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Effects of Particle Hydrophobicity, Surface Charge, Media pH Value and Complexation with Human Serum Albumin on Drug Release Behavior of Mitoxantrone-Loaded Pullulan Nanoparticles Xiaojun Tao 1 , Shu Jin 2 , Dehong Wu 3 , Kai Ling 1 , Liming Yuan 1 , Pingfa Lin 4 , Yongchao Xie 1 and Xiaoping Yang 1, * Received: 3 November 2015; Accepted: 17 December 2015; Published: 25 December 2015 Academic Editor: Andrea Danani 1

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Department of Pharmacy, School of Medicine, Hunan Normal University, Changsha 410013, China; [email protected] (X.T.); [email protected] (K.L.); [email protected] (L.Y.); [email protected] (Y.X.) Department of Gastroenterology, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China; [email protected] Department of Radiology, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China; [email protected] Fujian Vocational College of Bioengineering, Fuzhou 350300, China; [email protected] Correspondence: [email protected]; Tel.: +86-158-7406-6132; Fax: +86-731-8891-2417

Abstract: We prepared two types of cholesterol hydrophobically modified pullulan nanoparticles (CHP) and carboxyethyl hydrophobically modified pullulan nanoparticles (CHCP) substituted with various degrees of cholesterol, including 3.11, 6.03, 6.91 and 3.46 per polymer, and named CHP´3.11 , CHP´6.03 , CHP´6.91 and CHCP´3.46 . Dynamic laser light scattering (DLS) showed that the pullulan nanoparticles were 80–120 nm depending on the degree of cholesterol substitution. The mean size of CHCP nanoparticles was about 160 nm, with zeta potential ´19.9 mV, larger than CHP because of the carboxyethyl group. A greater degree of cholesterol substitution conferred greater nanoparticle hydrophobicity. Drug-loading efficiency depended on nanoparticle hydrophobicity, that is, nanoparticles with the greatest degree of cholesterol substitution (6.91) showed the most drug encapsulation efficiency (90.2%). The amount of drug loading increased and that of drug release decreased with enhanced nanoparticle hydrophobicity. Nanoparticle surface-negative charge disturbed the amount of drug loading and drug release, for an opposite effect relative to nanoparticle hydrophobicity. The drug release in pullulan nanoparticles was higher pH 4.0 than pH 6.8 media. However, the changed drug release amount was not larger for negative-surface nanoparticles than CHP nanoparticles in the acid release media. Drug release of pullulan nanoparticles was further slowed with human serum albumin complexation and was little affected by nanoparticle hydrophobicity and surface negative charge. Keywords: pullulan nanoparticles; degree of substitution; HSA; surface charge

1. Introduction Many drugs with highly efficient action are dysfunctional as treatment because of the fast drug metabolism and a peak-valley reaction. With fast drug metabolism, the drug reaches peak concentrations quickly, which usually leads to severe drug toxicity, and the peak-valley reaction is inefficient or invalid. To avoid these problems, a slow-release drug strategy has been developed to

