Journal of Functional Biomaterials

Review The Use of Some Clay Minerals as Natural Resources for Drug Carrier Applications †

Marina Massaro 1,*, Carmelo Giuseppe Colletti 1 , Giuseppe Lazzara 2 and Serena Riela 1,*

1 Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), University of Palermo, Viale delle Scienze, Ed. 17, 90128 Palermo, Italy; [email protected] 2 Dipartimento di Fisica e Chimica (DiFC), University of Palermo, Viale delle Scienze, Ed. 17, 90128 Palermo, Italy; [email protected] * Correspondence: [email protected] (M.M.); [email protected] (S.R.); Tel.: +39-09123897546 (S.R.) † Dedicated to Prof. Giuseppe Musumarra for his brilliant scientific career.



Received: 20 September 2018; Accepted: 17 October 2018; Published: 19 October 2018


Abstract: The goal of modern research is to use environmentally preferable materials. In this context, clay minerals are emerging candidates for their bio- and ecocompatibility, low cost and natural availability. Clay minerals present different morphologies according to their layer arrangements. The use of clay minerals, especially in biomedical applications is known from ancient times and they are regaining attention in recent years. The most representative clay minerals are kaolinit, montmorillonite, sepiolites and halloysite. This review summarizes some clay minerals and their derivatives for application as nanocontainer for biologically active species.

Keywords: clay minerals; drug carrier; halloysite

1. Introduction Clay minerals are a well-known class of compounds which have been used for pharmacological applications since ancient times. In prehistory, clay minerals were used for the treatment of minor ailments including infections, pains, aches, and food poisoning. In this context, several documents [1,2] indicate that Homo erectus and Homo Neanderthalensis usually used mixtures of clay minerals to treat wounds, while, in ancient Greek, they were used as antiseptic materials for skin affliction treatments, cicatrizes or to cure snake bites. The medicinal use of clay minerals became more and more prominent when, during the Renaissance, the Pharmacopoeia classified clay minerals as drug. Up to now, clay minerals are widely employed in the pharmaceutical industry as common additives. They are for example used as oral treatment of diarrhea or as gastrointestinal protector; or for topical dermatological applications [3–5]. Besides, they have found application as diluents, lubricants, flavor correctors, carriers of active ingredients in pharmaceutical products and so on [1,4,6]. The formation of clay occurs mainly in geological environment following the action of atmospheric agents on other silicate minerals giving rise to layer-type aluminosilicate [7]. The arrangement of atoms in the structure gives these minerals a platy morphology; the further arrangement of the tetrahedral and octahedral sheets allows the classification of clay minerals in three categories: 1:1, 2:1 and 2:1:1. The phyllosilicates such as kaolinite and halloysite 1:1 have per each clay layer one tetrahedral and one octahedral sheet. The 2:1 clay minerals are made up of one octahedral sheet between two tetrahedral ones. In this class of clay minerals belong montmorillonite, laponite, and illite. Finally, phyllosilicates such as cloisite, composed of 2:1:1 succession of the sheets, consist of a succession of the octahedral sheet adjacent to a 2:1 layer. The different chemical composition and

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J. Funct. Biomater. 2018, 9, x FOR PEER REVIEW 2 of 22 structure usually cause clay minerals to possess structural or surface charge which allow properties suchstructure as swelling usually or cause cations clay exchangeminerals to capacity. possess structural The first or one surface is permanent charge which and allow exists properties due to ion substitutions,such as swelling whereas or cations the latter exchange usually capacity. depends The on the first pH one value is permanent [8]. and exists due to ion substitutions,Currently, kaolinwhereas (Si/Al the latter oxide, usually 1:1), montmorillonitedepends on the pH and value sepiolite [8]. (Si/Al oxide, 2:1), where the 1 nm-thickCurrently, clay aluminosilicate kaolin (Si/Al oxide, sheets 1:1), are montmorillonite stacked in bulky and platelets sepiolite or (Si/Al processed oxide, with2:1), where exfoliation the 1 to bilayernm-thick sheets clay (Figure aluminosilicate1), are the mostsheets used are stacked clay minerals in bulky for platelets the research or processed and applications with exfoliation for medical to purpose.bilayer Moresheets recently, (Figure another 1), are claythe most mineral used has clay gained minerals attention, for the allophaneresearch and one applications (Si/Al molar for ratio ofmedical 0.5 and 1,purpose. corresponding More recently, to Al-rich another and clay Si-rich minera allophane,l has gained respectively) attention, [the9]. Commonly,allophane one allophane (Si/Al molar ratio of 0.5 and 1, corresponding to Al-rich and Si-rich allophane, respectively) [9]. appearsmolar asratio nano- of or0.5 micro-aggregates and 1, corresponding (clusters) to differingAl-rich and in size Si-rich and shapeallophane, [9]. These respectively) characteristics [9]. Commonly, allophane appears as nano- or micro-aggregates (clusters) differing in size and shape endowCommonly, allophane allophane with abundant appears as porosity nano- or and micro-aggregates high specific surface (clusters) area. differing In addition, in size allophane and shape clay [9]. These characteristics endow allophane with abundant porosity and high specific surface area. In does[9]. not These show characteristics any cytotoxicity endow effects allophane [10] and with can abun be useddant asporosity carrier and system high for specific biological surface applications. area. In addition, allophane clay does not show any cytotoxicity effects [10] and can be used as carrier system Indeed, allophane has been used as carrier for cisplatin to minimize the drug side effects for application for biological applications. Indeed, allophane has been used as carrier for cisplatin to minimize the in anticancer therapy [11]. drug side effects for application in anticancer therapy [11].

FigureFigure 1. 1. CrystalCrystal structure of of the the pharmaceutical pharmaceutical used used clay clay minerals: minerals: (a) kaolinite; (a) kaolinite; (b) talc; (b )(c talc;) (c)montmorillonite; montmorillonite; (d (d) )palygorskite; palygorskite; and and (e ()e )sepiolite. sepiolite. Dashed Dashed lines lines represent represent the the unit unit cell. cell. Reproduced Reproduced withwith permission permission from from [12 [12].].

HalloysiteHalloysite (Si/Al (Si/Al oxide, oxide, 1:1)1:1) isis aa clayclay mineral of of the the kaolin kaolin group group with with particular particular properties properties due due to theirto their tubular tubular morphology morphology (HNT) (HNT) that that is is caused caused by by the the intercalation intercalation in ininterlayer interlayer regionregion ofof aa layerlayer of waterof water molecules molecules (Figure (Figure2). 2).

(a)(b)

(c)

FigureFigure 2. 2.(a )(a Schematic) Schematic representation representation ofof thethe rolledrolled structure of of halloysite. halloysite. (b (b) Transmission) Transmission electron electron microscopymicroscopy (TEM) (TEM) and and atomic atomic force force microscopymicroscopy (AFM)(AFM) images of of HNT HNT ed edges.ges. Reproduced Reproduced with with permissionpermission from from [13 [13].]. (c ()c FE-SEM) FE-SEM image image ofof halloysitehalloysite on Si-wafer Si-wafer (Left) (Left) and and schematic schematic illustration illustration of of crystallinecrystalline structure structure of of halloysite halloysite (Right). (Right). ReproducedReproduced with permission permission from from [14]. [14].

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The wide use of clay minerals is mainly due to their versatile properties such as high adsorption capacity and specific surface area, ion exchange capacity, colloid and thixotropy, chemical inertness swellingJ. property, Funct. Biomater. and,2018, 9 ,above 58 all, low toxicity for oral administration. Among the plethora3 of 22 of clay minerals, the most used for pharmacological applications are kaolinite, talc, smectites (montmorillonite,The wide saponite, use of clay mineralsand hectorite), is mainly due palygorskite, to their versatile and properties sepiolite such as high[5,15,16] adsorption (Figure 3). Palygorskite,capacity sepiolite, and specific and surface smectites area, ion possess exchange the capacity, peculiarity colloid to and adsorb thixotropy, protons chemical in gastric inertness acid and release non-toxicswelling property, ions such and, as above Mg2+ all, and low Al toxicity3+ and for therefore oral administration. they might Among act as theantacids plethora by of reducing clay the gastric minerals,acidity. theTheir most usedphysicochemical for pharmacological features applications have are kaolinite,been also talc, smectitesexploited (montmorillonite, for application as saponite, and hectorite), palygorskite, and sepiolite [5,15,16] (Figure3). Palygorskite, sepiolite, gastrointestinaland smectites protectors possess thesince peculiarity they can to adsorb efficie protonsntly increase in gastric the acid andbarrier release thickness non-toxic and, ions due to adhesionsuch on asto Mgintestinal2+ and Al and3+ and gastric therefore membrane, they might clay act as minerals antacids bydecrease reducing the the gastric gastric acidity.secretion and irritation.Their physicochemical features have been also exploited for application as gastrointestinal protectors Othersince uses they canare efficiently related increase to the the application barrier thickness as and,antidiarrheals due to adhesion in on which to intestinal they and can gastric absorb the enteropathogensmembrane, clayand minerals reduce decrease the liquid the gastric in secretionthe colon. and irritation.Although their use in pharmaceutical Other uses are related to the application as antidiarrheals in which they can absorb the formulation is advantageous, there are some risks associated to a prolonged administration of clay enteropathogens and reduce the liquid in the colon. Although their use in pharmaceutical formulation minerals.is advantageous,The long-term there oral are someadministration risks associated caused to a prolonged kidney administrationstone formation of clay [17–19] minerals. and the adsorptionThe of long-term some nutritive oral administration elements caused and enzyme, kidney stone leading formation to their [17–19 elimination] and the adsorption from the of some body [5,20]. Severalnutritive formulations elements and enzyme,based leadingon clay to theirminerals elimination for fromdermatological the body [5,20 ].applications have been developed, sinceSeveral clay formulations minerals, based such on clayas kaolinite, minerals for talc, dermatological and smectite, applications show have nonfibrous been developed, morphology since clay minerals, such as kaolinite, talc, and smectite, show nonfibrous morphology and high and high adsorption capacity. adsorption capacity.

