Cyclodextrin Nanosponges Inclusion Compounds Associated with Gold Nanoparticles for Potential Application in the Photothermal Release of Melphalan and Cytoxan

International Journal of Molecular Sciences

Article Cyclodextrin Nanosponges Inclusion Compounds Associated with Gold Nanoparticles for Potential Application in the Photothermal Release of Melphalan and Cytoxan

Sebastián Salazar 1,2,3,* , Nicolás Yutronic 1, Marcelo J. Kogan 2,3,* and Paul Jara 1,*

1 Departmento de Química, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Ñuñoa, Santiago 7800003, Chile; [email protected] 2 Departamento de Química, Farmacológica y Toxicológica, Universidad de Chile, Sergio Livingstone 1007, Santiago 8380492, Chile 3 Advanced Center for Chronic Diseases (ACCDiS), Universidad de Chile, Santos Dumont 964, Independencia, Santiago 8380494, Chile * Correspondence: [email protected] (S.S.); [email protected] (M.J.K.); [email protected] (P.J.); Tel.: +56-229-787-396 (P.J.)

Abstract: This article describes the synthesis and characterization of β-cyclodextrin-based nanosponges (NS) inclusion compounds (IC) with the anti-tumor drugs melphalan (MPH) and cytoxan (CYT), and the addition of gold nanoparticles (AuNPs) onto both systems, for the potential release of the drugs by means of laser irradiation. The NS-MPH and NS-CYT inclusion compounds were characterized using scanning electron microscopy (SEM), X-ray powder diffraction (XRPD), energy Citation: Salazar, S.; Yutronic, N.; dispersive spectroscopy (EDS), thermogravimetric analysis (TGA), UV–Vis, and proton nuclear Kogan, M.J.; Jara, P. Cyclodextrin magnetic resonance (1H-NMR). Thus, the inclusion of MPH and CYT inside the cavities of NSs Nanosponges Inclusion Compounds was conﬁrmed. The association of AuNPs with the ICs was conﬁrmed by SEM, EDS, TEM, and Associated with Gold Nanoparticles β for Potential Application in the UV–Vis. Drug release studies using NSs synthesized with different molar ratios of -cyclodextrin Photothermal Release of Melphalan and diphenylcarbonate (1:4 and 1:8) demonstrated that the ability of NSs to entrap and release the and Cytoxan. Int. J. Mol. Sci. 2021, 22, drug molecules depends on the crosslinking between the cyclodextrin monomers. Finally, irradiation 6446. https://doi.org/10.3390/ assays using a continuous laser of 532 nm showed that photothermal drug release of both MPH and ijms22126446 CYT from the cavities of NSs via plasmonic heating of AuNPs is possible.

Academic Editors: Keywords: β-cyclodextrin; cyclodextrin polymers; anti-tumor; drug delivery; photothermal therapy; Antonino Mazzaglia, Lajos Szente plasmon surface resonance and Francesco Trotta

Received: 29 May 2021 Accepted: 14 June 2021 1. Introduction Published: 16 June 2021 In recent years, nanotechnology has received increasing attention and consideration

Publisher’s Note: MDPI stays neutral due to its potential to improve the effectiveness of various drugs that are currently being with regard to jurisdictional claims in used in cancer treatment. An inappropriate drug concentration at the tumor site and published maps and institutional affil- toxicity complications are the main causes of inadequate chemotherapeutic efficiency [1,2]. iations. Localized delivery is imperative in order to develop efficient treatments for aggressive types of cancer, such as melanoma, ovarian cancer, neuroblastoma, and multiple myeloma, among others [3–5]. Melphalan (MPH) and cytoxan (CYT) (Figure1) are anti-cancer chemotherapy drugs classified as alkylating agents [5,6]. Alkylating agents work by damaging the DNA of Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. cancer cells. Unfortunately, alkylating agents do not differentiate between cancerous cells This article is an open access article and normal cells. Common side effects for patients taking MPH and CYT include low distributed under the terms and blood counts, nausea, vomiting, and hair loss, to name a few [7–9]. conditions of the Creative Commons To overcome the limitations associated with anti-cancer agents and to minimize the Attribution (CC BY) license (https:// adverse side effects, nano-materials and nano-porous systems (such as cyclodextrin-based creativecommons.org/licenses/by/ nano-sponges (NS) have been synthesized and characterized, as they have great potential 4.0/). for applications in nano-therapies and drug delivery systems [10–15]. Cyclodextrins (CD)

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are water soluble oligosaccharides, most commonly consisting of six, seven, or eight glucopyranose units which are linked by α-1,4 glycosidic bonds. Among CDs, β-CD is interesting due to its cavity dimensions and its stability with crosslinking agents. As such, β-CD has been used to form nano-porous polymers [15]. The rising popularity of NSs as the material of choice over native CD is mainly due to their higher entrapment capacities, control over particle size and their ability to be regenerated and reutilized [16–19]. NSs can Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 19 also improve the physicochemical properties of the guest molecules, such as solubility and stability, and absorb liquids into their nano-porous structure to convert liquid materials into a solid form [20].