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preserve the drug-efficient concentration in the blood by a sustained release effect and has become a Nanomaterials 2016,field 6, 2 [1,2]. major research Nanoparticles have novel nano-size structure with the capacity of capturing small molecule novel preparations nano-size structure with the[3–5]. capacity of capturing small molecule drugsNanoparticles to form novel have nano-drug or formulations Nano-drug formulations improve drugs to form novel nano-drug preparations or formulations [3–5]. Nano-drug formulations improve slow-drug release capacity, which is vital for enhancing the drug curative effect and reducing toxic slow-drug release capacity, which is vital for enhancing the drug curative effect and reducing toxic side effects [6–9]. side effects [6–9]. Polymeric amphiphiles such as polysaccharides modified with hydrophobic groups can Polymeric form amphiphiles such as structure polysaccharides modified can spontaneously a nano-spherical in aqueous media with with ahydrophobic hydrophobicgroups micro-core spontaneously formshell a nano-spherical structure in aqueous mediacore withcan a hydrophobic micro-coredrug and and a hydrophilic [10–12]. A nanoparticle hydrophobic load a hydrophobic a hydrophilic shell [10–12]. A nanoparticle hydrophobic core can load a hydrophobic drug via via a hydrophobic interaction [13,14]. The hydrophobic group of nano-materials affects the amounta hydrophobic interaction [13,14]. hydrophobic affects of drug loading and the drug The release efficiencygroup [15]. ofItnano-materials plays a major role the in amount the size ofofdrug the loading and the drug release efficiency [15]. It plays a major role in the size of thehas formed formed nanoparticles, related to the final drug function [16]. The carboxyl group been nanoparticles, related to amphiphiles the final drug [16]. The carboxyl group has been conjugated to conjugated to polymeric to function obtain self-aggregated nanoparticles with negative surface polymeric to obtain negative surface charge aqueous charge in amphiphiles aqueous solution to self-aggregated confer uniquenanoparticles stability andwith liver targeting ability as in compared solution to confer unique stability and liver targeting ability as compared with counterparts [17,18]. with counterparts [17,18]. Adding the carboxyl group can affect the self-assembly process of Adding the carboxyl group can affect the self-assembly process of formed nanoparticles by affecting formed nanoparticles by affecting the sizes formed and the drug-loading amount [17]. Therefore, the sizes formed and the adrug-loading [17]. Therefore, nano-drug release with a nanoparticle nano-drug release with nanoparticleamount negative-surface charge should be studied for in vivo drug negative-surface charge should be studied for in vivo drug efficiency function. efficiency function. Besides the Besides the nanoparticle nanoparticle hydrophobicity hydrophobicity and and surface surfacecharge, charge,the thepH pHvalue valueininthe themedium mediumhas hasa critical role in nano-drug release [18,19]. The drug release amount is greater with pH < 7 than ≥ 7 [20]. a critical role in nano-drug release [18,19]. The drug release amount is greater with pH < 7 than ě 7 [20]. This finding finding is is vital vital for for cancer cancer treatment treatment because because nano-drug nano-drug release release is is better better with with an an acidic acidic This environment [21,22]. [21,22]. environment Human serum albumin (HSA) (HSA)isisaamajor majorsoluble solubleglobular globularprotein proteinininthe the circulatory system and Human serum albumin circulatory system and is is often coated on the surfaces of many nanoparticles with nano-drug in vivo treatment [23,24]. often coated on the surfaces of many nanoparticles with nano-drug in vivo treatment [23,24]. It affects It affects nano-drug also tissue, the targeted leading to altered nano-drug nano-drug release andrelease also theand targeted therebytissue, leadingthereby to altered nano-drug efficiency [25,26]. efficiency [25,26]. Therefore, nano-carriers with precise properties should be designed according to Therefore, nano-carriers with precise properties should be designed according to certain biologic certain biologicfor environments for the desired slow-drug environments the desired slow-drug release function.release function. Pullulan is a linear polysaccharide with maltotriose repeating unites unites jointed jointed by by α-1, α-1, 6-linkages Pullulan is a linear polysaccharide with maltotriose repeating 6-linkages (Figure 1), that is safe, non-toxic, hydrophilic, and biodegradable [27,28]. Its many hydroxyl (Figure 1), that is safe, non-toxic, hydrophilic, and biodegradable [27,28]. Its many hydroxyl groups groups can be modified with hydrophobic groups such as a long-chain alkyl group and cholesterol to form can be modified with hydrophobic groups such as a long-chain alkyl group and cholesterol to form monodispersed self-aggregated pullulan nanoparticles [29–31]. Pullulan nanoparticles include a monodispersed self-aggregated pullulan nanoparticles [29–31]. Pullulan nanoparticles include a hydrophobic core with cholesterol moiety and a hydrophilic surface with pullulan chain (Figure 2). hydrophobic core with cholesterol moiety and a hydrophilic surface with pullulan chain (Figure 2). Pullulan nanoparticles can be used as a small-molecule carriers, or macromolecule carriers such as Pullulan nanoparticles can be used as a small-molecule carriers, or macromolecule carriers such as protein and gene carriers, widely studied for medical applications [32–34]. However, besides their protein and gene carriers, widely studied for medical applications [32–34]. However, besides their superior targeting function and drug efficiency, pullulan nanoparticles have not been studied for superior targeting function and drug efficiency, pullulan nanoparticles have not been studied for their effect on nano-drug slow release. their effect on nano-drug slow release. Therefore, we investigated the effect of nanoparticle properties, release-media pH value, and Therefore, we investigated the effect of nanoparticle properties, release-media pH value, HSA complexation on drug-release behaviors of these nano-drug carriers in a biological application. and HSA complexation on drug-release behaviors of these nano-drug carriers in a biological Especially, the drug release of pullulan nanoparticles with HSA complexation is important to explore application. Especially, the drug release of pullulan nanoparticles with HSA complexation is because these data will be helpful for further in vivo application. important to explore because these data will be helpful for further in vivo application.