Figure 3. Comparison of the number of scientific publications on “Halloysite”, “Kaolinite”, Figure “Montmorillonite”3. Comparison andof “Sepiolite”the number refined of each scientific with the term publications “drug”. Data analysison “Halloysite”, of publications, “Kaolinite”, as “Montmorillonite”of June 2018, wasand done “Sepiolite” using the refined SciFinder each Scholar with search the system. term “drug”. Data analysis of publications, as of JuneBesides 2018, thewas pharmaceutical done using th application,e SciFinder claySchola mineralsr search have system. been extensively used as excipients in some formulation; as lubricants in manufacturing pills; disintegrants; anticaking and thickening Besidesagents; the binders pharmaceutical and diluents; emulsifiers;application, and clay carriers mine ofrals biologically have been active extensively molecules for used improving as excipients in some drugsformulation; bioavailability. as lubricants in manufacturing pills; disintegrants; anticaking and thickening agents; bindersHowever, and diluents; some limitations emulsifiers; hinder and the use carriers of clay of minerals biologically as they active are, because molecules of the for strong improving interactions which can exist between clay mineral and drugs that may influence the rate release of drugs bioavailability.the drug, and therefore could limit the molecule bioavailability. This aspect represents a problem However,when drugs some such limitations as antihistamines hinder are the associated use of with clay clay minerals minerals as since they they are, need because an immediate of the strong interactionstherapeutic which concentration can exist between in the blood. clay To mineral overcome and all these drugs drawbacks, that may several influence attempts the have rate been release of the drug,made and over therefore the years, could and thelimit most the successful molecule strategy bioavailability. is to functionalize This theaspect carrier. represents For example, a problem when drugsone approach such as consistsantihistamines of coat the are drug–clay associated hybrids with with clay cationic minera polymersls since to slow they down need the an drug immediate release and ensure a prolonged administration of the drug over time. therapeutic concentrationThis review summarizes in the blood. the development To overcome in the all use these of claydrawbacks, minerals inseveral the pharmaceutical attempts have been made overfield, the focusing years, on and kaolinite, the most montmorillonite, successful sepiolite, strategy and is halloysite.to functionalize In particular, the thecarrier. clay mineral For example, one approachproperties, consists their application of coat the as drugdrug–clay carriers hybrid and theirs functionalizationwith cationic polymers are discussed. to slow down the drug release and ensure a prolonged administration of the drug over time. This review summarizes the development in the use of clay minerals in the pharmaceutical field, focusing on kaolinite, montmorillonite, sepiolite, and halloysite. In particular, the clay mineral properties, their application as drug carriers and their functionalization are discussed.

J. Funct. Biomater. 2018, 9, x FOR PEER REVIEW 4 of 22 J. Funct. Biomater. 2018, 9, 58 4 of 22 2. Kaolinite

2. KaoliniteThe kaolin group comprises predominantly kaolinite; thus, kaolin is usually wrongly used to replace the more accurate one of kaolinite, a 1:1 clay mineral, with an ideal formula of Al2Si2O5(OH)4, featuredThe by kaolin a basic group unit comprises of a two-dimensional predominantly layer kaolinite; of silicate thus, groups kaolin bonded is usually to a layer wrongly of aluminate used to groups.replace the more accurate one of kaolinite, a 1:1 clay mineral, with an ideal formula of Al2Si2O5(OH)4, featuredThe interlayer by a basic space unit of of1:1 a clay two-dimensional mineral is about layer 7.2 Å, of a value silicate lowe groupsr than bondedthe 2:1 clay to amineral. layer ofIt isaluminate little expandable groups. due to the strong hydrogen bond in the interlayer space, allowing only few water moleculesThe interlayer or cations space to enter of 1:1the clay structure mineral (Figure is about 4). The 7.2 Å,more a value available lower surface than the is the 2:1 external clay mineral. one, withIt is little an overall expandable surface due area to thelimited strong to hydrogen10–30 m2 g bond−1 [21,22]. in the interlayer space, allowing only few water moleculesFurthermore, or cations its tocation enter exchange the structure capacity (Figure is 4very). The low, more which available explain surface the low is the fertility external of one,soils 2 −1 withthat are an overallrich in kaolinite surface area minerals. limited to 10–30 m g [21,22].

Figure 4. MolecularMolecular simulation simulation model model of of kaolinite kaolinite structure structure (1(1 × 2× ×2 2 ×unit2 unit cells) cells) showing showing siloxane siloxane and aluminoland aluminol surfaces. surfaces. Reproduced Reproduced with withpermission permission from from [23]. [23].

InFurthermore, recent years, its kaolinite cation exchange is extensively capacity used is very in several low, which pharmacological explain the low preparations fertility of soilswhere that it canare richbe considered in kaolinite both minerals. an excipient which helps in the controlling the efficiency of the dosage form, and asIn recentan active years, component. kaolinite isIn extensivelyaddition, in used a kind in several of formulation, pharmacological the presence preparations of kaolinite where can it improvecan be considered the drug bioavailability. both an excipient If the which kaolinite helps inis used the controlling as excipient, the it efficiency has to meet of thesome dosage important form, requisites:and as an it active must component.possess high Inchemical addition, purity, in a keep kind the of formulation,stability and consistency the presence of of its kaolinite final dosage can forms,improve and the drugimprove bioavailability. bioavailability If the kaoliniteand controll is useded asrelease excipient, of active it has tocomponents meet some importantin drug administrationrequisites: it must and possess delivery. high In chemical this context, purity, keepit is theused stability as diluent; and consistency binder; disintegrant; of its final dosage and pelletizing,forms, and improvegranulating, bioavailability amorphizing, and controlled particle f releaseilm coating, of active emulsifying components and in drugsuspending administration agents (Tableand delivery. 1). In this context, it is used as diluent; binder; disintegrant; and pelletizing, granulating, amorphizing,Besides the particle commonly film coating, known emulsifying properties, and such suspending as sorption agents capacity, (Table1). high specific area, favorableBesides rheological the commonly and swelling known properties, properties, such iner astness, sorption etc., capacity, the kaolinite highspecific clay can area, induce favorable and acceleraterheological blood and swelling clotting. properties,Thanks to inertness,this feature, etc., it thehas kaolinite been used clay as can activating induce andagent accelerate for clotting blood in physician’sclotting. Thanks routine to this practices feature, and, it has from been 2013, used it as was activating recognized agent by for FDA clotting as inadditive physician’s in ordinary routine practicesgauze due and, to its from efficiency 2013, it in was hemorrhage recognized control by FDA wi asthout additive risk inof ordinary thermal gauzeinjury due(it is to commercialized its efficiency in ashemorrhage QuikClot Combat control withoutGauze). risk of thermal injury (it is commercialized as QuikClot Combat Gauze).

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Table 1. Kaolinite functionalities as pharmaceutical excipient. Adapted with permission from [23].

Excipient Functionality Kaolin Grade/Composites Commercial/Excipient Dosage Form References Herbal slimming tablet: products (Slimwell® and Quantrim®). Gastro-resistant tablets: Mecysteine Hydrochloride® 100 mg. Granules for oral suspension: Riclasip® and Co-amoxiclav DST Grünenthal®. Light a and heavy b kaolin Diluent Riboflavin (vitamin B2) hard gelatin capsules. [24–28] Heavy kaolin Pyridoxine hydrochloride (vitamin B6) kaolin capsules. Thiamine (vitamin B1) and ascorbic acid (vitamin C) tablets and capsules. Binder Kaolin/Eudragit® E30D mixture Tablets and capsules [29,30] Disintegrant Heavy kaolin Tablets and pellets [31–33] Heavy kaolin Granules: (10% sodium chloride). Granulating agent [32–35] Light kaolin Granules: (20% calcium chloride, with polyethylene glycol and polyvinyl alcohol). Microcrystalline cellulose and hydrochlorothiazide (HCT) pellets. Chitosan/kaolin Pellets (5% sodium lauryl sulfate) of size range (850–1180 µm). Pellets (25% kaolin and 5% Pelletizing agent [36,37] Heavy kaolin crospovidone). Pellets (kaolin with microcrystalline cellulose and lactose). Pellets (45% kaolin, 5% aerosil® 200, 39.5% lactose, 2.5% liquid paraffin and 8% hydroxypropylmethylcellulose phthalate). Amorphizing agent Light kaolin kaolin-ibuprofen solid dosage forms. [38] - Hypericon® and Metformin® tablets. Koallicoat IR system and Film-coating additive - Pseudoephedrine hydrochloride, theophylline, and diphenhydramine hydrochloride pellets. [39,40] Kaolin/Eudragit® E30D dispersion - Dyphylline® coated tablets. Wetting and emulsifying agent Light kaolin Sulfur ointment. Oil-in-water Pickering emulsions. Non-aqueous oil-in-oil emulsions. [41,42] Suspending and anticaking agent Heavy kaolin Toxiban®, Kaolin-Pectin®, Kapect®, Kaolin, and Morphine mixture BP® suspensions. Heavy kaolin and light kaolin Kaolin powder (high and low kaolinite crystallinity) loaded by sodium amylobarbitone. Light kaolin Porous kaolin-based pellets loaded by Diltiazem HCl. Pellets loaded by highly potent opioids. Drug carrier Metakaolin [43–48] The derivative powder loaded by anticancer 5-fluorouracil drug and herbicide amitrole. Methoxy-modified kaolinite Powder loaded by α-lactalbumin, bovine serum albumin and β-lactoglobulin protein. Kaolin and metakaolin a Light, white powder, free from gritty particles, odorless and unctuous to the touch. A native hydrated aluminum silicate, selectively sourced and dried and milled to a fine powder. It contains a suitable dispersing chemical and is tested for chemical and physical properties in the manner defined by the specification of the British Pharmacopoeia 2013. b Fine, white powder, odorless and smooth to the touch and practically insoluble in water and in organic solvents. A refined, natural hydrated aluminum silicate. It is tested for chemical and physical properties in the manner defined by the specification of British Pharmacopoeia 2013. J. Funct. Biomater. 2018, 9, 58 6 of 22 J. Funct. Biomater. 2018, 9, x FOR PEER REVIEW 6 of 22

