008; Huang and Wang, 2006). Another interesting application of liposomally encapsulated protein is the treatment of lung carcinoma with IL-2. The pulmonary application of IL-2 loaded liposomes, but not of free IL-2 in solution, induced a substantial immune response in dogs because of an increased cellular uptake of liposomally encapsulated IL-2 (Khanna et al., 1997). The activation of pulmonary leukocytes inhibited tumor proliferation and progression and significantly reduced pulmonary metastasis from osteosarcoma (Khanna et al., 1997). Patients tolerated the liposomal IL-2 formulation well, but unfortunately the treatment did not provide significant benefit (Ten et al., 2002). Kaipel et al. tested liposomes for pulmonary delivery of superoxide dismutase (SOD) (Kaipel et al., 2008). The decrease of reactive oxygen species achieved by SOD treatment is beneficial for patients suffering from rheumatoid arthritis or ischemia–reperfusion injury. But SOD has a short circulation half-life making sustained delivery desirable. The liposomal formulation was able to increase the SOD half-life with persisting higher blood levels without affecting the physiological function of the lung (Kaipel et al., 2008). Various studies considered the inhalative delivery of peptides encapsulated in liposomes. For example, pulmonary delivery of antimicrobial



peptides enables local treatment of lung infections at reduced systemic side effects. An antimicrobial peptide could be successfully encapsulated and nebulized with liposomes of different lipid combinations; dimyristoylphosphatidyl choline/dimyristoyl phosphatidylglycerol 3:1 being the most promising combination. The formulations should allow for adequate peptide levels in adults and even more so in children (Lange et al., 2001). Systemic peptide delivery was tested with the GnRH analog leuprolide used in cancer and endometriosis treatment. Leuprolide acts as an agonist at pituitary GnRH receptors. By desensitizing the GnRH receptors, it indirectly down-regulates the secretion of luteinizing hormone and follicle-stimulating hormone. GnRH loaded liposomes promoted an increased half-life when compared with inhalation of solution or subcutaneous administration (Shahiwala and Misra, 2005). Stark et al. developed liposomes containing the 28 amino acid long vasoactive intestinal peptide (VIP) for pulmonary application which is highly sensitive to enzymatic cleavage. In an ex vivo lung arterial model, liposome associated VIP induced sustained vasodilatory effects with the same efficacy as free VIP (Stark et al., 2007). Murata et al. linked wheat germ agglutinin, which specifically interacts with cells of the lung epithelium, to the mucoadhesive polymer carbopol. They used this conjugate to prepare surface-modified liposomes which better interact with lung epithelial cells and thus improved the efficiency of calcitonin (Murata et al., 2013). Similar enhancement and prolongation of the therapeutic efficacy of elcatonin after pulmonary administration resulted from decorating liposomes with chitosan oligosaccharide and polyvinyl alcohol with a hydrophobic anchor (Murata et al., 2012). So far, the applications described focused on local or systemic delivery without specific cell targeting. In order to achieve specific effects, Briscone et al. used pH-sensitive liposomes which change their structure in an acidic environment and hence facilitated the intracellular delivery of SOD to epithelial cells (Briscone et al., 1995). Another approach is to change general physicochemical properties, like surface charge or hydrophobicity. Furthermore, many ligands coupled to the liposomal surface can trigger cell uptake including lectins (Brueck et al., 2001), the human immunodeficiency virus I transcriptional activator (HIV-TAT) (Kawabata et al., 2012; Kleemann et al., 2005), antiintercellular adhesion molecule-1 (ICAM-1) antibody (Azarmi et al., 2008), protein transduction domains, or peptides like LHRH (Taratula et al., 2011). These approaches, however, have not yet been exploited in detail for nanoparticulate systems carrying peptides or proteins. 5.5. Polymeric nanoparticles for pulmonary delivery A high number of different nanoparticulate materials has been investigated for pulmonary delivery of biomacromolecules including

