Strategies to load therapeutics into polysaccharide-based nanogels with a focus on microfluidics: A review

Nowadays nanoparticles are increasingly investigated for the targeted and controlled delivery of therapeutics, as suggested by the high number of research articles (2400 in 2000 vs 8500 in 2020). Among them, almost 2% investigated nanogels in 2020. Nanogels or nanohydrogels (NGs) are nanoparticles formed by a swollen threedimensional network of synthetic polymers or natural macromolecules such as polysaccharides.

Carbohydrate Polymers 266 (2021) 118119 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Review Strategies to load therapeutics into polysaccharide-based nanogels with a focus on microfluidics: A review N Zoratto a, E Montanari b, *, M Viola a, J Wang a, T Coviello a, C Di Meo a, *, P Matricardi a a b Department of Drug Chemistry and Technologies, Sapienza University of Rome, 00185 Roma, Italy Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland A R T I C L E I N F O A B S T R A C T Keywords: Nanogels Polysaccharides Drug loading methods Microfluidics Nanogels-based vaccines Nowadays nanoparticles are increasingly investigated for the targeted and controlled delivery of therapeutics, as suggested by the high number of research articles (2400 in 2000 vs 8500 in 2020) Among them, almost 2% investigated nanogels in 2020 Nanogels or nanohydrogels (NGs) are nanoparticles formed by a swollen threedimensional network of synthetic polymers or natural macromolecules such as polysaccharides NGs represent a highly versatile nanocarrier, able to deliver a number of therapeutics Currently, NGs are undergoing clinical trials for the delivery of anti-cancer vaccines Herein, the strategies to load low molecular weight drugs, (poly) peptides and genetic material into polysaccharide NGs as well as to formulate NGs-based vaccines are summa­ rized, with a focus on the microfluidics approach Introduction In 1999 Alexander V Kabanov and Serguei V Vinogradov intro­ duced the term NanoGel™ referring to an innovative nano drug delivery system formed by a hydrophilic polymer network of cross-linked poly­ ethyleneimine and carbonyldiimidazole-activated polyethylene glycol (PEG), using an emulsification/solvent evaporation technique (Vinog­ radov et al., 1999) This chemically cross-linked NG was used to deliver antisense oligonucleotides (Kabanov & Vinogradov, 2009) However, already few years before, Junzo Sunamoto and Kazunari Akyioshi described the phenomenon of the physical cross-linking (self-assembly) of cholesterol (Ch)-modified polysaccharides, such as pullulan (Pul), mannan (Man) and hyaluronic acid (HA), which resulted in the forma­ tion of hydrogels with a nano-scale size (Akiyoshi et al., 1993; Lee & Akiyoshi, 2004; Nakai et al., 2012; Yamane et al., 2009) NGs are nano-sized three-dimensional networks (Fig 1) able to absorb a large amount of water and to easily swell and de-swell in aqueous media NGs are generally soft, hydrophilic, biocompatible and represent a highly versatile nano-system able to deliver a variety of bioactive Abbreviations: AA, asiatic acid; Alg, alginate; Alg-CHO, aldehyde-functionalized alginate; Alg-PDEA, alginate-poly(2-(diethylamino)ethyl methacrylate); ALN, alendronate; AmPs, antimicrobial peptides; APCs, antigen-presenting cells; BoHc/A, botulinum type-A neurotoxin subunit antigen Hc; BSA, bovine serum albumin; CDDP, cisplatin-based HA nanocomplexes; CDs, cyclodextrins; CMD-SS-LCA, carboxymethyl dextran-lithocholic acid; Cs, chitosan; CSLNs, cationic solid lipid nanoparticles; DA, desoxycholic acid; DCs, dendritic cells; DD, deacetylation degree; DEAE, diethyl amino ethyl amine; DEX, dexamethasone; Dex, dextran; DHA, 1,4dihydroxyanthraquinone; DOX, doxorubicin; DSB, di-strylbenzene derivative; dsDNA, double-stranded DNA; E.E., encapsulation efficiency percentage; FA, folic acid; FNC, flash nanocomplexation; Gel, gellan; Gel-Ch, gellan-cholesterol; Gel-Rfv, gellan-riboflavin; GSH, glutathione; HA, hyaluronic acid; HA-AT, thiolated alkyl derivative of hyaluronic acid; HA-APBA, hyaluronan‑boronic acid; HA-Ch, hyaluronan-cholesterol; HA-Rfv, hyaluronan-riboflavin; HBsAg, surface protein of Hep­ atitis B virus; HCPT, hydroxycamptothecin; IDA, iminodiacetic acid; MA, malonic acid; Man, mannan; MIC, minimum inhibitory concentration; miRNA, microRNA; MIVM, multi-inlet vortex mixer; mRNA, messenger RNA; MW, molecular weight; NGs, nanogels; OVA, ovalbumin; PDI, polydispersity index; pDNA, plasmid DNA; PAA, poly(acrylicacid); PEI, polyethylenimine; PEG, polyethylene glycol; PIR, piroxicam; PPZ, perphenazine; PTX, paclitaxel; Pul, pullulan; Pul-Ch, pullulancholesterol; pβ-CD, poly-β-cyclodextrin; RA, retinoic acid; RGD, Arg-Gly-Asp; rHBsAg, recombinant hepatitis B surface antigen; siRNA, short interfering RNA; SODB1, superoxide dismutase; SpAcDEX, spermine-modified acetalated dextran; ssDNA, single-strended DNA; TA, tannic acid; TOPSi, thermally oxidized porous silicon particles; TPP, pentasodium triphosphate; TT, tetanus taxoid * Corresponding authors E-mail addresses: nicole.zoratto@uniroma1.it (N Zoratto), elita.montanari@pharma.ethz.ch (E Montanari), viola.1943830@studenti.uniroma1.it (M Viola), ju wang@uniroma1.it (J Wang), tommasina.coviello@uniroma1.it (T Coviello), chiara.dimeo@uniroma1.it (C Di Meo), pietro.matricardi@uniroma1.it (P Matricardi) https://doi.org/10.1016/j.carbpol.2021.118119 Received 11 February 2021; Received in revised form April 2021; Accepted 15 April 2021 Available online 28 April 2021 0144-8617/© 2021 The Authors Published by Elsevier Ltd This is an (http://creativecommons.org/licenses/by-nc-nd/4.0/) open access article under the CC BY-NC-ND license N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 swelling and de-swelling nature in response to external stimuli such as solvent composition, light, temperature, pH, pressure, magnetic and electric fields, NGs have attracted attention as functional smart mate­ rials for biotechnological and biomedical applications (Eckmann et al., 2014; Zha et al., 2011) NGs can be prepared from natural (i.e., poly­ saccharides, polypeptides) and/or synthetic polymers (i.e., poly lacticco-glycolic acid, PEG, polyglycolic acid, polycaprolactone, poly(Nisopropylacrylamid), poly(methylmethacrylate), poly(acrylicacid), pol­ yacrilamide, poly(N-vinyl-pyrrolidone) and depending on the kind of network linkages, NGs are classified into two groups: physically or ´ & Etrych, 2018) Herein, only chemically cross-linked NGs (Kousalova polysaccharide NGs are described Polysaccharides are biopolymers consisting of chains of monosaccharide or disaccharide units joined by glycosidic bonds (Fig 2) (Coviello et al., 2007) Polysaccharides are usually non-toxic, biocompatible and biodegradable (Mizrahy & Peer, 2012) Both hydrophilic and hydrophobic therapeutics have been entrapped into polysaccharide NGs with a significant enhancement of both the drug bioavailability and pharmacological activity (Kabanov & Vinogradov, 2009; Vinogradov, 2010) Herein, the strategies to load molecular or macromolecular therapeutics into NGs and to formulate polysaccharide-based vaccines are reported, with a focus on microfluidics Microfluidics is emerging as a promising strategy dealing with the Fig Schematic representation of a hydrogel, microgel and NG molecules such as hydrophobic as well as hydrophilic drugs, (poly) peptides and genetic material (Choi et al., 2009; Ganguly et al., 2014; Montanari et al., 2013; Montanari et al., 2018) Indeed, the porosity of the NGs network provides a reservoir for loading molecular and macromolecular therapeutics as well as protecting them from the envi­ ronmental degradation Furthermore, because of their inherent rapid Fig Average structures and/or repeating units of the various reported polysaccharides: A) Pul; B) Man; C) HA; D) Cs; E) Alg; F) Gel; G) Dex; H) β-1,3-D-glucan; I) heparin N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 manipulation of small volumes of fluids (from pico-to-nanoliter) inside a miniaturized device, with a millisecond mixing time and a real-time monitoring Microfluidic devices are made of a number of materials, such as silicon, glass borosilicate and polydimethylsiloxane which are patterned into micrometer-sized channels, whilst syringe pumps provide the driving force for the fluid flow in the microchannels (Fig 3) Fluid manipulation occurs by an active or passive control in the microfluidic device Active control means that external forces (e.g., magnetic or electric fields, heat) are responsible for the flow movement, whilst in the passive control the fluidic movement is governed by channel geometries and/or by liquid flow rates In a microfluidic device, the nucleation and growth stages of nanoparticles can be spaced from the position where the solution mixing takes place, leading to a precise control in the par­ ticle size and morphology, hence to a polydispersity index (PDI) reduction (Hung & Lee, 2007; Ma et al., 2017) Furthermore, the particle size can be finely tuned by modifying the flow rate and ratio of the phases All these features make microfluidics a cost-effective, repro­ ducible and scalable technology (Shrimal et al., 2020) The drug loading into NGs is usually achieved by polymer emulsification or by exploiting other approaches such as solvent extraction, solvent diffusion, solvent evaporation or coacervation within the microfluidic