Advances on the formulation of proteins using nanotechnologies

Accepted Manuscript Advances on the formulation of proteins using nanotechnologies Irene Santalices, Andrea Gonella, Dolores Torres, María José Alonso PII: S1773-2247(17)30251-4 DOI: 10.1016/j.jddst.2017.06.018 Reference: JDDST 416 To appear in: Journal of Drug Delivery Science and Technology Received Date: 23 March 2017 Revised Date: 22 June 2017 Accepted Date: 23 June 2017 Please cite this article as: I. Santalices, A. Gonella, D. Torres, Marí.José. Alonso, Advances on the formulation of proteins using nanotechnologies, Journal of Drug Delivery Science and Technology (2017), doi: 10.1016/j.jddst.2017.06.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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This manuscript version is made available under the CC-BY-NC-ND 4.0 license (http://creativecommons.org/licenses/by-nc-nd/4.0/) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 Advances on the formulation of proteins 1 using nanotechnologies 2 Irene Santalices a* , Andrea Gonella a* , Dolores Torres b , María José Alonso a,b# 3 4 a Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Campus Vida 5 University of Santiago de Compostela, Santiago de Compostela 15782, Spain. 6 7 b Department of Pharmaceutics and Pharmaceutical Technology, School of Pharmacy, Campus 8 Vida, University of Santiago de Compostela, Santiago de Compostela 15782, Spain. 9 10 *These authors contributed equally to this work. 11 12 # Corresponding author e-mail address: [email protected] 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 2 Table of contents 35 1. Introduction 36 2. Formulation technologies (nanocarriers, preparation techniques, characterization, drug 37 loading and release profile) 38 2.a. Lipid-based nanocarriers 39 2.a.1. Liposomes 40 2.a.2. Solid Lipid Nanoparticles (SLN) 41 2.a.3. Microemulsions and nanoemulsions 42 2.a.4. Nanocapsules (NCs) 43 2.b. Polymer-based nanocarriers 44 2.b.1. Polyesters-based nanocarriers 45 2.b.2. Acrylic polymers-based particles 46 2.b.3. Polysaccharide-based particles: 47 2.b.4. Protein-based particles: 48 3. Current status of peptide/protein-loaded nanotechnologies 49 4. Conclusions 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 3 Abstract 76 Therapeutic proteins and peptides are very attractive from the pharmaceutical point of view due 77 to their high potency and selectivity. Nonetheless, their instability and low bioavailability make 78 their administration through non parenteral routes very difficult, a fact that hampers their 79 efficient exploitation in therapeutics. Since the 70´s, significant amount of research in the area of 80 drug delivery and nanotechnology has been done with the final goal of overcoming those hurdles. 81 In particular, biodegradable and biocompatible lipid and polymer-based nanocarriers have 82 emerged as promising delivery platforms to enable the administration of proteins and peptides. 83 This review provides an overview of the mostly explored nanotechnologies to date intended to 84 produce lipidic and polymeric nanocarriers for protein/peptide delivery. The basic principles of the 85 different techniques are discussed, and the main factors involved in the drug association and 86 release, are analyzed. Finally, a brief overview of the potential applications of these 87 protein/peptide-loaded nanocarriers, highlighting the nanomedicines that have reached the 88 market or the clinical development phase, is provided. 89 90 Keywords: protein delivery, peptide delivery, lipid formulation, polymeric formulation, 91 nanocapsule, nanoparticle, liposome, microemulsion 92 93 Graphical abstract: 94 95 *X-ray structure of Human Recombinant insulin . Image from the RCSB PDB (www.rcsb.org) of PDB ID 5E7W. 96 97 1. Introduction 98 99 During the last decades, important efforts have been oriented to the commercialization of 100 therapeutic proteins and peptides. Unfortunately, despite the well-known advantages of these 101 drugs in terms of potency and selectivity, their exploitation is being limited by their instability, 102 restricted bioavailability and intrinsic immunogenicity (specially for high molecular weight 103 proteins) [1]. These draw-backs have stimulated the research in the area of drug delivery and 104 nanotechnology with the final goal of making the administration of these powerful drugs more 105 efficient [2–4]. 106 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 4 107 The possibility of including peptides and proteins in nanovehicles that are able to protect and 108 deliver them at the adequate site has generated increasing expectation during last decades (Fig. 1) 109 [5]. Liposomes were the first nanocarriers proposed for protein delivery in the early 70’s [6,7]. 110 Meanwhile, Speiser and co-workers investigated the possibility to encapsulate drugs or antigens 111 into polyacrylic nanoparticles using micelle polymerization techniques [8]. A decade later, 112 poly(alkylcyanoacrylate) nanocapsules were proposed as carriers for the oral administration of 113 insulin [9]. Finally, over the 90’s Gasco et al. produced for the first time peptide-loaded solid lipid 114 nanoparticles [10–12] and our group pioneered the development of nanoparticles made of PLGA 115 [13], PLA-PEG [14] and chitosan [15] for the delivery of proteins and antigens. As illustrated in 116 Figure 1, the interest around the use of all these nanocarriers for protein/peptide delivery has 117 progressively increased in the past decades, being liposomes and polysaccharide-based 118 nanoparticles the ones receiving the greatest attention. Noteworthy, the use of inorganic 119 nanoparticles in the peptide/protein delivery field has grown-up during the past decade, as well. In 120 particular, nanoparticles made of gold, iron oxide [16,17], calcium phosphate and silica likewise 121 carbon nanotubes [18,19] have received a certain attention. However, overall, the tendency has 122 been towards the use of biodegradable and biocompatible biomaterials that can form 123 nanostructures based on friendly and easily scalable techniques. This tendency is expected to 124 change the translational prospective of these delivery vehicles. Indeed, still nowadays the 125 development of efficacious and cost–effective nano-based protein products remains a challenge 126 and this justifies the limited number of protein/peptide-loaded nanoparticulate products in the 127 market [20–22]. The necessity for these nanomedicines to exhibit important quality attributes 128 such as significant drug loading, maintenance of the loaded peptide/protein activity and controlled 129 drug release, are just some examples of the bottlenecks to be overcame [23]. 130 131 Figure 1. Trend of reported experimental works concerning nanocarriers for peptide/protein delivery from the 70’s up to 132 date. Data taken from Scopus (1971–2017) using protein/peptide delivery and the type of system as searching criteria. 133 ME: microemulsion; NCs: nanocapsules; NE: nanoemulsion; NPs: nanoparticles; SEDDS: self-emulsified drug delivery 134 system; SMEDDS: self-microemulsified drug delivery system; SNEDDS: self-nanoemulsified drug delivery system; SLN: 135 solid lipid nanoparticles. 136 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 5 137 This review aims to analyze the main technologies employed until today to produce lipid and 138 polymer-based nanoparticulate carriers for peptide/protein delivery. Additionally, a brief overview 139 of state of the art of the protein loading, protein structural stability and release properties from 140 these nanocarriers, as well as, their final applications, is discussed. The analysis of the technologies 141 to produce inorganic particles and their characterization was considered to be beyond the scope 142 of this review. 143 144 2. Formulation technologies 145 146 2.a. Lipid-based nanocarriers 147 148 In the last decades, lipid-based nanocarriers (Fig. 2) have emerged as potential nanocarriers for 149 macromolecular delivery. This has been mainly due to the absorption enhancing properties of the 150 lipids and the nanocarrier’s ability to improve the drug stability. Furthermore, the biocompatible 151 character of these biomaterials and the low cost of the production techniques have increased the 152 interest in these nanocarriers [24]. Despite of this, the inclusion of hydrophilic macromolecules 153 into these systems has been so far limited by their solubility. In order to improve their 154 incorporation into these systems, many innovative strategies, which are summarized below, have 155 been described as promising approaches for the formulation of peptide lipid-based delivery 156 nanosystems (Fig. 3). 157 158 Figure 2. Illustration of the main lipid-based nanosystems explored for protein/peptide delivery. Adapted with permission 159 from [25]. 