Recently, YangYang etet al.al.[ [49]49]developed developed anan ironiron oxide–kaolinite oxide–kaolinite nanocomposite nanocomposite ( α(α–Fe–Fe2O2O33–kaolin–kaolinKAcKAc) for hemorrhagehemorrhage control.control. TheThe authors authors used used a a combined combined approach approach where where they they exploited exploited the the properties properties of αof–Fe α–Fe2O32Oand3 and kaolinite kaolinite to assemble to assemble a composite a composite with excellent with excellent wound healingwound performances healing performances (Figure5). In(Figure vivo tests5). In show vivo theretests show exists there a synergism exists a betweensynergism kaolinite between and kaoliniteα–Fe2 Oand3: theα–Fe former2O3: the absorbed former fluidabsorbed to concentrate fluid to concentrate blood platelets, blood RBCs, platelets, and clottingRBCs, and factors clotting and factors activated and the activated intrinsic the coagulation intrinsic cascade,coagulation whereas cascade, the whereas latter facilitated the latter RBC facilitated aggregation RBC andaggregation clotting. and clotting.

Figure 5.5. ((aa)) ζζ–potential–potential curves curves of of kaolin, kaolin, FeOOH–kaolin FeOOH–kaolinKAcKAc, α, –Feα–Fe2O23O–kaolin3–kaolinKAcKAc,,γ γ–Fe–Fe2O2O33–kaolin–kaolinKAcKAc,,

and FeFe33O4–kaolin–kaolinKAcKAc. .((b)b )Platelet Platelet aggregation aggregation induced induced by by the the samples, samples, normal normal saline, and ADPADP represented as as the the maximal maximal percentage percentage aggregat aggregationion of ofsodium sodium citrated citrated treated treated whole whole blood. blood. (c)

(Illustrationc) Illustration of ofthe the presumed presumed synergism synergism of of αα–Fe–Fe22OO33–kaolin–kaolinKAcKAc compositecomposite in in hemostasis. hemostasis. Reproduced Reproduced with permission fromfrom [[49].49].

Similarly, WangWang et et al. [al.50 ][50] synthesized synthesized a new a hemostatic new hemostatic sponge withsponge kaolin with powders kaolin embedded powders intoembedded graphene into sheet. graphene The authors sheet. The found authors that the found obtained that spongethe obtained can stop sponge bleeding can instop approximately bleeding in 73approximately s in rabbit artery 73 s in injury rabbit model. artery Thisinjury outstanding model. This result outstanding is also due result to theis also kaolinite due to embedded the kaolinite in theembedded graphene in sheets.the graphene The clay sheets. indeed The will clay activate indeed hemostatic will activate factors, hemostatic such as factors, Factor XXII,such as Factor Factor X, FactorXXII, Factor V, and X, platelets, Factor V, favoring and platel theets, hemostasis favoring process.the hemostasis process. The modificationmodification ofof kaolinite kaolinite clay clay opens opens up up several severa strategiesl strategies to loadto load and and release release in an in efficient an efficient way differentway different drugs. drugs. For example, For example, by functionalization by functionalization of kaolinite of kaolinite with with a cationic a cationic surfactant, surfactant,Li et Li al. et [51 al.] observed[51] observed an increase an increase in the in loading the loading of diclofenac. of diclofenac. The introduction The introduction of the organicof the organic molecule, molecule, indeed, increasesindeed, increases the clay the mineral clay mineral surface surface hydrophobicity hydrophobi andcity confers, and confers, to the clay, to the a positiveclay, a positive surface surface which betterwhich interactionbetter interaction with the with drug. the drug. 3. Montmorillonite 3. Montmorillonite Montmorillonite (MMT) is a dioctahedral 2:1 phyllosilicate constituted by two tetrahedral sheets Montmorillonite (MMT) is a dioctahedral 2:1 phyllosilicate constituted by two tetrahedral (T:O:T) and one octahedral sheet. The main characteristic of montmorillonite is the interlayer space sheets (T:O:T) and one octahedral sheet. The main characteristic of montmorillonite is the interlayer which is present between each triple-sheet-layer (Figure6). Furthermore, the triple sheet layers consist space which is present between each triple-sheet-layer (Figure 6). Furthermore, the triple sheet of isomorphic substitutions in the tetrahedral sheet of Si4+ by Al3+ and Al3+ by Mg2+ in the octahedral layers consist of isomorphic substitutions in the tetrahedral sheet of Si4+ by Al3+ and Al3+ by Mg2+ in ones. This arrangement confers to montmorillonite a negative residual charge compensated by cations the octahedral ones. This arrangement confers to montmorillonite a negative residual charge in the interlayer space. The interlayer space constitutes about the 90% of the total clay surface and, thus, compensated by cations in the interlayer space. The interlayer space constitutes about the 90% of the it confers to the mineral the possibility to absorb water molecule or other compounds, which increase total clay surface and, thus, it confers to the mineral the possibility to absorb water molecule or other the adsorption capacity of the clay for polar molecules [2,52]. compounds, which increase the adsorption capacity of the clay for polar molecules [2,52].

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Figure 6. Molecular structure of montmorillonite. Reproduced with permission fromfrom [[53].53].