device In all these conditions, drug-loaded NGs are formed in a single step, improving both the NGs drug loading efficiency and the ability to release drugs in a controlled fashion (Chiesa et al., 2018) However, some shortcomings still need to be overcome: for example, organic solvents should be avoided since they may have poor biocompatibility and may affect the activity of the encapsulated molecules Moreover, the drug-loaded NGs production should be further optimized in terms of fabrication process and drug delivery efficacy (Ma et al., 2020) The next sections describe the strategies that can be adopted in the formulation of drug-loaded NGs, with a focus on the microfluidic approach or physical cross-linking In this respect, T Thambi et al loaded the poorly water-soluble anticancer drug doxorubicin (DOX) into carbox­ ymethyl Dex-lithocholic acid-based NGs (CMD-SS-LCA) An organic so­ lution of DOX was added to the aqueous polymer solution, forming an oil-in-water emulsion followed by a dialysis against water that led to the pure drug-loaded NGs formation (Thambi et al., 2014) R Guo et al prepared both chemically and physically cross-linked Alg-poly(2(diethylamino)ethyl methacrylate) (PDEA) NGs for the delivery of hydroxycamptothecin (HCPT) At neutral pH, HCPT exhibits the lactone-ring-opened structure which is water soluble Therefore, a HCPT aqueous solution (at pH = 8) was firstly added to a mixture of Alg-DEA, followed by the chemical polymerization of DEA monomers, initiated by K2S2O8, and the physical crosslinking of Alg chains by CaCO3 As the chemical polymerization proceeded, the lower pH led to the formation of the HCPT into its water-insoluble lactone, thus allowing the drug entrapment in the hydrophobic core of the Alg-PDEA NGs (Guo et al., 2007) Inorganic compounds were also loaded into polysaccharide NGs M C Coll Ferrer and colleagues synthetized NGs based on a lysozyme core and a Dex shell in which AgNO3 was loaded by the autoclaving process The high temperature allowed the reduction of Ag+ to Ag0, in a process in which lysozyme contributed to the in situ reduction and stabilization of Ag/NGs The amount of embedded Ag increased with the increase of lysozyme content Unfortunately, the loss or retention of the lysozyme activity was not shown after the NGs formation (Coll Ferrer et al., 2014) The in bulk loading methods might take long incubation time (i.e., overnight) (Pedrosa et al., 2014; Thambi et al., 2014) and might require the use of organic solvents (Bertoni et al., 2018; Stefanello et al., 2017; Thambi et al., 2014) Moreover, drug encapsulation is often achieved by a two-step procedure: NGs are firstly synthetized and then the payload is loaded (Pedrosa et al., 2014; Stefanello et al., 2017) Furthermore, the sterilization process represents another critical issue In order to over­ come some of these disadvantages, the autoclaving process was exploi­ ted (Manzi et al., 2017) The aqueous suspension of the amphiphilic hyaluronan-riboflavin (HA-Rfv) polymer was added to the drug film and then autoclaved to form sterile and drug loaded NGs Piroxicam (PIR), dexamethasone (DEX) and PTX were efficiently loaded into HA-Rfv NGs by exploiting this approach (Manzi et al., 2017) In other works, auto­ claving was used to achieve drug-loaded Gel-Ch (Musazzi et al., 2018), Gel-riboflavin (Gel-Rfv) (Musazzi et al., 2018) and HA-Rfv NGs (Di Meo et al., 2015), which were loaded with a number of hydrophobic mole­ cules in a single step, confirming the versatility of this method However, the autoclaving process cannot be used for encapsulating thermosensitive drugs (Montanari et al., 2019) Moreover, the molecular weight (MW) of the polysaccharide may decrease after autoclaving, thus producing new chemical species Taking into account the limits of the autoclaving approach and considering that all the described strategies may lead to high batch variability (i.e., large size distribution and high polydispersity) and to the formulation of low yield of nano-systems, a robust procedure for a scalable production of NGs is still under investigation Loading of low molecular weight drugs into NGs 2.1 Physical loading by hydrophobic forces A number of poorly water-soluble drugs have been loaded into NGs, offering the advantage to enhance their apparent water solubility (Table IA) For example, the chemical functionalization of the poly­ saccharide chains with hydrophobic moieties allows the formation of amphiphilic derivatives able to self-assemble into NGs with internal hydrophobic residues, which can host hydrophobic drugs Typically, the increase of NGs hydrophobicity enables higher loading capability, as well as longer sustained release profiles (Bewersdorff et al., 2019) One strategy for loading poorly-water soluble drugs into NGs is the incuba­ tion of the preformed nanoparticle suspension with a concentrated organic solution of the bioactive molecule (Pedrosa et al., 2014; Stefa­ nello et al., 2017) Hydrophobic molecules can also be loaded mean­ while the NGs are formed This can be achieved by adding the bioactive molecules in the polymer solution before the gelation process that refers to the formation of a polymeric three-dimensional network by chemical Fig An example of a microfluidic setup for the preparation of drug-loaded NGs N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 Table I Summary of the physical A) and chemical B) loading strategies employed in the preparation of polysaccharide-based NGs loaded with low molecular weight drugs A Physical loading Class of therapeutics Starting material Loading driving forces Loading strategy Advantages/Disadvantages References Low-molecular weight hydrophobic drugs Amphiphilic polysaccharides Hydrophobic forces NGs incubation with the drug solution - Long incubation time - Organic solvents may be required - Drug encapsulation achieved by a two step procedure - Low efficiency - Organic solvents may be required - Pedrosa et al., 2014 - Stefanello et al., 2017 - Montanari et al., 2019 - Yang et al., 2011 - Sterile and drug-loaded NGs formed in a single-step procedure - High reproducibility - Incompatible with thermosensitive drugs - Change of the polymer Mw - High reproducibility - Control over the size, PDI and compactness of NGs - Coll Ferrer et al., 2014 - Manzi et al., 2017 - Musazzi et al., 2018 - Di Meo et al., 2015 - Majedi et al., 2013 - Majedi et al., 2014 - Wannasarit et al., 2019 - Kłodzi´ nska et al., 2019 - Liu et al., 2015 - Bongiovì et al., 2020 - Gref et al., 2006 Addition of the drug solution to the polymer suspension, followed by NGs formation Autoclaving process Microfluidics/Millifluidics Low-molecular weight hydrophobic drugs CDs/polysaccharide mixtures or polysaccharides containing chelating moieties Complexation or coordination NGs incubation with the drug solution CDs incubation with the drug, followed by NGs formation Low-molecular weight drugs Charged polysaccharides Electrostatic interactions Incubation of the chelating polymer with the drug, followed by NGs formation Addition of drug to the polymer solution, followed by NGs formation - Long incubation time - Drug encapsulation achieved by a two/three step procedure - Long incubation time - Drug encapsulation achieved by a two step procedure - Drug loaded NGs formed in a single step procedure - Low versatility - Typically good E.E.% - Possible interference with the NGs formation NGs incubation with the drug - Possible low stability in human fluids Autoclaving process - Sterile and drug-loaded NGs formed in a single-step procedure - High reproducibility - Incompatible with thermosensitive drugs - Change of the polymer Mw - High reproducibility - Control over the size, PDI and compactness of NGs Microfluidics/Millifluidics Thambi et al., 2014 - Gref et al., 2006 - Thiele et al., 2011 - Ohta et al., 2016 - Deacon et al., 2015 Rossi et al., 2017 - Rajaonarivony et al., 1993 - Curcio et al., 2015 - Yang et al., 2011 - Zhang et al., 2006 - Schmitt et al., 2010 - Curcio et al., 2015 - Montanari et al., 2018 - Moradikhah et al., 2020 - Dong & Hadinoto, 2017 (continued on next page) N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 Table (continued) B Chemical loading Stimuli responsiveness Ligand Starting material Loading strategy References pH-responsive NGs DOX Aldehyde-functionalised Alg Dihydrazide-modified HA HA‑boronate Heparin Cystamine-modified HA Cystamine-modified Pul Schiff base condensation Hydrazone linkage Cyclic boronic ester formation Amide bond formation Disulphide linkage Disulphide linkage - Redox-responsive NGs Tannic acid Aminated-RA and aminated-FA DOX Protoporphyrin IX Among the investigated approaches, microfluidics appears to offer a number of advantages: I) the possibility to finely tune the size of the nanoparticles and, hence, the nanoparticle compactness by modifying the polymer concentration and the flow ratio of the dispersed and continuous phase; II) the significant reduction of the PDI; III) the high reproducibility; IV) the lack of user talent variability F S Majedi et al., prepared PTX-loaded hydrophobically modified Cs-based NGs by using a flow focusing microfluidic device (Fig 4A) (Majedi et al., 2013) The Tshape microfluidic device was provided with two inlets for aqueous buffer (pH = 9), to achieve two water streams at the flow-focusing Tjunction, and one inlet for the mixture of palmitoyl-Cs and PTX