160 161 - Reverse micellization. This strategy involves the use of amphiphilic molecules able to self-162 organize as reverse micelles exposing their hydrophobic chains to the exterior and their 163 hydrophilic head groups to the inner part of the structure [26]. This inner cavity facilitates the 164 incorporation of hydrophilic macromolecules prior to its inclusion in the final system [27]. 165 166 - Double emulsion method. This technique consists on the formation of a W/O emulsion in which 167 the hydrophilic drug is confined within its internal aqueous phase prior to its inclusion in the final 168 system [28–30]. 169 170 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 6 - Hydrophobic ion paring. This approach has been used to enhance the hydrophobicity of the 171 drug, thereby improving its lipid solubility. It is based on the ionic complexation of a 172 peptide/protein with a molecule, often an amphiphilic compound, with an opposite surface charge 173 [11,31,32] or even with complex structures such as liposomes [33]. 174 175 - Hydrophobic – hydrophilic interactions. This approach involves the dispersion of an aqueous 176 solution of the hydrophilic drug into an amphiphilic compound, followed by the addition of the 177 formed dispersion into the oily phase [34,35]. 178 179 180 Figure 3. Illustration of the main strategies employed to improve the incorporation of hydrophilic macromolecules into 181 lipid-based delivery nanosystems. Adapted with permission from [25]. 182 183 2.a.1. Liposomes 184 185 Since their discovery in 1964 [36,37], liposomes have been the most extensively drug delivery 186 vehicles investigated. To date, 13 liposome-based products have been approved for human use by 187 the FDA [25]. Briefly, liposomes are defined as vesicles with an aqueous core in the inner cavity, 188 surrounded by one or more bilayers of amphiphilic phospholipids. Their sizes range from 20 nm (if 189 unilamellar) up to microns (if multilamellar) [38]. Among the wide variety of lipids, those 190 amphiphilic able to self-assembly, such as phospholipids, phosphatidylglycerol derivatives and 191 both saturated and unsaturated fatty acids, are the most commonly used for producing liposomes 192 [25]. Additionally, it is also possible the inclusion of polymers and surfactants into their structure 193 [39–42]. Finally, the use of special lipids has led to the formation of nanostructures named as 194 archeosomes (i.e., diether or tetraether lipids) [43] and niosomes (i.e., polyoxyethylene alkyl 195 ethers) [44], which were supposed to facilitate the entrapment of peptides and proteins [45]. 196 197 2.a.1.a. Preparation techniques 198 199 Overall, the technologies to prepare liposomes are relatively similar. The main difference among 200 the variety of techniques described so far relies on the way of drying the lipids from the organic 201 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 7 solvents and rehydrating them in aqueous media [46]. The main liposomes preparation methods 202 used for protein/peptide association are those described below. 203 204 i) Film hydration 205 This technique was introduced by Bangham and coworkers to produce liposomes by the first time 206 (Fig. 4) [36,37]. This technique involves the dissolution of the phospholipids in an organic solvent, 207 followed by the solvent evaporation and the deposition of the phospholipids forming a lipid film. 208 Then, an aqueous solution containing the protein is added over the lipidic film to hydrate it, 209 usually with the help of sonication, thus leading to the formation of liposomes [47,48]. 210 211 212 Figure 4. Schematic view of the film hydration technique to produce liposomes 213 214 ii) Reverse-phase evaporation 215 This technique simply involves the formation of reverse micelles by mixing an organic solution of 216 the phospholipids with a small volume of an aqueous phase containing the peptide/protein, 217 usually using sonication. The evaporation of the solvent results in the formation of large 218 unilamellar or multilamellar liposomes (Fig. 5) [49]. 219 220 221 222 Figure 5. Schematic view of the reverse-phase evaporation technique to produce liposomes 223 224 2.a.1.b. Characterization, peptide/protein loading, activity and release profile 225 226 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 8 - Particle size distribution: generally, a homogenization step is necessary in order to obtain a 227 narrow particle size distribution. The homogenization of the system can be achieved using 228 extrusion [50,51], freeze-thawing [52,53], dehydration-rehydration [54,55], sonication or high 229 pressure. Likewise, the ratio between the different components will influence the final liposomes 230 particle size distribution. 231 232 - Peptide/protein loading and activity: liposomes have the ability to encapsulate hydrophilic (in 233 the inner aqueous core), lipophilic (within the lipid bilayer) or amphiphilic drugs (partitioned 234 between the lipid bilayer and the aqueous core) [56]. In general, the driving force for the 235 encapsulation relies on the interaction between the protein/peptide and the lipids and also on the 236 bilayer rigidity. For example, liposomes with insulin association efficiency (AE) values varying from 237 10 up to 90 % could be obtained by changing the phosphatidylcholine:phosphatidylethanol ratio 238 [57]. To date, a variety of peptides and proteins have been efficiently entrapped into liposomes 239 using the preparation methods disclosed in Table 1. Unfortunately, the loading capacity of the 240 resulting formulations has not been described or has been low (< 1 %) [58]. Therefore, the loading 241 capacity could be considered as a limitation of these delivery carriers. 242 243 Additionally, the loaded protein must remain active once encapsulated into the liposomes. The 244 sources of peptide/protein instability differ depending on the production method considered, 245 being the film hydration the less stressful for the integrity of the protein, even if sonication could 246 affect its structure [59]. On the other hand, the reverse phase evaporation technique directly 247 exposes the peptide/protein to organic solvents, with the subsequent possibility of suffering 248 denaturation [59,60]. Homogenization, extrusion and freeze thaw cycles can also cause protein 249 denaturation/aggregation in both methods [59]. The integrity of the loaded peptide/protein has 250 been studied using different methods, such as electrophoresis-based techniques (e.g. Western 251 blot, SDS-PAGE, etc.), protein activity (particularly if the encapsulated protein is an enzyme) or 252 directly through in vivo experiments [48,57,59,61]. 253 254 - Peptide/protein release: the physicochemical properties of the phospholipids are known to 255 determine the fluidity of the lipid bilayer and, as a consequence, influence the peptide/protein 256 release profile. In this sense, a more sustained release is obtained when increasing the rigidity of 257 the bilayer by the inclusion of cholesterol or long hydrophobic chains in the liposome [62]. 258 Strategies to control the release of peptides/proteins from liposomes, such as the surface 259 modification with polyethylene glycol (PEG) or other polymers, as well as their inclusion in other 260 nanostructures, have been developed [58,63–66]. For example, a lower insulin release was 261 showed after 4 hours in simulated intestinal fluids from layer-by-layer coated liposomes (20 %) 262 compared to those uncoated (60 %) [48]. 263 264 In conclusion, both film hydration and reverse phase evaporation methods are suitable for 265 encapsulating peptides/proteins in liposomes, allowing both, good association efficiencies and 266 sustained release profiles. The bilayer rigidity and the electrostatic interactions between the 267 peptide and the liposomes components are the main factors conditioning the loading capacity of 268 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 15 As indicated, the protein/peptide stability is generally influenced by the presence of organic 416 solvents (e.g. double emulsion/solvent evaporation or nanoprecipitation methods), high shear 417 mechanical agitation and pressure (e.g. HPH method), high temperatures (e.g. 418 microemulsification/solidification and HPH methods) or sonication processes (double 419 emulsion/solvent evaporation method) [11,29,30,88]. The protein integrity and activity has been 420 usually analyzed using the same techniques described for liposomes in the previous 2.a.1.b. 421 section (i.e., SDS-PAGE, capillary electrophoresis, enzymatic assays and in vivo studies) [11,28–422 30,76]. 423 424 - Peptide/protein release: data indicated in Table 3 highlight the high variability in the release 425 profiles observed for different peptides/proteins entrapped in a variety of SLN [28,78,79]. 