Figure 1. Chemical structure of MPH (left) and CYT (right). Figure 1. Chemical structure of MPH (left) and CYT (right). NSs main advantages also include that they can be chemically modified and decorated withTo nanoparticles, overcome the thus limitations improving associated the usefulness with anti-cancer and properties agents of and the polymerto minimize [21]. the NSs adversehave been side proven effects, to be nano-materials safe, non-toxic, biodegradable,and nano-porous and ofsystems low cost (such [22–24 ].as As cyclodex- such, NSs trin-basedhave gained nano-sponges attention in (NS) targeted have drug been delivery synthesized research and aimed characterized, at improving as they the efficacy have greatof chemotherapies potential for applications and reducing in the nano-t adverseherapies effects and related drug to delivery the drugs systems [16–18,20 [10–15].,25–29]. CyclodextrinsGold nanoparticles (CD) are water (AuNP) soluble also oligosacch show promisingarides, most characteristics commonly consisting in drug delivery of six, seven,systems, or eight as they glucopyranose present high units solubility, which highare linked stability by inα-1,4 living glycosidic systems bonds. and improved Among CDs,kinetics β-CD for is drug interesting distribution due [to30 its–33 cavity]. AuNPs dimensions can be conjugated and its stability to inclusion with complexes crosslinking (IC) agents.of CDs As if the such, guests β-CD have has functional been used groups, to form such nano-porous as thiols or amines polymers [34,35 [15].]. NSs The associated rising popularitywith AuNPs of NSs might as promotethe material the release of choice of theover included native CD drugs is mainly by means due of to irradiation, their higher due entrapmentto the plasmon capacities, effect ofcontrol AuNPs. over A controlledparticle size drug and delivery their ability system to usingbe regenerated AuNPs and and CD reutilizedhas been [16–19]. reported NSs previously can also [36 improve,37]; however, the physicochemical there are no such properties studies usingof the systems guest molecules,with NSs assuch the as matrix. solubility and stability, and absorb liquids into their nano-porous structureThe to objectives convert liquid of this materials study were intoto a solid synthesize form [20]. and characterize NS inclusion com- poundsNSs (IC)main with advantages the anti-tumor also include drugs MPHthat th andey can CYT be and chemically to conjugate modified the ICs and to AuNPsdeco- ratedto study with thenanoparticles, migration ofthus the improving guest molecules the usefulness by laser and irradiation. properties This of articlethe polymer reports [21].drug NSs release have studiesbeen proven using NSsto be inclusion safe, non-to compoundsxic, biodegradable, associated and to AuNPs of low viacost plasmonic [22–24]. Asphotothermia such, NSs have for the gained first time. attention These resultsin targeted could drug be of delivery great interest research as they aimed contribute at im- to provingthe understanding the efficacy ofof thechemotherapies potential utility and of reducing NSs associated the adverse to AuNPs effects for related the controlled to the drugsrelease [16–18,20,25–29]. of drug molecules via local photothermic effects. Gold nanoparticles (AuNP) also show promising characteristics in drug delivery systems,2. Results as andtheyDiscussion present high solubility, high stability in living systems and improved kinetics2.1. Characterization for drug distribution of the ICs [30–33]. AuNPs can be conjugated to inclusion complexes (IC)2.1.1. of 1CDsH-NMR if the Spectra guests of have the ICsfunctional groups, such as thiols or amines [34,35]. NSs associatedTo confirm with AuNPs the formation might promote of the NS-MPHthe release and of the NS-CYT included complexes, drugs by1 H-NMRmeans of spec- ir- radiation,troscopy due was to used. the plasmon The changes effect in of the AuNP chemicals. A controlled shifts of the drug drugs delivery provide system evidence using for AuNPsthe formation and CD ofhas the been ICs. reported Figure2 previouslyshows the acquired[36,37]; however, spectra ofthere the NSs,are no the such anti-tumor studies usingdrugs systems and the with ICs. NSs as the matrix. TheThe objectives proton signals of this of study both guestwere moleculesto synthesize show and high-field characterize chemical NS inclusion shifts, possibly com- poundsdue to (IC) the screening with the anti-tumor effects caused drugs by theMPH spatial and restrictionCYT and to and conjugate the change the ofICs environment to AuNPs todue study to thethe drugsmigration being of entrappedthe guest molecules inside the by cavities laser ofirradiation. the NSs. This The article protons reports within drugthe hydrophobicrelease studies cavities using NSs of the inclusion NSs (H co3 andmpounds H5), as associated well as the to hydroxylAuNPs via groups plasmonic (OH 2 photothermiaand OH3) displayed for the first the time. most These pronounced results chemicalcould be of shifts, great which interest strongly as they suggests contribute the tocomplexation the understanding of the drugs.of the potential The protons utility on theof NSs outside associated cavities to of AuNPs the NSs for (H 2the,H 4,con-and trolledH6) also release experienced of drug molecules chemical shifts. via local It is photothermic possible that effects. the drugs were included not only inside the cavities of the CD monomers, but also on the multiple interstices of the NSs that 2. Results and Discussion 2.1. Characterization of the ICs 2.1.1. 1H-NMR Spectra of the ICs To confirm the formation of the NS-MPH and NS-CYT complexes, 1H-NMR spectroscopy was used. The changes in the chemical shifts of the drugs provide evidence for the formation of the ICs. Figure 2 shows the acquired spectra of the NSs, the anti-tumor drugs and the ICs.

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were produced by polymerization. The differences in the chemical shifts of the drugs upon inclusion with NSs conﬁrm the complexation of the drugs, as the occurrence of changes in Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 19 the micro-environment between the free and entrapped drugs was expected, as reported in previous studies [11,21,38–40].

1 FigureFigure 2.2. 1H-NMR spectra of NSs, MPH, CYT, and the ICs.

ProtonThe proton assignments signals of for both the guest NSs and molecules the anti-tumor show high-field drugs are chemical shown in shifts, Figure possibly3. The chemical shifts for MPH and CYT after inclusion are shown in Tables1 and2, respectively. due to the screening effects caused by the spatial restriction and the change of environment due to the drugs being entrapped inside the cavities of the NSs. The protons within Table 1. Proton assignments and chemical shifts for MPH and the NS-MPH complex. the hydrophobic cavities of the NSs (H3 and H5), as well as the hydroxyl groups (OH2 and OHSystem3) displayed H1 the most H2 pronounced H3 H4chemicalH5 shifts, which H6 strongly OH2 suggests OH3 the OH6 complexation of the drugs. The protons on the outside cavities of the NSs (H2, H4, and H6) also NS 4.827 3.300 3.627 3.361 3.579 3.655 5.705 5.673 4.440 experienced chemical shifts. It is possible that the drugs were included not only inside NS- the cavities4.825 of the CD 3.298 monomers, 3.618 but 3.358also on the 3.573 multiple 3.652 interstices 5.718 of the 5.680 NSs that 4.445 were MPH produced by polymerization. The differences in the chemical shifts of the drugs upon inclusion∆δ with0.002 NSs confirm 0.002 the 0.009 complexation 0.003 of 0.006 the drugs, 0.003 as the−0.013 occurrence−0.007 of changes−0.005 in the micro-environment between the free and entrapped drugs was expected, as re- System H’1 H’2 H’3 H’4 H’5 H’6 H’7 ported in previous studies [11,21,38–40]. MPHProton assignments 3.738 for 3.447 the NSs 7.135and the anti 6.798-tumor drugs 2.835 are shown 3.117 in Figure 11.035 3. The chemicalNS-MPH shifts 3.735 for MPH 3.444and CYT after 7.130 inclusion 6.793 are shown 2.830 in Tables 3.115 1 and 2, 11.029 respectively.∆δ 0.003 0.003 0.005 0.005 0.005 0.002 0.006

Table 1. Proton assignments and chemical shifts for MPH and the NS-MPH complex.

System H1 H2 H3 H4 H5 H6 OH2 OH3 OH6 NS 4.827 3.300 3.627 3.361 3.579 3.655 5.705 5.673 4.440 NS-MPH 4.825 3.298 3.618 3.358 3.573 3.652 5.718 5.680 4.445 Δδ 0.002 0.002 0.009 0.003 0.006 0.003 −0.013 −0.007 −0.005

System H’1 H’2 H’3 H’4 H’5 H’6 H’7 MPH 3.738 3.447 7.135 6.798 2.835 3.117 11.035 NS-MPH 3.735 3.444 7.130 6.793 2.830 3.115 11.029

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ΔδΔδ 0.003 0.003 0.0030.003 0.0050.005 0.0050.005 0.0050.005 0.0020.002 0.0060.006 Table 2. Proton assignments and chemical shifts for CYT and the NS-CYT complex.