at acidic pH (pH 5.5) In the microfluidic device, the pH increase induces the simultaneous deprotonation of the Cs hydrophobic side chains and the Cs amine groups, leading to self-aggregation, thus the NGs formation The mixing time was in the millisecond scale; the flow ratio of Cs (pH = 5.5) and aqueous buffer (pH = 9.0) streams was changed from 0.03 to 0.2 By controlling the flow ratio, it was possible to finely tune the size, the surface charge and the density of the NGs Compared to the con­ ventional mixing method, this approach allowed the formation of more stable NGs with high encapsulation efficiency percentage (E.E., up to 95% and 60% for microfluidic-formed and bulk mixing-formed NGs, respectively) and a remarkably lower PDI (PDI < 0.2 for the microfluidic-formed and PDI > 0.6 for the bulk mixing-formed NGs) Moreover, the E.E of NGs increased by increasing the functionalization degree of the hydrophobically modified Cs, thanks to the hydrophobic nature of the drug Furthermore, by reducing the mixing times in the microfluidic device, higher E.E were obtained, since more hydrophobic moieties could interact with the PTX molecules during the NGs forma­ tion (Majedi et al., 2013; Majedi et al., 2014) Similarly, drug-loaded hydrophobically modified Dex NGs were obtained by grafting poly (lauryl methacrylate-co-methacrylic acid) onto acetylated Dex and were prepared by nanoprecipitation in a glass-capillary microfluidic device, as shown in Fig 4B Specifically, an ethanolic solution containing the polymer and asiatic acid (AA, a pentacyclic triterpenoid with anticancer activity) with a flow rate of mL/h was used as inner phase, whilst an Pei et al., 2018 Yin et al., 2018 Montanari et al., 2016 Tran et al., 2012 Yin et al., 2018 Xia et al., 2017 aqueous solution at pH 7.4 with a flow rate of 20 mL/h was selected as an outer phase NGs formation and loading occurred in a single step when the polymer solution was quickly mixed with the outer fluid The resulting NGs, exhibited a quite low PDI value (0.16) and a high E.E (~ 80%) (Wannasarit et al., 2019) The same nanoprecipitation method was also exploited by S Kłodzinska et al for the preparation of octenyl succinic anhydride-modified HA NGs loaded with azithromycin The polymer solution was injected into the outer streams of a three-inlet microfluidic chip at a flow rate of 5.4 mL/min, whilst the azi­ thromycin acidic solution was injected into the centre stream of the device at a flow rate of 1.2 mL/min, yielding a combined flow of 12.1 mL/min By optimizing the working parameters, the highest azi­ ´ ska et al., 2019) Other hydrophobic thromycin E.E was 45% (Kłodzin drugs, such as imatinib and a mixture of PTX and sorafenib were encapsulated into HA-based NGs and hybrid porous silicon-acetylated Dex NGs, respectively, via microfluidic, obtaining E.E of almost 50% (Bongiovì et al., 2020; Liu et al., 2015) 2.2 Physical loading by electrostatic interactions The basic principle of electrostatic forces is that oppositely charged polymer derivatives and bioactive molecules give rise to strong in­ teractions in aqueous phase By using this approach, the loading of the guest molecules can occur during the NGs formation (Table IA) E Montanari et al., loaded the highly hydrophilic drug gentamicin, into self-assembled HA-Ch-based NGs, by exploiting the electrostatic in­ teractions between the positively charged antibiotic molecules and the negatively charged polymer chains, at a suitable pH value Although gentamicin is highly hydrophilic, E.E ~ 36% and good sustained-release were achieved (Montanari et al., 2018) A number of antibiotics have a net positive charge under physiological pH and, hence, negatively charged polysaccharides may represent suitable materials for their de­ livery (Deacon et al., 2015; Rossi et al., 2017) Tobramycin, an amino­ glycoside antibiotic, was encapsulated in physically crosslinked Alg/Csbased NGs by J Deacon et al Since tobramycin and Alg can strongly Fig Schematic illustration of A) PTX loaded HMCS, reprinted with permission from (Majedi et al., 2013) Copyright (2013) The Royal Society of Chemistry; and B) AA loaded ADMAP NGs preparation via microfluidics, reprinted with permission from (Wannasarit et al., 2019) Copyright (2019) John Wiley & Sons N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 interact via electrostatic interactions, tobramycin-loaded NGs were prepared by mixing the drug with the Alg solution, followed by the addition of Cs, with the aim to form self-assembled polyelectrolytes NGs The binding energy of tobramycin with Alg was investigated by isothermal titration calorimetry demonstrating that the association be­ tween the drug and the polymer was enthalpically driven (ΔH = − 40.33 kcal/mol), with a resulting free energy (ΔG) of − 7.98 kcal/mol (Deacon et al., 2015) The anticancer drug DOX was physically entrapped into Alg-based NGs during the ionotropic gelation process by M Rajaonarivony et al In fact, a solution of calcium chloride was added to Alg solutions con­ taining various concentrations of DOX, followed by the addition of a solution of poly-lysine The electrostatic interactions between the cal­ cium ions and the oligopolyguluronic sequences of Alg led to the for­ mation of the so called “egg-box structure”, as evidenced by the presence of polymer aggregates The further addition of the poly-lysine solution resulted in the formation of a polyelectrolyte complex thanks to its interaction with the mannuronic residues of the Alg chains, trans­ forming the Alg‑calcium aggregates in small and well-defined NGs The loading efficiency values were in the range of 93–97% (Rajaonarivony et al., 1993) DOX can exhibit both hydrophobic moieties and ionizable groups: in fact, DOX is positively charged at physiological pH (pKa 8.6) whilst in its deprotonated form it is hydrophobic Consequently, depending on the pH of the formulation, as well as on the physicochemical properties of NGs, DOX might be encapsulated via electro­ static or hydrophobic forces (Yang et al., 2011) The physical entrapment by electrostatic interactions is usually simple and leads to relatively high E.E (Curcio et al., 2015; Schmitt et al., 2010; Zhang et al., 2006) However, this approach might suffer of some limitations: the physical entrapment into preformed NGs may result in an initial burst release of the cargo since part of the drug molecules might be absorbed onto the NGs surface and, on the other hand, the simultaneous incubation of the drug molecules with the polymer chains may interfere with the NGs formation (Vrignaud et al., 2011) Microfluidics was exploited for loading alendronate (ALN) into Cs/pentasodium triphosphate (TPP) NGs, by a hydrodynamic flow focusing method in a cross-junction microfluidic device Specifically, a solution of Cs/ALN (pH 6.5) at a flow rate of μL/min and two TPP solutions (pH = 3) at a flow rate of 5, or 10 μL/min were injected in the core flow and lateral flows of the microfluidic device, respectively At these pH values, the zwitterionic ALN interacted with the positively charged Cs, forming NGs with a narrow size distribution (Moradikhah et al., 2020) Also millifluidics represents a synthetic platform for the continuous preparation of NGs with tuneable sizes, lower susceptibility to particle fouling, and higher production throughput (Dong & Hadi­ noto, 2017) Millifluidics allows the use of a larger amount of fluids than microfluidics as well as the fluid manipulation in larger channels (~ mm) As a result, millifluidic chips are usually easier and cheaper to manufacture than the microfluidic ones (Lohse et al., 2013) A direct comparison between the millifluidic and the bulk mixing approaches for the formation of drug-loaded polysaccharide NGs was reported by B Dong et al which employed the antipsychotic perphenazine (PPZ) and Dex sulphate PPZ and DXT solutions were separately injected into a millifluidic reactor containing a Y-junction connector, in order to pro­ mote the mixing between the two phases The driving force for the NGs formation was the electrostatic interaction between the positively charged PPZ and the negatively charged Dex Although the two ap­ proaches exhibited similar trends in terms of particle sizes, pH depen­ dence, zeta potential values and stability data, some remarkable differences were reported In fact, NGs produced via millifluidic showed a smaller size distribution and higher PPZ E.E values than those found in the samples prepared in bulk (87 ± 11 nm vs 73 ± 40 nm for the particle size, whilst 85% vs 64% for the E.E.) (Dong & Hadinoto, 2017) 2.3 Loading by complexation or coordination The drug encapsulation into polysaccharide NGs can be also ach­ ieved through the formation of an inclusion complex between the drug and the nanocarrier (Table IA) Drug entrapment via complexation or coordination offers the advantage to avoid the use of surfactants or organic solvents In this respect, polysaccharides should be properly modified with molecules able to complex the drug as, for example, cy­ clodextrins (CDs) The covalent bonds of CDs to the polysaccharide backbone may allow the CDs to: I) retain their ability to form inclusion complexes between poor water-soluble drugs and the hydrophobic cavity of CDs, without decreasing the hydrophilicity of the overall structure and; II) enable NGs to entrap certain drugs and to release them in a controlled fashion (Moya-Ortega et al., 2012; Yuan et al., 2013) R Gref et al., prepared self-assembled NGs based on hydrophobically modified Dex (MD) and poly-β-cyclodextrin (pβ-CD) Two different drugs, benzophenone and tamixifen, were loaded into NGs Benzophe­ none was incorporated following two strategies: by the formation of an inclusion complex of the drug with the pβ-CD before the mixing with MD or by loading the drug directly within the NGs (Fig A and B), whilst tamoxifen was incorporated by exploiting only the first strategy Both drugs were selected thanks to their ability to form inclusion complexes with β-CD The hydrophobic cavity of β-CD fulfils two requirements: the capability to form complexes with the hydrophobic moieties of MD, leading to stable self-assemblies via ‘lock and key’ mechanism, and the possibility to entrap lipophilic drugs NMR spectra of benzophenone-pβCD solutions showed the shift of the ortho, para and metha protons of the benzophenone, suggesting the formation on an inclusion complex between the drug and the pβ-CD (Gref et al., 2006) C Thiele et al developed self-assembled NGs based on negative oxidized starch chains and positive CD derivative molecules 1,4-dihydroxyanthraquinone (DHA) was loaded into oxidized starch- β-CD NGs through the forma­ tion of an inclusion complex with the hydrophobic cavity of the β-CD The drug loading increased with the increasing of the particle sizes of NGs, up to a maximum value of 86% (Thiele et al., 2011) The drug loading by coordination was reported by S Ohta et al Cisplatin (CDDP)incorporated HA nanocomplexes were prepared by using a chelating ligand-metal coordination cross-linking reaction HA was previously chemically modified with two chelating moieties, namely iminodiacetic acid (IDA) and malonic acid (MA) Then, CDDP was loaded by mixing HA-IDA or HA-MA derivatives with CDDP, followed by heating In this way, spherical and CDDP loaded NGs were formed in a single step The ligand-conjugated HA was possibly cross-linked via bridging of ligands by CDDP or via the hydrophobic forces of CDDP with the coordinated ligands that lose their hydrophilicity through coordination (Ohta et al., 2016) To the best of our knowledge, the microfluidics approach was never employed, for loading low molecular weight drugs into poly­ saccharide NGs by complexation or coordination 2.4 Chemical loading by smart linkages The conjugation of drugs to polysaccharide NGs via chemical bonds leads to higher drug stability; however, it is not feasible with every kind of molecule and it is usually more time and cost consuming Further­ more, the drug degradation may occur once hard conditions are required Last, but not least, the drug should be linked to NGs with co­ valent linkages which can be cleaved in vivo (and possibly in situ) in order to perform its therapeutic activity In this respect, a number of linkages responsive to a wide range of stimuli (i.e., pH, light, tempera­ ture, enzymatic or redox reactions) were investigated in the last decades (Wang et al., 2019) Among the pH-responsive linkages, those based on imines or boronic esters have been studied to load drugs into polysaccharide NGs (Table IB) Imine bonds can be hydrolysed under very slightly acidic conditions (pH ~ 6.8) which are, for example, typical of solid tumours In a work of M Pei et al Alg was oxidized with sodium periodate into N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 Fig Schematic representation of NGs formation from MD and a cross-linked pβ-CD, redrawn from R Gref et al., 2006 The drug was incorporated into the nanoassemblies by A) the formation of an inclusion complex of the drug with pβ-CD before the mixing with MD and B) by the drug loading within the preformed NGs aldehyde-functionalized Alg (Alg-CHO) before the conjugation with DOX (E.E., 37%), via direct Schiff base reaction Such work highlighted a reasonable loading efficacy, responsiveness, and physiological stability of the nano-formulations (Pei et al., 2018) Boronic acids bind to diols, forming cyclic boronic esters which are pH-responsive, being cleavable under acidic conditions In fact, B − O bonds show different hydrolytic stability when involving tricoordinated boron atoms (at low pH, easily hydrolyzable) or the quaternarized ones (at neutral or basic pH, more stable against hydrolysis) (Springsteen & Wang, 2002) Diols show a number of different structures, including sugars and catechols and typically, the affinity for boronates of sugar diols is markedly lower than that of aromatic diols (Gennari et al., 2017) Such pH-responsiveness has been exploited by E Montanari and co-workers to develop HA‑boronic acid (HA-APBA)-based NGs loaded with the poly-catechol tannic acid (TA) TA worked both as a drug and as a bi-functional cross-linker, for the NGs formation HA-APBA spontaneously reacted with TA at neutral pH, yielding NGs with a size that decreases with decreasing HA MW (e g., 200 nm for 4.4 × 104 g/mol, 400 nm for 7.37 × 105 g/mol) The boronate esters made NGs stable at physiological pH, but their hydro­ lysis in an acidic environment (pH = 5) led to swelling/solubilization, potentially allowing TA release in endosomal compartments (Montanari et al., 2016) (Fig 6) A similar approach was also explored by F Abdi and co-workers (Abdi et al., 2020) Boronic esters also show a redox responsive behaviour In fact, C − B bonds can be easily cleaved by oxidants (i.e., hydrogen peroxide), possibly working as a tool to release payloads under oxidative conditions (e.g., sites of inflammation) (de Gracia Lux et al., 2012) Redox responsive linkages can be also degraded by glutathione (GSH) Since in tumour cells GSH concentration can reach values four time higher than those in normal cells (Bansal & Simon, 2018), GSH-responsive nano­ particles were engineered to improve the delivery and the release of therapeutics into cancer cells (Alejo et al., 2019) In this respect, the most studied linkage is the disulphide bridge which is cleaved by GSH especially in cells, leading to the intracellular drug release T Yin et al coupled adryamicin/DOX to a disulphide-hydrazine-functionalized HA (E.E., 75%), forming dual responsive (redox and pH) NGs (Yin et al., 2018) J Xia et al exploited the 4-dimethylami-nopyridine activation of Pul followed by cystamine functionalization and protoporphyrin IX photosensitizer conjugation through amide bond (Xia et al., 2017) A novel microfluidic approach was proposed by T.H Tran et al with the Fig Schematic representation of pH-responsive HA‑boronic acid-based NGs, chemically cross-linked with TA through reversible boronate esters Reprinted with permission from (Montanari et al., 2016) Copyright (2016) John Wiley & Sons N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 partially folded or misfolded proteins typically expose hydrophobic domains on their surface which might cause irreversible protein ag­ gregation; molecular chaperones reversibly bind the protein hydro­ phobic regions, thus preventing misfolding and/or aggregation and preserving the protein activity (Eichner et al., 2011) In this respect, NGs formed by amphiphilic polysaccharides, such as Pul-Ch, were exten­ sively investigated (Nomura et al., 2003; Takahashi et al., 2011) In­ teractions between NGs and proteins arise from complex mechanisms, which may be predominantly electrostatic, hydrophobic, as well as being complemented by Van der Waals forces (Salmaso & Caliceti, 2013) These forces can be optimized in order to accommodate specific proteins, by modifying the NGs structure and the external medium during the loading For example, NGs based on amphiphilic poly­ saccharides, are able to encapsulate proteins, predominantly through the orientation of the hydrophobic residues on the protein surface to­ wards the hydrophobic moieties within the NGs (Akiyama et al., 2007) Typically, a higher number of protein hydrophobic residues show rather strong forces with hydrophobized polysaccharide-based NGs, leading to both, high E.E values and stability of the nano-formulations (Takahashi et al., 2011) Moreover, the extent of the protein interactions with NGs, also depends on the size and MW of the protein K Akiyoshi and col­ leagues demonstrated that the loading of small proteins, like insulin, increased with an increase of the hydrophobic moieties linked to the polysaccharide chains, whilst larger proteins, like bovine serum albumin (BSA) showed a different behaviour (Akiyoshi et al., 1998) (Poly)peptides can be physically or chemically entrapped into NGs: in fact, the loading of (poly)peptides mainly occurs through either passive diffusion into the NGs (already formed) or through in-situ crosslinking of the NGs in the presence of the protein molecules (Table II) aim to develop heparin-based NGs delivering retinoic acid (RA) and folic acid (FA) RA and FA were coupled via acid cleavable bonds to NGs The modified heparin was previously synthesized in a solvent-resistant lab on-a-chip microreactor, mixing heparin, FA and ethyl dimethylamino­ propyl carbodiimide in formamide and RA in dimethyl formamide, at different flow rates to modulate the coupling ratio of RA to heparin The used organic phase ratios were 1:1 (v/v) Successively, the modified heparin was able to self-assemble in aqueous medium into NGs in which RA and FA were covalently loaded (E.E., 94% and 40% for RA and FA, respectively) (Tran et al., 2012) This approach