426 Although in some works, no burst release was reported, normally there is a variable amount of 427 peptide accumulated at the O/W interface during the production process that is released 428 prematurely [72]. This burst effect and the subsequent release profile has been modulated 429 following specific formulation approaches. In particular, the overall release profile is highly 430 dependent on the SLN composition, since it is mainly governed by the peptide diffusion through 431 the channels, originally present in the matrix, and enlarged in the course of the lipase-mediated 432 lipids degradation [78]. These findings suggest that a selection of the lipidic components is 433 important in order to modulate the protein/peptide release. 434 435 It is important to highlight the possibility of an interaction between the peptide/protein and the 436 lipid components and their degradation products. For example, in a work intended to encapsulate 437 leuprolide acetate into SLN using the nanoprecipitation technique, the use of a hydrophobic ion 438 pairing complex between leuprolide and sodium stearate led to a considerable reduction of the 439 burst effect (1 h burst release: 10 % vs. 45 %). Following this initial fast release, the peptide was 440 slowly released for up to 2 days [80]. In another example the sustained release of sCT (40 - 45 % in 441 6 h) from chitosan-coated SLN produced by the double emulsion-solvent evaporation method was 442 attributed to the high affinity of the positively charged sCT for the negatively charged lipids 443 (lecithin and tripalmitin) [28]. 444 445 The incorporation of PEG into the lipid matrix has also been proposed as a strategy to modulate 446 the release profile. For example, the release of insulin from SLNs produced by the supercritical 447 fluid technology, could be controlled by incorporating 5 kDa PEG in the lipid mixture as a pore-448 forming agent [89]. Indeed, the total amount of insulin associated to PEG-containing SLNs was 449 released in 3 days, whereas PEG-free SLNs needed 5 days to deliver their content. 450 451 From the results in literature up to date (Table 3 shows some examples), we can conclude that the 452 release of peptides from SLN is affected by the composition of the lipidic matrix (governing the 453 degradation of the particles) and by the affinity of the peptide/protein towards the formulation 454 components. Normally, the in vitro release of the proteins/peptides is prolonged for a few days, 455 however, it could be expected that in an in vivo situation the process could be accelerated 456 depending on the degradation rate of the lipidic matrix. 457 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 16 458 Table 3. Examples of peptide/protein-loaded SLN obtained through the different preparation methods: drug loading and 459 release properties. 460 Preparation method Specific strategy Peptide/ Protein AE (%) LC (%) ≤1h burst / cumulative release (time) – pH medium Ref. Microemulsion- based technique - CyA n.a. 6 - 13 <4 % / <4 % (2 h) pH 7.4 [ 84] Double emulsion [D-Trp-6] LHRH 90 n.a. <3 % / 10 % (8 h) pH 6.5 [10] Thymopentin 2 n.a. <5 % / 10 % (6 h) pH 6.5 [11] Hydrophobic ion pairing Thymo - pentin 5 n.a. <5 % / 10 % (6 h) pH 6.5 Hot HPH - CyA 95 - 98 0.5 - 2 theor. n.a. [75] 96 1.9 n.a. [90] Cold HPH - CyA 79 - 94 0.5 - 2 theor. n.a. [75] Lysozyme 43 - 59 0.03 n.a. [76] Emulsion – solvent evaporation Double emulsion sCT 31->90 n.a. <30 % / <45 % (6 h) pH 4 [28, 29] Double emulsion / Reverse micellization sCT 88 - 95 5 - 11 60 - 100 % / 100 % (2 h) pH 6.8* [86] Insulin 76-100 19 0 - 35 % / 60 - 90 % (6 d) pH 7.4 [87] Nanoprecipitation - Gonado - relin 50 - 69 n.a. <30 % / <80 % (14 d) pH 6.8 [79] Leupro - lide 28 0.3 <45 % / 100 % (2 d) pH 6.8 [80] Hydrophobic ion pairing Leupro - lide 46 0.5 <10 % / 100 % (2 d) pH 6.8 Supercritical fluid technology - Insulin 20 - 80 1 - 4 0 - 17 % / 100 % (6 d) pH 7.4 [89] 57 2.9 <10 % / 100 % (4 d) pH 7.4 [91] rh-GH 48 2.4 <5 % / 100 % (4 d) pH 7.4 AE: association efficiency (100 x associated peptide mass / total peptide mass); CyA: cyclosporine A; [D-Trp-6] LHRH: 461 agonist triptorelin - luteinizing hormone-releasing hormone; HPH: high pressure homogenization; LC: loading capacity 462 (100 x peptide mass / total formulation mass); n.a.: not applicable; Ref.: references; rh-GH: recombinant human growth 463 hormone; sCT: salmon calcitonin; theor.: theoretical; *Enzyme supplemented. 464 465 2.a.3. Microemulsions and Nanoemulsions 466 467 Both, water-in-oil (W/O) and oil-in-water (O/W) microemulsions are usually considered as 468 thermodynamically stable and isotropic systems, displaying sizes below 100 nm. The 469 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 17 microemulsion formation has been described as a spontaneous process that occurs after mixing 470 the oil and the water phases containing a certain amount of surfactants, in order to achieve a low 471 interfacial tension between the two phases [92]. Nanoemulsions have also been described as 472 colloidal dispersions that generally display sizes below 200 nm. In contrast with microemulsions, 473 these systems are not isotropic. The nanoemulsion formation requires an external energy input in 474 order to overcome their positive free energy and increase their contact area, leading to the 475 formation of a kinetically stable colloidal dispersion [93]. A special type of emulsions is the one 476 present in the self-emulsifying drug delivery systems (SEDDS) and the self-micro-emulsifying drug 477 delivery systems (SMEDDS) which typically consist of mixtures of oil, surfactant and co-surfactants. 478 Recently, many of these SMEDDS have been classified as self-nanoemulsifying systems (SNEDDS) 479 [94]. Among the wide variety of lipids, the long and medium chain glycerides and fatty acids are 480 the most commonly used for the preparation of self-emulsifying systems, microemulsions and 481 nanoemulsions containing peptides [25]. Medium chain fatty acids are known to improve the 482 peptide solubility and facilitate the emulsification process since their mixture with the aqueous 483 phase is easier. 484 485 2.a.3.a. Preparation techniques 486 487 A wide variety of methods have been developed to produce micro/nanoemulsions. These 488 techniques can be classified depending on the procedure used to supply energy to the system [95–489 97], being broadly categorized into the following two groups: i) High-energy processes, which 490 imply the application of mechanical and intensive disruptive forces to the different phases of the 491 system. Special devices are necessary in order to intermingle the oily and the aqueous phases, 492 leading to the formation of nanodroplets (homogenization, microfluidization and ultrasonication) 493 [98–100]; ii) Low-energy processes (spontaneous emulsification and phase inversion), which are 494 based on the spontaneous formation of nanoemulsions either by changing the composition (i.e., 495 ratio surfactant:oil:water, addition of salts, etc.) or the process conditions (i.e., temperature-time 496 profile, stirring, addition speed, etc.) [97,101–104]. Among the wide variety of techniques, those 497 based on the spontaneous emulsification are, so far, the most commonly used for the association 498 of peptides/proteins. This is mainly due to the fact that this method avoids the peptides/proteins 499 being exposed to any temperature or pressure stress. 500 501 i) Spontaneous Emulsification. 502 Through this method, the nanoemulsion is spontaneously formed upon the mixture of the oily and 503 the aqueous phases (Fig. 11) [105,106]. The protein/peptide is included in one of them depending 504 on its hydrophilicity or incorporated into the oily phase in a small amount of water. Both phases 505 are immiscible in each other; however, one of the components present in one of them (i.e., an 506 organic solvent, a surfactant) is partially miscible in both. Once the two phases are in contact, a 507 non-equilibrium state is formed, causing the rapid shifting of the miscible component from its 508 original phase into the other. This fact will lead to an increase in the oil-water interfacial area and 509 turbulence, promoting the spontaneous formation of the nanoemulsion [107]. 510 511 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 18 512 Figure 11. Schematic view of the spontaneous emulsification technique to produce nanoemulsions 513 514 2.a.3.b. Characterization, peptide/protein loading, activity and release profile 515 516 - Particle size distribution: by selecting appropriately the ingredients and the preparation method, 517 emulsions showing a wide range of sizes, charges and physical properties can be obtained. The 518 final size distribution of the emulsion can be modulated by optimizing its composition 519 (concentration of the components, ratio surfactant:oil:water, interfacial tension, viscosity, 520 emulsifier adsorption kinetics, etc.) and the operating conditions (temperature-time profile, 521 stirring rate, pressure, amplitude of sonication and number of cycles, etc.) [108–111]. The use of 522 ternary phase diagrams is an useful tool to predict the optimum conditions for the formation of 523 the nanoemulsion [112,113]. 