European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Review

Protamine–oligonucleotide-nanoparticles: Recent advances in drug delivery and drug targeting B. Scheicher, A.-L. Schachner-Nedherer, A. Zimmer ⇑ University of Graz, Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Universitätsplatz 1, 8010 Graz – member of: BioTechMed-Graz, Austria

a r t i c l e

i n f o

Article history: Received 12 January 2015 Received in revised form 9 April 2015 Accepted 10 April 2015 Available online xxxx Keywords: Protamine Antisense siRNA CpG – oligonucleotides

a b s t r a c t Application of oligonucleotides as active compounds has become a crucial field of pharmaceutical research in recent years. In order to improve inadequate transfection rate and to avoid rapid enzymatic degradation of antisense oligonucleotides (AS-ODNs) a novel nanoparticulate delivery system was reported by our group at the beginning of 2000. AS-ODNs are condensed by the polycationic peptide protamine into solid particles in the size range of 100–200 nm. Nanoparticle formation is driven by a selfassembling process based on electrostatic interactions between the oppositely charged biomolecules. This new delivery system was named ‘‘proticles’’ and showed very efficient protection against enzymatic digestion, high transfection rates and significant antisense effects in vitro. Throughout broader research, this promising approach was enlarged, and AS-ODNs were replaced by siRNA or CpG-oligonucleotides to address the aspect of immune-modulation and vaccination. More recent studies on proticles verified upscaling of the self-assembling process as well as the potential of proticle formulations for active drug targeting, like tumor- or atherosclerotic plaque targeting. Thereby also the application for diagnostic purposes was emphasized. This review will focus on the characterization of the nucleoprotein protamine as well as on the variety of possible nucleotides/peptides which were already assembled into the proticle matrix. Furthermore it will provide an insight into the broad area of application where proticles can present a valuable tool for successful oligonucleotide delivery. Ó 2015 Elsevier B.V. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Protamine: a DNA binding peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Protamine: a pharmaceutical excipient and drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proticles as drug delivery systems: manufacturing and application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. First generation proticles – introducing a binary system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Modification of the initial binary system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Upscaling of assembling process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Surface modified proticles for drug targeting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Lipid coated protamine–oligonucleotide-complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Peptide or protein ligand targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proticles as immune-modulator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AlPrO, albumin–protamine–oligonucleotide; Apo A-1, apolipoprotein A-1; AS-ODNs, antisense oligonucleotides; AS-PTOs, antisense phosphorothioate oligonucleotides; CpG-ODNs, cytosine-phosphate-guanine-oligonucleotides; HSA, human serum albumin; LPD, lipid–peptide-DNA; LPH, lipid–peptide–hyaluronic acid; ODNs, oligonucleotides; PEG, polyethylene glycol; PTOs, phosphorothioate oligonucleotides; VIP, vasoactive intestinal peptide. ⇑ Corresponding author. Tel.: +43 316 380 888 0. E-mail address: [email protected] (A. Zimmer). http://dx.doi.org/10.1016/j.ejps.2015.04.009 0928-0987/Ó 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Scheicher, B., et al. Protamine–oligonucleotide-nanoparticles: Recent advances in drug delivery and drug targeting. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.04.009

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B. Scheicher et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx

1. Introduction

2.2. Protamine: a pharmaceutical excipient and drug

In the year 2000 a new method for preparing solid nanoparticles from antisense oligonucleotides (AS-ODNs) together with the cationic peptide protamine was invented by our research group (Junghans et al., 2000a). In general, drug delivery of AS-ODNs by nanoparticles based on partly cationically charged viral proteins was evaluated by the group of Bertling in 1991 and was continued by our group in 1999 (Bertling et al., 1991; Braun et al., 1999). Before these innovations were published, the aggregation into compact structures with short segments of single-stranded DNA was reported with the polycation poly(L-lysine) by the group of Lebleu for the first time in 1987 (Lemaitre et al., 1987). Comparing the polycations protamine, spermine and spermidine in terms of their potential to condense different types of oligonucleotides and antisense drugs, protamine was found to be most efficient to form nanoparticles in the size range of 100– 200 nm (Junghans et al., 2001). Consequently, the new complex between protamine and oligonucleotides resulting into nanoparticles was called ‘‘proticles’’ and was also protected as trademark (DPMAregister, 1999). Today, depending on the concentration of protamine, oligonucleotides or further excipients/drugs like peptides or proteins as well as the applied manufacturing method, the size range can be controlled between 80 and 1000 nm. During the last 15 years several applications of proticles in the field of drug delivery and drug targeting were described and patents were filed (Junghans et al., 2000b). The present review will summarize the latest knowledge in this field of research.