Several studiesstudies havehave shown shown that that MMT MMT possesses possesses low low cytotoxicity cytotoxicity both bothin vitroin vitroand andin vivo in vivo.. It has It beenhas been demonstrated demonstrated that that this this clay clay mineral mineral does does not not possess possess any any relevant relevant cytotoxic cytotoxic effectseffects inin mice, −1 eveneven atat thethe highhigh dosagedosage ofof 10001000 mgmg kgkg−1.. On On the the ground ground of of these findings, findings, MMT can bebe efficientlyefficiently used asas carriercarrier systemsystem forfor severalseveral drugdrug molecules.molecules. According toto the the British, British, European European and United and StatesUnited Pharmacopeias, States Pharmacopeias, the use of montmorillonite the use of ismontmorillonite mainly as auxiliary is mainly material as auxiliary in the pharmaceuticalmaterial in the pharmaceutical industry for topical industry or oral for topical dosage or forms. oral MMTdosage indeed, forms. thanksMMT indeed, to its physicochemical thanks to its physicoche propertiesmical (such properties as high swelling (such as ratio), high swelling can intercalate ratio), activecan intercalate compounds active between compounds the layers between generating the layers a host generating for oralor a topicalhost for drug oral deliveryor topical [54 drug–66]. Fordelivery montmorillonite [54–66]. For topicalmontmorillonite use, it possesses topical use, beneficial it possesses effects beneficial in cosmetic effects and in dermatological cosmetic and applicationsdermatological (paleotherapy applications and (paleotherapy geotherapy) and [1,67 geotherapy)]. [1,67]. InIn thisthis context,context, IannuccelliIannuccelli etet al.al. [[68]68] developeddeveloped aa novelnovel systemsystem basedbased onon organo-modifiedorgano-modified bentonite, for the topicaltopical deliverydelivery ofof gentamicin,gentamicin, which may represent aa safersafer alternativealternative andand moremore effectiveeffective wayway ofof gentamicingentamicin utilization.utilization. This drug possesses a low systemic absorption, probably due toto thethe difficultdifficult penetration penetration through through the the deep deep layers layers of of the the skin, skin, and and therefore therefore its useits use is restricted is restricted to the to localthe local effect effect that that involves involves mainly mainly the most the most superficial superficial skin layers.skin layers. The authors The authors highlighted highlighted that the that drug the wasdrug intercalated was intercalated in montmorillonite in montmorillonite clay, which clay, favorswhich itsfavors skin its permeation, skin permeation, as shown as byshown means by ofmeans tape strippingof tape stripping test. test. Turco LiveriLiveri et et al. [al.69 ][69] reported reported the intercalation the intercalation of metronidazole of metronidazole (MNE), an(MNE), antibiotic an commonlyantibiotic usedcommonly for amoebiasis used for treatment,amoebiasis on treatment, MMT-K10 on clay MMT-K10 (a montmorillonite clay (a montmorillonite clay with a specific clay with surface a specific area 2 −1 ofsurface 250 m areag of). 250 The m authors2 g−1). The found authors that thefound drug that adsorption the drug isadsorption strictly related is strictly to pH, related and this to pH, process and involvesthis process a multistep involves mechanism, a multistep where mechanism, the drug canwh interactere the withdrug the can clay interact by protonation with the of clay the clay by activeprotonation sites, ionicof the exchange, clay active and sites, electrostatic ionic exchan interactions.ge, and electrostatic Furthermore, intera the organic–inorganicctions. Furthermore, hybrid the composedorganic–inorganic by MNE/MMT-K10 hybrid composed allows by for MNE/MMT-K10 a sustained release allows of thefor a drug sustained in the gastrointestinalrelease of the drug tract, in protectingthe gastrointestinal it by degradation, tract, protecting as highlighted it by degrada by thetion, in vitro as highlighted kinetic study. by the in vitro kinetic study. Praziquantel/montmorillonite complexes, complexes, with with the the drug drug intercalated in thethe montmorillonitemontmorillonite interlayerinterlayer space,space, werewere successfullysuccessfully developed by Viseras etet al.al. [[70].70]. To improveimprove thethe bioavailabilitybioavailability and the percent percent loading loading of of the the drugs drugs into into the the clay, clay, the the supramolecular supramolecular complex complex was wasobtained obtained in a innon-aqueous a non-aqueous polar polar medium medium (ethanol). (ethanol). This This strategy strategy is ispromising promising alternative alternative to to improveimprove bioavailability of such drugs that show low solubilitysolubility in aqueousaqueous medium, such as praziquantel, sincesince owing to the presence of the clay the dissolution rate of the drug has been increased. Montmorillonite waswas also also used used to improveto improve the organolepticthe organoleptic properties properties of some of drugs some (see, drugs for example,(see, for Sildenafilexample, Sildenafil [18] and Aripiprazole [18] and Aripiprazole [19]). Furthermore, [19]). Furthermore, the clay is usefulthe clay to is stabilize useful to piroxicam, stabilize piroxicam, a common photolabilea common photolabile compound compound [20]. [20]. MMT hashas alsoalso been been used used to to overcome overcome the the extrapyramidal extrapyramidal side side effects effects of chlorpromazine of chlorpromazine (CPZ) (CPZ) [71]. The[71]. intercalation The intercalation of CPZ of into CPZ the into clay, the indeed, clay, indeed, is advantageous is advantageous since the since drug the is released drug is in released a sustained in a mannersustained for manner a longer for period a longer and, period therefore, and, thetherefor side effectse, the side could effects potentially could potentially be reduced. be reduced. The effectseffects of of the the functionalization functionalization of MMTof MMT with Tween-20with Tween-20 surfactant surfactant on the loadingon the ofloading cinnamic of acidcinnamic were acid evaluated were evaluated by Sciascia by etSciascia al. [72 et]. al. Adsorption [72]. Adsorption isotherms isotherms of the of drug the ondrug both on both MMT MMT and and MMT-Tween 20 hybrid confirm that the introduction of the non-ionic surfactant increases the adsorption rate of the cinnamic acid onto the clay.

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MMT-TweenJ. Funct. Biomater. 202018 hybrid, 9, x FOR confirm PEER REVIEW that the introduction of the non-ionic surfactant increases 8 of the 22 adsorption rate of the cinnamic acid onto the clay. InIn anotheranother workwork [[73],73], thethe MMTMMT clayclay waswas functionalizedfunctionalized withwith aa thiolatedthiolated chitosan–polyethylenechitosan–polyethylene glycol blend,blend, obtainingobtaining a a biodegradable biodegradable composite composite for for the the insulin insulin oral oral delivery. delivery. The The authors authors found found that thisthat novelthis novel system system limits limits the release the release of insulin of insulin in acidic in mediumacidic medium (Figure (Figure7), thus avoiding7), thus avoiding the protein the inactivationprotein inactivation in the gastrointestinal in the gastrointestinal tract. Furthermore, tract. Furthermore, biological in biological vitro studies in onvitro the studies carrier vehicleon the aftercarrier the vehicle complete after release the complete of the protein release show of the that protein it possesses show that antimicrobial it possesses activity, antimicrobial thereby activity, making thethereby system making potentially the system biocompatible. potentially biocompatible.

Figure 7.7.Schematic Schematic illustration illustration for for the the release release of Insulin of Insulin from MMTfrom clayMMT functionalized clay functionalized with a thiolated with a chitosan–polyethylenethiolated chitosan–polyethylene glycol blend. glycol Reproduced blend. Reproduced with permission with permission from [73]. from [73].

InIn thisthis context,context, Ruiz-HitzkyRuiz-Hitzky etet al.al. [[74]74] incorporated,incorporated, byby anan ion-exchangeion-exchange mechanism,mechanism, metforminmetformin (MF),(MF), aa drugdrug usuallyusually usedused forfor thethe treatmenttreatment ofof typetype 22 diabetesdiabetes intointo Na-montmorillonite.Na-montmorillonite. The physicochemicalphysicochemical characterizationcharacterization of thethe hybridhybrid MMT/MFMMT/MF shows that the intercalatedintercalated metformin is arranged in the form of monoprotonatedmonoprotonated molecules disposed as a mono-layer covering thethe interlayerinterlayer surfacesurface ofof montmorillonite.montmorillonite. The useuse of of montmorillonite montmorillonite as a platformas a platform for the controlledfor the controlled release of MFrelease could of be MF advantageous could be toadvantageous reduce the doses to reduce currently the administereddoses currently to patients admini withstered type to 2patients diabetes, with to minimize type 2 diabetes, side effects. to Pureminimize metformin side effects. hydrochloride, Pure metformin indeed, hydrochloride, is commonly indeed administered, is commonly at high administered doses and produces at high collateraldoses and unsuitable produces collateral gastrointestinal unsuitable effects, gastrointestinal which may be effects, minimized which with may abe better minimized control with of the a drugbetter administration. control of the drug administration. InIn aa compositecomposite system, system, constituted constituted by by MMT MMT and and polymers, polymers, drug drug molecules molecules can interactcan interact both withboth thewith clay the and clay the and polymer the polymer matrix; matrix; therefore, therefore, it is interesting it is interesting to figure to outfigure whether out whether drug–polymer drug–polymer matrix ormatrix drug–clay or drug–clay interactions interactions are more are favorable. more favorable. To answer To to answer this, computational to this, computational tools such tools as molecular such as dynamicsmolecular (MD)dynamics and quantum(MD) and chemistry quantum arechemistry quite helpful. are quite Tekin helpful. et al. Tekin [75] studied et al. [75] the studied interaction the betweeninteraction curcumin, between chosencurcumin, asanticancer chosen as druganticancer model, drug and model, a PLGA/MMT and a PLGA/MMT composite composite by means by of differentmeans of computationaldifferent computational tools ranging tools from ranging cluster, from periodic cluster, DFT periodic and MDDFT simulations. and MD simulations. All methods All showedmethods that showed curcumin that stronglycurcumin interacts strongly with interact both PLGAs with andboth MMT PLGA and and the MM presenceT and of the MMT presence is useful of sinceMMT it is makes useful easier since theit makes release easier of curcumin the release due of to curcumin the increased due to diffusion the increased rate. diffusion rate. Finally, MMTMMT waswas alsoalso usedused toto developdevelop anan ecologicecologic andand economiceconomic fertilizer system, for the slow releaserelease ofof urea.urea. ToTo dodo this,this, severalseveral approachesapproaches areare currentlycurrently presentpresent inin thethe scientificscientific literature literature [ 76[76].]. Pereira et al. [[77]77] reportedreported the exfoliationexfoliation of MMT clay into urea matrix, by extruded methods. Conversely, WanikaWanika [[78]78] couldcould efficientlyefficiently intercalateintercalate ureaurea inin thethe MMTMMT byby aa simplesimple immersionimmersion of thethe clay inin ureaurea solution.solution.

4. Sepiolite The mostmost usedused clayclay mineralsminerals forfor pharmacologicalpharmacological applications are the onesones derivedderived fromfrom montmorillonite, eveneven if, if, in in the the last last years, years, sepiolites sepiolites have have attracted attracted increasing increasing attention attention since since they they lead tolead an to improvement an improvement of the of properties the proper ofties biodegradable of biodegradable polymers polymers [79]. [79]. Sepiolite (Si12O30Mg8(OH)4(H2O)4 × 8H2O) is a naturally occurring fibrous clay mineral constituted by fine microporous channels, of 3.5 × 10.6 Ų in cross section [80], running parallel to the length of the fibers. The structure is in some aspects similar the common 2:1 trioctahedral silicates, but it is different from, for example, montmorillonite since it possesses channels due to inversion

J. Funct. Biomater. 2018, 9, 58 9 of 22

Sepiolite (Si12O30Mg8(OH)4(H2O)4 × 8H2O) is a naturally occurring fibrous clay mineral constituted by fine microporous channels, of 3.5 × 10.6 Å2 in cross section [80], running parallel

J.to Funct. the lengthBiomater. of2018 the, 9, x fibers. FOR PEER The REVIEW structure is in some aspects similar the common 2:1 trioctahedral 9 of 22 silicates, but it is different from, for example, montmorillonite since it possesses channels due to andinversion discontinuities and discontinuities of the silica of thesheets silica (Figure sheets 8). (Figure Some8 ).of Some the Si of atoms the Si atomsat the atcorners the corners of the ofouter the blocksouter blocks are bound are bound to hydroxyls to hydroxyls (Si–OH) (Si–OH) [81]. [81].