524 525 - Peptide/protein loading and activity: the combination of the spontaneous emulsification 526 technique with several specific strategies, such as, double emulsification, reverse micellization, 527 hydrophobic ion paring or peptide-lipid/surfactant interaction (section 2.a.) has been effective for 528 the loading of hydrophilic peptides (Table 4) such as insulin, with AEs higher than 85 % 529 [27,35,114,115]. Among the factors influencing this association, it has been found that small 530 variations in the final pH (from 6.5 to 6.8), may lead to sharp decreases in the AEs from 79 to 30 %. 531 This result was attributed to the different ionization degree of both, the peptide and the polymer 532 at the selected pHs, and their electrostatic and/or hydrophobic interactions [116]. Despite the 533 good association efficiencies achieved, the loading capacity of these systems is usually lower than 534 1 % [31,117]. 535 536 After system preparation, the loaded peptide/protein must be able to keep its activity. In fact, 537 there are some operation conditions, i.e. the use of organic solvents and surfactants, which can 538 lead to protein denaturation and/or aggregation. High shear agitations, temperatures or pressures 539 can affect the integrity of the protein, as well [31,118,119]. In this regard, the use of ELISA assays 540 has been reported as an efficient method to understand if the activity of the encapsulated protein 541 is kept. However, in the specific case of micro- and nanoemulsions, direct in vivo evaluation of the 542 formulation is the main approach reported to evaluate the efficacy of the loaded therapeutic 543 agent [118,120]. 544 545 - Peptide/protein release: only a few papers have been published dealing with the mechanism 546 behind the release of the protein/peptide drugs from micro/nanoemulsions. In general, the 547 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 19 release of the drug has been related to its partition between the emulsion and the surrounding 548 medium and also to the alteration/degradation of the lipidic components. For example, when they 549 are orally administrated, their contact with the gastrointestinal fluids can cause a phase inversion 550 or separation of the emulsion phases, that may lead to a premature drug release [120,121]. The 551 conversion of these liquid systems into solid forms through freeze drying, spray drying, melt 552 granulation, melt extrusion or adsorption over solid carriers has been proposed as a way to 553 overcome the colloidal instability of these systems [122]. Further improvements of this technology 554 in order to optimize the delivery of hydrophilic drugs from self-emulsifying systems are still 555 needed. However, for lipophilic peptides, some formulations, such as Neoral® (SMEDDS containing 556 cyclosporine) have already been marketed [1]. 557 558 Table 4. Examples of peptide/protein-loaded micro/nanoemulsions and SEDDS/SMEDDS/SNEDDS obtained through the 559 spontaneous emulsification method: drug loading and release properties. 560 System Specific strategy Peptide/ Protein AE (%) LC (%) ≤1h burst/ cumulative release (time) – pH medium Ref. O/W - sCT > 90 n.a. n.a. [119, 123] Pliti - depsin 95-98 0.54 n.a. [124] W/O - TAT 97 0.006 TAMRATAT 90 % (1 h) pH 6.8* [120] rhPTH1 - 34 83 n.a. (45 mg/mL) 100 % (50 min) pH 8* 65 % / 80 % (2 h) pH 2* [125] Insulin + aprotinin 97 0.1 (30 IU/g) 0 % (1 h) pH 1.2* [118] Reverse micelles Insulin > 85 n.a. (2.2 % w/v theor.) n.a. [27] Hydrophobic ion pairing Insulin 30 - 79 (complexation) n.a. <10 % (1 h) pH 1.2* [116] W/O/W Double emulsion Insulin 96 - 97 n.a. (18 IU/g) 0 - 80 % / 0 - 80 % (1.5 h) pH 7 [114] Insulin + aprotinin 88 - 97 0.075 20 - 30 % / 20 - 30 % (2 h) pH 7 [115] sCT + aprotinin n.a. n.a. (400 IU/g theor.) 90 % / 100 % ( 2 h ) pH 6.4/1.2* 80 % / 80 % (2 h) pH 7.5* [126] SMEDDS SNEDDS Hydrophobic ion pairing Insulin 64 - 71 0.3 - 1.1 15 % / 30 % (8 h) pH 7.4 [117] Leuprorelin 59 (complexation) 0.4 theor. complex <20 % / 40 % of complex (30 h) pH 6.8 [31] Hydrophilic - hydrophobic Insulin 85 - 99 n.a. 1 % / 14 % (24 h) pH 7.4 [35] MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 20 interactions AE: association efficiency (100 x associated peptide mass / total peptide mass); LC: loading capacity (100 x peptide mass 561 / total formulation mass); n.a.: not applicable; Ref.: references; rhPTH1-34: recombinant 1-34 N-terminal fragment of 562 endogenous human parathyroid hormone; sCT: salmon calcitonin; TAMRA: tetramethylrhodamine; TAT: HIV 563 transactivator of transcription; theor.: theoretical; *Enzyme supplemented. 564 565 2.a.4. Nanocapsules 566 567 Nanocapsules are core-shell structured drug delivery carriers. They consist of an oily core which is 568 stabilized by surfactants and it is surrounded by one or more polymer shells [127]. Both, core and 569 outer shell layers, play a crucial role in the outcome of the formulation: whereas the core usually 570 works as a drug reservoir, the polymer coating helps the associated drug to overcome biological 571 barriers and modulate its release profile. Among the wide variety of lipids, the long chain fatty 572 acids and the medium chain glycerides (mono-, di- and tri-), both showing penetration enhancer 573 properties, are the most commonly used for producing nanocapsules [25]. 574 575 2.a.4.a. Preparation techniques 576 577 The preparation of nanocapsules involves the emulsification of an oily phase into an aqueous 578 phase. The polymer forming the shell can be incorporated into the organic phase or the aqueous 579 phase [128,129]. Additionally, two different polymers can be incorporated one in each phase 580 [130,131]. The shell is formed due to its precipitation at the interphase or to an ionic interaction 581 between the oily core and the polymer. In a different situation, i.e. poly(alkylcyanoacrylates), the 582 polymer shell is formed due to an interfacial polymerization process [9,132]. The main factors 583 driving the choice of the appropriate nanocapsules production technique are the nature of the 584 polymer as well as that of the peptide/protein to be encapsulated (Table 5). 585 586 Table 5. Main characteristics of the most commonly used preparation methods for nanocapsules 587 Technique Principle Stress Exposure Organic solvents Simpli - city “In situ” polymerization / Interfacial polymerization Oily core nanocapsules Monomers polymerization “in situ” at the interface of an emulsion Undesirable reactions drugmonomers / Vigorous stirring Yes + Aqueous core nanocapsules No necessarily Polymer precipitation/ deposition Solvent displacement Solvent diffusion to the aqueous phase and polymer precipitation/deposition Moderate stirring Yes ++ Self-emulsifi- cation Surfactant shifting from the oily to the aqueous phase and polymer deposition High surfactant concentration / Moderate stirring No ++ 588 i) “In situ” polymerization. 589 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 21 In this method, which is also named as interfacial polymerization, the polymer formation occurs 590 “in situ” at the interface of an emulsion through a fast polymerization among reactive monomers. 591 Due to their rapid and easy polymerization, alkylcyanoacrylates have been the monomers of 592 choice for this purpose [133,134]. Unfortunately, the potential reaction between the drug and the 593 reactive monomers during the process constitutes a limitation of this approach [135]. 594 595 - Interfacial polymerization in oily core nanocapsules. In this case, the organic phase is composed 596 by the peptide/protein, the oil, the monomers and an organic solvent. The solvent needs to be 597 water-miscible in order to promote its diffusion towards the aqueous phase, allowing the 598 spontaneous formation of nanometric oily droplets [136]. The organic phase is usually injected 599 into the aqueous phase, which contains at least a hydrophilic surfactant. This process is usually 600 performed under vigorous stirring, leading to the instantaneous formation of the nanocapsules 601 (Fig. 12). An additional final step to remove the organic solvents can be performed [9,134]. 602 603 604 Figure 12. Schematic view of the interfacial polymerization technique to produce oily core nanocapsules 605 606 - Interfacial polymerization in aqueous core nanocapsules. In this method, the aqueous phase, 607 which contains the protein/peptide, water and sometimes water-miscible solvent, is emulsified 608 into an organic phase consisting of an oil and a lipophilic surfactant using sonication or vigorous 609 stirring. Once the W/O emulsion is formed, the monomers are added under mechanical stirring. 