allowed to overcome the solubility issues related to the bulk reactions, but it did not avoid the use of organic solvents Strategies to load (poly)peptides into NGs Cytokines, growth factors and antibodies are examples of biologi­ cally active proteins which represent a promising class of macromolec­ ular therapeutics of the last decades (Desai & Brightling, 2009; Martino et al., 2015; Scott et al., 2012) However, proteins are often unstable and quickly degrade in the human body, because of the activity of enzymes (e.g., proteases), the side-products of cell metabolism (i.e., radicals) or acidic pH conditions (Lecker et al., 2006; Uzman et al., 2000) It is therefore necessary to find strategies which allow protecting the struc­ ture, controlling the release and localising the delivery of proteins in the human body, thus guaranteeing the effectiveness of the therapy with less side effects (Arnfast et al., 2014) This might be achieved by using nanocarriers, which can improve the biological half-life of proteins as well as their effectiveness in situ (Ray et al., 2017; Solaro et al., 2010) However, the protein encapsulation into nanocarriers represents a crucial step which should avoid the aggregation of the macromolecules, hence, the loss of protein activity Self-assembled Pul-Ch NGs show a peculiar ability, the so called ‘artificial chaperone activity’, that offers a number of advantages, like the prevention of protein aggregation and precipitation during the entrapment step (Hashimoto et al., 2018) In fact, in living systems, molecular chaperons regulate the protein folding: 3.1 Physical loading T G Van Thienen and collaborators prepared protein-loaded Dex NGs by using liposomal vesicles as reactors (Van Thienen et al., 2007) Table II Summary of the physical A) and chemical B) loading strategies employed in the preparation of poly(peptides)-loaded NGs A Physical loading Class of therapeutics Starting material Loading driving forces Loading strategy References Poly(peptides) Pul-Ch Dex-derivative Hydrophobic forces Van der Waals forces Alg Van der Waals forces NGs incubation with the cargo Protein addition to the polymer solution/suspension, followed by NGs formation Microfluidics Octenyl succinic anhydridemodified HA Cs Both hydrophobic forces and electrostatic interactions Electrostatic interactions Microfluidics - Akiyoshi et al., 1998 - Van Thienen et al., 2007 - Bazban-Shotorbani et al., 2016 - Water et al., 2015 Flash nanocomplexation - He et al., 2017 B Chemical loading Stimuli responsiveness Ligand Starting material Loading strategy References Redox-responsive Synthetic antigenic peptides Cationic Dex containing methacylamidedisulphide linker Cationic Dex containing methacylamidedisulphide linker Anionic methacrylated Dex Disulphide conjugation after NGs formation Disulphide conjugation after NGs formation Disulphide conjugation after NGs formation Disulphide conjugation before NGs formation Schiff base condensation after NGs formation Schiff base condensation before NGs formation - Kordalivand et al., 2019 - Li et al., 2016 S-acethylthioacetate OVA pH-responsive RNase A modified with Traut's reagent Cysteinylated exendin-4 (Pyridyldithio)-propionate Cs Bovine haemoglobin Aldeide-functionalized Dex OVA Oxidazed Alg - Kordalivand et al., 2018 - Ahn et al., 2013 - Wei et al., 2017 - Zhang et al., 2017 N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 Same NGs were coated with a lipid layer (coated-NGs or naked-NGs, respectively) and the effect of the cross-link density of the NGs network was investigated by following the release of BSA and lysozyme The cross-link density had a clear effect both on BSA and lysozyme release; in fact, higher cross-link density leads to a slower release of the two proteins Moreover, compared to naked-NGs, coated-NGs released BSA more slowly In contrast, the release of lysozyme from coated- and naked-NGs occurred similarly Furthermore, lysozyme was released faster than BSA from NGs, independently from the cross-link density This may be ascribed to the different protein size, being lysozyme much smaller than BSA (14.7 kDa and 66.7 kDa for lysozyme and BSA, respectively) The encapsulated lysozyme retained 75% of its biological activity after the loading process Pore size and density of the NGs network are important parameters which should be finely controlled, both for an efficient loading and for a sustained release of (poly)pep­ tides In this respect, S Bazban-Shotorbani and co-workers used a crossjunction flow focusing microfluidic chip for developing Alg-based NGs with controlled pore sizes, dimensions and density (Fig 7A) (BazbanShotorbani et al., 2016; Hasani-Sadrabadi et al., 2012) Specifically, Alg solution was used as inner phase at a flow rate of 0.5 μL/min, whilst a CaCl2 solution was injected into the two lateral streams at the flow rate in the range of 24.0–2.8 μL/min The relationship between the “on-chip” time of mixing and the average pore size was studied: the increase in the flow ratio led to an increase in the pore size and dimensions of the NGs and to a decrease of their density This approach was then employed for studying both the loading and release of a model protein, BSA There was a direct relationship between pore size of NGs and the release rate NGs formed by bulk mixing showed the fastest release rate of BSA, probably due to the lack of control in the NGs structure, which leads to the formation of pores with the largest average sizes, whereas lower release profiles were obtained by decreasing the flow rate of the NGs formation, and hence the pore size (Bazban-Shotorbani et al., 2016) Although microfluidics seems to finely tune the BSA release from NGs, no data regarding the BSA biological activity retention are reported, after its encapsulation into Alg-based NGs Another important param­ eter that should be considered is the NGs tortuosity, which is the path that molecular or macromolecular therapeutics should cross to diffuse throughout the network (Saltzman, 2001) Tortuosity can also be tuned with a microfluidic apparatus, by changing the polymeric content in the chip: high polymer concentration increases tortuosity leading to a slower release rate (Bazban-Shotorbani et al., 2016) The microfluidicbased system was also used by J.J Water and collaborators to formu­ late self-assembled Novicidin-loaded octenyl succinic anhydride-HA NGs (Water et al., 2015) Novicidin belongs to the group of antimicrobial peptides (AmPs) AmPs are an emergent class of antimi­ crobial agents, consisting of 10–50 amino acids, typically having overall positive charge and amphiphilic three-dimensional structure (Zasloff, 2002) In this work, the aqueous polymer solution was injected into the lateral streams of a three-channelled microfluidic platform, whilst the novicidin solution was injected in the central one The polymernovicidin ratio was fixed at 9:1 The study evidenced that the flow rate was the main determinant for both ζ-potential and E.E of NGs: in fact, by increasing the flow rates an increase in ζ-potential values was observed, whereas the E.E was inversely related to the flow rate By contrast, the mean hydrodynamic diameter of NGs was not significantly affected by any parameter in the microfluidic chip, suggesting that the flow rate manly has an impact on the internal structure and organization of NGs, without affecting the sizes (Water et al., 2015) Moreover, to assess whether novicidin encapsulation into NGs leads to lower anti­ microbial activity, a standard minimum inhibitory concentration (MIC) test was performed, demonstrating no reduction of the antimicrobial activity against S aureus (Water et al., 2015) Another novel approach that offers a high degree of control over particle size and distribution is the flash nanocomplexation (FNC) (Santos et al., 2016) FNC is a technique that allows the continuous and scalable production of uniform polyelectrolyte nanocomplexes, thanks to the kinetically controlled and rapid mixing of aqueous polycation and polyanion streams, which collide in the jet mixer (Lee et al., 2019) Despite the bulk mixing or pipetting procedures, which are widely used in laboratory-scale preparations, but often lead to low reproducibility of the samples, FNC allows preparing highly reproducible nanostructures in a continuous flow operation process, which is amenable to the scaleup production (Santos et al., 2016) In this respect, Z He and coworkers, developed insulin-entrapped Cs/tripolyphosphate (Cs/TPP)based NGs (He et al., 2017) After adding Cs, TPP, insulin and water into the four inlets of a multi-inlet vortex mixer (MIVM) device (Fig 7B), three essential parameters were controlled: flow rate, Cs/TPP/insulin mass ratio and pH In fact, the average size of NGs decreased from 115 to 45 nm as the flow rate increased from to 25 mL/min; the loading content of insulin increased with the Cs/TPP ratio and it was strongly dependent by the final pH of the mixture in the MIVM chamber, reaching E.E of ~90% at pH 6.5 On the other hand, Cs/ TPP NGs prepared by drop-wise addition and bulk mixing, exhibited larger size and PDI, lower E.E (62 and 42% for drop-wise addition and bulk mix­ ing, respectively) and released the double amount of insulin within the first h, compared to NGs prepared by the FNC method Fig Schematic representation of A) microfluidic-based system, reprinted with permission from (Bazban-Shotorbani et al., 2016) Copyright (2016) American Chemical Society; and B) flash nanocomplexation, for producing (poly)peptides-loaded NGs, reprinted with permission from (He et al., 2017) Copyright (2017) Elsevier N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 3.2 Chemical loading by smart linkages intracellular localization and release In 2018, the first nanoformulation (Onpattro), based on ionizable lipids delivering siRNA, was approved by the Food and Drug Administration for the treatment of polyneuropathies (Akinc et al., 2019; Kulkarni et al., 2018) Two years later PfizerBioNtech and Moderna exploited a similar technology for formulating the anti-Covid 19 vaccines, delivering mRNA encoding genetic variants of the SARS-CoV-2 spike protein (Nature Nanotechnology, 2020; Shin et al., 2020) Despite lipid nanoparticles, also polysaccharide-based NGs have been investigated for loading and delivering several RNA- and DNA-based materials (Cevher et al., 2012; Khan et al., 2012; Kumari & Badwaik, 2019; Raemdonck et al., 2013), as reported in this chapter Strategies for loading (poly)peptides into NGs, by exploiting covalent linkages have been also investigated, with the aim to achieve more stable nano-systems capable to release the cargo in situ, in a responsive fashion In this respect, N Kordalivand et al developed NGs loaded with antigenic peptides via disulphide bonds (Kordalivand et al., 2018) A number of synthetic peptides, with the MW of ~2.5 × 103 g/mol were synthesized via fluorenylmethyloxycarbonyl solid phase approach, with the aim to introduce CTL and CD4+ T-helper epitopes, for the induction of T-cell response The employed polysaccharide was a methacrylatederivatized Dex which was functionalized with a methacrylamidedisulphide linker NGs were obtained by inverse mini-emulsion tech­ nique, photo-polymerized and, finally, the cys-ending peptides were covalently conjugated to NGs This procedure allowed to obtain a redoxresponsive nano-system with high peptide content (E.E., 86–96%) and an average size of ~200 nm A similar strategy was adopted by D Li et al that linked the functionalized S-acethylthioacetate ovalbumin (OVA) to Dex-methacrylate-based NGs, after a deacetylation step (Li et al., 2016) N Kordalivand et al linked RNase A (1.4 × 104 g/mol) through disulphide bonds to Dex-methacrylate-based NGs, with the aim to trigger the protein release under reductive conditions (Kordalivand et al., 2018) The nano-complex was modified in situ with the Traut's reagent, in order to obtain responsive covalent linkages A yeast RNA digestion assay showed that 86% of the RNase A biological activity was retained after conjugation, whilst the RNase A E.E was 72% Even the peptide Exendin was conjugated via responsive disulphide bonds to Csbased NGs, by S Ahn et al (Ahn et al., 2013) After the reaction, NGs exhibited an average size of 100 nm, whilst the conjugated Exendin retained its starting biological activity, which was assessed by a glucoseinduced insulin secretion study carried out on INS-1 pancreatic β-cells X Wei et al linked bovine haemoglobin (6.4 × 104 g/mol) to Dex-based NGs by exploiting the imine bond, via a Schiff-base reaction, which was carried out in three steps (Wei et al., 2017) Dex was modified with a succinic-dopamine moiety and subjected to spontaneous self-assembly under acidic conditions NGs were then oxidized with sodium period­ ate to obtain both crosslinking and ring-opening formation of aldehyde moieties, available for the subsequent haemoglobin conjugation through Schiff-base reaction NGs showed dimensions of approximately 350 nm that were reduced to 260 nm after the haemoglobin conjugation (E.E., 34%) Authors claimed the oxygen affinity of loaded haemoglobin was higher than that of free haemoglobin; however, data regarding the retention of the biological activity of the protein were not reported, after loading A similar strategy was explored by C Zhang et al., that developed pH-responsive NGs by ionic crosslinking of two types of functionalized Alg (Zhang et al., 2017) The first played the targeting role, bearing an aminophenyl-α-D-mannopyranoside moiety (MAN-Alg), the second worked as a drug-carrier being conjugated to the model OVA protein via iminic bond (OVA-Alg), through the oxidation step, followed by a Schiffbase reaction NGs showed an average size of 310 nm and an E.E of 51% 4.1 Physical loading Genetic material was loaded into NGs (Table IIIA) mainly using two encapsulation strategies (Barclay et al., 2019) The first is named ‘presynthetic loading’ and is based on the mixing of nucleic acids with the polymer chains during the NGs formation (Fig A), whilst the second is defined as ‘post-synthetic loading’ and refers to the nucleic acid adsorption on the already formed NGs (Fig B) 4.1.1 Pre-synthetic loading: The pre-synthetic loading allows the one-step preparation of geneloaded NGs and usually ensures good E.E and protection of the nucleic acids from degradation (Kandil & Merkel, 2019; Vauthier et al., 2013) Typically, Cs is widely used for engineering gene material-based polysaccharide NGs (Lee et al., 2009; Wang et al., 2017) thanks to its polycationic nature that allows to establish electrostatic interactions with the negatively charged nucleic acids H.D Han et al entrapped siRNA into Arg-Gly-Asp (RGD) peptide modified Cs via ionic gelation RGD peptide was previously conjugated with Cs by thiolation reaction and then TPP and siRNA were mixed with the RGD-Cs polymer solution siRNA/RGD-Ch NGs were spontaneously formed under stirring at 25 ◦ C The NGs size was around 200 nm and the presence both of RGD and siRNA in NGs was confirmed by fluorescence microscopy, using FITClabeled RGD (green) and Alexa555-labeled siRNA Unfortunately, the E.E of siRNA in the formulation was not reported (Han et al., 2010) A similar strategy was employed by C He et al who modified Cs with methyl iodide, mannose and cysteine forming the mannose-modified trimethyl Cs-cysteine (MTC) derivative Subsequently, siRNA and TPP were dissolved in water and added dropwise to the MTC solution under stirring at 37 ◦ C for 30 min, with the aim to allow the NGs formation via ionic gelation The NGs size was around 150 nm and the nanosystem was tested in vivo via oral administration Unfortunately, even in this work the E.E of siRNA in the formulation was not reported (He et al., 2013) The MW and deacetylation degree (DD) of Cs might influence the gene encapsulation capacity and the transfection efficiency of NGs, in relation to the number of available cationic moieties In this respect, E Lallana and co-workers formulated Cs/HA NGs loaded with mRNA or siRNA and studied the effects of parameters, such as the Cs MW and DD on the E.E and on the transfection efficiency mRNA- and siRNA-loaded Cs/HA NGs were produced with a two-step process, consisting of an initial RNA/Cs complexation, followed by the addition to HA NGs with a size between 200 and 300 nm were obtained The different Cs MW and DD did not affect the ability of NGs to entrap mRNA or siRNA: in fact, both RNAs were quantitatively entrapped (E.E >95%) into NGs Moreover, they did not even affect the ability of NGs in protecting both the loaded siRNA and mRNA On the other hand, the molecular size of the payload affected the NGs size, with siRNA providing smaller NGs than mRNA Furthermore, siRNA was more easily released from NGs than mRNA and better mRNA transfection was observed with larger MW Cs, whereas no clear influence of Cs MW was seen on siRNA activity (Lallana et al., 2017) Although its polyanionic nature, HA has been investigated as a material for gene delivery, thanks to its ability to target specific re­ ceptors (e.g., CD44) (Lee et al., 2007) J.S Park and co-workers prepared HA-shielded polyethylenimine (PEI)/pDNA NGs in HEPES-buffered Loading of genetic material into NGs Gene transfer refers to the insertion of one or multiple foreign genes or genetic sequences in a specific and identified cell population, by using a selected gene delivery system (Doudna, 2020; Remaut et al., 2007) Messenger RNA (mRNA), short interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), doublestranded DNA (dsDNA) can be introduced in the human body with the aim to treat a number of diseases (Cullis, 2015; Friedmann & Roblin, 1972; Hao et al., 2017; Verma & Weitzman, 2005) However, unpro­ tected RNA- and DNA-based materials are quickly degraded in the body fluids, therefore nanoparticles play a fundamental role in shielding the cargo from degradation and in offering control over its biodistribution, 10 N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 Table III Summary of the physical A) and chemical B) loading strategies employed in the preparation of gene-loaded polysaccharide NGs A Physical loading Class of therapeutics Starting material Loading driving forces Loading strategy References mRNA, siRNA, miRNA, pDNA, ssDNA, ds-DNA Cs or Cs derivatives Electrostatic interactions - Han et al., 2010 - He et al., 2013; - Lallana et al., 2017 HA derivatives or HA/PEI mixtures Electrostatic interactions Cationic Pul derivatives Electrostatic interactions Addition of genetic material to the polymer suspension or solution, followed by NGs formation Addition of genetic material to the polymer solution, followed by NGs formation Microfluidics Addition of genetic material to the polymer solution, followed by NGs formation