Figure Chemical structure structure of Figure 1. 1. Chemical of pullulan. pullulan.

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Figure 2. 2. Structural pullulan nanoparticle. nanoparticle. Figure Structural graph graph of of pullulan

We grafted cholesterol to hydroxyl groups of pullulan by esterification and changed the degree We grafted cholesterol to hydroxyl groups of pullulan by esterification and changed the degree of cholesterol substitution to obtain different types of hydrophobically modified pullulan (CHP). of cholesterol substitution to obtain different types of hydrophobically modified pullulan (CHP). Figure 2.nanoparticle, Structural graph of pullulan nanoparticle. To design a negative-surface charge carboxyethyl pullulan hydrophobically-modified To design a negative-surface charge nanoparticle, carboxyethyl pullulan hydrophobically-modified with cholesterol (CHCP) was synthesized via an esterification reaction. CHP and CHCP conjugates We grafted cholesterol to hydroxyl groups pullulan by esterification changed the degree with cholesterol (CHCP) was synthesized via anofesterification reaction. and CHP and CHCP conjugates can self-aggregate with different differenttypes hydrophobicity and modified negativepullulan surface(CHP). charge by a of cholesterol nanoparticles substitution to obtain of hydrophobically can self-aggregate nanoparticles with different hydrophobicity and negative surface charge by dialysisTomethod. The preparedcharge pullulan nanoparticles were characterized to analyze particle design a negative-surface nanoparticle, carboxyethyl pullulan hydrophobically-modified a dialysis method. The prepared pullulan nanoparticles were characterized to analyze particle with cholesterol was synthesized esterification reaction. CHP and CHCP conjugates group composition including(CHCP) surface charge, size via andanshape, and illustrate the effect of cholesterol composition including surface charge, size and shape, and illustrate the effect of charge cholesterol group can self-aggregate nanoparticles with different hydrophobicity and negative surface by a and carboxyethyl groups on drug release. We measured drug encapsulation, loading capacity and and carboxyethyl groups on drug release. We measured drug encapsulation, loading capacity method. The prepared pullulan nanoparticles were characterized to analyze particle sizes ofdialysis pullulan nanoparticles with different properties and observed the morphology via and sizes of pullulan nanoparticles with properties and the morphology via composition including surface charge, sizedifferent and shape, and illustrate theobserved effect of cholesterol group transmission electron microscopy (TEM). The effect of nanoparticle hydrophobicity and surface and carboxyethyl groups on drug release.The We measured encapsulation, loading capacityand and surface transmission electron microscopy (TEM). effect ofdrug nanoparticle hydrophobicity charge on the drug loading amount and drug release in phosphate buffered saline (PBS) was studied sizes pullulan nanoparticles withdrug different properties and observed morphology viastudied charge on theofdrug loading amount and release in phosphate bufferedthe saline (PBS) was to clarify the application asmicroscopy a drug carrier. We collected release rate of pullulan transmission electron (TEM). The effect of cumulative nanoparticle drug hydrophobicity and(%) surface to clarify the application as a drug carrier. We collected cumulative drug release rate (%) of pullulan charge on the different drug loading amount and drug release in phosphate buffered saline (PBS) release was studied nanoparticles with properties and charge surface in weak acid and strong media and nanoparticles with different as properties andWe charge surface in weak acid and strong release media to clarify the application a drug carrier. collected cumulative drug release rate (%) of pullulan studied alterations in drug release amount with acid intensity by nanoparticle properties. We also and studied alterations in drug releaseand amount with in acid intensity by nanoparticle properties. nanoparticles with different properties charge surface weak acid and strong release media and studied HSA complexation with CHP nanoparticles. This complexation can be divided into two We alsostudied studied HSA complexation CHP complexation alterations in drug release with amount withnanoparticles. acid intensity by This nanoparticle properties.can We be alsodivided processes: HSAHSA fast-surface coating and slow inter-insertion (Figure 3)can [35]. HSAinto complexation studied complexation with CHP nanoparticles. This complexation be For divided into two processes: HSA fast-surface coating and slow inter-insertion (Figure 3) [35]. two For HSA with nano-carrier processes, drug release from the loaded pullulan nanoparticles was further processes: HSA fast-surface coating and slow inter-insertion (Figure 3) [35]. For HSA complexation complexation with nano-carrier processes, drug release from the loaded pullulan nanoparticles was sustained [36]. We studied the nano-drug release pullulan nanoparticles with different properties with nano-carrier processes, drug release fromofthe loaded pullulan nanoparticles was further further sustained [36]. We studied the nano-drug release of pullulan nanoparticles with different sustained [36]. We studied the nano-drug release of pullulan nanoparticles with different properties on HSA complexation and discuss whether HSA complexation is vital for nano-drug release. Finally, properties on complexation HSA complexation and discuss HSA complexation is release. vital for nano-drug on HSA and discuss whether HSAwhether complexation is vital for nano-drug Finally, we demonstrate a drug delivery paradigm with CHP and mitoxantrone as a model drug. Our nanorelease.we Finally, we demonstrate a drug delivery with CHPasand mitoxantrone as a model demonstrate a drug delivery paradigm with paradigm CHP and mitoxantrone a model drug. Our nanodrug slow preparation can be used for further in vivo efficacy study. drugnano-drug slow preparation be used for further vivo efficacy study.in vivo efficacy study. drug. Our slowcan preparation can beinused for further