610 This last step, triggers the polymerization at the W/O interface and leads to a final system 611 consisting of aqueous core nanocapsules dispersed in oil (Fig. 13) [137,138]. The nanocapsules are 612 finally isolated by ultracentrifugation followed by their resuspension in water [139,140]. 613 614 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 22 615 Figure 13. Schematic view of the interfacial polymerization technique to produce aqueous core nanocapsules 616 617 ii) Polymer precipitation/deposition 618 Contrarily to the “in situ” polymerization, the use of preformed polymers allows a good control of 619 the final polymer molecular weight, avoiding undesirable reactions between the drug and 620 monomers. In this case, the polymer coating can be formed by either polymer precipitation or 621 polymer deposition/interaction. 622 623 - Polymer precipitation. This technology was first reported by Fessi and coworkers [141,142]. This 624 method involves the use of an organic polar phase containing a lipophilic surfactant, an oil, and 625 the polymer, and an aqueous phase, that may contain hydrophilic surfactants. The usual 626 procedure can be summarized as follows (Fig. 14): the organic phase is added dropwise over the 627 aqueous phase under moderate stirring leading to the instantaneous diffusion of the water-628 miscible solvent from the lipophilic solution to the aqueous phase. As a consequence, the polymer 629 precipitates at the interface of the formed oily droplets, stabilizing them. In a final step, solvents 630 can be removed by evaporation under vacuum [128,130,142]. 631 632 - Polymer deposition/interaction. Alternatively, nanocapsules can be produced using water 633 soluble polymers according to a deposition/interaction technique. In this case, the polymer shell is 634 formed due to its ionic interaction with the lipophilic components of the oily core. This interaction 635 may occur during the solvent displacement process or after the incubation of the preformed 636 nanoemulsion with the water-soluble polymer [123,129,143,144]. Additionally, the possibility of 637 obtaining multi-layer nanocapsules has been reported. This layer by layer approach is based on the 638 adsorption of different polymeric layers onto a colloidal template. The addition of each polymeric 639 layer should invert the overall charge of the system in all the absorption steps [127]. Our group 640 has reported the possibility of obtaining protein-loaded nanocapsules by triggering the polymer 641 deposition by a self-emulsification method avoiding the use of organic solvents. The principle of 642 this technique is the same described in section 2.a.3.a. for the spontaneous formation of 643 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 23 micro/nanoemulsions (Fig. 11), including, additionally, a water-soluble polymer into the aqueous 644 phase [145] or in a subsequent incubation step [146,147]. 645 646 647 Figure 14. Schematic view of the solvent displacement - polymer precipitation/deposition technique to produce 648 nanocapsules 649 650 2.a.4.b. Characterization, peptide/protein loading, activity and release profile 651 652 - Particle size distribution: the main factors affecting the final particle size distribution of 653 nanocapsules are the ratio and the mixing conditions between the two phases, as well as the 654 physicochemical properties and concentration of the different components 655 [119,128,129,148,149]. Overall, nanocapsules have been produced so far with a size between 30 656 and 400 nm. 657 658 - Peptide/protein loading and activity: Couvreur and coworkers were the first reporting the 659 possibility of using nanocapsules as delivery vehicles for proteins [9]. Since their contribution 660 through the encapsulation of insulin in poly(alkylcyanoacrylate) nanocapsules, several authors 661 have demonstrated the capability of nanocapsules to entrap different peptides/proteins (Table 6). 662 Despite the high AEs attained, the LC values reported so far are below 2 %, which is usually due to 663 the hard solubilization of hydrophilic peptides into the lipidic phase and their tendency to diffuse 664 to the outer aqueous phase [139]. When nanocapsules are obtained by interfacial polymerization, 665 the monomer concentration has been proved to be one of the main factors influencing the 666 peptide association efficiency [150]. The pH of the peptide solution has also been shown to 667 influence the AE of peptides to PACA nanocapsules. This effect is attributed to the influence of the 668 pH on the polymerization rate of the polymer [148]. 669 670 Our group has also shown the possibility to attach proteins to preformed polymer nanocapsules. 671 For example, we have efficiently associated the recombinant hepatitis B surface antigen (rHBsAg) 672 onto preformed chitosan nanocapsules. In this situation the attachment of the protein was found 673 to be dependent on both protein and nanocapsules concentration and the mechanism of 674 attachment was based on ionic/hydrophobic interactions [151–153]. 675 676 Different formulation parameters could influence the peptide/protein structure. With “in situ” 677 polymerization the drug could work as a monomer during the polymerization procedure, being 678 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 24 denatured and losing its activity. However, ethanol can be used to preserve the peptide/protein 679 structure [154]. Furthermore, in all the techniques described above for nanocapsules production, 680 the presence of organic solvents and surfactants, as well as the vigorous stirring, could also affect 681 the structure of the encapsulated peptide/protein [139,143,148]. Electrophoresis-based 682 techniques (e.g. native SDS-PAGE), HPLC-based methods or circular dichroism have been reported 683 to study the structural stability of nanoencapsulated peptides/proteins [139,148,154]. However, in 684 the majority of the works, the activity of the encapsulated drug was evaluated after its in vivo 685 administration [128,143,155]. 686 687 - Peptide/protein release: the mechanism driving the release of peptides/proteins entrapped into 688 nanocapsules has been defined as a combination of two main processes: the partition of the drug 689 between the nanocarrier and the external release medium and the degradation of the polymer 690 shell and the lipid core. Both processes can be affected by different factors, such as the pH of the 691 release medium, the nature of the lipidic cores, the type and molecular weight of the polymer, as 692 well as the thickness of the polymer shell [138,143,149,156]. BSA cumulative releases ranging 693 from 35 % up to 90 % were reported for poly(butylcyanoacrylate) nanocapsules after 8 h in release 694 media with different pHs (from 2.5 to 8.5) and different profiles were showed when 695 poly(butylcyanoacrylate) of 4, 7 or 10 kDa was used. Likewise, the loading and the molecular 696 weight confer the protein with different diffusion capacities and specific interactions with the 697 components of the system. High loadings increase the protein gradient between the nanocapsule 698 core and the outer phase, and proteins with high molecular weights diffuse more slowly through 699 the polymeric wall [139]. On the other hand, when the protein is attached to the polymer shell, 700 the mechanism of release is based on its disassociation [157] and this process is normally 701 dependent on the pH and ionic strength of the release medium. 702 703 From the results in literature up to date, we can conclude that the solvent displacement technique 704 is the most advantageous for encapsulating hydrophilic peptides in nanocapsules. Apart from its 705 simplicity, and the possibility of controlling the exact molecular weight of the polymer and 706 avoiding undesirable cross-reactions, high association efficiencies can be attained. 707 708 Table 6. Examples of peptide/protein-loaded nanocapsules obtained by the different preparation methods: drug loading 709 and release properties. 710 Preparation method Peptide / Protein AE (%) LC (%) ≤1h burst / cumulative release (time) – pH medium Ref. Interfacial polymerization (Oily / Aqueous core) Insulin 55 - 98 n.a. n.a. [9,158, 159] 90 n.a. (0.45 mg/mL) 10 % / 13 % (5 h) pH 7.4/1-2* 77 % / 80 % (5 h) pH 6-7* [154, 160, 161] 57 - 95 n.a. n.a. [148, 162] 100 n.a. n.a. [163] Human 35 - 79 n.a. (0.0067 - 40 - 60 % (20 min) pH 7.4* [164] MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 31 mechanism of release is mainly driven by the degradation of the polymer, the nature of the 872 protein may also influence its solubility, its interaction with the polymer and its diffusion across 873 the channels generated in the polymer degradation process [171,183,188,190,194]. 