Protamine represents a well-established pharmaceutical ingredient and is used on the market in several pharmaceutical products for many years. For example, insulin therapy for the treatment of diabetes mellitus is a well-known application area. Thereby protamine is combined with insulin to formulate protamine zinc insulin and neutral protamine Hagedorn insulin to reach a prolonged effect of lowering blood glucose levels. These intermediate-acting insulin preparations are popular as once- or twice daily subcutaneous injection (Owens, 2011). In addition, protamine is used as drug and antidote for neutralization of the anticoagulant heparin. Electrostatic binding between polycationic protamine and polyanionic heparin forms a stable complex. Because of these antagonizing properties, protamine is often applied after cardiac or vascular surgeries to prevent postoperative bleeding and reverse the anticoagulant activity of heparin (He et al., 2014). Next to these particularly pharmacological effects, the heterogenic group of protamines has become of major interest in the field of molecular biology and delivery-systems for biomolecules, such as non-viral vectors for in vivo gene transfer. The interest is mainly laid on the cell penetrating and nucleus targeting properties of protamine (Lindgren et al., 2000; Mitchell et al., 2000; Sorgi et al., 1997; Stewart et al., 2008). In various studies protamine was also used as penetration enhancer in combination with different nanoparticles (Apaolaza et al., 2014; Delgado et al., 2012) or liposomes, which was introduced by the group of Huang (Gao and Huang, 1996; Li et al., 1998; Sorgi et al., 1997). By applying protamine with different macromolecules also new possibilities for self-assembling of nanoplexes were established in recent years, as example: hyaluronic acid (Umerska et al., 2014) and carrageenan (Dul et al., 2015).

2. Protamine 2.1. Protamine: a DNA binding peptide Protamine, a peptide well known as pharmaceutical excipient, is derived from the sperm of different fish-species and has a molecular mass of approximately 4000 Da with about 70 mol% of the basic amino acid arginine. Basically, protamines belong to a diverse protein family of arginine rich peptides. Using protamine for DNA complexation was observed from nature where this principle is very efficient condensing the spermatid genome. Discovery and isolation of protamine is attributed to Friedrich Miescher in 1874. He found out that DNA of Rhine salmon is associated with highly basic substances (Miescher, 1874). Structural elements have been identified in all vertebrate protamines consisting of arginine-rich DNA-anchoring domains and multiple phosphorylation sites. In mammals and humans two classes of sperm protamines, named P1 and P2, were found (Balhorn, 2007; Bianchi et al., 1994). P1 is responsible for the packaging of sperm DNA in all mammals, whereas the zinc binding P2 appears only in the sperm of primates, many rodents and in part of other placental mammals. However, human and fish protamines show differences in their amino acid composition and molecular weight. During spermatogenesis, nuclear histones are replaced by protamines acting as a nucleoprotein. Their functions comprise the compact condensation and protection of DNA against enzymatic degradation. The stability of the DNA–protamine-complex is attained through the combination of hydrogen bonds, van der Waals and electrostatic interactions between the positively charged arginine residues of protamine and the negatively charged phosphate groups of DNA. Binding of protamine in the grooves of DNA leads to neutralization of the phosphodiester backbone. Each protamine molecule serves as a link between several DNA molecules and therefore a close packing of DNA is enabled. A historical overview and more detailed information about the progress of structural analysis concerning protamine and the DNA–protamine-complex are highlighted in the review of Raukas and Mikelsaar (1999).

3. Proticles as drug delivery systems: manufacturing and application The formation of proticles is based on previously described strong electrostatic interactions between the positively charged protamine and the negatively charged backbone of DNA/RNA oligonucleotides. Junghans et al. have shown in 2000 that mixing aqueous solutions of protamine and oligonucleotides in well-defined mass ratios induces spontaneous self-assembling into solid nanoparticles (Junghans et al., 2000a). The transparent solution converts into opaque indicating the presence of nanoparticles. It could be shown that particle building is possible for phosphodiester as well as phosphorothioate oligonucleotides, but a minimum chain length of nine nucleotides is necessary (Junghans et al., 2001). Within several studies a variety of oligonucleotides were assembled into proticles and further additional peptides were implemented into the particle matrix. An optimized mass ratio of the components had to be developed for each proticle composition, because the concentration of the components plays a crucial role concerning particle size, loading, zeta potential and transfection efficiency. As these specifications have to be customized for each application of a drug delivery system an individual mass ratio is necessary for the applied proticle formulation. Different formulations which were evaluated throughout the years for various application areas in pharmaceutical science are highlighted in Table 1. 3.1. First generation proticles – introducing a binary system As mentioned above, the initial idea behind protamine–oligonucleotide-nanoparticles was the development of a suitable delivery system for AS-ODNs. These are short single-stranded nucleic acid fragments of 13–25 nucleotides which are able to regulate gene

Please cite this article in press as: Scheicher, B., et al. Protamine–oligonucleotide-nanoparticles: Recent advances in drug delivery and drug targeting. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.04.009

B. Scheicher et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx

3

Table 1 Summary of proticle research ordered by composition and possible areas for application. Composition of proticles

Purpose/effect

References



" ODN transfection " ODN protection from degradation

Dinauer et al. (2004), Junghans et al. (2001)

HSA

" stabilization in physiological media

Lochmann et al. (2005), Weyermann et al. (2005)

Cationic lipids

" ODN loading ; toxicity of cationic lipids

Junghans et al. (2005)



" immune response of CpG-ODNs

Kerkmann et al. (2006)

Ara h 2

; undesired Th-2 immune response " IgG2a levels (Th-1)

Pali-Scholl et al. (2013)



Carrier system for siRNA

Reischl and Zimmer (2009)

Apolipoprotein A-1

" permeability through blood–brain barrier

Kratzer et al. (2007)

Adiponectin IL-10

Targeting atherosclerotic plaques Improving atherosclerotic plaques imaging

Almer et al. (2014,2011)

VIP

Depot formulation for peptides Active tumor targeting (lung)

Wernig et al. (2008), Ortner et al. (2010)