Figure 8. CrystallineCrystalline structurestructure and and needle needle structure structure of sepiolite.of sepiolite. Reproduced Reproduced with with permission permission from from [82]. [82]. Turco Liveri et al. [83] loaded Vitamin A (VitA) in Sepiolite (SPT) and MTM by impregnation. The studiesTurco Liveri of in vitro et al.release [83] loaded kinetics Vitamin showed A that(VitA) the in release Sepiolite of VitA (SPT) is relatedand MTM to the by type impregnation. of support Theas well studies as on of the in pH. vitro Besides release the kinetics controlled showed release, that it was the demonstrated release of VitA that is the related SPT is ableto the to preventtype of supportthe oxidative as well degradation as on the pH. of the Besides VitA the [83]. controlled release, it was demonstrated that the SPT is able to preventSepiolite the clayoxidative was efficiently degradation dispersed of the VitA in gelatin-egg [83]. white biodegradable films for active food packagingSepiolite applications clay was efficiently by Guimerez dispersed et al. in [84 gelati]. Then-egg introduction white biodegradable of the clay films mineral for active showed food a packagingremarkable applications enhancement by in theGuimerez films mechanical et al. [84]. properties The introduction and, after of incorporation the clay mineral of clove showed essential a remarkableoil, an improvement enhancement of their in antimicrobialthe films mechanical and antioxidant properties activities and, after was observed.incorporation The of authors clove essentialexplained oil, these an resultsimprovement by a clay of effect their on antimicrobial the controlled and release antioxidant of eugenol activities from the was film, observed. by hindering The authorsthe interactions explained between these results the protein by a clay polymeric effect on matrix the controlled and the essential release oil.of eugenol from the film, by hinderingChitosan/Poly the interactions vinyl between alcohol the (PVA)/sepiolite protein polymeric hybrid matrix hydrogel and the films essential were oil. prepared for the loadingChitosan/Poly of cefazolin vinyl [85]. Thealcohol hydrogels (PVA)/sepiolite were made hybrid by crosslinking hydrogel chitosan films were with prepared PVA by means for the of loadingfreezing–thawing of cefazolin cycles. [85]. Afterward,The hydrogels to this, were sepiolite made by was crosslinking added as reinforcing chitosan with agent, PVA and by it wasmeans used of freezing–thawingto load and release cycles. the drug. Afterward, The antibacterial to this, sepiolite property was of the added novel as films reinforcing against gram-negativeagent, and it was and usedgram-positive to load bacteriaand release was finally the studied.drug. The The antibacterial cefazolin-loaded property films showed of the wider novel area films of inhibition against gram-negativeagainst Bacillus and cereus gram-positivebacterium. bacteria was finally studied. The cefazolin-loaded films showed widerAnother area of inhibition work reports against the useBacillus of sepiolite cereus bacterium. for the adsorption and release of nitric oxide (NO), an endogenousAnother work molecule reports with the use an importantof sepiolite biological for the adsorption action [86 and]. The release toxicity of nitric of the oxide sepiolite/NO (NO), an endogenoushybrid was investigatedmolecule with towards an important HeLa cells biological and the obtainedaction [86]. results The showedtoxicity aof high the survivalsepiolite/NO rate, −1 hybrideven for was material investigated concentrations towards higher HeLa thancells 450 and mg the mL obtainedmainly results due toshowed the use a of high materials survival that rate, are evenprepared for material from a naturally concentrations occurring higher clay. than These 450 findings mg mL assert−1 mainly the sepiolite due to the can use be used,of materials similar that to other are prepareduniversally from recognized a naturally clay, occurring for biological clay. applicationsThese findings since assert it ensures the sepiolite low toxicity can be at used, higher similar doses. to otherSepiolite universally toxicity recognized was investigated clay, for biological by several applications research group. since it Sepiolite, ensures low with toxicity its microfibrous at higher doses.structure, has raised safety concerns, since the SPT fibers can stick in the human body after their adsorption,Sepiolite causing toxicity the was same investigated damages ofby asbestos. several research The SPT group. safety wasSepiolite, thoroughly with its investigated microfibrous by structure,Ruiz-Hitzky has et raised al., who safety showed concerns, that some since deposits the SPT of fibers sepiolite can reportedstick in the previously human body [87] possess after their the adsorption,optimal dimensions causing the to besame excluded damages by of cells asbestos. without The toxicity. SPT safety Therefore was thoroughly it is possible investigated that sepiolite by Ruiz-Hitzkymay pose a low et al., health who risk, showed especially that some when deposits it possesses of sepiolite fiber with reported a <5 mm previously length [88 [87]]. Based possess on the optimalaforementioned dimensions features, to be theexcluded International by cells Agency withou oft toxicity. Research Therefore on Cancer it (IARC)is possible has that classified sepiolite the maysepiolites pose asa low non-hazardous health risk, especially and non-carcinogenic when it posse materialsses fiber [88 with]. a <5 mm length [88]. Based on the aforementioned features, the International Agency of Research on Cancer (IARC) has classified the sepiolites as non-hazardous and non-carcinogenic material [88].

5. Halloysite Nanotubes

Halloysite nanotubes (HNTs), with chemical formula Al2Si2O5(OH)4·nH2O, are 1D natural inorganic clay nanotubes belonging to kaolin group. It is a dioctahedral 1:1 clay mineral present in soils especially in wet tropical and subtropical regions and weathered igneous and non-igneous rocks. Each deposit is characterized by different purity grade, characteristic sizes, and hydration state.

J. Funct. Biomater. 2018, 9, x FOR PEER REVIEW 10 of 22

J. Funct.The Biomater. name 2018of “halloysite”, 9, 58 is due to a mineralogist, M. Berthier, who first used it in 1826 in honor10 of 22 of Omalius d’Halloy, who found the mineral in Angleur, Liége, Belgium. Typically, the halloysite clay possesses a hollow tubular structure in the sub-nanometer range with5. Halloysite an aspect Nanotubes ratio of ca. 20; the wall is constituted of 10–15 bilayers, with a spacing of 3 approximatelyJ. Funct. Biomater. 20180.72, 9nm, x FOR and PEER has REVIEWa density of 2.53 g/cm . The external surface is composed of siloxane 10 of 22 Halloysite nanotubes (HNTs), with chemical formula Al2Si2O5(OH)4·nH2O, are 1D natural (Si–O–Si)inorganic groups, clay nanotubes while the belonging inner one to consists kaolin of group. a gibbsite-like It is a dioctahedral array of aluminol 1:1 clay mineral(Al–OH) present groups, in andsoils Al–OHThe especially name and of inSi–OH “halloysite” wet tropical groups isandat due the subtropical toedges a mineralogist, of the regions tube. and M.The Berthier, weathered sequence who of igneous thefirst layers used and givesit non-igneous in 1826 to the in tubeshonor rocks. Si–Oof Omalius groupsEach deposit d’Halloy, at the isouter characterized who surface found and the by Al–OHmineral different groups in purity Angleur, at grade, the Liége, inner characteristic Belgium.surface (Figure sizes, 9) and [89–91]. hydration At pH state. 3– 8 [92],Typically,The the nameHNT the ofsurfaces “halloysite” halloysite are oppositely clay is due possesses to charged a mineralogist, a hollow according tubular M. Berthier,to the structure different who in first thechemical used sub-nanometer it incomposition 1826 in honorrange of theirwithof Omalius groups an aspect d’Halloy,which ratio undergo who of foundca. ionization 20; the the mineral [93].wall is in Angleur,constituted Lié ge,of Belgium.10–15 bilayers, with a spacing of 3 approximatelyTheTypically, halloysite 0.72 the halloysitenmlength and ishas claygenerally a density possesses in of the2.53 a hollow rangeg/cm tubular. ofThe 0.2–1.5 external structure μm, surface while in the is thecomposed sub-nanometer inner andof siloxane outer range diameters(Si–O–Si)with an aspect ofgroups, tubes ratio whileare of in ca. thethe 20; innerranges the wall one of is consists1 constituted0–30 nm of and a ofgibbsite-like 40–70 10–15 nm, bilayers, respectivelyarray with of aluminol a spacing [94–96] (Al–OH) of, depending approximately groups, on theand0.72 extraction Al–OH nm and and site has Si–OH and a density purification groups of 2.53at theprocesses. g/cm edges3. of The In the so external metube. deposits, The surface sequence halloysite is composed of the tubes layers with of siloxanegives length to the (Si–O–Si)up totubes 3– 5Si–Ogroups, μm groupswere while found at the the inner butouter in one surfacethe consists size and distribution ofAl–OH a gibbsite-like groups curve at arraythey the innerhave of aluminol surfacea minor (Al–OH) (Figure fraction. 9) groups, [89–91].However, and At Al–OH pHthese 3– shorter8and [92], Si–OH thetubes HNT groups are surfacesthe at most the are edgesattractive oppositely of the from tube. charged a biologic The sequence accordingal point of toof the viewthe layers different since gives they chemical to theare tubesmore composition suitable Si–O groups for of compositestheirat the groups outer with surface which sustained andundergo Al–OH delivery ionization groups of at[93]. chemicals the inner surfaceand drugs (Figure in9 )[comparison89–91]. At pHto the 3–8 longer [92], the ones. HNT Besides,surfacesThe clay arehalloysite oppositelytubes 1 μlengthm charged in lengthis generally according may have in to theanthe additional differentrange of chemical advantage0.2–1.5 compositionμm, because while itthe of is their ainner safe groups dimensionand whichouter fordiametersundergo macrophage ionization of tubes removal are [93 ].in of the the ranges nanopa ofrticles 10–30 from nm and living 40–70 organisms nm, respectively [96]. [94–96], depending on the extraction site and purification processes. In some deposits, halloysite tubes with length up to 3– 5 μm were found but in the size distribution curve they have a minor fraction. However, these shorter tubes are the most attractive from a biological point of view since they are more suitable for composites with sustained delivery of chemicals and drugs in comparison to the longer ones. Besides, clay tubes 1 μm in length may have an additional advantage because it is a safe dimension for macrophage removal of the nanoparticles from living organisms [96].