3. Sketch Sketch illustration of human serum albumin complexation with pullulan Figure Figure 3. illustration of human serum (HSA) albumin (HSA) complexation with nanoparticles. pullulan nanoparticles. Figure 3. Sketch illustration of human serum albumin (HSA) complexation with pullulan 2. Results and Discussion nanoparticles. 2. Results and Discussion

2.1. Properties of Pullulan Nanoparticles

2. Results andofDiscussion 2.1. Properties Pullulan Nanoparticles

Pullulan self-aggregated nanoparticles with three kinds of hydrophobic properties were

preparedself-aggregated and collected at thenanoparticles volume (10 mL)with of the concentration 10−6 mol/L. Figure 4 shows were Pullulan three kinds 8.28 of ×hydrophobic properties 2.1. Properties of Pullulan Nanoparticles prepared and collected at the volume (10 mL) of the concentration 8.28 ˆ 10´6 mol/L. Figure 4 shows 3/14 Pullulan self-aggregated nanoparticles with three kinds of hydrophobic properties were prepared and collected at the volume (10 mL) of the concentration 8.28 × 10−6 mol/L. Figure 4 shows

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the optical appearance of pullulan nanoparticles and Figure 5 shows that pullulan nanoparticles were regularly in shape. The milky light of CHP particles with a high cholesterol substitution the opticalspherical appearance of pullulan nanoparticles and−6.91 Figure 5 shows that pullulan nanoparticles were Nanomaterials 2016, 6, 2 was stronger than that of CHP −6.03milky , CHPlight −3.11, and CHCP nanoparticles by the order of light reduction. regularly spherical in shape. The of CHP particles with a high cholesterol substitution ´6.91 The appearance the nanoparticles depended degree of by on the cholesterol was optical stronger than that of CHPpullulan , CHP , and CHCP nanoparticles orderwere ofgroup light the optical appearance of pullulan nanoparticles Figure 5 shows that pullulan nanoparticles ´6.03 ´3.11and substitution: the more substitution tolight more We measured size to explore reduction. The optical of the pullulan depended degree ofsubstitution on cholesterol regularly spherical inappearance shape. The lead milky of milky-like. CHPnanoparticles −6.91 particles with a highnanoparticle cholesterol was stronger than that of CHPsubstitution −6.03 , CHP−3.11 , and nanoparticles by the order of light agroup relationship with the cholesterol group degree oftosubstitution. Figure 6We showed that reduction. peak size was substitution: the more leadCHCP more milky-like. measured nanoparticle The optical appearance of the pullulan nanoparticles depended degree of on cholesterol group 92.62, and a129.1 nm for CHP , CHP −6.03 and group CHP−6.91degree , respectively. However,Figure the average size size to85.20 explore relationship with−3.11 the cholesterol of substitution. 6 showed substitution: the 92.62, more substitution lead to more milky-like. We nanoparticle size to explore was 83.11, 101.9 nm and nm, andfor decreased with the ´ increasing of degree of cholesterol that successively peak size was 85.20 and110 129.1 nm CHP´3.11 , measured CHP and CHP , respectively. 6.03 ´6.91 a substitution. relationship with the cholesterol group degree of substitution. Figure 6 showed that peak was group CHP nanoparticles carried a83.11, fractional (about easesize in adhering However, the average size was successively 101.9charge nm and 110−2–0 nm, mV) and for decreased with the 92.62, 85.20 and 129.1 nm for CHP−3.11, CHP−6.03 and CHP−6.91, respectively. However, the average size to interparticles, which thegroup average particle size. Figure 7 compares zeta potential of CHP −3.11 increasing of degree of enlarged cholesterol substitution. CHP nanoparticles carried a fractional charge was successively 83.11, 101.9 nm and 110 nm, and decreased with the