Scale up of self-assembling process

Eitzlmayr et al. (2011), Petschacher et al. (2013)

Protamine

Antisense-ODNs

CpG-ODNs

siRNA

Control-ODNs

Thiomers

expression at posttranscriptional or posttranslational level through mRNA binding due to sequence complementarity. Therefore, they show potential to act as promising therapeutic compounds in the treatment of various diseases. Unfortunately, they possess some limiting characteristics like rapid degradation by nucleases, low bioavailability and poor penetration rate through cell membranes. To overcome these limitations, the development of a potent drug delivery system is of great importance. In addition, modified oligonucleotides like phosphorothioates (PTOs) with increased enzymatic stability may be used. Crucial aspects for successful antisense delivery include the penetration of proticles into the targeted cells as well as the release of the antisense compound at target site. The reviews of Dias and Stein about AS-ODNs and Levin (AS-PTOs) provide a detailed and comprehensive overview about these oligonucleotides describing their chemistry and mode of action (Dias and Stein, 2002; Levin, 1999). In our studies we were able to show that proticles serve as successful carrier systems for AS-ODNs. The self-assembled nanoparticles protected oligonucleotides very efficiently against enzymatic digestion by nucleases. Both phosphodiester and phosphorothioate ODNs spontaneously built complexes with protamine. We pointed out that oligonucleotide chain length, protamine/ODN mass ratio and ionic strength of buffer solutions have an impact on nanoparticle formation, number and surface charge of particles (Junghans et al., 2000a, 2001; Lochmann et al., 2004). An excess of protamine in proticle formulations was necessary for complete binding and sufficient enzymatic protection of ODNs. With increasing amounts of protamine particle number increased and zeta potential changed to positive values. Size measurements indicated that the nanoparticles composed of PTOs seem to be more stable and show less aggregation tendency than ODNs after storage of several days. Further, the early research in this field demonstrated an improved cellular uptake of oligonucleotides. The highest uptake efficiencies were detected with increasing mass ratios of protamine and positive zeta potential, which leads to an enhanced electrostatic interaction with the negatively charged cell membranes (Junghans et al., 2000a, 2001). In subsequent studies we explored the transfection efficiency and antiviral activity of proticles loaded with AS-ODNs and ASPTOs directed against human immunodeficiency virus type 1 (HIV-1) tat mRNA in HIV-1 target cells (Dinauer et al., 2004). Protamine was able to complex AS-ODNs as well as AS-PTOs to form nanoparticles with diameters between 100 and 200 nm and

surface charges up to +30 mV. Again cellular uptake of proticles was significantly enhanced compared to naked oligonucleotides. However, the release behavior of proticles with AS-ODNs differed from that with AS-PTOs. The former showed liberation of the complexed antisense compound leading to a specific inhibition of tat mediated HIV-1 transactivation. In contrast, antisense activity of proticles with AS-PTOs was not significant. This finding was attributed to a lack of release of AS-PTOs due to strong electrostatic interactions within the nanoparticles, resulting in a very stable complex. It was concluded that dissociation of the antisense compound from the nanoparticle complex is an essential prerequisite for antisense activity. As the first proticle generation has shown some disadvantages like the inability to release AS-PTOs and aggregation tendency under physiological conditions an optimization of the binary system had to be acquired. 3.2. Modification of the initial binary system The physicochemical properties of nanoparticles are a meaningful characteristic for their application potential in vitro and in vivo as well as for prospective pharmaceutical developments. As mentioned above, particular properties of first generation proticles minimized their efficiency as potent drug-delivery system. Therefore, we studied in more detail the modification of the binary system to overcome known disadvantages. As already noted, AS-PTOs were superior to unmodified ODNs because of their higher stability towards nucleases and their slower increase in size with time. However, proticles with PTOs showed instability in physiological salt solutions which led to particle aggregation. To solve this problem, polyethylene glycol 20 000 (PEG 20 000) was integrated into the assembling process. The results indicated an increased stability in cell medium, but further modifications in nanoparticle composition were essential because PEG 20 000 is physiologically not compatible (Lochmann et al., 2004). It is well documented that the non-toxic macromolecule human serum albumin (HSA) which is widely used in nano- and microparticle preparation also shows oligonucleotide-binding behavior (Faneca et al., 2004; Fischer et al., 2001; Simoes et al., 2000; Wartlick et al., 2004). HSA was found to be a potent stabilizer of proticles and in addition to the initial binary system of protamine and ODNs, ternary systems including HSA showed superior properties in terms of cellular uptake and intracellular ODN distribution.

Please cite this article in press as: Scheicher, B., et al. Protamine–oligonucleotide-nanoparticles: Recent advances in drug delivery and drug targeting. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.04.009

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B. Scheicher et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx

Albumin–protamine–oligonucleotide (AlPrO) nanoparticles were prepared by mixing modified or unmodified ODNs with an aqueous mixture of HSA and protamine. The studies showed that AlPrO nanoparticles are able to achieve sufficient stability in salt solutions, high intracellular uptake and a diffuse distribution over the whole cytoplasm indicating an increased release of the antisense compound, especially for PTOs. To a large extent, the striking differences of PTO release in ternary proticles in contrast to the binary systems are attributable to the conformational change of HSA at endosomal pH value. The fusogenic properties of albumin under acidic conditions may lead to destabilization of endosomes, thus improving intracellular gene delivery (Lochmann et al., 2005; Simoes et al., 2000; Vogel et al., 2005; Weyermann et al., 2005). Another modification in proticle composition came from the application of protamine sulfate to modify the particle diameter in the lower nanometer range more efficiently. The substitution of protamine free base by protamine sulfate caused a drastic size reduction, however no differences were found concerning particle uptake in murine fibroblasts and intracellular release. This modification can be advantageous when size-dependent particle uptake is observable (Mayer et al., 2005). Though, the reason for the difference in particle size using protamine sulfate is still unknown. Continuing research led to a comparison between different cationic transfection reagents, demonstrating lowest cytotoxicity in vitro for proticles but highest efficacy for cationic lipids (Lochmann et al., 2004). Our previous investigations exhibited that proticles aggregate under physiological conditions. As a consequence, aggregated particles stuck into the cell membranes and were concentrated in a certain area. The addition of HSA resulted in an improvement of the proticle formulation avoiding particle aggregation in isotonic solution like cell media. To conclude, ternary AlPrO nanoparticles represent an optimized new delivery system for AS-ODNs. The cellular uptake was significantly enhanced (up to 12 times) in comparison to free ODNs. Regarding transfection efficiencies AlPrO nanoparticles were found to be comparable to commercial available transfection reagents based on cationic lipids (Weyermann et al., 2005). In addition, no considerable cytotoxic side effects were observed for the proticle formulations. After cellular uptake the dissociated oligonucleotides were distributed within the cytoplasm and showed a desired antisense effect (Weyermann et al., 2004).

3.3. Upscaling of assembling process More recent research on proticles included methods to simulate self-assembling and to establish a microreactor technology to scale-up the manufacturing process. This plays an essential step for controlling particle quality and bringing the formulation closer to the market. Major advantages of microreactors include more controlled fluid transport, rapid chemical reaction and low cost productions, enabling enhanced reproducible processing accuracy and efficiency. Principles and progression in microreactor processing are highlighted in several reviews (Chang et al., 2008; Hung and Lee, 2007). The self-assembling process of nanoparticles is a spontaneous particle formation and thus hard to control. Mixing effects highly influence particle size distribution and external influences such as shear forces may result in secondary effects like aggregation or disaggregation. Our investigations demonstrated that for obtaining particles with a narrow size distribution higher flow rates had to be disposed which resulted in a stronger mixing effect with higher binding efficiencies of the anionic and cationic component (Eitzlmayr et al., 2011; Petschacher et al., 2013).

4. Surface modified proticles for drug targeting 4.1. Lipid coated protamine–oligonucleotide-complexes A combination of proticles with liposomes was reported in 2005. Junghans et al. showed the possibility to coat the initial protamine– oligonucleotide-particle with a lipid film (Junghans et al., 2005). In brief, negatively charged proticles containing an equal mass ratio of ODN:protamine were merged with a mix of anionic or neutral and cationic lipids (Zimmer et al., 1999), which are commonly used non-viral transfection enhancers. Ultrasonication was used to induce reorganization of the bilayer and partial proticle incorporation into the liposomes. The major advantages of this procedure were reduced toxicity of the applied lipids and higher ODN loading efficiencies. ODN binding of pure proticles was not as high as in previous studies, because of a non-adjusted ODN:protamine mass ratio. As mentioned above, the addition of protamine into the formation process of delivery vectors is very common, especially for non-viral vectors like lipid-peptide-DNA-complexes (LPDs). In contrast to the study of Junghans et al. (2005) where ODNs were encapsulated, LPDs were basically developed for transfection of plasmid DNA (Gao and Huang, 1996). Comparable to proticles, LPDs showed higher loading efficiencies and higher transfection rates than complexes without precondensation by protamine. In recent years research on LPDs was expanded towards active tumor targeting (Chen et al., 2010; Xu et al., 2014) and the integration of hyaluronic acid (LPH complexes) to reduce immunotoxicity (Chono et al., 2008; Wang et al., 2013). Even without lipid coating hyaluronic acid is used to form nanoplexes with protamine in order to act as drug delivery system for peptides. Umerska et al. recently reported an oral delivery system for salmon calcitonin which showed prolonged release and successful protection against enzymatic digestion (Umerska et al., 2014).

4.2. Peptide or protein ligand targeting Besides high loading efficiencies, successful protection and sufficient release of an active compound, a potent drug delivery system must offer the possibility of drug targeting. Therefore, recent steps in proticle research also addressed the question of active drug targeting by coating or co-assembling of targeting sequences. Consequently, in 2010 it was demonstrated for the first time to target proticles loaded with vasoactive intestinal peptide (VIP) to tumor cells which overexpressed VPAC receptor (Ortner et al., 2010). It was shown that VIP loaded proticles accumulated at the surface of VPAC receptor expressing cells and internalization of physiological active VIP occured. Furthermore, this approach was also demonstrated in human lung tumor tissue ex vivo. An observable depot effect of proticles advanced cell uptake by VIP protection while receptor recycling process occurred. Depot effect of VIP loaded proticles was already investigated in a former study using VIP as pulmonary vasodilator (Wernig et al., 2008). High VIP loading efficiencies and prolonged vasodilatation pointed out that assembling within proticle matrix is a promising approach for sustained delivery systems of peptides. The ability to overcome the blood–brain barrier and achieving CNS uptake by proticles was investigated by apolipoprotein A-1 (Apo A-1) coating (Kratzer et al., 2007). The approach of apolipoprotein coating in order to mimic lipoprotein particles for receptor mediated endocytosis was reported by Kreuter et al. earlier (Kreuter et al., 2002). Coated proticles showed a markedly enhanced transcytosis through brain capillary endothelial cells, compared to uncoated proticles. Results of this study showed the ability of a proticle formulation to improve permeability of the blood–brain barrier reaching deeper regions of the brain. Also the

