Use of Protamine in Nanopharmaceuticals—A Review

nanomaterials Review Use of Protamine in Nanopharmaceuticals—A Review Ivana Ruseska †, Katja Fresacher †, Christina Petschacher and Andreas Zimmer * Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmaceutical Sciences, Karl-Franzens-University Graz, Universitätsplatz 1, 8010 Graz, Austria; [email protected] (I.R.); [email protected] (K.F.); [email protected] (C.P.) * Correspondence: [email protected] † Both authors are contributed equally. Abstract: Macromolecular biomolecules are currently dethroning classical small molecule therapeu- tics because of their improved targeting and delivery properties. Protamine-a small polycationic peptide-represents a promising candidate. In nature, it binds and protects DNA against degradation during spermatogenesis due to electrostatic interactions between the negatively charged DNA- phosphate backbone and the positively charged protamine. Researchers are mimicking this technique to develop innovative nanopharmaceutical drug delivery systems, incorporating protamine as a carrier for biologically active components such as DNA or RNA. The first part of this review high- lights ongoing investigations in the field of protamine-associated nanotechnology, discussing the self-assembling manufacturing process and nanoparticle engineering. Immune-modulating properties of protamine are those that lead to the second key part, which is protamine in novel vaccine technologies. Protamine-based RNA delivery systems in vaccines (some belong to the new class of mRNA-vaccines) against infectious disease and their use in cancer treatment are reviewed, and we provide an update on the current state of latest developments with protamine as pharmaceutical Citation: Ruseska, I.; Fresacher, K.; excipient for vaccines. Petschacher, C.; Zimmer, A. Use of Protamine in Nanopharmaceuticals— Keywords: protamine; proticles; nanoparticles; novel vaccine technologies A Review. Nanomaterials 2021, 11, 1508. https://doi.org/10.3390/ nano11061508 Academic Editors: Ruth Prassl, 1. Introduction Angelina Angelova and Horacio Protamines are a group of polycationic peptides present in spermatids of many ani- Cabral mals and plants. Their history started with the discovery of the water-soluble protamine Salmine, extracted from the sperm of salmons in 1874 by Friedrich Miescher. In those Received: 19 April 2021 days, protamines were already recognized to form insoluble salts with nucleic acids in the Accepted: 27 May 2021 sperm [1,2]. Some years later, several other protamines were discovered, like Clupeine in Published: 7 June 2021 the sperm of herrings and Scombrine in the sperm of mackerels [3]. Today, we know that two protamines, P1 and P2, can also be found in mammals. These two are said to be the Publisher’s Note: MDPI stays neutral most studied protamines thus far [4]. with regard to jurisdictional claims in At the end of the 19th century, Albrecht Kossel proved that all protamines have published maps and institutional affil- one thing in common: they are strongly basic proteins, containing a high amount of the iations. amino acid arginine (up to 70%) assembled in clusters [3,5,6]. Most of the protamines show a molecular weight of 4000–5000 Da. They are rather short proteins, comprising 50–110 amino acids and are classified into three groups according to the number of different kinds of basic amino acids they include. Monoprotamines exhibit a very simple amino acid Copyright: © 2021 by the authors. composition incorporating only arginine as basic amino acid. Diprotamines additionally Licensee MDPI, Basel, Switzerland. contain either the basic amino acid lysine or histidine, and triprotamines include all of This article is an open access article the three basic amino acids [6]. The basic amino acid clusters, especially the arginine distributed under the terms and residues, represent the DNA-binding domains of protamines. These enable the formation conditions of the Creative Commons of DNA-protamine-complexes, leading to condensation and stabilization of the spermatid Attribution (CC BY) license (https:// genome. Protamines replace histones in this function during spermatid maturation and creativecommons.org/licenses/by/ protect the DNA from degradation. These DNA-protamine-complexes are held together 4.0/). Nanomaterials 2021, 11, 1508. https://doi.org/10.3390/nano11061508 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021, 11, x FOR PEER REVIEW 2 of 44 by an electrostatic linkage between the negatively charged phosphate ions of the nucleic Nanomaterials 2021, 11, 1508 acids and the cationic arginine moieties of protamine. The complexes are soluble in 2high of 42 salt concentration and show a minimum solubility in isotonic salt solutions [4,6]. At the beginning of this