increasing of degree of cholesterol and CHCP nanoparticles. The degree oftocholesterol group substitution was similar, but the surface (about ´2–0 mV) for ease in adhering interparticles, which enlarged the average particle size. group substitution. CHP nanoparticles carried a fractional charge (about −2–0 mV) for ease in adhering charge greater (−19.9 mV). The of mean size was larger for CHCP than CHP −3.11 nanoparticles (160 vs. Figure 7 compares zeta potential and CHCP nanoparticles. The degree ofofCHP cholesterol ´3.11 towas interparticles, which enlarged theCHP average particle size. Figure 7 compares zeta potential −3.11 110 nm). In fact, nanoparticles. the was difference would be larger if we considered the effect of adhesion interparticles group substitution similar, but the surface charge wassubstitution greater (´19.9 mV). The size was and CHCP The degree of cholesterol group was similar, butinmean the surface on size. Therefore, the cholesterol group was vital for the formed nanoparticle size, which larger for CHCP than CHP nanoparticles (160 vs. 110 nm). In fact, the difference would be charge was greater (−19.9 mV). ´3.11The mean size was larger for CHCP than CHP−3.11 nanoparticles (160 vs. was In fact, the difference would be larger ifin weinterparticles considered the on effect of adhesion in interparticles affected by surface charge. Hydrophobic interaction among hydrophobic-dependant larger110 if nm). wenanoparticle considered the effect of adhesion size. Therefore, the cholesterol size.vital Therefore, the cholesterol grouphydrophobic was forwas the formed nanoparticle size, which was groups results infor thethe formation of compact cores that can a hydrophobic drug into grouponwas formed nanoparticle size,vital which affected byload nanoparticle surface charge. affected by nanoparticle surface charge. Hydrophobic interaction among hydrophobic-dependant aHydrophobic nanoparticle core [29,37,38]. We found that pullulan conjugates with different hydrophobic and interaction among hydrophobic-dependant groups results in the formation of compact groups results in the formation of compact hydrophobic cores that can load a hydrophobic drug into carboxyethyl affected nanoparticle size because nanoparticle size core affected nano-drug tissue hydrophobic groups cores that can load a hydrophobic drugthe into a nanoparticle [29,37,38]. We found a nanoparticle core [29,37,38]. We found that pullulan conjugates with different hydrophobic and distribution final drug efficiency. and chargeaffected amountnanoparticle depending that pullulanand conjugates with differentNanoparticle hydrophobichydrophobicity and carboxyethyl groups carboxyethyl groups affected nanoparticle size because the nanoparticle size affected nano-drug tissue on and carboxyl size groups, also nano-drug affected the protein coating, and which is related to drug sizehydrophobic because theand nanoparticle affected tissue distribution final drug efficiency. distribution final drug efficiency. Nanoparticle hydrophobicity and charge amount depending release Nanoparticle hydrophobicity andgroups, chargealso amount depending oncoating, hydrophobic carboxyl groups, on[39–41]. hydrophobic and carboxyl affected the protein which and is related to drug also affected the protein coating, which is related to drug release [39–41]. release [39–41].

Figure 4. The optical appearance pullulannanoparticles nanoparticles with hydrophobicity and and surface Figure 4.4.The optical appearance ofof pullulan withdifferent different hydrophobicity surface Figure The optical appearance of pullulan nanoparticles with different hydrophobicity and charge. charge. surface charge.

Figure 5. Transmission electron microscopy (TEM) of CHP−6.91 (a), CHP−6.03 (b), CHP−3.11 (c) and CHCP (d) nanoparticles.