Please cite this article in press as: Scheicher, B., et al. Protamine–oligonucleotide-nanoparticles: Recent advances in drug delivery and drug targeting. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.04.009

B. Scheicher et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx

cell penetrating properties of protamines are used to enhance CNS uptake of nanoparticles. As example, PEG-PLA nanoparticles were functionalized with covalently bound low molecular weight protamine to facilitate brain delivery after bypassing the blood–brain barrier via intranasal application (Xia et al., 2011). Applying proticles as targeted delivery system was also established for diagnostic purposes. Signal emitting molecules were linked to proticles for detection of atherosclerotic plaques (Almer et al., 2011), a field of growing interest for prevention of cardiovascular diseases. Formation and progression of atherosclerotic plaques play a major role in the inflammatory pathway of atherosclerosis (Mangge et al., 2014; Rocha and Libby, 2009). It could be shown that through coating of adiponectin as targeting sequence onto the surface of proticles an enhanced non-invasive imaging of atherosclerotic plaques was possible. Later, an improved IL-10 mediated targeting was developed and differences in localization between proticles and targeted liposomes were evaluated ex-vivo in mice (Almer et al., 2014). Beyond the diagnostic approach an additional therapeutic potential of anti-inflammatory cytokine IL-10 was considered. Comparison of the two delivery systems pointed out an accumulation of signal emitting particles in different regions of atherosclerotic plaques which offers new possibilities to visualize different stages of plaque scenario.

5. Proticles as immune-modulator Within the last years, proticles were investigated for their use in the field of vaccination. The first publication which evaluated the immunogenic properties of proticles showed the possibility to enhance immune-modulation using CpG-oligonucleotides (CpGODNs) (Kerkmann et al., 2006). The cytosine-phosphate-guanine motif of these oligonucleotides is prevalently present in bacterial DNA (Krieg et al., 1995) and triggers strong immunostimulatory effects via recognition by toll-like receptor 9 (Bauer et al., 2001). It was shown that the incorporation of type B CpG-ODNs into proticles resulted in a 20-fold higher IFN-alpha production of plasmacytoid dendritic cells compared to pure CpG-ODNs, most probably due to the presence of a particulate structure. Normally, type B CpG-ODNs strongly activate B cells, but they only show weak IFN-alpha secretion (Hartmann et al., 2003). Most successfully, proticles with non-immunogenic CpG-control-oligonucleotides were found to be not immunogenic at all. Recently, an enhanced proticle-CpG-ODN formulation was applied to study its possibility as immunoadjuvant and modulator in vivo. Therefore, major peanut allergen Ara h 2 was integrated into proticle matrix as model allergen (Pali-Scholl et al., 2013). Compared to commonly used immunoadjuvant aluminum hydroxide, proticles with CpG-ODN effectively suppressed undesired Th2-dominated immune response. In accordance with other studies CpG-ODNs drove the immune response towards favorable Th1 (Chu et al., 1997) indicated by highest IgG2a levels when Ara h 2 was incorporated in proticles. Similar Th1-driven response was found when protamine was co-encapsulated with CpG-ODNs in PLGA microparticle formulations with bee venom allergen phospholipase A2 (Martinez Gomez et al., 2007). In that case the addition of protamine sulfate also strengthened the immune response which was related to ODN stabilization and higher CpG loading of microparticles. Another advantage for the use of proticles in immunotherapy could be an observed depot effect after subcutaneous injection verified by real-time optical fluorescent imaging (Pali-Scholl et al., 2013). A recently published work from Xu et al. addresses the application of siRNA as well as CpG-ODNs as immunoadjuvant for vaccination against Melanoma (Xu et al., 2014). In this work a synergistic combination of two modified carrier systems (LPD