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cyanoacrylate based particles for insulin delivery (Zhang et al., 2001), solid-lipid nanoparticles (SLNs) which can provide rather high loading and efficient delivery (Liu et al., 2008), or gelatin nanoparticles which require crosslinking for stabilization and are limited in loading capacity (Zhao et al., 2012). Chitosan and PLGA, however, are the most studied materials. Menon et al. directly compared gelatin, chitosan, alginate, PLGA, PLGA–chitosan, and PLGA–PEG nanoparticles for pulmonary protein delivery. All nanoparticles except PLGA–PEG and alginate were below 300 nm in size with a biphasic release profile. PLGA and natural polymer based nanoparticles exhibited the highest biocompatibility and dose dependent accumulation in human alveolar type-1 epithelial cells. Following a single inhalation of rhodamine-labeled erythropoietin, widespread protein distribution persisted for up to 10 days (Menon et al., 2014). It has to be kept in mind that drug release from polymeric particulate drug delivery systems is usually controlled by diffusion through the polymer matrix and its degradation. Due to the short path length, drugs can rapidly diffuse out of polymeric nanoparticles (Beck-Broichsitter et al., 2012). Consequently, additional retention mechanisms are required including stable mechanical entrapment, strong charge interactions, covalent linkage, controlled and reversible protein aggregation, or coating of the nanoparticles to increase the diffusion distance. 5.5.1. Chitosan nanoparticles Another widely used biodegradable polymer for pulmonary delivery is chitosan and its derivatives. Grenha et al. prepared 2–4 μm sugar particles containing insulin-loaded chitosan nanoparticles by spray-drying with high encapsulation efficiency (Grenha et al., 2008). Similar insulinloaded chitosan nanoparticles resulted in a pronounced and prolonged hypoglycemic effect (Fig. 5) (Al-Qadi et al., 2012). Whereas the release of insulin from these particles occurred rather fast, the release can be sustained by including phospholipids into the formulation (Grenha et al., 2008). PLGA nanospheres coated with the mucoadhesive chitosan for pulmonary delivery of the peptide elcatonin have been mentioned above as an example of the advantages of decoration of PLGA nanocarriers (Murata et al., 2012). Nebulization of chitosancoated PLGA nanoparticles improved the absorption of calcitonin, delayed their elimination and thus the effect of loaded calcitonin was prolonged as compared to non-coated particles (Yamamoto et al., 2005). The improved adsorption was attributed to two chitosan effects, mucoadhesion and opening of epithelial tight junctions. A similarly enhanced particle retention, sustained release, and pharmacological hypoglycemic effect could be demonstrated for inhaled chitosancoated PLGA-nanoparticles loaded with modified exendin-4 (Lee et al., 2013). Chitosan loaded particles have also been successfully employed for pulmonary vaccination (Bivas-Benita et al., 2009).

Fig. 5. (a) Transmission electron microscopy picture: insulin-loaded chitosan nanoparticles (chitosan/pentasodium tripolyphosphate/insulin = 5/1/1.5). (b) Scanning electron microscopy picture: insulin-loaded and microencapsulated chitosan nanoparticles (mannitol/nanoparticle ratio = 80:20 (w/w)). (c) Confocal microscopy: deposition of FITC–bovine serum albuminloaded chitosan nanoparticles incorporated in mannitol microspheres in alveoli following intratracheal application. Based on and reprinted with permission from Al-Qadi et al. (2012).


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5.5.2. PLGA nanoparticles PLGA copolymers are frequently used for nanoparticle preparation due to their high biocompatibility, their biodegradability, and the approved status of various PLGA containing parenteral depot products and frequent use in medicinal products. Fast degrading PLGA types are preferred for inhalation and their pulmonary biocompatibility has been shown (d'Angelo et al., 2014). Drug encapsulation in PLGA matrices via spray or emulsion processes forms the basis for sustained release. At the nanosize level, particle surface modification with hydrophilic polymers, e.g. PEG or chitosan, has been found useful to modulate carrier interactions with mucus and cells. Kawashima et al. loaded insulin onto PLGA nanospheres using a modified emulsion solvent diffusion method. An aqueous dispersion of the PLGA nanospheres was nebulized and 3.9 IU insulin/kg was administered to guinea pigs. Administration of the PLGA nanospheres significantly reduced the blood glucose level and hypoglycemia sustained for over 48 h, compared to 6 h after inhalation of free insulin (Kawashima et al., 1999). Yang et al. adsorbed salmon calcitonin on the surface of PLGA-nanoparticles. Subsequently, the nanoparticles were lyophilized and loaded onto inhalable carriers. They achieved high drug loading and inhalation efficiency as well as improved hypocalcemic action compared to calcitonin solution (Yang et al., 2012). Alternatively, PLGA-nanoparticles can be embedded in sugar microparticles via spraydrying (Yamamoto et al., 2007). 5.6. Outlook In order to translate the nanoparticle based protein and peptide pulmonary delivery systems into therapy and patient benefit various critical aspects have to be considered. Protein instability is a major obstacle for any controlled delivery system and protein integrity needs to be thoroughly characterized (Jiskoot et al., 2012). Furthermore, the drug to carrier and vehicle ratio needs to be maximized when aiming for safe and effective delivery. The use of highly complex particle architectures limits the transfer to a marketable product and simple strategies are sought after. 6. Conclusion There are several promising studies describing the successful use of soft matter nanocarriers for the systemic delivery of proteins and peptides. Only a few publications, however, focus on the topical delivery of biomacromolecules which are overall in an early stage of development. More advanced studies are only found for the topical delivery of vaccines or vaccine adjuvants and for pulmonary delivery. Several reasons account for these facts which are mainly attributed to general limitations of nanoparticles such as low loading efficiencies, limited stability, or nanotoxicological aspects. In contrast to nanoparticles, the generation of microparticulate carrier systems for the delivery of biomacromolecules is more advanced but this is beyond the scope of this review. Nevertheless, protein and peptide delivery via the nonparenteral route are highly appealing due to their non-invasive nature and needs to be further addressed in the future. For other applications and indications intensive research efforts are already conducted resulting in highly sophisticated soft matter nanocarriers like charge conversional polyionic micelles (Lee et al., 2009) or stimuli-responsive polymeric micelles which release the payload depending on the environmental pH (Koyamatsu et al., 2014), temperature (Cuggino et al., 2011) or ionic strength (Lu et al., 2014). Acknowledgment This work was supported by grants from the German Research Foundation (DFG): KU 2904/2-1 S.H. and Collaborative Research Center 1112 (Nanocarriers: Architecture, Transport, and Topical Application of Drugs for Therapeutic Use, project C02).

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