Figure 5. 5. Transmission Transmissionelectron electronmicroscopy microscopy(TEM) (TEM)ofofCHP CHP CHP (b); −3.11 CHP and ´6.91 Figure −6.91 (a),(a); CHP −6.03 ´6.03 (b), CHP (c)´3.11 and (c) CHCP CHCP (d) nanoparticles. (d) nanoparticles. 4/14

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Figure 6. Size distribution of of CHP (b);CHP CHP−3.11 and CHCP nanoparticles. Figure 6. Size distribution CHP −6.91(a); (a),CHP CHP´6.03 −6.03 (b), (c)(c) and CHCP (d)(d) nanoparticles. ´6.91 ´3.11 Figure 6. Size distribution of CHP−6.91 (a), CHP−6.03 (b), CHP−3.11 (c) and CHCP (d) nanoparticles.

Figure 7. Zeta potential of CHP −6.91(a); (a),CHP CHP´6.03 −6.03 (b), (c)(c) and CHCP (d)(d) nanoparticles. Figure 7. Zeta potential of CHP (b);CHP CHP−3.11 and CHCP nanoparticles. ´6.91 ´3.11 Figure 7. Zeta potential of CHP −6.91 (a), CHP−6.03 (b), CHP−3.11 (c) and CHCP (d) nanoparticles.

2.2. Drug-Loading Drug-Loading of of Pullulan Pullulan Nanoparticles Nanoparticles 2.2. 2.2. Drug-Loading of Pullulan Nanoparticles CHP can can self-aggregate self-aggregate to to form form nanoparticles nanoparticles in in aqueous aqueous media media with with a hydrophobic hydrophobic micro-core micro-core CHP CHP can self-aggregate to form nanoparticles in aqueous media with aa hydrophobic micro-core because of the hydrophobic interactions among modified cholesterol groups, and hydrophilic sugar because of of the the hydrophobic hydrophobic interactions interactions among among modified modified cholesterol cholesterol groups, groups, and and hydrophilic hydrophilic sugar sugar because chain as as an an external external shell shell [35,37]. [35,37]. Mitoxantrone Mitoxantrone as as an an anticancer anticancer model model drug drug with with benzene benzene and and cyclic cyclic chain chain as an external shell [35,37]. Mitoxantrone as an anticancer model drug with benzene and cyclic structure show a strong hydrophobic property. It can be adsorbed to cholesterol groups to form structure show It can can be be adsorbed adsorbed to to cholesterol cholesterol groups groups to to form form structure show aa strong strong hydrophobic hydrophobic property. property. It nanoparticle hydrophobic core in the nanoparticle self-aggregating process [35,37]. Mitoxantrone with nanoparticle hydrophobic hydrophobiccore coreininthe the nanoparticle self-aggregating process [35,37]. Mitoxantrone nanoparticle nanoparticle self-aggregating process [35,37]. Mitoxantrone with manymany hydrophilic groups, such such as OHasand NH group, can be also adsorbed to the external sugar with hydrophilic groups, OHNH andgroup, NH group, also adsorbed the external many hydrophilic groups, such as OH and can becan alsobeadsorbed to the to external sugar chain of pullulan nanoparticles by intermolecular force. Hydrophobic adsorption has a stronger force sugar of chain of pullulan nanoparticles by intermolecular Hydrophobic adsorption has a stronger chain pullulan nanoparticles by intermolecular force. force. Hydrophobic adsorption has a stronger force and plays a main role in drug loading, and the intermolecular force is weak, for a role in force plays and plays a main in drug loading, intermolecular forceis isweak, weak,for for aa role role in in and a main role role in drug loading, andand thethe intermolecular force supplementary drug drug loading. We loaded mitoxantrone into pullulan pullulan nanoparticles nanoparticles by by the the dialysis dialysis supplementary We loaded loaded mitoxantrone mitoxantrone into into supplementary drug loading. loading. We pullulan nanoparticles by the dialysis method. 4 mg mitoxantrone, 40 mg pullulan conjugates, and 3 mL triethylamine were dissolved in method. 44 mg mg mitoxantrone, mitoxantrone, 40 40 mg mg pullulan pullulan conjugates, conjugates, and and 33 mL mL triethylamine triethylamine were were dissolved dissolved in in method. 20 mL mL dimethyl sulfoxide sulfoxide with triethylamine, triethylamine, and the the mixture was was put into into a dialysisbag. bag. In In the the 20 20 mL dimethyl dimethyl sulfoxide with with triethylamine, and and the mixture mixture was put put into aa dialysis dialysis bag. In the dialysis process, cholesterol groups can self-assemble to form a nanoparticle core via a hydrophobic dialysis process, process, cholesterol cholesterol groups groups can self-assemble to to form form aa nanoparticle nanoparticle core core via via aa hydrophobic hydrophobic dialysis can self-assemble force with with the the mitoxantrone mitoxantrone adsorption. adsorption. It was assumed that the more cholesterol groups groups of of the the force It was assumed that the more cholesterol force with the mitoxantrone adsorption. It was assumed that the more cholesterol groups of the prepared conjugates, conjugates, the greater greater the drug drug loading for for the formed formed pullulan nanoparticles. nanoparticles. Table prepared Table 111 prepared conjugates, the the greater the the drug loading loading for the the formed pullulan pullulan nanoparticles. Table proved our assumption, showing encapsulation efficiency from 52.4% to 90.2% and loading capacity proved our our assumption, assumption, showing showing encapsulation encapsulation efficiency efficiency from to 90.2% 90.2% and and loading loading capacity capacity proved from 52.4% 52.4% to from 4.45% to 10.1% with cholesterol substitution from 3.11 to 6.91. However, CHCP conjugates with from 4.45% to 10.1% with cholesterol substitution from 3.11 to 6.91. However, CHCP conjugates from 4.45% to 10.1% with cholesterol substitution from 3.11 to 6.91. However, CHCP conjugates with cholesterol substitution 3.46, greater than for CHP−3.11, can form negative-charge nanoparticles with cholesterol substitution 3.46, greater than for CHP−3.11, can form negative-charge nanoparticles with 5/14 5/14