TableTableSystem 2. 2. Proton Proton H1 assignments assignments H2 and and H3chemical chemical shifts H4shifts for for CYT H5CYT and and the the H6 NS-CYT NS-CYT OH2 complex. complex. OH3 OH6 SystemSystemNS 4.827H1H1 3.300H2H2 3.627H3H3 3.361H4H4 3.579H5H5 3.655H6H6 OH2 5.705OH2 OH3 5.673OH3 OH6 4.440OH6 NSNS-NS 4.827 4.827 3.3003.300 3.6273.627 3.3613.361 3.5793.579 3.6553.655 5.7055.705 5.6735.673 4.4404.440 4.822 3.297 3.621 3.359 3.572 3.652 5.712 5.678 4.443 NS-CYTNS-CYTCYT 4.8224.822 3.2973.297 3.6213.621 3.3593.359 3.5723.572 3.6523.652 5.7125.712 5.6785.678 4.4434.443 Δδ∆Δδδ 0.0050.005 0.005 0.003 0.0030.003 0.006 0.0060.006 0.002 0.0020.002 0.007 0.0070.007 0.003 0.0030.003 −−−0.0070.007 −−−0.0050.005 −−0.0030.0030.003

System H’1 H’2 H’3 H’4 H’5 H’6 SystemSystem H’1H’1 H’2H’2 H’3H’3 H’4H’4 H’5H’5 H’6H’6 CYT 3.380 3.735 4.228 1.728 3.465 3.319 CYTCYT 3.380 3.380 3.7353.735 4.2284.228 1.7281.728 3.4653.465 3.3193.319 NS-CYTNS-CYT 3.378 3.3783.378 3.733 3.7333.733 4.225 4.2254.225 1.725 1.7251.725 3.460 3.4603.460 3.316 3.3163.316 Δδ∆Δδδ 0.0020.002 0.002 0.002 0.0020.002 0.003 0.0030.003 0.003 0.0030.003 0.005 0.0050.005 0.003 0.0030.003

FigureFigureFigure 3. 3.3. ProtonProton Proton assignments assignmentsassignments for forfor the thethe NSs, NSs,NSs, MPH, MPH,MPH, and andand CYT. CYT.

2.1.2.2.1.2.2.1.2. TGA TGATGA of ofof the thethe ICs ICsICs TheTheThe TGA TGA provides provides informationinformation information aboutabout about the the the thermal thermal thermal stability stability stability of of theof the the ICs ICs ICs in in comparisonin comparison comparison to totheto the the free free free drugs. drugs. drugs. Figure Figure Figure4 shows 4 4 shows shows the the TGAthe TGA TGA of MPH, of of MPH, MPH, CYT, CYT, CYT, NSs NSs andNSs and theand NSsthe the NSs loadedNSs loaded loaded with with thewith theanti-tumorthe anti-tumor anti-tumor compounds. compounds. compounds.

FigureFigureFigure 4. 4. TGATGA TGA of ofof MPH, MPH,MPH, CYT, CYT,CYT, NS, NS, and and the the ICs. ICs.

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MPH, CYT, NSs, and the ICs showed a weight loss at 100 ◦C due to the presence of water.MPH, NSs CYT, main NSs, degradation and the ICs process showed occurs a weight at 345 loss◦C, whichat 100 is°C an due indicator to the presence of its good of water.thermal NSs stability. main degradation The main degradation process occurs step for at MPH345 °C, occurred which atis 250an indicator◦C, whereas of its for good CYT, thermalthat weight stability. loss was The shown main atdegradation 271 ◦C. The st ICs’ep for thermogram MPH occurred shows at two 250 degradation °C, whereas steps. for ◦ CYT,The ﬁrstthat weightweight lossloss atwas 230 shownC can at be 271 related °C. The to ICs’ the degradationthermogram of shows the drugs. two degradation The second ◦ steps.and main The weightfirst weight loss steploss wasat 230 observed °C can be at 350relatedC, whichto the degradation corresponds of to the degradation drugs. The of secondNSs [38 and,41]. main The thermogramsweight loss step show was that obse therved inclusion at 350 of °C, MPH which and corresponds CYT in the cavitiesto degra- of dationthe NSs of might NSs [38,41]. increase The the thermograms thermal stability show of thethat drugs the inclusion [21,29,41 of–44 MPH]. The and decomposition CYT in the cavitiestemperatures of the forNSs MPH, might CYT increase and the the ICs thermal are summarized stability of in the Table drugs3. [21,29,41–44]. The decomposition temperatures for MPH, CYT and the ICs are summarized in Table 3. Table 3. Decomposition temperatures and weight loss for MPH, CYT, and the ICs. Table 3. Decomposition temperatures and weight loss for MPH, CYT, and the ICs. System Decomposition Temperature (◦C) Weight Loss (%) SystemNS Decomposition 345.7 Temperature (°C) Weight 65.1 Loss (%) MPHNS 249.5345.7 25.165.1 MPH 249.5 25.1 NS-MPH 233.9 39.7 NS-MPH 233.9 39.7 NS-MPH 348.1 48.3 NS-MPH 348.1 48.3 CYT 271.5271.5 27.127.1 NS-CYT 235.1235.1 41.1 41.1 NS-CYT 350.7350.7 49.9 49.9

2.1.3.2.1.3. XRPD XRPD of of the the ICs ICs TheThe XRPD XRPD analyses analyses for for MPH, MPH, CYT, CYT, and and th thee ICs ICs are shown in Figure 55.. TheThe XRPDXRPD resultsresults of of the the free free drugs indicate strong diffraction peaks. The The disappearance disappearance of of these these peakspeaks after after complexation complexation of of the the drugs drugs on on the the NSs NSs indicates indicates a a reduction reduction in in the the crystallinity crystallinity ofof the the drugs drugs molecules. molecules. In In the the ICs ICs diffractograms, diffractograms, a a widened widened peak peak appeared appeared due due to to a a differentdifferent crystal arrangementarrangement of of the the ICs. ICs. None None of theof characteristicthe characteristic peaks peaks of the of anti-tumor the anti-tumordrugs are drugs preserved are preserved in the NS-MPH in the NS-MPH and NS-CYT and NS-CYT diffraction diffraction patterns. patterns. This conﬁrms This con- the firmsencapsulation the encapsulation of the drugs of the inside drugs the inside cavities th ofe cavities the NSs of and the the NSs lack and of freethe drugslack of in free the drugsICs, as in reported the ICs, previouslyas reported [ 21previously,38,45–47 ].[21,38,45–47].