FigureFigure 9. SchematicSchematicrepresentation representation of theof the rolled rolled structure structure of halloysite of halloysite (left) and (left schematic) and schematic illustration illustrationof the crystalline of the crystalline structure of structure halloysite of (halloysiteright). Reproduced (right). Reproduced with permission with permission from [89]. from [89].

InThe the halloysite past, the length main isuse generally of thisin clay the rangemineral of 0.2–1.5had beenµm, only while in the ceramics, inner and as outeralternative diameters to kaolinite.of tubes areSince in the2005, ranges however, of 10–30 with nm the and growing 40–70 nm, interest respectively in biomedical [94–96], clay depending materials, on theseveral extraction uses ofsite the and clay purification have been taken processes. into account. In some The deposits, increa halloysitesing interest tubes in the with halloysite length up is testified to 3–5 µ mbywere the found but in the size distribution curve they have a minor fraction. However, these shorter tubes are numberFigure of patents 9. Schematic which hasrepresentation virtually equaled of the therolled number structure of publications of halloysite on ( leftthe) subjectand schematic (Figure 10). the most attractive from a biological point of view since they are more suitable for composites with Basedillustration on these offindings, the crystalline the interest structure in of the halloysite industry (right seems). Reproduced evident withwhich permission can be translatedfrom [89]. into importantsustained new delivery technologies of chemicals that and could drugs in the in comparisonfuture become to the more longer prominent ones. Besides, with respect clay tubes to other 1 µm clays.in lengthIn the may past, have the an main additional use of advantage this clay becausemineral it had is a safebeen dimension only in ceramics, for macrophage as alternative removal to of kaolinite.the nanoparticles Since 2005, from however, living organisms with the [growing96]. interest in biomedical clay materials, several uses of theIn clay the have past, thebeen main taken use into of this account. clay mineral The increa hadsing been interest only in in ceramics, the halloysite as alternative is testified to kaolinite. by the numberSince 2005, of patents however, which with has the virtually growing equaled interest the in biomedicalnumber of publications clay materials, on severalthe subject uses (Figure of the clay10). Basedhave beenon these taken findings, into account. the interest The increasing in the indu intereststry inseems the halloysiteevident which is testified can be by translated the number into of importantpatents which newhas technologies virtuallyequaled that could the in number the futu ofre publications become more on prominent the subject with (Figure respect 10). Based to other on clays.these findings, the interest in the industry seems evident which can be translated into important new technologies that could in the future become more prominent with respect to other clays.

Figure 10. Distribution (%) of scientific publications in “patent” and “journal” for halloysite. Data analysis of publications, as of June 2018, was performed using the SciFinder Scholar search system using as “Document type” the “Journal, letter, book, reviews” and “Patent”, respectively.

The most interesting aspect of the halloysite clay is its different surface chemical composition whichFigure allows 10. theDistribution selective (%) modification of scientific publicationsleading to inthe “patent” synthesis and “journal” of different for halloysite. appealing nanomaterials.FigureData analysis 10. Distribution of publications, (%) of scientific as of June publications 2018, was performedin “patent”using and “journal” the SciFinder for halloysite. Scholar search Data analysissystem using of publications, as “Document as of type” June the 2018, “Journal, was pe letter,rformed book, using reviews” the SciFinde and “Patent”,r Scholar respectively. search system using as “Document type” the “Journal, letter, book, reviews” and “Patent”, respectively.

The most interesting aspect of the halloysite clay is its different surface chemical composition which allows the selective modification leading to the synthesis of different appealing nanomaterials.

J. Funct. Biomater. 2018, 9, 58 11 of 22 J. Funct. Biomater. 2018, 9, x FOR PEER REVIEW 11 of 22

The mostmodification interesting of aspectthe halloysi of thete halloysite lumen is clay mainly is its based different on surfacethe electrostatic chemical compositioninteractions whichbetween allows negatively the selective charged modification molecules leading and the to po thesitive synthesis aluminum of different surface. appealing Pioneering nanomaterials. work by Lvov,The Price, modification and Veerabradan of the halloysitehighlighted lumen the possibility is mainly to based include on in the the electrostatic lumen drug interactions molecules betweensuch as tetracyclines negatively charged[97]. Since molecules then, many and diff theerent positive molecules aluminum have surface. been successfully Pioneering loaded work on by Lvov,halloysite Price, lumen, and Veerabradan which benefits highlighted of a sustained the possibility release to includeover th ine time the lumen and improved drug molecules biological such asactivity tetracyclines [97–104]. [97 ]. Since then, many different molecules have been successfully loaded on halloysite lumen,Insulin, which a benefits protein ofwhich a sustained shows releaseseveral over drawback the times for and its improved oral administration, biological activity such [as97 –rapid104]. degradation,Insulin, adigestion, protein whichand inactivation shows several by proteolytic drawbacks enzymes for its oral [105] administration,, was efficiently such loaded as rapid on degradation,HNT nanomaterial digestion, by exploiting and inactivation the different by proteolytic surfaces enzymescharge of [ 105the], clay was [106]. efficiently It was loaded found onthat HNT the nanomaterialmaximum loading by exploiting is achieved the in different acidic medium, surfaces chargewhere the of theprotein clay [can106 ].interact It was mostly found with that the maximumHNT lumen loading and isalso achieved with the in acidicexternal medium, surface where as verified the protein by canζ–potential interact mostlymeasurements. with the HNTThe lumenHNT/insulin and also hybrid with thepres externalents a more surface negative as verified ζ–potential by ζ–potential (−45.9 mV) measurements. compared to The that HNT/insulin of pristine hybridHNTs ( presents−34.4 mV), a more in agreement negative ζ–potential with the (selective−45.9 mV) electrostatic compared tointeractions that of pristine between HNTs the (− 34.4positive mV), inhalloysite agreement inner with surface the selective and insulin. electrostatic The loading interactions of insulin between into the HNT positive led halloysiteto the synthesis inner surface of an andefficient insulin. nanocarrier The loading which of allows insulin a intoprolonged HNT ledand to sustained the synthesis release of of an the efficient protein nanocarrier over seven which days. allowsNoteworthy, a prolonged dichroism and circular sustained experiments release of highlight the protein that over the protein seven days. is released Noteworthy, from the dichroism carrier in circularits native experiments form (Figure highlight 11). that the protein is released from the carrier in its native form (Figure 11).

Figure 11.11. Schematic representation ofof thethe HNT/insulinHNT/insulin hybrid, release, and stability. Reproduced with permission from [[106].106].