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withNanomaterials cholesterol substitution 3.46, greater than for CHP´3.11 , can form negative-charge nanoparticles 2016, 6, 2 with encapsulation efficiency and loading capacity of 50.1% and 4.25% as compared with 52.4% and encapsulation efficiency and loading capacity of the 50.1% and 4.25% asforce compared with 52.4% and 4.45% 4.45% for CHP´3.11 nanoprticles. Thus, besides hydrophobic contribution to drug loading, for CHP −3.11 nanoprticles. Thus, besides the hydrophobic force contribution to drug loading, the the carboxyethyl group can improve the hydrophobic drug-loading behavior. The mean size of carboxyethyl group can improve the hydrophobic drug-loading behavior. The mean size of mitoxantrone-loaded pullulan nanoparticles was larger than their nano-carrier, from 128.5 to 162.3 nm mitoxantrone-loaded pullulan nanoparticles was larger than their nano-carrier, from 128.5 to 162.3 nm for CHP´3.11 to CHP´6.91 , respectively. The drug-loaded size of pulllan nanoparticles depended on for CHP−3.11 to CHP−6.91, respectively. The drug-loaded size of pulllan nanoparticles depended on degree of degree of cholesterol substitution. CHCP nanoparticles had the largest mean size, 213.5 nm. TEM cholesterol substitution. CHCP nanoparticles had the largest mean size, 213.5 nm. TEM graph (Figure 8) graph (Figure 8)that, demonstrated that, mitoxantrone-loaded nanoparticles had a regularly demonstrated mitoxantrone-loaded pullulan nanoparticlespullulan had a regularly spherical shape. spherical shape. Table 1. Characteristics of self-aggregated nanoparticles loading mitoxantrone.

Table 1. Characteristics of self-aggregated nanoparticles loading mitoxantrone. Sample C/D (w/w) a EE (%) b LC (%) c Dh (nm) d CHP−3.11 C/D1/10 52.4 ± 2.03b 4.45LC ± 0.26 162.3 ± 4.6 d Sample (w/w) a (%) c EE (%) Dh (nm) CHCP 1/10 50.1 ± 1.92 4.25 ± 0.23 213.5 ± 6.2 CHP 1/10 52.4 ˘ 2.03 8.82 4.45 ˘ 0.26 142.6 162.3 ˘ 4.6 ´3.11 CHP −6.03 1/10 85.1 ± 1.92 ± 0.23 ± 4.2 CHCP 1/10 50.1 ˘ 1.92 4.25 ˘ 0.23 213.5 ˘ 6.2 CHP−6.91 1/10 90.2 ± 2.43 10.1 ± 0.32 128.5 ± 3.6 CHP´6.03 1/10 85.1 ˘ 1.92 8.82 ˘ 0.23 142.6 ˘ 4.2 a b Data are mean standard deviation CHP±´6.91 1/10 (SD). mitoxantrone/CHP 90.2 ˘ 2.43 10.1or˘CHCP 0.32 (mg/mg); 128.5 ˘ encapsulation 3.6

capacity efficiency determined by ultraviolet (UV) spectrophotometry at 608 nm; c loading Data are mean ˘ standard deviation (SD). a mitoxantrone/CHP or CHCP (mg/mg); b encapsulation d Size and size distribution determined by the determined by UV spectrophotometry at 608 nm; c efficiency determined by ultraviolet (UV) spectrophotometry at 608 nm; loading capacity determined by UV dynamic laser light-scattering. spectrophotometry at 608 nm; d Size and size distribution determined by the dynamic laser light-scattering.