FigureFigure 5. 5. XRPDXRPD of of MPH, MPH, CYT, CYT, and and the the ICs. ICs. 2.1.4. SEM Analyses of the ICs 2.1.4. SEM Analyses of the ICs The formation of the ICs was conﬁrmed using SEM and EDS analyses. Figure6 shows The formation of the ICs was confirmed using SEM and EDS analyses. Figure 6 SEM micrographs of free NSs, MPH, CYT, and the ICs. The images reveal the highly shows SEM micrographs of free NSs, MPH, CYT, and the ICs. The images reveal the porous morphology of the NSs, whereas MPH and CYT show rectangular crystals and rods, highly porous morphology of the NSs, whereas MPH and CYT show rectangular crystals and rods, respectively. After drug loading, changes in the surface of the samples were

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observed, as NSs show a homogeneous phase, with the absence of microcrystals of the drugs, confirming the inclusion of the guest molecules in the NSs cavities and excluding the possibility of co-precipitation of the individual phases [16,19,38,48].

Int. J. Mol. Sci. 2021, 22, 6446 2.1.5. UV–Vis of MPH and CYT after Contact with NSs 6 of 19 UV–Vis analyses were carried out in chloroform to determine the maximum absorbance of the drugs after different incubation times with the NSs. Figure 7 shows that the absorbance of both MPH and CYT decreased as the incubation time with the NSs increased,respectively. thus Afterconfirming drug loading, the entrapment changes of in the the anti-tumor surface of thedrugs samples inside were the cavities observed, of the as polymer.NSs show The a homogeneous decrease in phase,drug absorbance with the absence with increasing of microcrystals contact of time the drugs, of NSs conﬁrming indicates the inclusionformation ofof theinclusion guest moleculescomplexes, in which the NSs are cavities in a suspension, and excluding according the possibility to previous of studiesco-precipitation [21,28,49]. of the individual phases [16,19,38,48].

Figure 6. SEM micrographs of NSs (A,,B),), MPHMPH ((C),), NS-MPHNS-MPH ((DD),), CYTCYT ((EE),), andand NS-CYTNS-CYT ((FF).).

2.1.5. UV–Vis of MPH and CYT after Contact with NSs UV–Vis analyses were carried out in chloroform to determine the maximum absorbance of the drugs after different incubation times with the NSs. Figure7 shows that the absorbance of both MPH and CYT decreased as the incubation time with the NSs increased, thus conﬁrming the entrapment of the anti-tumor drugs inside the cavities of the polymer. The decrease in drug absorbance with increasing contact time of NSs indicates the formation of inclusion complexes, which are in a suspension, according to previous studies [21,28,49]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 19 Int. Int. J. Mol. Sci. 2021, 22, 6446x FOR PEER REVIEW 77 of of 19

Figure 7. UV–Vis spectra of MPH (left) and CYT (right) after different contact times with NSs. Figure 7. UV–Vis spectra of MPH (left) and CYT (right)(right) afterafter differentdifferent contactcontact timestimes withwith NSs.NSs. 2.2. Characterization Characterization of ICs Associated with the AuNPs 2.2. Characterization of ICs Associated with the AuNPs 2.2.1. SEM SEM and and EDS EDS Analyses of the ICs Associated with the AuNPs 2.2.1. SEM and EDS Analyses of the ICs Associated with the AuNPs Figures 88 andand9 9 show show SEM SEM micrographs micrographs of of the the AuNPs AuNPs attached attached to to the the ICs’ ICs’ surface. surface. It It Figures 8 and 9 show SEM micrographs of the AuNPs attached to the ICs’ surface. It isis evident that the ICs retained their porous morphology after the deposition of the gold is evident that the ICs retained their porous morphology after the deposition of the gold nanoparticles. nanoparticles.

Figure 8. SEM micrographs of NS-MPH associated with the AuNPs. Figure 8. SEM micrographs of NS-MPH associated withwith thethe AuNPs.AuNPs.

Figure 9. SEM micrographs of NS-CYT associated with the AuNPs. Figure 9. SEM micrographs of NS-CYT associated with the AuNPs. Figure 9. SEM micrographs of NS-CYT associated with the AuNPs.

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TheThe EDSEDS analysis results results are are shown shown in in Figures Figures 10 10 and and 11 11 for for NS-MPH-AuNP NS-MPH-AuNP and and The EDS analysis results are shown in Figures 10 and 11 for NS-MPH-AuNP and NS-CYT-AuNP,NS-CYT-AuNP, respectively. These These analyses analyses provide provide information information about about thethe elemental elemental NS-CYT-AuNP, respectively. These analyses provide information about the elemental compositioncomposition of the ICs. The The graphs graphs show show the the weight weight percentages percentages and and presence presence of C, of O, C, N O, N composition of the ICs. The graphs show the weight percentages and presence of C, O, N andand ClCl inin the NS-MPH complex. complex. N N and and Cl Cl correspond correspond to tothe the amine amine and and chloroethylamine chloroethylamine and Cl in the NS-MPH complex. N and Cl correspond to the amine and chloroethylamine functionalfunctional groups of of MPH. MPH. The The EDS EDS result result of of NS-CYT NS-CYT shows shows the the detection detection of C, of O, C, N, O, P, N, P, functional groups of MPH. The EDS result of NS-CYT shows the detection of C, O, N, P, andand ClCl duedue to the amine, phosphate phosphate and and chlo chloroethylroethyl functional functional groups groups of CYT. of CYT. The The EDS EDS and Cl due to the amine, phosphate and chloroethyl functional groups of CYT. The EDS spectraspectra inin FiguresFigures 99 andand 10 also show show the the detection detection of of Au, Au, which which confirms conﬁrms the the association association spectra in Figures 9 and 10 also show the detection of Au, which confirms the association ofof thethe ICsICs toto the AuNPs. of the ICs to the AuNPs.

Figure 10. EDS analysis of NS-MPH associated with the AuNPs. FigureFigure 10.10. EDS analysis of of NS-MPH NS-MPH associated associated with with the the AuNPs. AuNPs.