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and LPH) is described. On the one hand a mannose modified LPD complex is applied carrying tumor antigen Trp2 and CpG-ODNs to induce a systemic immune response against the tumor. Additionally, a PEGylated LPH with siRNA is used to silence immune suppressive TGF-beta in tumor cells. The aspect of siRNA delivery by proticles was outlined in a review by Reischl and Zimmer (2009). 6. Summary Summarizing the present knowledge in the field of protamineoligonucleotide-nanoparticles, this drug delivery system provides remarkable flexibility in terms of composition and application. Oligonucleotides were just a starting point of the drug application followed by peptides, proteins, lipids and lipoproteins used as drugs and excipients. In general, it was found that the oligonucleotides can be replaced by other polyanionic macromolecules with similar mass and charge. At the beginning of this research field drug loading, enzymatic protection and cellular uptake were key issues followed by more complex applications such as pulmonary drug delivery or immune-modulation and vaccination. Clearly in the future, drug targeting will be a more challenging issue, not only for cancer application, but also for metabolic disorders. Diagnostics as well as in combination with drug therapy will open the field of theranostics with a huge demand of targeted transport vehicles. In this context, from the last 15 years, we can conclude that proticles will be a valuable tool in this field of research in the future. References Almer, G., Summers, K.L., Scheicher, B., Kellner, J., Stelzer, I., Leitinger, G., Gries, A., Prassl, R., Zimmer, A., Mangge, H., 2014. Interleukin 10-coated nanoparticle systems compared for molecular imaging of atherosclerotic lesions. Int. J. Nanomed. 9, 4211–4222. Almer, G., Wernig, K., Saba-Lepek, M., Haj-Yahya, S., Rattenberger, J., Wagner, J., Gradauer, K., Frascione, D., Pabst, G., Leitinger, G., Mangge, H., Zimmer, A., Prassl, R., 2011. Adiponectin-coated nanoparticles for enhanced imaging of atherosclerotic plaques. Int. J. Nanomed. 6, 1279–1290. Apaolaza, P.S., Delgado, D., Pozo-Rodríguez, A.d., Gascón, A.R., Solinís, M.Á., 2014. A novel gene therapy vector based on hyaluronic acid and solid lipid nanoparticles for ocular diseases. Int. J. Pharm. 465, 413–426. Balhorn, R., 2007. The protamine family of sperm nuclear proteins. Genome Biol. 8, 227. Bauer, S., Kirschning, C.J., Hacker, H., Redecke, V., Hausmann, S., Akira, S., Wagner, H., Lipford, G.B., 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. U.S.A. 98, 9237– 9242. Bertling, W.M., Gareis, M., Paspaleeva, V., Zimmer, A., Kreuter, J., Nurnberg, E., Harrer, P., 1991. Use of liposomes, viral capsids, and nanoparticles as DNA carriers. Biotechnol. Appl. Biochem. 13, 390–405. Bianchi, F., Rousseaux-Prevost, R., Bailly, C., Rousseaux, J., 1994. Interaction of human P1 and P2 protamines with DNA. Biochem. Biophys. Res. Commun. 201, 1197–1204. Braun, H., Boller, K., Lower, J., Bertling, W.M., Zimmer, A., 1999. Oligonucleotide and plasmid DNA packaging into polyoma VP1 virus-like particles expressed in Escherichia coli. Biotechnol. Appl. Biochem. 29 (Pt 1), 31–43. Chang, C.-H., Paul, B., Remcho, V., Atre, S., Hutchison, J., 2008. Synthesis and postprocessing of nanomaterials using microreaction technology. J. Nanopart. Res. 10, 965–980. Chen, Y., Bathula, S.R., Yang, Q., Huang, L., 2010. Targeted nanoparticles deliver siRNA to melanoma. J. Invest. Dermatol. 130, 2790–2798. Chono, S., Li, S.D., Conwell, C.C., Huang, L., 2008. An efficient and low immunostimulatory nanoparticle formulation for systemic siRNA delivery to the tumor. J. Controlled Release: Official J. Controlled Release Soc. 131, 64–69. Chu, R.S., Targoni, O.S., Krieg, A.M., Lehmann, P.V., Harding, C.V., 1997. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. J. Exp. Med. 186, 1623–1631. Delgado, D., del Pozo-Rodriguez, A., Solinis, M.A., Aviles-Triqueros, M., Weber, B.H., Fernandez, E., Gascon, A.R., 2012. Dextran and protamine-based solid lipid nanoparticles as potential vectors for the treatment of X-linked juvenile retinoschisis. Hum. Gene Ther. 23, 345–355. Dias, N., Stein, C.A., 2002. Antisense oligonucleotides: basic concepts and mechanisms. Mol. Cancer Ther. 1, 347–355. Dinauer, N., Lochmann, D., Demirhan, I., Bouazzaoui, A., Zimmer, A., Chandra, A., Kreuter, J., von Briesen, H., 2004. Intracellular tracking of protamine/antisense