874 875 Table 8 shows examples of proteins associated to PLGA nanoparticles produced by different 876 techniques. 877 878 Table 8.Examples of peptide/protein-loaded PLGA nanoparticles obtained through different preparation methods: drug 879 loading and release properties. 880 Preparation method Peptide/ Protein AE (%) LC (%) ≤ 1 h burst / cumulative release (time) - pH medium Ref. Double emulsion - solvent evaporation BSA 70 - 80 0.7 - 0.8 n.a. / 80 % ( 28 d ) pH 7.4 [190] Insulin 70 - 80 3. 5 - 4 n.a. / 20 % ( 28 d ) pH 7.4 Cyclosporine A 60 - 90 n.a. 15 - 25 % / 70 - 90 % ( 24 h ) pH 7.4 [195] BSA 28 - 88 n.a. n.a. / 40 - 100 % (28 d) pH 7.4 [178] HSA 22 - 33 1.3 - 2.6 n.a. [196] Tetanus toxoid 31 - 37 n.a. n.a. / 7 - 18 % (1 d) pH 7.4 <7 / 4 - 15 % (4 h) pH 1.2*/7.5* [170, 171] L - Aspara ginase 15 - 40 1.8 - 4.9 n.a./ 15 - 95 % (21 d) pH 7.4 [189] Insulin n.a. n.a. n.a. / 70 % (40 d) pH n.a. [197] IGF - 1 22 - 43 n.a. n.a. / 78 % (40 d) pH n.a. Emulsion - solvent diffusion BSA 4 - 60 1 - 4 theor. 60 - 80 % / 80 - 90 % ( 14 d ) pH 7.4 [188] IgG n.a. 1 - 4 theor. 5 - 25 % / 10-30 % (14 d) pH 7.4 Insulin 20 - 40 0.2 - 0.4 20 % / 80 % ( 14 h ) pH 7.4 [181] PDGF - BB 87 0.01 40 % / 80 % ( 40 d ) pH 7.4 [182] FGF - 2 68 0.01 40 % / 80 % ( 40 d ) pH 7. 4 Nanoprecipitation Insulin 14 - 23 0.3 - 0.5 n.a. [184] Lysozyme 35 - 91 0.7 - 1.8 n.a. α-chymotrypsin 11 - 71 2 - 5 theor. n. a. [185] Cyt - c 72 3.6 n. a. / 100 % (120 d) pH 7.3 AE: association efficiency (100 x associated peptide mass / total peptide mass); BSA: bovine serum albumin; Cyt-c: horse 881 heart cytochrome c; FGF-2: fibroblast growth factor; HAS: human serum albumin; IGF-1: insulin-like growth factor; IgG: 882 immunoglobulin G; LC: loading capacity (100 x peptide mass /total formulation mass); n.a.: not applicable; PDGF-BB: 883 platelet-derived growth factor; Ref.: references; theor.: theoretical; *Enzyme supplemented. 884 885 2.b.2. Acrylic polymers-based nanoparticles 886 887 Following the pioneering work of P. Speiser and co-workers on the association of antigens (human 888 immunoglobulin G and tetanus toxoid) to polyacrylamide nanoparticles in 1976 [8], different types 889 of acrylic polymers have been used to produce nanoparticles, including polyacrylic acid, 890 polyacrylamides, polymethylmethacrylates and poly(alkylcyanoacrylates) [198]. These synthetic 891 polymers are considered to be biocompatible and, in some cases, biodegradable polymers 892 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 32 [199,200]. Among them, poly(alkylcyanoacrylates) (PACA) are the most commonly used for 893 preparing nanoparticulate systems and, in particular, for the delivery of proteins. Their nitrile and 894 ester groups are electron attractive functional groups and this property makes the vinyl carbon of 895 the monomer really reactive, hence, able to polymerize in the presence of an initiator. Free 896 radical, anionic or zwitterionic polymerization are the main approaches adopted so far for the 897 production of PACA nanoparticles [200–202]. Overall, despite the early development and 898 attention that these particles received in the past, only a few papers describing their use for 899 protein delivery have been found in the literature. 900 901 2.b.2.a. Preparation techniques 902 903 Apart from the interfacial polymerization method, which has been mainly used for oily core 904 nanocapsules production, and as such, it was described in the previous section (Section 2.a.4.a.), 905 two main strategies (summarized in Table 9) have been described to synthesize polyacrylate-based 906 nanostructures: the anionic polymerization and the free radical dispersion polymerization 907 techniques. In both cases, the use organic solvents is avoided, being the main source of protein 908 instability its potential reactivity with the monomer. 909 910 Table 9. Main characteristics of the most commonly used techniques to form peptide/protein-loaded polyacrylate-based 911 nanoparticles 912 Technique Principle Stress exposure Organic solvents Simplicity Anionic polymerization Monomers polymerization due to OH - groups in the medium Undesirable reactions drug-monomers No + Free radical dispersion polymerization Monomers polymerization due to the generation of free radicals and crosslinking Undesirable reactions drug-monomers- crosslinking agent / Free radicals / UV / Heat No + 913 i) Anionic polymerization 914 In this technique, the acrylic monomers, a stabilizer and an initiator (OH - in water) are necessary to 915 form the nanoparticles. The monomers, which are poorly soluble in water, are emulsified into an 916 acidic water solution (pH 2 - 4) containing the stabilizer (typically dextran). Once the droplets are 917 formed, the monomer starts to polymerize thanks to the hydroxyl ions (OH - ) present in the water 918 phase (Fig.19). The acidic pH slows down the polymerization rate, thereby controlling the process 919 of particles formation [133,201]. Proteins can be attached onto the surface of the particles, or 920 simply incorporated into the reaction mixture during particles formation [203–207]. 921 922 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 33 923 Figure 19. Schematic representation of the anionic-polymerization technique to produce polyacrylate-based 924 nanoparticles 925 926 ii) Free radical dispersion polymerization. 927 Peppas and co-workers used this technique to obtain gel nanospheres through a photo- or 928 thermal-initiated polymerization (Fig. 20). This technology involves the use of specific initiators as 929 well as a crosslinking agent. The monomers (i.e. methacrylic acid, MAA and monomethylether 930 monomethacrylate, PEGMA), the crosslinking agent (i.e. tetra (ethylene glycol) dimethacrylate) 931 and the initiator (i.e., 1-Hydroxylcyclohexyl phenyl ketone) are solubilized in an aqueous phase. 932 Once the initiator is activated (UV, heat), the formation of oligomers and crosslinks starts. Finally, 933 since the polymer is not soluble in water, nuclei of polymerization are created leading to the 934 formation of nanospheres (i.e. P(MAA-g-PEG)). Once the polymerization is completed, 935 nanospheres are purified by repeated washing steps to remove the unreacted monomers and the 936 association of the protein (i.e. insulin, OVA) is carried out in a subsequent incubation step 937 [208,209]. 938 939 940 Figure 20. Schematic representation of the free radical dispersion polymerization technique to produce polyacrylate- 941 based gel nanospheres 942 943 2.b.2.b. Characterization, peptide/protein loading, activity and release profile 944 945 - Particle size distribution: in general, polyacrylate-based nanoparticles described in the literature 946 have a size in the range of 50 nm and 500 nm and a negative surface charge [210–212]. Different 947 parameters can affect the polymerization process and, as a consequence, the physicochemical 948 properties of PACA nanoparticles. The most important parameter, which allows the control of the 949 polymerization rate and, hence the particle formation is the pH, however, the monomer 950 concentration also has a significant influence in this process. Finally, the temperature and the 951 addition of surfactants have also been described as a way to modulate the particle size [203,212–952 215]. 953 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 34 954 - Peptide/protein loading and activity: Table 10 gives an overview of the properties of some 955 protein/peptide-loaded polyacrylate-based nanoparticles formulations. The AE and LC values 956 described in the literature are very variable, ranging between 3.5 and 95 % AE and up to 26 % LC 957 [205,206,216]. Among the factors influencing the AE, the time at which the protein is added during 958 the polymerization process has been found to be critical. For example, both insulin and GRF 959 (growth hormone releasing factor) reached around 85 % AE when they were added to the 960 polymerization medium 30 minutes after the process started [135,205]. 961 962 As for the “in situ” polymerization method, the peptide/protein could undesirably work as a 963 monomer during the polymerization procedure, which may result in its inactivation [206,217]. 964 Apart from techniques like HPLC or enzymatic assays [203,217], direct in vivo efficacy of the 965 formulation has often been used to test the integrity and activity of the loaded peptides/proteins 966 [209]. 