Figure 11. EDS analysis of NS-CYT associated with the AuNPs. FigureFigure 11.11. EDS analysis of of NS-CYT NS-CYT associated associated with with the the AuNPs. AuNPs. 2.2.2. TEM Analyses of the ICs Associated with the AuNPs 2.2.2.2.2.2. TEM Analyses of of the the ICs ICs Associated Associated with with the the AuNPs AuNPs TEM micrographs of the ICs conjugated to the AuNPs are shown in Figure 11. The TEM micrographs of the ICs conjugated to the AuNPs are shown in Figure 11. The AuNPsTEM are micrographs distributed on of the the ICs, ICs both conjugated isolated toand the aggregated, AuNPs are contained shown inin Figurethe organic 11. The AuNPs are distributed on the ICs, both isolated and aggregated, contained in the organic AuNPsmaterial. are The distributed micrographs on theshow ICs, three both componen isolated andts in aggregated,the ICs: the containedspherical AuNPs, in the organic the material. The micrographs show three components in the ICs: the spherical AuNPs, the material.organic phase The micrographscorresponding show to the three ICs, and components a nano bar-shaped in the ICs: component the spherical associated AuNPs, to the organic phase corresponding to the ICs, and a nano bar-shaped component associated to organicthe AuNPs. phase The corresponding nano bars are toof theorganic ICs, natu andre, anano which bar-shaped can probably component be associated associated to the to the AuNPs. The nano bars are of organic nature, which can probably be associated to the thecrystallization AuNPs. The of nanothe anti-tumor bars are of drugs, organic due nature, to a partial which disintegration can probably of be the associated ICs during to the crystallization of the anti-tumor drugs, due to a partial disintegration of the ICs during crystallizationpreparation of ofthe the TEM anti-tumor samples with drugs, ethanol. due to Selected a partial area disintegration electron diffraction of the ICs(SAED during preparationpreparation of the TEM samples samples with with ethanol. ethanol. Selected Selected area area electron electron diffraction diffraction (SAED (SAED (Figure 12D)) conﬁrms a single metallic crystalline phase which can be indexed to the (220), (311), and (422) crystallographic planes of AuNPs [50].

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Int. J. Mol. Sci. 2021, 22, 6446 9 of 19 (Figure(Figure 12D))12D)) confirmsconfirms aa singlesingle metallicmetallic crystallinecrystalline phasephase whichwhich cancan bebe indexedindexed toto thethe (220),(220), (311),(311), andand (422)(422) crystallographiccrystallographic planesplanes ofof AuNPsAuNPs [50].[50].

FigureFigure 12. TEMTEM micrographs micrographs of of NS-MPH NS-MPH associated associated with with AuNPs AuNPs (A (),),A NS-CYTNS-CYT), NS-CYT associatedassociated associated withwith with AuNPsAuNPs AuNPs ((B),), ( BNS-CYTNS-CYT), NS-CYT as-as- associatedsociated with with AuNPs AuNPs selected selected area area for for SAED SAED (C (),C), ),andand and SAEDSAED SAED patternpattern pattern ofof of AuNPsAuNPs AuNPs ((D (D).). ).

2.2.3. UV–Vis UV–Vis SpectraSpectra ofof thethe ICsICs AssociatedAssociated withwith thethe AuNPsAuNPs UV–Vis andand diffusediffuse reﬂectancereflectance areare usefuluseful techniquestechniques toto conﬁrmconfirmconfirm thethe depositiondeposition ofof nanoparticles onto the surfacesurface ofof organicorganic substrates.substrates. Figure Figure 13 shows thethe absorptionabsorption spectra of the ICs conjugated to the AuNPs. The characteristic plasmon band ofof sphericalspherical gold nanoparticles was observed at 565 nm. The bathochromic shift could be due to gold nanoparticles was observed at 565 nm. The bathochromic shift could be due to the the aggregation of some of the AuNPs onto microcrystals, which is consistent with the aggregation of some of the AuNPs onto microcrystals, which is consistent with the broadening of the absorption band and with the TEM images shown in Section 2.2.2. The broadening of the absorption band and with the TEM images shown in Section 2.2.2. The presence of the plasmon band in the ICs conjugated to the AuNPs provides evidence of the presence of the plasmon band in the ICs conjugated to the AuNPs provides evidence of nanoparticles’ stability, as indicated by previous studies [36,37]. thethe nanoparticles’nanoparticles’ stability,stability, asas indicatedindicated byby previousprevious studiesstudies [36,37].[36,37].

Figure 13. UV–Vis spectraspectra ofof thethe AuNPsAuNPs depositeddeposited onon thethe ICs’ICs’ surface.surface.

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2.2.4. DLS and Z-Potential of the ICs Associated with the AuNPs Table4 shows the hydrodynamic diameter and Z-Potential of the AuNPs and the ICs conjugated to the AuNPs.

Table 4. DLS and Z-Potential of AuNPs and ICs associated with the AuNPs.

System DLS (nm) Z-Potential (mV) PDI AuNPs 19 ± 2 −46 ± 0.4 0.21 AuNP-NS-MPH 633 ± 43 −31 ± 0.5 0.47 AuNP-NS-CYT 618 ± 38 −35 ± 0.5 0.45

The AuNPs showed a Z-Potential of −46 mV. The negative surface charge can be attributed to the stabilization by the citrate ions. Colloidal AuNPs show a hydrodynamic diameter of 19 nm, which is different than the diameter of 12 nm shown by TEM, as the latter technique indicates the diameter of the metallic phase. DLS also provides information of the hydrodynamic diameter of the ternary systems, with depicted values of 633 for AuNPs- NS-MPH and 618 for AuNPs-NS-CYT. Upon association, the Z-Potential of the AuNPs decreased due to the stabilization of the AuNPs by the neutral ICs. Nano-suspensions with Z-Potential values over ±30 mV are considered stable according to the literature [20,29,51], thus conﬁrming that the supramolecular systems will not undergo aggregation over time. Polydispersity index (PDI) values are used to describe the size distribution of nanoparticles. PDI values over 0.7 indicate that the samples have broad particle size distribution [13]. Thus, the obtained PDI values for AuNPs and the AuNPs associated to the ICs show that the nano formulations are stable and homogeneous in nature.

2.3. Guest Photothermal Release by Laser Irradiation 2.3.1. Drug Loading and Encapsulation Efficiencies MPH and CYT were loaded in NSs with different β-CD and DPC molar ratios (1:4 and 1:8). The complexes’ stoichiometry was determined using the 1H-NMR spectra of the ICs and the free drugs, with the integration of H1 of the NSs as a reference. This allowed for the determination of host numbers per drug [52]. The observed NS/drug ratios were 1:7 for the drugs loaded in NSs (1:4) and 1:4 for the drugs loaded in NSs (1:8). Drug loading and encapsulation efficiencies were calculated using Equations (1) and (2), respectively. Among the NSs, the drug loading and encapsulation efficiency was higher in NSs (1:4) than in NSs (1:8), as shown in Table5. This indicates that the degree of crosslinking in the polymer affected the complexation capacity of the NSs.

Table 5. Drug loading and encapsulation efﬁciencies for NS-MPH and NS-CYT.

