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.

Available online 28 April 2021

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Review

Strategies to load therapeutics into polysaccharide-based nanogels with a

focus on microfluidics: A review

N Zorattoa, E Montanarib,*, M Violaa, J Wanga, T Covielloa, C Di Meoa,*, P Matricardia

aDepartment of Drug Chemistry and Technologies, Sapienza University of Rome, 00185 Roma, Italy

bInstitute 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

Keywords:

Nanogels

Polysaccharides

Drug loading methods

Microfluidics

Nanogels-based vaccines

A B S T R A C T 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 three- dimensional 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

1 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, 1,4- dihydroxyanthraquinone; 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, pullulan- cholesterol; 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

(P Matricardi)

Contents lists available at ScienceDirect Carbohydrate Polymers

journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2021.118119

Received 11 February 2021; Received in revised form 4 April 2021; Accepted 15 April 2021

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

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 lactic-

co-glycolic acid, PEG, polyglycolic acid, polycaprolactone, poly(N-

isopropylacrylamid), 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 chemically cross-linked NGs (Kousalov´a & Etrych, 2018) Herein, only 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 1 Schematic representation of a hydrogel, microgel and NG

Fig 2 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

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

2 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

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 soso-lution, 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 thermo- sensitive 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

Fig 3 An example of a microfluidic setup for the preparation of drug-loaded NGs

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

-

- Pedrosa et al.,

2014

- Stefanello et al.,

2017

- Montanari et al.,

2019

- Yang et al., 2011 Addition of the drug solution to the

polymer suspension, followed by NGs formation

- Organic solvents may be required Thambi et al., 2014 Autoclaving process - Sterile and drug-loaded NGs

formed in a single-step procedure

- High reproducibility

- Incompatible with thermo- sensitive drugs

- Change of the polymer Mw

- Coll Ferrer et al.,

2014

- Manzi et al., 2017

- Musazzi et al.,

2018

- Di Meo et al.,

2015 Microfluidics/Millifluidics - High reproducibility

- Control over the size, PDI and compactness of NGs

- 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 Low-molecular

weight

hydrophobic drugs

CDs/polysaccharide mixtures or polysaccharides containing chelating moieties

Complexation or coordination NGs incubation with the drug solution - Long incubation time - Drug encapsulation

achieved by a two/three step procedure

- Gref et al., 2006

CDs incubation with the drug, followed by NGs formation - Long incubation time - Drug encapsulation

achieved by a two step procedure

- Gref et al., 2006

- Thiele et al., 2011

Incubation of the chelating polymer with the drug, followed by NGs formation

- Drug loaded NGs formed in a single step procedure

- Low versatility

- Ohta et al., 2016

Low-molecular

weight drugs Charged polysaccharides Electrostatic interactions Addition of drug to the polymer solution, followed by NGs

formation

- Typically good E.E.%

- Possible interference with the NGs formation

- Deacon et al.,

2015 Rossi et al., 2017

- Rajaonarivony

et al., 1993

- Curcio et al.,

2015 NGs incubation with the drug - Possible low stability in

human fluids - - Zhang et al., 2006 Yang et al., 2011

- Schmitt et al.,

2010

- Curcio et al.,

2015 Autoclaving process - Sterile and drug-loaded NGs

formed in a single-step procedure

- High reproducibility

- Incompatible with thermo- sensitive drugs

- Change of the polymer Mw

- Montanari et al.,

2018

Microfluidics/Millifluidics - High reproducibility

- Control over the size, PDI and compactness of NGs

- Moradikhah

et al., 2020

- Dong &

Hadinoto, 2017

(continued on next page)

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 T-

shape microfluidic device was provided with two inlets for aqueous

buffer (pH = 9), to achieve two water streams at the flow-focusing T-

junction, 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 2 mL/h was used as inner phase, whilst an

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-thromycin E.E was 45% (Kłodzi´nska et al., 2019) Other hydrophobic 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/Cs- based NGs by J Deacon et al Since tobramycin and Alg can strongly

B

Chemical loading

Stimuli responsiveness Ligand Starting material Loading strategy References

pH-responsive NGs DOX Aldehyde-functionalised Alg Schiff base condensation - Pei et al., 2018

Dihydrazide-modified HA Hydrazone linkage - Yin et al., 2018 Tannic acid HA‑boronate Cyclic boronic ester formation - Montanari et al., 2016 Aminated-RA and aminated-FA Heparin Amide bond formation - Tran et al., 2012 Redox-responsive NGs DOX Cystamine-modified HA Disulphide linkage - Yin et al., 2018

Protoporphyrin IX Cystamine-modified Pul Disulphide linkage - Xia et al., 2017

Fig 4 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

Table 1 (continued)

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 physico-

chemical 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 1 μL/min and two TPP

solutions (pH = 3) at a flow rate of 5, 7 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 (~ 1

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 5 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-pβ-

CD 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

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 5 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 nano- assemblies 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

Fig 6 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

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

3 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:

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)

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 Hydrophobic forces NGs incubation with the cargo - Akiyoshi et al., 1998

Dex-derivative Van der Waals forces Protein addition to the polymer solution/suspension,

followed by NGs formation - 2007 Van Thienen et al.,

et al., 2016 Octenyl succinic anhydride-

modified HA Both hydrophobic forces and electrostatic interactions Microfluidics - Water et al., 2015

Cs Electrostatic interactions Flash nanocomplexation - He et al., 2017

B

Chemical loading

Stimuli

Redox-responsive Synthetic antigenic peptides Cationic Dex containing methacylamide-

disulphide linker Disulphide conjugation after NGs formation - 2019 Kordalivand et al., S-acethylthioacetate OVA Cationic Dex containing methacylamide-

disulphide linker Disulphide conjugation after NGs formation - Li et al., 2016 RNase A modified with Traut's

reagent Anionic methacrylated Dex Disulphide conjugation after NGs formation - 2018 Kordalivand et al., Cysteinylated exendin-4 (Pyridyldithio)-propionate Cs Disulphide conjugation before NGs

formation - Ahn et al., 2013 pH-responsive Bovine haemoglobin Aldeide-functionalized Dex Schiff base condensation after NGs

formation - Wei et al., 2017

before NGs formation - Zhang et al., 2017

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 cross-

junction flow focusing microfluidic chip for developing Alg-based NGs

with controlled pore sizes, dimensions and density (Fig 7A) (Bazban-

Shotorbani 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 microfluidic-

based 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 polymer- novicidin 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 scale-

up production (Santos et al., 2016) In this respect, Z He and co- workers, 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 1 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 2 h, compared to NGs prepared by the FNC method

Fig 7 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

3.2 Chemical loading by smart linkages

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 methacrylate-

derivatized Dex which was functionalized with a methacrylamide-

disulphide 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 redox-

responsive 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 4 was conjugated via responsive disulphide bonds to Cs-

based 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 4

retained its starting biological activity, which was assessed by a glucose-

induced 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 Schiff-

base reaction NGs showed an average size of 310 nm and an E.E of

51%

4 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), double-

stranded 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,

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 Pfizer- BioNtech 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

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 ‘pre- synthetic loading’ and is based on the mixing of nucleic acids with the polymer chains during the NGs formation (Fig 8 A), whilst the second is defined as ‘post-synthetic loading’ and refers to the nucleic acid adsorption on the already formed NGs (Fig 8 B)

4.1.1 Pre-synthetic loading:

The pre-synthetic loading allows the one-step preparation of gene- loaded 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 FITC- labeled 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