Microfluidics Mixing of genetic material with the polymer solution, followed by NGs formation Adsorption of genetic material on the NGs shell Cationic Dex derivatives Electrostatic interactions NGs incubation with genetic material - Ho et al., 2009 - Park et al., 2016 - Agnello et al., 2017 - Wang et al., 2014 - San Juan et al., 2009 - Chen et al., 2017 - Zhao et al., 2015; - Hu et al., 2019; Liu et al., 2019; - Zink et al., 2019 B Chemical loading Stimuli responsiveness Ligand Starting material Loading strategy References Redox-responsive NGs Thiolated siRNA Reducible HA derivative Disulphide conjugation before NGs formation Disulphide-linked poly-siRNA Thiolated glycol Cs Disulphide conjugation before NGs formation - Park et al., 2013 - Lee, Kong, et al., 2014 - Lee, Lee, et al., 2014 solutions Firstly, the polycationic PEI was mixed with the polyanionic pDNA forming complexes which were then coated with HA, producing NGs PEI:HA ratio ranged from 1:1 to 1:10 leading to an increase of the NGs size from 70 to 150 nm, respectively Unfortunately, the E.E of PEI/ pDNA complexes in NGs was not reported (Park et al., 2016) Also, Pul has been exploited for formulating NGs for gene delivery For example, FA- and PEI-modified Pul derivatives were used to encapsulate pDNA and siRNA, leading to P-PEI/pDNA and P-PEI-FA/pDNA NGs (Wang et al., 2014) Microfluidics has been used for developing RNA- or DNA-loaded nanomaterials with the aim to make the formulations scalable and to allow the simultaneous evaluation of several transfection conditions (Giupponi et al., 2018; Kim et al., 2011; Leung et al., 2012) Y.P Ho et al prepared Cs/pDNA nanocomplexes by controlling the rapid mixing of labeled-pDNA with Cs in a microfluidic T-junction device at the flow rate of 12 or 20 nL s− The formation kinetics of the nanocomplexes was confirmed by quantum-dot-mediated FRET, even though the NGs size and DNA loading were not reported (Ho et al., 2009) S Agnello and colleagues developed HA-EDA-C18 NGs by using a hydrophilic splitand-recombine micromixer containing 12 mixing stages (Agnello et al., 2017) Different solutions were pumped in different microfluidic channels, leading to the formation of NGs by modulating their selfassembly into a micromixer chip Four different flow ratios, equal to 0.05, 0.1, 0.25, and 0.5 (expressed as the ratio between the flow of the polymer dispersion and the external phase) were employed by keeping constant the flow rate of the polymer dispersion at 100 μL/min and varying the flow rate of the external phase between 2000 and 200 μL/ The particle size increased from 150 to 450 nm according to the increase of the flow ratio from 0.05 to 0.5, which was ascribed to the controlled nanoprecipitation of amphiphilic HA-EDA-C18 due to the diffusion of the polymer into the saline external phase and its nucleation The group employed a similar HA-based NGs for the complexation of siRNA (Palumbo et al., 2015) already formed NGs and DNA or RNA (Park et al., 2016) However, it should be taken into account that such approach might lead to several disadvantages: I) lower stability of the drug in the NGs network and, hence, in the body fluid; II) possible initial burst release of the cargo; III) less protection of the DNAs or RNAs by NGs For the post-synthetic loading, the polycationic Cs-based NGs have been extensively exploi­ ted (Bao et al., 2011; Edson et al., 2018; Yeo et al., 2010) Polycationic Pul derivatives were also synthetised by using, for instance, diethyl amino ethyl amine (DEAE) (San Juan et al., 2007; San Juan et al., 2009) or PEI (Mao et al., 2010; Rekha & Sharma, 2011; Ambattu & Rekha, 2015) These Pul derivatives might show high cationic charge density, contributing to enhance the condensation ability of nucleic acids and to improve the gene loading efficiency L Chen et al reported the Pulbased amphiphilic bifunctional polymer for the co-delivery of both Dox and pDNA for cancer therapy (Chen et al., 2017) In this study, Pul was grafted to desoxycholic acid (DA) and PEI DA was selected to form a hydrophobic core for the encapsulation of DOX, whilst positively charged PEI, located on the NGs hydrophilic shell, allowed the encap­ sulation of the negatively charged pDNA with high loading efficiency value, even though the exact E.E was not reported The NGs size was around 160 nm Cationic amino acids, such as arginine and histidine as well as PEI, were grafted to Dex for achieving NGs-based gene delivery systems (Hu et al., 2019; Liu et al., 2019; Zhao et al., 2015; Zink et al., 2019) Typically, cationic materials are essential for forming stable nanocomplexes with DNA or RNA, even though they might display higher cytotoxicity than neutral or negatively charged materials, due to the possible mitochondrial and lysosomal damages as well as to the strong interactions that can establish with the plasma membranes, leading to their disruption (Fră ohlich, 2012) This is why, long-term therapies based on these formulations are still not feasible, due to an insufficient compliance However, single or double injections of these materials did not report highly relevant side effects, so far (Feldman et al., 2019; Mulligan et al., 2020) 4.1.2 Post-synthetic loading Another approach for the gene loading into polysaccharide NGs, is the formation of nanocomplexes via electrostatic interactions between 11 N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 Fig Strategies for gene encapsulation: A) pre-synthetic loading and B) post-synthetic loading 4.2 Chemical loading by smart linkages our knowledge, works in which gene-based materials were covalently loaded into polysaccharide NGs via microfluidics were not found It is worth noting that even if NGs have been investigated for delivering DNAs and RNAs, so far the most efficient non-viral delivery vector is based on ionizable or cationic lipid nanoparticles (Buck et al., 2019; Jayaraman et al., 2012; Kulkarni et al., 2018) Therefore, further studies are necessary for improving the ability of NGs to maximize the potency of DNA- or RNA-based therapeutics The use of covalent crosslinking was recently investigated for loading genes into polysaccharide NGs (Table IIIB) Covalent linkages between NGs and the genetic material should allow higher stability and protection of the cargo, however two requirements are necessary: I) DNAs and RNAs should not be damaged during the crosslinking reaction that might require hard conditions; II) the linkage between DNAs or RNAs and NGs should be cleaved in the way that the cargo is released without any structural modification, in the appropriate intracellular compartment A common strategy is, for example, the formation of redox responsive linkages (i.e., disulphide bridge), which allow exploiting the redox processes that normally occur in the intracellular environments This approach was followed by K Park and co-workers for the preparation of HA-siRNA conjugates through chemical cross­ linking (Park et al., 2013) The thiolated siRNA was covalently coupled to the positive HA derivative that was previously functionalized with a bifunctional linker, achieving an E E of 75% Then, a tight complex was obtained by adding the polycationic linear-PEI that allowed the forma­ tion of NGs with an average size of 250 nm The linkage responsiveness was confirmed by gel electrophoresis, using tris(2-carboxyethyl) phosphine as a reducing agent The resulting NGs appeared to show higher in vitro gene silencing efficiency than the non-cleavable HA-based NGs (60–70% vs 30–40%, respectively) A similar strategy was employed by M.Y Lee et al who conjugated thiolated siRNA to disulphide-linker-functionalized HA (65% E.E.), followed by their complexation with lipoprotein-based cationic solid lipid nanoparticles (CSLNs) In this way, NGs with the average size of ~300 nm were ach­ ieved and were capable of an efficient liver-specific transfection and gene silencing (Lee, Kong, et al., 2014) Another approach was proposed by S.J Lee et al (Lee, Lee, et al., 2014) The system was based on sense and antisense strand couples of a thiol-modified siRNA annealed together and then linked to form a disulphide-linked poly-siRNA chain with thiol extremities under basic conditions These poly-siRNA chains were then assembled and cross-linked to thiolated glycol Cs, exhibiting up to 78% gene silencing The modification on the 5′ end-thiolated siRNA did not affect the RNA activity (Lee et al., 2010) To the best of Strategies to formulate vaccine-based NGs Recently, NGs have been engineered to stimulate or suppress im­ mune responses or to enhance antigen delivery in the prevention or treatment of infections (Cordeiro et al., 2015), cancer (Muraoka et al., 2014), allergies (Ferreira et al., 2013) and autoimmune diseases (Feng et al., 2019) Among them, Pul-Ch NGs have been evaluating in clinical ´ndez-Adame et al., 2019) The in­ trials as anti-cancer vaccines (Herna terest in polysaccharide NGs relies on their ability to deliver and to protect antigens in vivo (Han et al., 2018) as well as to mimic the composition of natural pathogens (Cordeiro et al., 2015; Neamtu et al., 2017) In this respect, in vitro studies have shown that NGs may activate the humoral immunity (Dacoba et al., 2019), i.e maturation of dendritic cells (DCs, a class of antigen-presenting cells), behaving as synthetic adjuvants (Dobrovolskaia & McNeil, 2007) The NGs interaction