System Drug Loading (%) Encapsulation Efﬁciency (%) NS (1:4)–MPH 83.1 ± 2.25 90.3 ± 0.25 NS (1:4)–CYT 85.2 ± 2.31 93.7 ± 0.22 NS (1:8)–MPH 63.1 ± 2.13 75.3 ± 0.15 NS (1:8)–CYT 67.2 ± 2.08 77.1 ± 0.35

Crosslinking on NSs with different molar ratios can be estimated using 1H-NMR. The technique allows to compare the integration of the hydroxyl groups of native β-CD with the integration of the hydroxyl group of NSs. Integration deltas of OH groups increase as the concentration of crosslinker increases, as they react with DPC to form links between the CD monomers. These results are shown in Table6, thus conﬁrming the higher degree of crosslinking on NSs (1:8). Int. J. Mol. Sci. 2021, 22, 6446 11 of 19 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 19

Table 6. Chemical shifts and integrations of NSs (1:4), NSs (1:8) and free β-CD. Table 6. Chemical shifts and integrations of NSs (1:4), NSs (1:8) and free β-CD. R R R ∆R ∆R Protons β-CD NSs 1:4 NSs 1:8 ∫ ∫ ∫ Δ∫ Δ∫ Protons β-CD NSs 1:4(β -CD) NSs 1:8 (NSs 1:4) (NSs 1:8) (NSs 1:4) (NSs 1:8) (β-CD) (NSs 1:4) (NSs 1:8) (NSs 1:4) (NSs 1:8) OH2 5.721 5.705OH2 5.721 5.701 5.705 7.035.701 7.03 6.33 6.33 5.675.67 0.7 0.7 1.36 1.36 OH3 5.670 5.673OH3 5.670 5.670 5.673 7.055.670 7.05 6.22 6.22 5.405.40 0.830.83 1.65 1.65 OH6 4.453 4.440OH6 4.453 4.437 4.440 7.154.437 7.15 6.18 6.18 5.335.33 0.970.97 1.82 1.82

2.3.2. Laser Irradiation Assays To studystudy drugdrug migrationmigration phenomenaphenomena byby laserlaser irradiation,irradiation, thethe ICsICs associatedassociated withwith thethe AuNPs were added toto a two-phase system consisting ofof an aqueous phase and an organic phase. Generation of local heat might promote the release ofof MPH and CYT molecules, asas shown in Figure 1414..

Figure 14. Schematic representation of guestguest moleculemolecule migrationmigration byby meansmeans ofof plasmonicplasmonic hyperthermia.hyperthermia.

The assays were performed by irradiation withwith a laser at intervals of 15 minmin untiluntil aa maximum time of 90 min was reached. Absorbance of the guest molecules was measured using UV–Vis inin chloroform.chloroform. The concentrationconcentration of the drugsdrugs waswas determineddetermined usingusing thethe Lambert–Beer equation. Molar attenuation of both drugs was determined using a setset ofof −1 −1 drug solutions using UV–Vis UV–Vis spectroscopy. The The molar molar attenuation attenuation was was 9.31 9.31 mM mM−1 cmcm−1 for −1 −1 forMPH MPH and and 7.27 7.27 mM mM−1 cm−1 cmfor CYT.for CYT. Drug release assays were carriedcarried outout usingusing NSsNSs synthesizedsynthesized withwith differentdifferent molarmolar ratios of β-CD and DPC (1:4 and 1:8) to determine which system providesprovides higherhigher loadingloading efficienciesefficiencies and drugdrug releaserelease percentages.percentages. Figures 15 and 16 show the percentagespercentages of drugdrug releasedreleased toto thethe organicorganic phasephase atat different irradiation times forfor MPHMPH andand CYT,CYT, respectively.respectively. Migration ofof thethe drugs drugs to to the the organic organic phase phase occurred occurred gradually, gradually, as the as releasethe release of both of drugsboth drugs was observed was observed from thefrom first the interval first interv at 15al min. at 15 The min. highest The highest drug release drug release percentage per- wascentage observed was observed at 60 min at 60 for min CYT for and CYT at and 75 minat 75 for min MPH, for MPH, suggesting suggesting that thosethat those are theare optimum times for irradiation. After the optimum irradiation time, the concentration of the optimum times for irradiation. After the optimum irradiation time, the concentration released drug increased only slightly. of released drug increased only slightly. Drug release profiles for both MPH and CYT take place in two steps. The first step Drug release profiles for both MPH and CYT take place in two steps. The first step could be related to the guest molecules that are entrapped at the surface of the NSs, making could be related to the guest molecules that are entrapped at the surface of the NSs, them the first to be released. The second step corresponds to the guest molecules that are making them the first to be released. The second step corresponds to the guest molecules included within the NS matrix and, thus, might have higher affinity to the NSs than the that are included within the NS matrix and, thus, might have higher affinity to the NSs surface molecules [21,28]. than the surface molecules [21,28].

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FigureFigure 15. 15.Drug Drug releaserelease percentagespercentages for MPH. Statistical analysis from from ANOVA–Tukey ANOVA–Tukey (* pp << 0.05 0.05 Figureversus 15. NS Drug 1:8). release percentages for MPH. Statistical analysis from ANOVA–Tukey (* p < 0.05 versusversus NS 1:8).

Figure 16. Drug release percentages for CYT. Statistical analysis from ANOVA-Tukey (* p < 0.05 FigureFigureversus 16. NS Drug Drug1:8). release release percentages percentages fo forr CYT. CYT. Statistical analysis analysis from from ANOVA-Tukey ANOVA-Tukey (* p < 0.05 versusversus NS 1:8). NSs (1:4) showed higher drug release percentages at all irradiation times in com- NSsNSs (1:4) showedshowed higher higher drug drug release release percentages percentages at all at irradiation all irradiation times times in comparison in comparison to NSs (1:8). The differences in the release patterns show that the migration of the parisonto NSs (1:8).to NSs The (1:8). differences The differences in the releasein the re patternslease patterns show thatshow the that migration the migration of the of guest the guest molecules and the ability of NSs to entrap the drug molecules depend on the guestmolecules molecules and the and ability the ofability NSs toof entrap NSs to the entrap drug moleculesthe drug dependmolecules on thedepend crosslinking on the crosslinking between the cyclodextrin monomers. A higher concentration of cross-linker crosslinkingbetween the between cyclodextrin the cyclodextrin monomers. monome A higherrs. concentration A higher concentration of cross-linker of cross-linker results in results in hindering of the interaction of MPH and CYT with the cavities of the NSs, thus resultshindering in hindering of the interaction of the interaction of MPH and of MPH CYT and with CYT the cavitieswith the of caviti the NSs,es of thusthe NSs, reducing thus reducing their effectiveness as a drug carrier [53,54]. reducingtheir effectiveness their effectiveness as a drug as carrier a drug [53 carrier,54]. [53,54]. The IC-AuNP system was compared with an IC-AuNP system without irradiation The IC-AuNP system was compared with an IC-AuNP system without irradiation and with a system consisting of ICs without nanoparticles in order to determine whether and with a system consisting of ICs without nanoparticles in order to determine whether

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The IC-AuNP system was compared with an IC-AuNP system without irradiation theand anti-tumor with a system drugs consisting migrate ofto ICsthe withoutorganic ph nanoparticlesase of the system in order by to means determine of diffusion. whether Figurethe anti-tumor 17 shows drugsthe drug migrate release to percentages the organic for phase MPH of and the systemCYT after by an means irradiation of diffusion. time ofFigure 90 min, 17 forshows the above-mentioned the drug release percentages ICs. for MPH and CYT after an irradiation time of 90 min, for the above-mentioned ICs.