Similarly, Lvov et al.al. immobilizedimmobilized on halloysite surfaces some proteins with globule diameters of 3–8 nm by meansmeans ofof electrostaticelectrostatic interactionsinteractions [102102].]. Therefore, proteins proteins such such as laccase, lipase, glucose oxidase, and pepsin can be efficientlyefficiently immobilized both in the lumen and on the external surface exploiting the different surfacesurface charges.charges. The differentdifferent chargescharges are are important important for for drug drug delivery delivery since since they they allow allow closing closing the halloysite the halloysite edges withedges suitable with suitable molecules. molecules. In this In way, this it way, is possible it is po tossible slow to down slow thedown release the re oflease a drug of a from drug the from lumen. the Inlumen. this context,In this Fakhrullincontext, Fakhrullin et al. reported et al. thereported immobilization the immobilization of brilliant of green brilliant into HNTgreen followed into HNT by coatingfollowed the by hybrid coating complex the hybrid with complex dextrin with [107 dextrin]. The choice [107]. ofThe similar choice coating of simila isr due coating to the is factdue thatto the it canfact bethat disrupted it can be oncedisrupted the cell once absorbs the cell these absorb nanocarrierss these nanocarriers where sugar where can be sugar cleavable can be by cleavable intracellular by glycosylintracellular hydrolases. glycosyl hydrolases. Due to the presenceDue to the of presen dextrin,ce theof dextrin, drug may the bedrug preferentially may be preferentially released in thereleased rapidly in the proliferating rapidly proliferating cells, such ascells, tumor such cells, as tumor which cells, are pronewhich to are internalizing prone to internalizing the nanotubes. the Ducenanotubes. et al. usedDuce HNT et al. as used carrier HNT for as salicylic carrier acidfor salicylic (SA) for acid application (SA) for in application the field of in active the field packaging of active for foodpackaging industry for [food108]. industry The authors [108]. thoroughly The authors investigated thoroughly the investigated hybrid HNT/SA the hybrid from a physicochemicalHNT/SA from a pointphysicochemical of view demonstrating point of view its demonstrating ability to stabilize its ability HNT suspensions.to stabilize HNT The suspensions. biological properties The biological of the hybridproperties were of investigatedthe hybrid were by studyinginvestigated the by antibacterial studying the properties antibacterial of the properties SA against of the Pseudomonas SA against fluorescensPseudomonas IMA fluorescens 19/5, highlighting IMA 19/5, an actualhighlighting application an actual as packaging application for food as protection.packaging for food protection.However, the application of pristine halloysite as nanocontainer, despite the several examples reported,However, is limited. the application Halloysite clayof pristine can establish halloysite on weakas nanocontainer, interaction with despite guest the molecules, several examples based on hydrogenreported, is bonds limited. or VanHalloysite der Waals clay forces, can establish resulting on in weak low capabilityinteraction for with tunable guest drugmolecules, release based [52,109 on]. hydrogen bonds or Van der Waals forces, resulting in low capability for tunable drug release [52,109].

J. Funct. Biomater. 2018, 9, 58 12 of 22

The covalent modification of the tubes with suitable moieties is a good strategy adopted to solve these drawbacks. For example, the external halloysite surface is efficiently covalently modified by grafting organosilane molecules via condensation with the hydroxyl groups of the edges or the defects of the surface. Owing to this, different materials of large interest were obtained with consequent increase of the HNT application fields [106,110]. Among the different organosilane molecules which can be used for the HNT modification, the most used one is for sure the 3-aminopropyltriethoxysilane (APTES). Due to the introduction of an amino group on the HNT external surface, the modification allows obtaining a material that can be further modified and therefore with interesting feature for application in the drug carrier and delivery field. For example, Kumar-Krishnan et al. used the HNT–APTES compound for enzyme loading, finding that glucose oxidase, for example, is better loaded on halloysite than p-HNT without loss of its biocatalytic performances. Noteworthy, the functionalization avoids the enzyme partial deactivation [111,112]. A similar strategy was used to increase, with respect to pristine halloysite, the ibuprofen (IBU) loading. The electrostatic interaction between the amino groups on the surface of the tubes and the carboxyl groups of IBU allowed to obtain better loading efficiency and also slows down the kinetic release of the drug, improving the performance of the material [109,113]. Similar results were obtained by Yang et al. immobilizing aspirin molecules on HNT–APTES. The authors found an increase in the drug loading, from 3.84 to 11.98 wt% compared to pristine halloysite. Furthermore, once again, the kinetic release of the drug was slowed down compared to the similar system based on pristine halloysite [114]. Halloysite–cyclodextrin nanosponge hybrid was used for the loading of clotrimazole (CLT), an antifungal drug commonly used for the buccal or vaginal treatment of candidiasis [115]. The HNT carrier was developed ad hoc by the covalent grafting of β-cyclodextrin on the HNT external surface [116]. In this way, a multicavity system was obtained which has shown enhanced loading performances. Once synthesized, the hybrid was further functionalized by cysteamine hydrochloride grafting to confer mucoadhesive properties to the overall system. Release studies showed that the hybrid materials reported in this study avoid the deactivation of CLT by preserving it from the hydrolysis of the imidazole ring. A similar approach was exploited to attach on the HNT external surface some cyclodextrin units (HNT–CDs) [117]. The obtained multifunctional carrier presents an enormous advantage with regard to the conventional carrier system; the two different cavities offer the remarkable possibility for a simultaneous loading of one or more drugs with different physicochemical properties, followed by different paths for drug release consistent with the different types of the cavities interacting with the molecules. To exploit this aspect, two different drugs were chosen as model: quercetin and silibinin, two flavonoids which possess different size and shape. The experimental findings show that the silibinin molecules interacts preferentially with the HNT lumen, while the quercetin with the cyclodextrin cavity. The different interaction site of the two drugs allows for a targeted release in different conditions. The in vitro release tests performed in physiological conditions by means of the dialysis membrane method showed that silibinin was better released in acidic solution, and therefore in a medium simulating the gastric environment, while quercetin was better released in a medium simulating the intestinal fluid (phosphate buffer pH 7.4). Cytotoxic studies performed on 8505C cell lines highlighted that the drugs delivered by halloysite hybrids showed improved antitumoral efficacy compared to the free drug. Finally, the interaction between cells and the carrier, analyzed by fluorescence microscopy, showed that the materials were taken up into cells surrounded the nuclei. If the same HNT–CDs system was further functionalized with some sugar moieties (such as mannose units, specific for binding cellular lectin) [118,119], an enhanced cellular internalization J. Funct. Biomater. 2018, 9, 58 13 of 22 was observed, as revealed by fluorescence microscopy, due to the carbohydrate-receptor-mediated endocytosis. In this case, the nanomaterial is mainly localized in the cell nucleus. A thermo-responsive polymer, such as poly-(N-isopropylacrylamide) (PNIPAAM), was grafted on HNT external surface by amide condensation mediated by DCC [120]. This nanomaterial presents some interesting features: (i) an empty HNT lumen, which can encapsulate biologically active molecules; and (ii) the polymer brushes which can interact with a suitable drug. In addition, PNIPAAM shows a low critical solution temperature (LCST) around 32 ◦C, after which the polymer brushes collapse on the HNT surface and therefore they could allow a targeted release of a drug. In this context, the interaction of HNT–PNIPAAM hybrid and curcumin was investigated. It was found that the curcumin molecules are released from the carrier only a temperature of 37 ◦C and mainly in a medium that mimics the intestinal fluid. Since the carrier retains the almost totality of the drug loaded in acidic medium, it represents a versatile drug carrier system for the targeted delivery of curcumin, preventing its degradation in the gastrointestinal tract. An interesting alternative to obtain hybrid HNT–Curcumin hybrid systems is the direct covalent grafting of the bioactive molecule on the surface of the tubes developing a pro-drug system [121]. The prodrug so-obtained has shown specific properties; the covalently linked curcumin, indeed, can be released by a specific external stimulus, in particular, the kinetic release is influenced by both glutathione concentration or pH conditions. Therefore, in this work, a novel system with a dual stimuli-responsive nature is obtained. The potential application of the system can be also increased by further modification of HNT. The presence on an empty cavity, in the clay, allows to load other molecules in the lumen obtaining a prodrug system with could possess synergistic effects. The synergistic effect between a drug loaded in the inner lumen and another covalently grafted on the external surface was also exploited by us [118]. In this context, we have synthesized, characterized and studied the antioxidant properties of HNT with complementary antioxidant functionalities based on Trolox and quercetin. Experimental findings showed that the Trolox molecules, covalently grafted on HNT external surface, were available to trap free radicals, whereas the quercetin, second co-antioxidant, supramolecularly linked on the lumen, were slowly released to generate prolonged synergistic protection. Similar to the HNT–PNIPAAM thermo-responsive carrier, a synergistic effect was also observed in a pH-sensitive HNT–triazolium salt carrier [122,123]. As reported for the HNT–Trolox/Que system, in this case, the presence of the triazole moiety on the external surface could exert some synergistic effects with a drug loaded onto the halloysite surfaces, due to the biological properties of triazolium. Based on this evidence, the halloysite based triazolium salts were used as carriers for curcumin and cardanol. The physicochemical characterization showed that the electron rich molecules can interact with the positively charged halloysite lumen and besides with the external surface by π–π interactions between the aromatic rings of cardanol and curcumin and that of triazolium salts. The coexistence of these two species enhances the loading efficiency of the clay compared to that with pristine halloysite and an increased cytotoxicity towards different tumoral cell lines. To enhance the loading of quercetin (Que) and obtain a slow release over the time of the drug, He at al. grafted on modified HNT, a six-arm (Poly Ethylene-Glycol) amine (HNTs-g-PEG) [124]. This system was further modified by biotin conjugation, to achieve a selective delivery into carrier cells. Finally, to image the Que delivery through the body, on the HNTs-g-PEG-Biotin were loaded fluorescent carbon dots (Figure 12). J. Funct. Biomater. 2018, 9, 58 14 of 22 J. Funct. Biomater. 2018, 9, x FOR PEER REVIEW 14 of 22

FigureFigure 12.12. SchematicSchematic illustration illustration of of the the chemical chemical structure structure and and preparation procedure of. HNTs-g-PEG-CDs-Biotin,HNTs-g-PEG-CDs-Biotin, followedfollowed byby QueQue adsorption.adsorption. ReproducedReproducedwith withpermission permission from from [ 124[124].].