with antigen-presenting cells (APCs) mainly depends on the polymer MW, size, shape, surface charge, hydrophobicity/hydrophilicity ratio of NGs ´ndez-Adame et al., 2019) Such properties also affect the (Herna entrapment efficiency and the release of the NGs cargos So far, NGsbased vaccines have been mainly formulated by using synthetic pep­ tides and full-length proteins containing one or several epitopes of a pathogen protein that might be recognized by B and T cells as well as by using genes (DNA or RNA) encoding a protein (Ferreira et al., 2013) Positively charged NGs-based vaccines have been widely exploited as they show high uptake by DCs and can be prepared without using organic solvents (which may alter the antigen immunogenicity) Spe­ cifically, Cs NGs have been investigated for intranasal vaccine design, thanks to the easy NGs formation via ionic gelation in aqueous 12 N Zoratto et al Carbohydrate Polymers 266 (2021) 118119 environments and the intrinsic positive charges that facilitate the interaction with the negatively charged mucins on the mucosal surfaces Physically crosslinked Cs NGs were used for the delivery of the recom­ binant hepatitis B surface antigen (rHBsAg) to induce immunization against hepatitis B infection (Prego et al., 2010) The formation and the encapsulation of the rHBsAg was obtained by dissolving the antigens in the TPP solution and adding it to a Cs solution Free amino groups of Cs were able to interact with the negatively charged antigen molecules The encapsulation of tetanus taxoid (TT) into Cs/TPP NGs was also investi­ gated, leading to high and long-lasting IgG immune responses, after mice nasal administration (Vila et al., 2002) The same Cs/TPP system and loading strategy were also used to encapsulate the enzymatic extract of Streptococcus equi (S equi), the recombinant disulphide isomerase protein (recNcPDI) of Neospora caninum and the superoxide dismutase (SODB1) of Leishmania with the aim to induce vaccination against S equi, Neospora caninum and Leishmaniosis infections, respectively (Danesh-Bahreini et al., 2011; Debache et al., 2011; Figueiredo et al., 2012) Cs/TPP NGs were decorated with the mannosylate derivative of HA (HA-Man) by A Gennari and co-workers with the aim to obtain a synergic DCs targeting: HA and mannose interact with CD44 receptors and mannose-binding lectins (typical DC pattern recognition receptors), respectively The use of low MW Cs enabled a better exposure of HAMan followed by a significant increase in NGs uptake, suggesting that the interactions with mannose-binding receptors require a correct ligand presentation (Gennari et al., 2016) Other polysaccharides, such as Dex (Kordalivand et al., 2019; Li et al., 2016), Man and beta glucans (Jin et al., 2018) were used as car­ riers for vaccines, thanks to their presence in the cell walls of a wide range of pathogens, i.e bacteria, yeast Such property enables their recognition by APCs and, hence, working as integrated adjuvant (Cor­ deiro et al., 2015) Cationic Pul-Ch NGs were loaded with botulinum type-A neurotoxin subunit antigen Hc (BoHc/A) and were evaluated as effective vehicles for adjuvant-free intranasal vaccines in mice NGs loading was achieved by incubating NGs suspension with vaccine antigen at 1:1 M ratio Cationic NGs were administered intranasally to mice and they were retained in the nasal mucosa thanks to the interactions with the anionic epithelial cell layer, whilst their cargo was taken up by DCs after its release The immunized mice showed high concentration of botulinic neurotoxin/A specific IgA and IgG antibodies and they survived without any clinical sign when infected with C botulinum-producing neurotoxin (BONT/A) intraperitoneally or intranasally, indicating that the NGsbased vaccine nanoformulation induced both systemic and mucosal protective immunity (Nochi et al., 2010) The same Pul-Ch or cationic Pul-Ch NGs were also exploited for the design of several anticancervaccines, most of which have been investigating in phase I or I + II clinical trials (Kawabata et al., 2007; Kitano et al., 2006; Kyogoku et al., 2016; Shimizu et al., 2008) In all these works, the antigen loading within Pul-Ch NGs was obtained by mixing NGs with the cargos, thus leading to the formation of hydrophobic forces between the Ch moieties of the NGs and the hydrophobic domains of the antigen molecules Several studies were focused on the use of Cs NGs to encapsulate DNA or RNA for both parental and mucosal vaccination (Bivas-Benita et al., 2004; Cambridge et al., 2013; Khatri et al., 2008) Positive Cs NGs loaded with pDNA encoding the surface protein of Hepatitis B virus (HBsAg) were prepared by K Khatri et al NGs were obtained via ionic gelation between Cs and pDNA/TPP mixture, showing E.E of 96% Fe­ male BALB/c mice were vaccinated intranasally with Cs/pDNA NGs, resulting in both systemic and mucosal humoral immune responses Specifically, Cs-based NGs induced a 9-fold increase of the anti-HBsAg IgG compared to the conventional alum-adsorbed vaccine, suggesting the adjuvant ability of Cs NGs (Khatri et al., 2008) Similarly, M BivasBenita et al., prepared Cs NGs loaded with pDNA encoding different epitopes of Mycobacterium tuberculosis by coacervation Authors showed that Cs NGs protected the payload from nuclease degradation, induced the maturation of DCs and increased the IFN-γ secretion from T-cells after pulmonary mucosal immunization (Bivas-Benita et al., 2004) The same coacervation approach was used by C D Cambridge et al with the aim to encapsulate pDNA encoding for the major outer membrane protein (MOMP) of Chlamydia trachomatis, into CS NGs After parental vaccination, the MOMP gene transcript was expressed locally and sys­ temically in mice tissues (Cambridge et al., 2013) Other cationic polysaccharide derivatives, i.e quaternized β-glucan or spermine-Man were investigated for developing genetic material-loaded NGs as vac­ cines (Ruan et al., 2014; Tahara & Akiyoshi, 2015; Wang et al., 2012) So far, only few works describe the use of microfluidics for the preparation of NGs-based vaccines F Fontana et al used nano­ precipitation in a glass-capillary device with a co-flow geometry, in order to prepare the initial two layers of the nanovaccine, based on the spermine-modified acetalated Dex (SpAcDEX) and on the thermally oxidized porous silicon particles (TOPSi) Specifically, TOPSi NGs were suspended into an ethanol solution of SpAcDEX and their mixture was used as the inner phase at the flow rate of mL/h, whilst 1% polyvinyl alcohol was selected as the outer phase at the flow rate of 40 mL/h (the used volume ratios were 1:20 for the inner and outer phases, respec­ tively) NGs showed an average size of ~240 nm and a low PDI value (0.08) Such NGs were then conjugated to a model antigen (Trp2) to form TOPSi@SpAcDEX@Trp2 NGs or coated with vesicles derived from cancer cells (CCM), to form TOPSi@SpAcDEX@CCM NGs After the Trp2 conjugation, both NGs average size and PDI increased up to ~400 nm and 0.29, respectively The obtained nanosuspension was reported to show immunostimulant properties in human cells, promoting the expression of co-stimulatory signals and the secretion of pro-inflam­ matory cytokines (Fontana et al., 2017) Conclusions Since their discovery, NGs have become an important nanocarrier for delivering a wide range of molecular and macromolecular therapeutics Since there is not a single universal method that allow the achievement of suitable drug-loaded NGs formulations, a number of strategies have been explored for loading NGs with biologically active compounds, ac­ cording to both the drug and NGs physico-chemical properties Con­ ventional bulk approaches such as ultrasonic, dialysis, spray drying and high-pressure homogenization methods, rely on the breakdown of bulk products (top-down approach) or the nanoprecipitation/self-assembling of monomers (bottom-up approach), (Hamdallah et al., 2020; Zhang et al., 2019), allowing the loading of a number of therapeutics into NGs (i.e., low MW drugs, (poly)peptides, gene-based materials) However, the bulk NGs production might be significantly affected by high batch variability, poor-reproducibility and lack of control over the experi­ mental variables Moreover, both the synthesis and sterilization steps of drug-loaded NGs are far from making these formulations scalable at the industrial level, also due to the high production costs This is why, the NGs clinical translation is still facing several challenges, even though some NGs formulations already reached the clinical trials as anti-cancer vaccines In this respect, the autoclaving process showed several ad­ vantages: sterile and drug-loaded NGs (both with low molecular weight hydrophobic and hydrophilic drugs) were produced in a single step (i.e., a sterile cycle at 121 ◦ C, for 20 min) and with high reproducibility (de Rugeriis et al., 2013) Autoclaving might enhance the hydrophobic forces and electrostatic interactions between the drug and the polymer chains, meanwhile NGs are formed possibly thanks to the reduction of the polymer MW The mechanism with which the autoclaving process forms drug-loaded NGs 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Table II Summary of the physical A) and chemical B) loading strategies employed in the preparation of poly(peptides)-loaded NGs A Physical loading Class of therapeutics Starting material Loading