FigureFigure 17. 17. DrugDrug release release (%) (%) for for MPH MPH and and CYT CYT after after 90 90 min min of of irradiation. irradiation. NAu-Ni NAu-Ni = = No No AuNPs AuNPs and and nono irradiation. irradiation. NAu-I NAu-I = = No No AuNPs AuNPs and and irradiation. irradiation. Au-I Au-I = = AuNPs AuNPs and and irradiation. irradiation. Statistical Statistical data data analysisanalysis from from ANOVA–Tukey ANOVA–Tukey (* (* pp << 0.05 0.05 versus versus Au-NI). Au-NI).

FigureFigure 16 16 shows shows that that the the amount amount of of drug drug released released from from the the ICs ICs without without AuNPs AuNPs was was drasticallydrastically reduced inin comparisoncomparison to to the the irradiated irradiated NSs NSs that werethat were conjugated conjugated to the AuNPs.to the AuNPs.This shows This thatshows the that local the hyperthermialocal hyperthermia produced produced by AuNPs by AuNPs causes causes the releasethe release of theof thedrugs drugs to beto fasterbe faster and moreand more efﬁcient. efficient. No burst No burst effect waseffect observed was observed on the releaseon the proﬁles,release profiles,indicating indicating that the that NSs the could NSs be could a potential be a pote solutionntial solution for dose-related for dose-related side effects. side effects.

3.3. Materials Materials and and Methods Methods 3.1. Materials 3.1. Materials All chemical reactants used in this study are commercially available and were used All chemical reactants used in this study are commercially available and were used as received: β-cyclodextrin (Sigma-Aldrich, Saint Louis, MO, USA), melphalan (Sigma- as received: β-cyclodextrin (Sigma-Aldrich, Saint Louis, MO, USA), melphalan (Sig- Aldrich), cytoxan (Sigma-Aldrich), diphenylcarbonate (Sigma-Aldrich), chloroauric acid ma-Aldrich), cytoxan (Sigma-Aldrich), diphenylcarbonate (Sigma-Aldrich), chloroauric (Sigma-Aldrich), sodium citrate (Sigma-Aldrich) and nano-pure water (Merck, Darmstadt, acid (Sigma-Aldrich), sodium citrate (Sigma-Aldrich) and nano-pure water (Merck, Germany). The glassware used for the synthesis and sorption studies was washed with Darmstadt, Germany). The glassware used for the synthesis and sorption studies was aqua regia (3 HCl:1 HNO3), and then rinsed repeatedly with Milli-Q water. washed with aqua regia (3 HCl:1 HNO3), and then rinsed repeatedly with Milli-Q water. 3.2. Synthesis of AuNPs 3.2. Synthesis of AuNPs AuNPs were synthesized according to the Turkevich method [55,56]. In a 200-milliliter AuNPs were synthesized according to the Turkevich method [55,56]. In a flask equipped with a condenser, 100 mL of a 1 mM HAuCl4 aqueous solution was brought 200-milliliterto boil under flask stirring equipped conditions. with Then,a condenser, 10 mL of 100 a 38.8 mL mMof a solution1 mM HAuCl of sodium4 aqueous citrate solu- was tion was brought to boil under stirring conditions. Then, 10 mL of a 38.8 mM solution of added to the HAuCl4 solution as quickly as possible. The solution was heated for 30 min sodiumand then citrate allowed was to added cool. to Then, the HAuCl the solution4 solution was filteredas quickly using as possible. a 0.45-micrometer The solution cellulose was heatedacetate for membrane 30 min filter,and then thus allowed obtaining to AuNPs cool. Then, of 12 nmthe in solution diameter. was filtered using a

Int. J. Mol. Sci. 2021, 22, 6446 14 of 19

3.3. Synthesis of the NSs The synthesis of the NSs was adopted from a published procedure [10] with slight modifications. The NSs were prepared using 1.5 g of β-CD/0.856 g of diphenylcarbonate (DPC) (molar ratio of 1:4) and 1.5 g of β-CD/1.72 g of DPC (molar ratio of 1:8). Homog- enized anhydrous β-CD and DPC were placed in a conical flask. The reactants were heated to 100 ◦C under magnetic stirring and left to react for 6 h. During the reaction, phenol crystals appeared in the neck of the flask. The reaction mixture was left to cool, and the obtained white powder was broken up roughly with an agate mortar. The solid was repeatedly washed with distilled water to remove unreacted CD and with acetone to remove unreacted DPC and phenol, which was a byproduct of the reaction. Afterwards, the solid was further washed by Soxhlet extraction with ethanol for 48 h. Finally, the solid was left to dry at 100 ◦C in an oven for 48 h and stored at 25 ◦C for further use. Complete characterization of NSs was carried out by FT-IR, SEM, TEM, TGA, 1H-NMR, DLS, XRPD, and BET analysis using previously reported methods [38].

3.4. Preparation of NS-MPH and NS-CYT ICs MPH and CYT were loaded in NSs using previously reported methods [57]. Brieﬂy, 1 g of NS was dispersed in 50 mL of double-distilled water using a magnetic stirrer, and then, 20 mL each of MPH and CYT was added. The mixture was sonicated for 10 min and then kept for 24 h under stirring. The suspensions were centrifuged at 3000 rpm for 15 min to separate uncomplexed drugs from the colloidal supernatant. The obtained supernatant was lyophilized at −81 ◦C and 0.001 mbar. The dried powder was stored in a desiccator for further use.

3.5. Association of AuNPs onto the ICs Brieﬂy, 0.3 g of the ICs was immersed in 600 µL of AuNPs. The suspension was allowed to settle for 20 min and then centrifuged at 20,000 rpm for 30 min. Finally, the AuNPs associated with the ICs were dried under vacuum for further use.

3.6. Proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy 1H-NMR spectra of the NSs, MPH, CYT and the ICs were obtained using a Bruker Advance 400 MHz spectrometer. Stock solutions of the NSs, pesticides and complexes were prepared in deuterated dimethyl sulfoxide (DMSO) with tetramethyl silane (TMS) as an internal reference.