review, information about the structure and function of protamines,by an electrostatic aiming especial linkagely on between the mammalian the negatively protamines charged P1 phosphate and P2 [4,7] ions, is ofsummarized the nucleic andacids protamine and the cationic derivatives arginine like moietiesprotamine of protamine.sulfate [8] and The complexeslow-molecular-weight are soluble inprota- high minesalt concentration (LMWP) [9] are and discussed. show a minimum After providin solubilityg an inintroduction isotonic salt into solutions the nature [4,6]. of protamines,At one the question beginning inevitably of this review, arises: informationwhat are the about main theapplication structure fields and functionof protamines? of pro- Thetamines, primary aiming use especiallyof protamines on the is mammaliansettled in the protamines field of medicine P1 and P2and [4 ,pharmacy,7], is summarized which buildsand protamine the central derivatives focus of this like review. protamine For sulfatemany years, [8] and protamines low-molecular-weight are established protamine as adjuvants(LMWP) in [ 9insulin] are discussed. preparations After to providing prolong their an introduction effect by the into complexation the nature of insulin protamines, due toone electrostatic question inevitably interaction arises: [10,11] what. Additionally, are the main protamines application fieldsare used of protamines? as an antidote The againstprimary the use anticoagulation of protamines iseffect settled of negatively in the field charged of medicine heparin, and pharmacy,again by building which builds com- plexesthe central with focusit [12–15]. of this review. For many years, protamines are established as adjuvants in insulinAfter addressing preparations these to prolong longstanding their effect applicat by theions complexation of protamine of(Table insulin 1), duewe will to elec- fo- custrostatic on the interaction use of protamine [10,11]. as Additionally, part of drug protaminesdelivery systems are used (Figure as an 1). antidote Protamines against are noninvasivethe anticoagulation cell-penetrating effect of pe negativelyptides, showing charged the heparin, ability to again target by drugs building to specific complexes mol- eculeswith it within [12–15 ].the cells [9,16–22]. Their penetration and targeting effect can be further en- hancedAfter by creating addressing innovative, these longstanding nanosized drug applications delivery systems of protamine [23–29]. (Table 1), we will focusThus, on the the use first of part protamine of this review as part will of espe drugcially delivery highlight systems the ongoing (Figure1 research). Protamines in the fieldare noninvasive of protamine-associated cell-penetrating nanotechnology, peptides, showing giving the details ability about to target the drugsself-assembling to specific manufacturingmolecules within processes, the cells the [9, 16properties–22]. Their of penetrationthe resulting and nanoparticles targeting effect and canhow be they further can beenhanced functionalized. by creating innovative, nanosized drug delivery systems [23–29]. FigureFigure 1. ApplicationApplication fields fields of protamine.of protamine. This This review revi willew especially will especially focus on focus protamine on protamine in nanophar- in nanopharmaceuticalsmaceuticals as well as as its well approach as its approach in vaccines. in vaccines. TheThus, second the first key part part ofof thisthis reviewreview willcomprises especially a currently highlight highly the ongoing topical application research in fieldthe field of protamines: of protamine-associated their use as RNA-delivery nanotechnology, systems giving in details vaccines about against the self-assembling infectious dis- easesmanufacturing and in cancer processes, treatment. the propertiesThe outbreak of theof the resulting COVID-19 nanoparticles pandemic and at the how beginning they can ofbe 2020 functionalized. demanded a quick development of vaccines. Today, about one year later, several vaccinesThe against second this key disease part of thisare already review comprisesapproved and a currently on the market. highly topical Some of application them be- longfield to of the protamines: rather new their class use of mRNA-vaccine as RNA-deliverys [30]. systems Due to in the vaccines prevailing against great infectious interest indiseases the subject and inof cancer immunization, treatment. the The second outbreak part of of the this COVID-19 review will pandemic take this at thetopic beginning further of 2020 demanded a quick development of vaccines. Today, about one year later, several vaccines against this disease are already approved and on the market. Some of them belong to the rather new class of

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