967 968 - Peptide/protein release: the release of proteins from polyacrylate-based nanoparticles is mainly 969 due to the bioerosion of the polymeric matrix [135]. Typically, these particles show an initial burst 970 release, which can be buffered using additives. The presence of dextran into the formulation 971 medium could, for example, delay the release of BSA from poly(α-butylcyanoacrylate) 972 nanoparticles [206]. Protein release has also been shown to be strongly influenced by the type of 973 PACA used. For example, the release of GRF was faster in the case of poly(isobutylcyanoacrylate), 974 as compared to the case of poly(isohexylcyanoacrylate) nanoparticles. This was due to the 975 different bioerosion rates of the two polymers [135]. In the particular case of the polyacrylate-976 based gel nanospheres (acrylic acid (AA) or methacrylic acid (MAA), they were specifically 977 designed to exhibit a pH-dependent swelling and, hence, release behavior [209]. This control could 978 be achieved by adjusting the polymerization and crosslinking conditions. 979 980 Table 10. Examples of peptide/protein-loaded polyacrylate-based nanoparticles prepared by anionic and free radical 981 dispersion polymerization: drug loading and release properties. 982 Preparation method Peptide/ Protein AE (%) LC (%) ≤ 1h burst / cumulative release (tim e) - pH medium Ref. Anionic polymerization Insulin 87 n.a. n.a. [205] BSA 3.5 n.a. 15 - 55 % / 70 - 90 % ( 14 d ) pH 7.4 [206] SOD 7 - 33 n.a. n.a. [203] NR1 6 - 10 n.a. n.a. GRF 80 n.a 70 % / 80 - 90 % ( 8 h ) pH 7.4* [135] Free radical dispersion polymerization Insulin 65 2.1 n.a. [208] 93 - 95 7 10 - 80 % / 100 % (3 h) 1h pH 3 + 2 h pH 7 [209] OVA 51 26 0 % / 90 - 100 % (3 h) 1.5 h pH 3 + 2 h pH 7.4 [216] AE: association efficiency (100 x associated peptide mass / total peptide mass); BSA: bovine serum albumin; GRF: growth 983 hormone releasing factor; LC: loading capacity (100 x peptide mass /total formulation mass); n.a.: not applicable; NR1: 984 anti-glutamate N-methyl D-aspartate receptor 1 antibody; OVA: ovalbumin; Ref.: references; SOD: superoxide 985 dismutase; *Enzyme supplemented. 986 987 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 35 2.b.3. Polysaccharide-based nanoparticles 988 989 The most commonly employed polysaccharides for protein delivery purposes are chitosan, 990 alginate, dextran and hyaluronic acid. Chitosan, a deacetylated form of chitin, is formed by 991 repeated units of D-glucosamine and N-acetylglucosamine [41,42,43]. Alginate is a block co-992 polymer made by α-guluronic acid (pKa 3.4) and β-D-mannuronic acid (pKa 3.6) residues linearly 993 linked [220]. Like chitosan, it can be chemically modified on the acidic functional groups to obtain 994 the desired properties [221,222]. Dextran is made by α (1→6) glucopyranoside units [223–225]. 995 The hydroxyl groups are the main sites used for chemical modifications, with dextran sulfate as 996 the most common modified form for drug delivery applications [226–228]. Finally, hyaluronic acid 997 is a linear polysaccharide made by repeated units of the disaccharide formed by N-acetyl D–998 glucosamine and D–glucuronic acid [229]. These natural polysaccharides have in common the 999 property of being water-soluble; however their distinct chemistry results in different pKa and 1000 functionality in terms of their potential interaction with different targets and their capacity to be 1001 modified with different ligands. Among the polysaccharide-based nanoparticles described so far, 1002 those made of chitosan were originally developed in our lab for the association of proteins 1003 [15,230]. Since this discovery until now, chitosan nanoparticles have been classified as the 1004 polymeric delivery nanoparticles that have received the greatest deal of attention. Overall, an 1005 advantage of the techniques for the production of polysaccharide nanoparticles relies in the 1006 mildness of the procedures [231–233], with the exception of the chemical crosslinking [234], 1007 which may lead to the denaturation of the protein. 1008 1009 Different techniques have been described until now to produce polysaccharide-based 1010 nanoparticles and nanocomplexes, being the most commonly employed the ionic gelation and the 1011 polyelectrolyte complexation. General specifications of the different preparation techniques are 1012 presented in Table 11. 1013 1014 Table 11. Characteristics of the most commonly used techniques to form polysaccharide-based nanoparticles containing 1015 peptides/proteins 1016 Technique Principle Stress exposure Organic solvents Simplicity Ionic gelation/crosslinking Gelation of the particles by ionic crosslinking I onic intera c tions with the protein / Crosslinking agent No + + Polyelectrolyte complexation Ionic interaction between polymers of opposite charge Ionic interactions with the protein No + + 1017 i) Ionic gelation/Ionic crosslinking 1018 Our lab pioneered the development of chitosan nanoparticles using the ionic gelation/ionic 1019 crosslinking technique [15,230], which has been later extended to other polysaccharides such as 1020 alginate and dextran [235,236]. This technique is based on the fact that some charged 1021 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 36 polysaccharides can gel in aqueous solution in the presence of small ions and crosslinking agents 1022 (Fig.21) [133,222,237]. The type of gelling agent is different based on the type of polysaccharide. 1023 For example, in the case of chitosan, tripolyphosphate (TPP) is the most commonly crosslinking 1024 agent employed, while in the case of alginates, the use of calcium salts (calcium chloride, calcium 1025 sulfate, or calcium carbonate) is the most common gelation approach [15,222,236,238–241]. 1026 1027 Alternatively, nanoparticles can be produced using a chemical cross-linking reaction. However, this 1028 technique has not been almost explored for the association of proteins [234] due to the potential 1029 chemical reactions with the loaded protein. 1030 1031 1032 Figure 21. Schematic view of the ionic gelation/crosslinking technique to produce polysaccharide-based nanoparticles 1033 1034 ii) Polyelectrolyte complexation 1035 Polyelectrolytes complexes (PECs) are complexes resulting from the mixing of two oppositely 1036 charged macromolecules (i.e., polyelectrolytes). A schematic representation of the procedure is 1037 shown in Figure 22 [242,243]. The density of the charges and the charge distribution over the 1038 polymeric chains, in addition to the concentration of the two polyelectrolytes are the main 1039 parameters influencing the properties of the particles formed. The control of the ionic strength 1040 and pH of the reaction medium, which influences the degree of ionization, is also fundamental for 1041 the nanoparticles formation [244]. 1042 1043 1044 Figure 22. Schematic view of the polyelectrolyte complexation technique to produce polysaccharide-based nanoparticles. 1045 1046 2.b.3.b. Characterization, peptide/protein loading, activity and release profile 1047 1048 - Particle size distribution: the ionic gelation/crosslinking is, among the techniques described 1049 above, probably the one allowing a better control of the size. Indeed, in a report by our group 1050 [245], intended to compare the ionic crosslinking vs. the ionic complexation of chitosan and pDNA, 1051 we showed that the nanoparticles prepared by crosslinking of chitosan with TPP had a more 1052 controllable size and a lower polidispersity than those produced by ionic complexation. This result 1053 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 37 was attributed to the fact that the crosslinking with TPP led to the formation of nanogelled 1054 particles with a round and more defined structure [246,247]. Overall, the main factors influencing 1055 the particle size distribution are the ratio and the concentration of the ionically interacting species 1056 [15,227]. 1057 1058 - Peptide/protein loading and activity: in general, particles produced by gelation or complexation 1059 are characterized by a high LC, which can reach values up to 50 % and AE values close to 100 % 1060 [15,227,247,248]. The protein association efficiency is mainly affected by the number of 1061 interacting species and their degree of ionization. For example the AE of insulin to chitosan 1062 nanoparticles reached values close to 90 %, however the value decreased to 37 % in the case of 1063 chitosan/glucomannan polyelectrolyte complexes [247]. This was attributed to the different pHs of 1064 the protein solution and also to a competition between the protein and glucomannan for the 1065 chitosan positive sites. A similar competition phenomenon was observed for the basic peptide 1066 salmon calcitonin, which was found to compete with protamine in its association to hyaluronic 1067 acid/protamine nanoparticles [249]. These affinity/ionic competition phenomena have been taken 1068 into account for the modulation of the LC. For example, the association efficiency of insulin to 1069 chitosan-based nanoparticles could be increased from 66 % to 94 % when the anionic interacting 1070 polymers were alginate and dextran sulfate respectively. This behaviour was explained due to the 1071 strong ionic interactions between the insulin and the sulfate groups of dextran [250]. 