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oligonucleotide nanoparticles and their inhibitory effect on HIV-1 transactivation. J. Controlled Release: Official J. Controlled Release Soc. 96, 497–507. DPMAregister, 1999. Available from: (Accessed 01.12.15). Dul, M., Paluch, K.J., Kelly, H., Healy, A.M., Sasse, A., Tajber, L., 2015. Self-assembled carrageenan/protamine polyelectrolyte nanoplexes—investigation of critical parameters governing their formation and characteristics. Carbohydr. Polym. 123, 339–349. Eitzlmayr, A., Petschacher, C., Radl, S., Suzzi, D., Zimmer, A., Khinast, J.G., 2011. Modeling and simulation of polyacrylic acid/protamine nanoparticle precipitation. Soft Matter 7, 9484–9497. Faneca, H., Simões, S., Pedroso de Lima, M.C., 2004. Association of albumin or protamine to lipoplexes: enhancement of transfection and resistance to serum. J. Gene Med. 6, 681–692. Fischer, D., Bieber, T., Brüsselbach, S., Elsässer, H.-P., Kissel, T., 2001. Cationized human serum albumin as a non-viral vector system for gene delivery? characterization of complex formation with plasmid DNA and transfection efficiency. Int. J. Pharm. 225, 97–111. Gao, X., Huang, L., 1996. Potentiation of cationic liposome-mediated gene delivery by polycations . Biochemistry 35, 1027–1036. Hartmann, G., Battiany, J., Poeck, H., Wagner, M., Kerkmann, M., Lubenow, N., Rothenfusser, S., Endres, S., 2003. Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-a induction in plasmacytoid dendritic cells. Eur. J. Immunol. 33, 1633–1641. He, H., Ye, J., Liu, E., Liang, Q., Liu, Q., Yang, V.C., 2014. Low molecular weight protamine (LMWP): a nontoxic protamine substitute and an effective cellpenetrating peptide. J. Controlled Release: Official J. Controlled Release Soc. 193C, 63–73. Hung, L.H., Lee, A.P., 2007. Microfluidic devices for the synthesis of nanoparticles and biomaterials. J. Med. Biol. Eng. 27, 1–6. Junghans, M., Kreuter, J., Zimmer, A., 2000a. Antisense delivery using protamineoligonucleotide particles. Nucleic Acids Res. 28, E45. Junghans, M., Kreuter, J., Zimmer, A., 2000. Synthetic nucleic acid particle. WO2000036131 A1. Junghans, M., Kreuter, J., Zimmer, A., 2001. Phosphodiester and phosphorothioate oligonucleotide condensation and preparation of antisense nanoparticles. Biochim. Biophys. Acta 1544, 177–188. Junghans, M., Loitsch, S.M., Steiniger, S.C.J., Kreuter, J., Zimmer, A., 2005. Cationic lipid-protamine-DNA (LPD) complexes for delivery of antisense c-myc oligonucleotides. Eur. J. Pharm. Biopharm. 60, 287–294. Kerkmann, M., Lochmann, D., Weyermann, J., Marschner, A., Poeck, H., Wagner, M., Battiany, J., Zimmer, A., Endres, S., Hartmann, G., 2006. Immunostimulatory properties of CpG-oligonucleotides are enhanced by the use of protamine nanoparticles. Oligonucleotides 16, 313–322. Kratzer, I., Wernig, K., Panzenboeck, U., Bernhart, E., Reicher, H., Wronski, R., Windisch, M., Hammer, A., Malle, E., Zimmer, A., Sattler, W., 2007. Apolipoprotein A-I coating of protamine-oligonucleotide nanoparticles increases particle uptake and transcytosis in an in vitro model of the bloodbrain barrier. J. Control. Release 117, 301–311. Kreuter, J., Shamenkov, D., Petrov, V., Ramge, P., Cychutek, K., Koch-Brandt, C., Alyautdin, R., 2002. Apolipoprotein-mediated transport of nanoparticle-bound drugs across the blood-brain barrier. J. Drug Target. 10, 317–325. Krieg, A.M., Yi, A.K., Matson, S., Waldschmidt, T.J., Bishop, G.A., Teasdale, R., Koretzky, G.A., Klinman, D.M., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546–549. Lemaitre, M., Bayard, B., Lebleu, B., 1987. Specific antiviral activity of a poly(Llysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site. Proc. Natl. Acad. Sci. U.S.A. 84, 648–652. Levin, A.A., 1999. A review of issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides. Biochim. Biophys. Acta (BBA) – Gene Struct. Expression 1489, 69–84. Li, S., Rizzo, M.A., Bhattacharya, S., Huang, L., 1998. Characterization of cationic lipid-protamine-DNA (LPD) complexes for intravenous gene delivery. Gene Ther. 5, 930–937. Lindgren, M., Hallbrink, M., Prochiantz, A., Langel, U., 2000. Cell-penetrating peptides. Trends Pharmacol. Sci. 21, 99–103. Lochmann, D., Vogel, V., Weyermann, J., Dinauer, N., von Briesen, H., Kreuter, J., Schubert, D., Zimmer, A., 2004. Physicochemical characterization of protaminephosphorothioate nanoparticles. J. Microencapsul. 21, 625–641. Lochmann, D., Weyermann, J., Georgens, C., Prassl, R., Zimmer, A., 2005. Albuminprotamine-oligonucleotide nanoparticles as a new antisense delivery system. Part 1: physicochemical characterization. Eur. J. Pharm. Biopharm. 59, 419–429.