MTTMTT testtest showedshowed thatthat thethe obtainedobtained hybridhybrid possessespossesses aa lowlow cytotoxicitycytotoxicity andand excellentexcellent biocompatibilitybiocompatibility and,and, aboveabove allall enhancedenhanced anti-tumoranti-tumor effects,effects, whenwhen loadedloaded withwith Que,Que, againstagainst HeLaHeLa cells.cells. HNTs grafted with with CDs CDs and and Biotin Biotin nanoparticles nanoparticles on on PEG PEG have have shown shown potential potential applicability applicability as astargeting targeting carriers carriers for fordelivery delivery of drugs of drugs and and in vitroin vitro andand in vivoin vivo abilityability of could of could be tracked be tracked by the by theimaging. imaging. ByBy meansmeans ofof similarsimilar strategy,strategy, LiuLiu etet al.al. covalentlycovalently conjugatedconjugated thethe PEGPEG polymerpolymer toto prolongprolong halloysitehalloysite circulationcirculation time time and and controlling controlling its dosingits dosing interval interval [125]. Conversely[125]. Conversely to the above to the example, above inexample, this case, in tothis obtain case, tumor to obtain targeting tumor systems, targeting the sy authorsstems, the covalently authors graftedcovalently folate grafted units folate to the units PEG functionalizedto the PEG functionalized halloysite. Thehalloysite. HNT–PEG–FA The HNT–PEG– hybrid wasFA hybrid used as was carrier used of as doxorubicin carrier of doxorubicin (DOX) for the(DOX) treatment for the oftreatment breast cancer. of breast The cancer.in vitro Therelease in vitro test release highlighted test highlighted that DOX that is released DOX is from released the carrierfrom the up carrier to 35 h up in anto 35 acidic h in environment an acidic environment (pH = 5.3), (pH while = 5.3), it is relativelywhile it is stable relatively in neutral stable conditions. in neutral Cytotoxicityconditions. Cytotoxicity assays showed assays that showed the system that can the restrain system proliferation can restrain and proliferation induce death and of induce FR+ MCF-7 death - cells,of FR+ while MCF-7 it shows cells, relativewhile it low shows cytotoxicity relative towardslow cytotoxicity the FR L02 towards cells. Furthermore, the FR-L02 cells. the DOXFurthermore, complex canthe produceDOX complex more ROScan produce in MCF-7 more cells ROS which in MCF-7 leads to cells apoptosis. which leads to apoptosis. TheThe samesame authorsauthors usedused aa similarsimilar approachapproach toto linklink onon carboxyliccarboxylic acidacid functionalizedfunctionalized halloysite,halloysite, anotheranother polymer,polymer, specificallyspecifically thethe chitosanchitosan one.one. ThisThis newnew systemsystem waswas efficientlyefficiently usedused forfor thethe loadingloading andand releaserelease of of doxorubicin doxorubicin and and curcumin curcumin [126, 127[126,127]. Since]. chitosanSince chitosan itself possesses itself possesses interesting interesting biological properties,biological properties, the system the developed system developed in this work, in this showed work, good showed hemocompatibility, good hemocompatibility, stability instability body fluidsin body and fluids enhanced and enhanced cytocompatibility. cytocompatibility. Furthermore, Furthermore, the modified the modified halloysite halloysite allows in allows a targeted in a release;targeted both release; drugs both were drugs released were in released their active in thei formr active and form in a sustained and in a sustained manner only manner in the only “tumoral in the environment”“tumoral environment” instead of ininstead the normal of in physiologicalthe normal physiological conditions, avoidingconditions, in thisavoiding way, possiblein this way, side effectspossible of side the antitumoraleffects of the therapy. antitumoral therapy. ChitosanChitosan isis widelywidely usedused inin thethe biologicalbiological fieldfield sincesince itit possessespossesses anti-infectionanti-infection andand hemostatichemostatic activities;activities; duedue to to these these properties, properties, a chitosan/HNT a chitosan/H nanocompositeNT nanocomposite could could be utilized be utilized for wound for healingwound sincehealing it was since found it was that found it helps that theit helps re-epithelialization the re-epithelialization and collagen and collagen deposition. deposition. MoreMore recently,recently, severalseveral polymerspolymers werewere usedused forfor thethe HNTHNT functionalization,functionalization, obtainingobtaining differentdifferent nanocompositenanocomposite whichwhich havehave showedshowed toto bebe efficientefficient inin thethe deliverydelivery ofof drugs.drugs. InIn thisthis context,context, FanFan et et al. al. prepared prepared halloysite–sodium halloysite–sodium alginate/hydroxyapatite alginate/hydroxyapatite nanocompositenanocomposite beadsbeads byby generation generation of hydroxyapatiteof hydroxyapatite (HA) (HA) in a nanosizedin a nanosized regime regime [128]. On [128]. these On beads these was beads efficiently was loadedefficiently the diclofenacloaded the sodiumdiclofenac drug. sodium Similarly, drug. ofloxacin Similarly, (OFL) ofloxacin was loaded (OFL) on was magnetic loaded microspheres, on magnetic 2-hydroxypropyltrimethylmicrospheres, 2-hydroxypropyltrimeth ammoniumyl chloride ammonium chitosan/Fe chloride3O chitosan/Fe4/halloysite.3O4 Halloysite/halloysite. once Halloysite again, isonce helpful again, in is improving helpful in the improving OFL bioavailability the OFL bioa invailability the gastrointestinal in the gastrointestinal tract [129]. tract [129]. InterestingInteresting nanocompositesnanocomposites areare thosethose obtainingobtaining byby thethe introductionintroduction ofof HNTHNT fillerfiller inin aa hydrogelhydrogel matrix.matrix. InIn thisthis context,context, toto increaseincrease thethe fillerfiller dispersiondispersion inin aa peptidepeptide hydrogel,hydrogel, wewe havehave modifiedmodified thethe externalexternal HNTHNT surfacesurface withwith aminoamino acidacid derivatives.derivatives. To reachreach thisthis goal,goal, startingstarting fromfrom aa HNT–NHHNT–NH22 nanomaterial,nanomaterial, wewe covalentlycovalently graftedgrafted thethe Fmoc–phenylalanineFmoc–phenylalanine moleculemolecule (f–HNT)(f–HNT) [[130].130]. TheThe presencepresence ofof thethe aminoamino acidacid functionalityfunctionality onon thethe halloysitehalloysite surface allows specificspecific π––ππ interactionsinteractions betweenbetween modifiedmodified halloysitehalloysite andand Fmoc-phenylalanineFmoc-phenylalanine (Fmoc-Phe)(Fmoc-Phe) generatinggenerating aa hybridhybrid hydrogel with affected physicochemical properties which was efficiently used for the loading and release of camptothecin molecule (CPT).

J. Funct. Biomater. 2018, 9, 58 15 of 22 hydrogel with affected physicochemical properties which was efficiently used for the loading and release of camptothecin molecule (CPT). CPT was also loaded on folic acid conjugated chitosan oligosaccharides assembled magnetic halloysite nanotubes (FA-COS/MHNTs) [131]. This hybrid showed excellent receptor-specific targeting effects for Caco-2 cells, high superparamagnetic properties, an outstanding usefulness in killing the cancer cells. Moreover, under acidic conditions, the FA-COS/MHNTs showed a sustained drug release up to 60 h. All example reported showed that HNT is a promising material for biological applications since it can deliver a given species in a sustained and controlled manner, avoiding, in same case, its degradation and deactivation. Unfortunately, the HNT application is limited to the oral administration since the human body does not have the biological mechanisms to efficiently dispose the aluminosilicate degradation. However, halloysite is a biocompatible material as testified by several studies. It was found that the clay is not toxic towards several cell lines such as mammalian cells and some living organisms. In this context, the interaction of HNT with microscopic algae Chlorella pyrenoidosa, fresh water ciliate protist Paramecium caudatum [132] and Escherichia coli bacteria [128] as well as in yeast cells [94], was studied by Lvov and Fakhrullin et al. The obtained results highlight that no penetration of the nanomaterial into cell interior occurs [133] and that it is also not toxic in a wide range of concentration. In vivo studies on living nematodes (worms), Caenorhabditis elegance [134], chickens and piglets have shown that, by feeding them with halloysite, there is no toxicity in the organism and in the case of superior organisms the clay can remove some mycotoxins potentially present in grain feed [135]. As far as halloysite accumulation after its oral administration is concerned, a recent study has shown by several investigations of target organs that HNTs are safe materials [136]. In this context, Hu et al. [137] demonstrated that the prolonged administration of the clay by an oral route up to 30 days can lead to an Al accumulation in the liver with consequent hepatic damage. Thanks to this study, it is possible to determine the maximum dose that can be safely used for HNT oral administration (ca. 20 mg kg−1 BW). Finally, in a recent study, we also investigated the effect of halloysite on Raphanus Sativus L. to evaluate any potential phytotoxic effects. Our study highlights that it not only possesses no negative effects, but it seems that HNT can help the growth of the seeds [138].

6. Conclusions Clay minerals have attracted great interest since they are biocompatible materials with appealing properties. The most representative examples of clay minerals are kaolinite, montmorillonite, sepiolites and halloysite. The present review reports the recent developments of the clay minerals research, focusing on application as carrier for the delivery and sustained release of biological active species. The functionalization of clays surfaces, by means of supramolecular interactions or covalent modifications, opens different ways to obtain interesting nanomaterials which show improved biological properties with respect to the unmodified ones.

Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.

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