1072 1073 The main source of instability for the loaded peptide/protein is, in both ionic gelation and 1074 polyelectrolyte complexation, the possible ionic interaction between the peptide/protein and the 1075 polymers/crosslinking agents, which could drive to protein denaturation [248,251,252]. 1076 Additionally, the acidic pH often necessary to produce nanoparticles by ionic gelation (e.g. 1077 chitosan nanoparticles) can destabilize or affect the peptide/protein activity (e.g. pH optimum of 1078 enzymes) [253]. Both electrophoresis-based techniques (i.e. SDS-PAGE and Western blot) and 1079 ELISA assays have been used to check if the peptide/protein integrity and activity were preserved 1080 once included in polysaccharide-based nanoparticles[251,252,254]. Likewise, spectroscopy-based 1081 techniques like FTIR have been used to study the interactions between the functional groups of 1082 the peptide/protein and the polyelectrolytes [255]. In the case of enzymes, the activity was simply 1083 evaluated through enzymatic activity assays [256]. Finally, in some cases, the activity was only 1084 assessed after their in vivo administration [248,257]. 1085 1086 - Peptide/protein release: from the point of view of drug release, nanoparticles produced by ionic 1087 gelation or complexation normally show an ionic strength-dependent release profile, with an 1088 initial burst release. In fact, the sensitivity of these systems to pH changes and to the presence of 1089 ions, is one of their main drawbacks [228,247]. An example of this behavior has been observed for 1090 insulin-loaded dextran sulfate/polyethylenimine (PEI) nanoparticles produced by complexation, 1091 which completely released the peptide in PBS 50 mM after 5 minutes, while just the 65 % of the 1092 peptide was released in PBS 5 mM [228]. 1093 1094 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 38 Among the formulation factors that can be modified in order to have a certain control of the 1095 release process, the combination of different counteracting polymers and surfactants can be 1096 highlighted. For example, we have shown that the release of BSA from chitosan nanoparticles 1097 produced by ionic crosslinking was affected by the presence of poloxamer 188 in the formulation 1098 [15,230]. Similarly, Sarmento et al compared the insulin release profile from alginate/chitosan and 1099 dextran/chitosan nanoparticles [250]. They showed that the release of insulin was strongly 1100 influenced by type of polymers used, being the interaction between the protein drug and the 1101 polymers fundamental to control the release. These chitosan/alginate nanoparticles were shown 1102 to have a pH-dependent release profile, suitable for the gastric and intestinal environment. In fact, 1103 these systems were able to retain the protein at the low pH of the stomach, and release it in the 1104 intestine, when the pH increased [236,258]. Swelling, dissociation, diffusion and erosion are 1105 reported as the main mechanisms behind protein release from the nanoparticles made by ionic 1106 gelation or polyelectrolyte complexation [227,259]. 1107 1108 Overall, it could be concluded that polysaccharide-based nanoparticles are those leading to the 1109 highest protein loading capacity, among those indicated in this review. The challenge that remains 1110 associated to these nanoparticles is related to their limited capacity to control the release in 1111 different physiologically relevant media. Nevertheless, the combination of different biomaterials 1112 and surfactants are now seen as approaches to overcome this hurdle. 1113 1114 Table 12 reports examples of peptides and proteins encapsulated into polysaccharide-based 1115 nanoparticles synthesized by different strategies. 1116 1117 Table 12. Examples of peptide/protein-loaded polysaccharide-based nanoparticles prepared by the different methods: 1118 drug loading and release properties 1119 Preparation method Peptide / Protein AE (%) LC (%) ≤ 1 h burst / cumulative r elease (time) - pH medium Ref. Ionic gelation/ crosslinking Insulin 87 - 97 19 - 55 100 % / 100 % ( 2 h ) pH 4/7 80 - 100 % / 100 % (2 h) pH 6.4 [248] 40 - 90 20 - 22 theor. 15 - 90 % / 15 - 90 % (2 h) pH 7.4 [247] Immuno - modulatory protein P1 10 - 30 16 - 21 theor. 10 -75 % / 10 - 75 % (2 h) pH 7.4 BSA 5 - 80 1 0 - 50 n.a. / 30 - 100 % ( 8 d ) pH 7 [15] Tetanus Toxoid 50 10 n.a. [246] VEGF 32 - 94 0.04 - 0.34 80 % / > 90 % ( 24 h ) pH 7 [254] PDGF 27 - 54 0.05 - 0.1 n.a. / > 90 % ( 7 d ) pH 7 Insulin 69 10 95 % / 95 % (2 h) pH 1.2 80 % / 80 % (2 h) pH 6.8 [250] Polyelectrolyte complexation BSA 70 n.a. 40 - 60 % / 40 - 60 % (7 h) pH 7.4 [255] Insulin 66 - 94 5 - 13 55 - 100 % / 55 - 100 % (2 h) pH 1.2 70 - 100 % / 70 - 100 % (2 h) pH 6.8 [250] MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 39 rHBsAg 90 - 95 2.5 - 5 n.a. [252] sCT 100 10 - 39 55 % / 70 - 80 % ( 24 h ) pH 7.4 [249] TRIAL n.a. n.a. n.a. [257] ARH peptide 36 - 72 11 - 13 n.a. / 15 - 60 % (6 d) pH 7.4 [260] OVA 80 - 85 7 - 38 n.a. [220] AE: association efficiency (100 x associated peptide mass / total peptide mass); BSA: bovine serum albumin; INF-α: 1120 interferon alpha; LC: loading capacity (100 x peptide mass /total formulation mass); n.a.: not applicable; OVA: 1121 Ovalbumin; PDGF: platelet-derived growth factor; Ref.: references; rHBsAg: recombinant hepatitis B surface antigen; 1122 sCT: salmon calcitonin; theor.: theoretical; TRIAL: tumor necrosis factor-related apoptosis inducing ligand; VEGF: 1123 Vascular endothelial growth factor. 1124 1125 2.b.4. Protein-based nanoparticles 1126 Protein nanoparticles have been proposed for a long time as drug delivery systems due to their 1127 low cost, easy production, low cytotoxicity and biodegradability [261,262]. A protein nanoparticle-1128 based product for the delivery of paclitaxel (Abraxane®) has been approved by FDA and EMA, 1129 generating a high interest around this kind of particles. Recent works related to protein 1130 nanoparticles for protein delivery have been reported in literature, using gelatin, HSA, BSA, green 1131 fluorescent protein (GFP) and silk fibroin as starting materials to produce the particles [261]. 1132 1133 2.b.3.a. Preparation techniques 1134 1135 The preparation method most commonly used to produce protein nanoparticles is the desolvation 1136 technique, described below. 1137 1138 i) Desolvation 1139 An aqueous solution of both the therapeutic protein and the one used as a starting material to 1140 produce the particles is prepared. A desolvating agent, like acetone, ethanol or dimethyl sulfoxide 1141 (DMSO), is then slowly added to the proteins solution. After the desolvation process, 1142 nanoaggregates of the proteins are formed and a crosslinking agent, usually glutaraldehyde, is 1143 added, causing the formation of stable particles (Fig.23) [262,263]. Alternatively to the chemical 1144 crosslinking, a coating with an ionic polymer (e.g., PEI) can be done to improve the stability of the 1145 particles [264]. 1146 1147 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 40 1148 Figure 23. Schematic view of the desolvation technique to produce protein nanoparticles 1149 1150 2.b.3.b. Characterization, peptide/protein loading, activity, and release profile 1151 1152 - Particle size distribution: the size of the protein-based nanoparticles, which usually ranges 1153 between 150 and 400 nm, depends on parameters like the type of crosslinker and the crosslinking 1154 time. Their surface charge depends on the pH of the media and the type of protein used to 1155 produce the particles [263–266]. 1156 1157 - Peptide/protein loading and activity: although the number of references describing the use of 1158 protein nanoparticles for protein delivery is very low, in general high AE values are reported in 1159 literature (Table 13). Furthermore, the presence of a polymer coating that helps to retain the 1160 protein drug can also enhance the AE values of protein nanoparticles, as demonstrated for 1161 albumin nanoparticles prepared by desolvation with PEI forming the polymer coating [264]. 1162 1163 The main drawback of the desolvation process is the use of organic solvents or crosslinking agents, 1164 which could denaturate the peptide/protein structure, leading to protein inactivation. In this 1165 regard, ELISA and enzymatic assays have been used to check if the peptide/protein activity was 1166 retained after the nanoparticle formation [262,264,265]. 1167 1168 - Peptide/protein release: a first burst release followed by a sustained release profile is usually 1169 observed. The sustained release phase is associated to the degradation and dissolution of the 1170 protein matrix. 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