Mangge, H., Almer, G., Stelzer, I., Reininghaus, E., Prassl, R., 2014. Laboratory medicine for molecular imaging of atherosclerosis. Clin. Chim. Acta; Int. J. Clin. Chem. 437, 19–24. Martinez Gomez, J.M., Fischer, S., Csaba, N., Kundig, T.M., Merkle, H.P., Gander, B., Johansen, P., 2007. A protective allergy vaccine based on CpG- and protaminecontaining PLGA microparticles. Pharm. Res. 24, 1927–1935. Mayer, G., Vogel, V., Weyermann, J., Lochmann, D., van den Broek, J.A., Tziatzios, C., Haase, W., Wouters, D., Schubert, U.S., Zimmer, A., Kreuter, J., Schubert, D., 2005. Oligonucleotide-protamine-albumin nanoparticles: protamine sulfate causes drastic size reduction. J. Control. Release 106, 181–187. Miescher, F., 1874. Das Protamin, eine neue organische Base aus den Samenfäden des Rheinlachses. Ber. Dtsch. Chem. Ges. 7, 376–379. Mitchell, D.J., Kim, D.T., Steinman, L., Fathman, C.G., Rothbard, J.B., 2000. Polyarginine enters cells more efficiently than other polycationic homopolymers. J. Pept. Res.: Official J. Am. Pept. Soc. 56, 318–325. Ortner, A., Wernig, K., Kaisler, R., Edetsberger, M., Hajos, F., Kohler, G., Mosgoeller, W., Zimmer, A., 2010. VPAC receptor mediated tumor cell targeting by protamine based nanoparticles. J. Drug Target. 18, 457–467. Owens, D.R., 2011. Insulin preparations with prolonged effect. Diabetes Technol. Ther. 13 (Suppl 1), S5–14. Pali-Scholl, I., Szollosi, H., Starkl, P., Scheicher, B., Stremnitzer, C., Hofmeister, A., Roth-Walter, F., Lukschal, A., Diesner, S.C., Zimmer, A., Jensen-Jarolim, E., 2013. Protamine nanoparticles with CpG-oligodeoxynucleotide prevent an allergeninduced Th2-response in BALB/c mice. Eur. J. Pharm. Biopharm. 85, 656–664. Petschacher, C., Eitzlmayr, A., Besenhard, M., Wagner, J., Barthelmes, J., BernkopSchnurch, A., Khinast, J.G., Zimmer, A., 2013. Thinking continuously: a microreactor for the production and scale-up of biodegradable, selfassembled nanoparticles. Polym. Chem. 4, 2342–2352. Raukas, E., Mikelsaar, R.H., 1999. Are there molecules of nucleoprotamine? BioEssays: News Rev. Mol. Cell. Dev. Biol. 21, 440–448. Reischl, D., Zimmer, A., 2009. Drug delivery of siRNA therapeutics: potentials and limits of nanosystems. Nanomed. Nanotechnol. Biol. Med. 5, 8–20. Rocha, V.Z., Libby, P., 2009. Obesity, inflammation, and atherosclerosis. Nat. Rev. Cardiol. 6, 399–409. Simoes, S., Slepushkin, V., Pires, P., Gaspar, R., Pedroso de Lima, M.C., Duzgunes, N., 2000. Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum. Biochim. Biophys. Acta 1463, 459–469. Sorgi, F.L., Bhattacharya, S., Huang, L., 1997. Protamine sulfate enhances lipidmediated gene transfer. Gene Ther. 4, 961–968. Stewart, K.M., Horton, K.L., Kelley, S.O., 2008. Cell-penetrating peptides as delivery vehicles for biology and medicine. Org. Biomol. Chem. 6, 2242–2255. Umerska, A., Paluch, K.J., Martinez, M.-J.S., Corrigan, O.I., Medina, C., Tajber, L., 2014. Self-assembled hyaluronate/protamine polyelectrolyte nanoplexes: synthesis, stability, biocompatibility and potential use as peptide carriers. J. Biomed. Nanotechnol. 10, 3658–3673. Vogel, V., Lochmann, D., Weyermann, J., Mayer, G., Tziatzios, C., van den Broek, J.A., Haase, W., Wouters, D., Schubert, U.S., Kreuter, J., Zimmer, A., Schubert, D., 2005. Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties, and intracellular’ distribution. J. Control. Release 103, 99–111. Wang, Y., Xu, Z., Guo, S., Zhang, L., Sharma, A., Robertson, G.P., Huang, L., 2013. Intravenous delivery of siRNA targeting CD47 effectively inhibits melanoma tumor growth and lung metastasis. Mol. Ther. 21, 1919–1929. Wartlick, H., Spankuch-Schmitt, B., Strebhardt, K., Kreuter, J., Langer, K., 2004. Tumour cell delivery of antisense oligonuclceotides by human serum albumin nanoparticles. J. Controlled Release: Official J. Controlled Release Soc. 96, 483– 495. Wernig, K., Griesbacher, M., Andreae, F., Hajos, F., Wagner, J., Mosgoeller, W., Zimmer, A., 2008. Depot formulation of vasoactive intestinal peptide by protamine-based biodegradable nanoparticles. J. Control. Release 130, 192–198. Weyermann, J., Lochmann, D., Georgens, C., Zimmer, A., 2005. Albumin-protamineoligonucleotide-nanoparticles as a new antisense delivery system. Part 2: cellular uptake and effect. Eur. J. Pharm. Biopharm. 59, 431–438. Weyermann, J., Lochmann, D., Zimmer, A., 2004. Comparison of antisense oligonucleotide drug delivery systems. J. Control. Release 100, 411–423. Xia, H., Gao, X., Gu, G., Liu, Z., Zeng, N., Hu, Q., Song, Q., Yao, L., Pang, Z., Jiang, X., Chen, J., Chen, H., 2011. Low molecular weight protamine-functionalized nanoparticles for drug delivery to the brain after intranasal administration. Biomaterials 32, 9888–9898. Xu, Z., Wang, Y., Zhang, L., Huang, L., 2014. Nanoparticle-delivered transforming growth factor-b siRNA enhances vaccination against advanced melanoma by modifying tumor microenvironment. ACS Nano 8, 3636–3645. Zimmer, A., Atmaca-Abdel Aziz, S., Gilbert, M., Werner, D., Noe, C.R., 1999. Synthesis of cholesterol modified cationic lipids for liposomal drug delivery of antisense oligonucleotides. Eur. J. Pharm. Biopharm. 47, 175–178.

Please cite this article in press as: Scheicher, B., et al. Protamine–oligonucleotide-nanoparticles: Recent advances in drug delivery and drug targeting. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.04.009

Protamine-oligonucleotide-nanoparticles: Recent advances in drug delivery and drug targeting.

Application of oligonucleotides as active compounds has become a crucial field of pharmaceutical research in recent years. In order to improve inadequ...
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