3.7. X-ray Powder Diffraction (XRPD) XRPD patterns of the NSs and the ICs were recorded using a high-power powder Siemens/Bruker D5000 diffractometer equipped with a Cu anode and a Ni target ﬁlter. The diffractometer had a current of 40 kV/40 mA and a scan speed of 0.05◦/s. The samples were analyzed over a 2θ angle range of 2–30◦.

3.8. Thermogravimetric Analysis (TGA) TGA of the NSs, MPH, CYT, and the ICs was performed on a TGA-4000 Pyris 6 thermogravimetric analyzer over a temperature range of 25 to 400 ◦C. Approximately 10 mg of the sample was placed on a pan, which was then subjected to the above-mentioned conditions under nitrogen atmosphere.

3.9. Scanning Electron Microscopy (SEM) The surface morphology of the NSs, MPH, CYT, the ICs and the AuNPs conjugated to the ICs was determined using a LEO VP1400 analytical scanning electron microscope equipped with an EDS. The samples were prepared by application to carbon ﬁlms coated on aluminum stubs. Int. J. Mol. Sci. 2021, 22, 6446 15 of 19

3.10. Ultraviolet and Visible Absorption (UV–Vis) Spectroscopy UV–Vis spectra of the antitumor drugs and the AuNPs conjugated to the ICs were recorded using a Jasco V-760 UV–Vis spectrometer. Measurements were carried out over a range of 300 to 700 nm, using deionized water as a reference and concentrations of 0.01 mol/L for each drug.

3.11. Transmission Electron Microscopy (TEM) TEM analyses were performed using a Zeiss model EM-109 microscope, operating at 50 kV. All the samples were prepared by depositing 20 µL of the solution on a copper grid with a ﬁlm of Formvar.

3.12. Determination of Drug Content on NSs The encapsulation efﬁciency and drug loading capacity were calculated using the following equations [20]:

weight of drug in NSs Drug loading = × 100% (1) weight of NSs

[Drug] in NSs Encapsulation efﬁciency = × 100% (2) [Drug] initially

3.13. DLS and Z-Potential Hydrodynamic radius and Z-Potential were calculated using a DLS Zetasizer NanoS series, Malvern. All samples were diluted with double-distilled water before each mea- surement. Z-Potential measurements were made at a constant temperature of 25 ◦C using disposable zeta cells. A total of 12 measurements were carried out, and their average is reported in the results.

3.14. Laser Irradiation Assays Irradiation assays were carried out in accordance with previous studies [36,58,59]. A continuous laser at 532 nm with a beam diameter of 1 mm and 80 mW of light power was used. Due to the low aqueous solubility of both drugs, a system consisting of two phases was formed, using quartz cuvettes with 0.3 mL of a chloroform solution and 0.3 mL of water. Then, the ICs conjugated to the AuNPs were added. The AuNPs on the surface of the ICs were exposed to laser irradiation at different times (intervals of 15 min until a maximum time of 90 min was reached). The absorbance of both MPH and CYT was measured on the organic phase using UV–Vis. ICs without AuNPs were used for laser irradiation analyses in order to determine whether AuNPs are responsible for the drugs’ release. The maximum absorbances for the guest molecules were converted to drug release percentages and then compared with the total amount of drug that was aggregated in the system. All independent experiments were performed in triplicate. Percentages of released drug on the organic phase were calculated using the following equation:

[Drug] after irradiation Drug Release (%) = × 100% (3) [Drug] added initially

3.15. Temperature Control of the Samples after Irradiation The ICs conjugated to the AuNPs (20 mg) were added to 0.5 mL of a chloroform solution. Afterwards, the mixture was irradiated with a continuous laser beam at 532 nm. The temperature of the system after irradiation was measured at different times (intervals of 15 min until a maximum time of 120 min was reached) using a digital thermometer. The highest temperature reached was 31 ◦C at 120 min, whereas the minimum temperature was 26 ◦C at 0 min (no irradiation). This conﬁrmed that the temperature of the solution does not signiﬁcantly increase after irradiation, which is in agreement with previous studies [59]. Int. J. Mol. Sci. 2021, 22, 6446 16 of 19

3.16. Data Analysis The experiments were performed in triplicate, and the results are reported as mean ± SD. Statistical measurements were performed using GraphPad Prism 5 Software Inc. (San Diego, CA, USA). A one-way ANOVA with Tukey’s test was used, and results were considered to be significant if p < 0.05.

4. Conclusions In this work, we successfully promoted the release of MPH and CYT from the cavities of NSs via plasmonic heating of the AuNPs that were conjugated to the ICs. The β-CD- based NSs were able to efficiently form inclusion complexes with MPH and CYT, as proven using SEM, TGA, 1H-NMR, XRPD and UV–Vis characterization. The SEM, EDS, TEM, DLS, Z-Potential, and UV–Vis analyses showed that the ICs were a great substrate for stabilizing the AuNPs, thus giving the polymer additional and useful properties, as decorated NSs clearly outperform native NSs in terms of drug release efficiency. It is important to note that the guest molecule release not only depends on the conjugation of the ICs to the AuNPs, but also on the degree of crosslinking of the polymer, as a higher concentration of cross-linker could result in reduced availability of the inclusion sites. NS materials conjugated to nanoparticles could eventually be considered an improved technology for drug delivery, as they are cheap, efficient, and non-toxic materials.

Author Contributions: Conceptualization, S.S., M.J.K. and P.J.; methodology, S.S., M.J.K. and P.J.; validation, S.S., M.J.K. and P.J; investigation, S.S., N.Y., M.J.K. and P.J.; resources, P.J., M.J.K. and N.Y.; data curation, S.S.; writing—original draft preparation, S.S., N.Y., M.J.K. and P.J.; writing—review and editing, S.S., M.J.K. and P.J.; visualization, S.S., M.J.K. and P.J.; supervision, S.S., M.J.K., N.Y. and P.J.; project administration, M.J.K. and P.J. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: The authors would like to acknowledge the FONDECYT project 1171611, FON- DAP 15130011, 1211482, PhD scholarship to S.S.; PEEI, Facultad de Ciencias, Universidad de Chile. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

DMSO Dimethyl sulfoxide TMS Tetramethylsilane NSs Nanosponges DPC Diphenylcarbonate β-CD Beta cyclodextrin IC Inclusion compound MPH Melphalan CYT Cytoxan AuNPs Gold nanoparticles 1H-NMR Proton nuclear magnetic resonance TGA Thermogravimetric analysis DLS Dynamic light scattering XRPD X-ray powder diffraction SEM Scanning electron microscopy TEM Transmission electron microscopy EDS Energy dispersive spectroscopy SAED Selected area electron diffraction PDI Polydispersity index Int. J. Mol. Sci. 2021, 22, 6446 17 of 19

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