Figure 8. TEM of CHP´6.03 nanoparticles loading mitoxantrone. Figure 8. TEM of CHP−6.03 nanoparticles loading mitoxantrone.

2.3. 2.3. Effect of Nanoparticle Properties Effect of Nanoparticle PropertiesononDrug DrugRelease Release Pullulan nanoparticlesload loadmitoxantrone mitoxantrone in in two patterns: adsorption and and Pullulan nanoparticles patterns: drug drugsurface surface adsorption hydrophobic core embedding, which determines whether nano-drug release is fast or slow [36]. hydrophobic core embedding, which determines whether nano-drug release is fast or slow [36]. A weak molecule adsorptioncauses causesfast fast drug drug separation separation and nano-drug release. A weak molecule adsorption andresults resultsininfast fast nano-drug release. However, a strong hydrophobic force force and shell obstruction fromfrom the loading drug indrug the in However, a strong hydrophobic andsugar-chain sugar-chain shell obstruction the loading hydrophobic nanoparticle core induces a slow drug release. These two patterns are shown in the drug the hydrophobic nanoparticle core induces a slow drug release. These two patterns are shown in release graphs in Figure 9. Most free drug was released within 12 h, which indicates a fast-release the drug release graphs in Figure 9. Most free drug was released within 12 h, which indicates a pattern of the nude drug. In contrast, drug release amounts were 42.6%, 46.1%, 60.5% and 64.8% for fast-release pattern of the nude drug. In contrast, drug release amounts were 42.6%, 46.1%, 60.5% CHP−6.91, CHP−6.03, CHP−3.11 and CHCP−3.46 nanoparticles, respectively, at 48 h. Besides drug loading and amount, 64.8% for CHP , CHP´was CHP´3.11with andnanoparticle CHCP´3.46hydrophobicity. nanoparticles, Nanoparticles respectively, with at 48 h. 6.03 ,associated ´6.91amount drug release Besides drug loading amount, drug release amount was associated with nanoparticle hydrophobicity. greater cholesterol substitution (higher hydrophobicity) showed slower drug release. This rule was Nanoparticles with greaternanoparticles cholesterol with substitution hydrophobicity) slower not observed for CHCP degree of (higher substitution 3.46. The total showed drug released wasdrug release. This was not observed for CHCP with degree ofrelease substitution 3.46.slower The total 64.8% for rule CHCP nanoparticles and 60.5% fornanoparticles CHP−3.11 nanoparticles, so the effect was drugfor released wasCHCP 64.8%nanoparticles. for CHCP nanoparticles and 60.5% for CHP´3.11 nanoparticles, so the release CHP than

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Figure 9. The mitoxantrone pullulannanoparticles nanoparticlesin in phosphate buffered saline Figure 9. The mitoxantrone(MTO) (MTO) release release of pullulan phosphate buffered saline (PBS) at˝37 in vitro freemitoxantrone, mitoxantrone, ▼: , ◄:, đ CHCP, ●: CHP , ▲: CHP ). ´6.91 ). (PBS) at 37 C °C in vitro ((■: : free İ : CHP CHP−3.11 : CHCP, ‚ : −6.03 CHP , N−6.91 : CHP ´6.03 ´3.11

Effect of 9. Nanoparticle Property Drug Release with Different pH 2.4. 2.4. Effect of Nanoparticle Property ononDrug Release with Different pH in phosphate buffered saline Figure The mitoxantrone (MTO) release of pullulan nanoparticles (PBS) at 37 °C in vitro hydrophobicity (■: free mitoxantrone, ▼: CHPaffected −3.11, ◄: CHCP, ●: CHP −6.03, ▲: −6.91). Since nanoparticle regularly the drug release asCHP described in above Since nanoparticle hydrophobicity regularly affected the drug release as described in above experiments, we chose CHCP and CHP−3.11 nanoparticles to examine the effect of nanoparticle surface experiments, we chose CHCP and CHP to examine the effect of nanoparticle ´ 3.11 nanoparticles 2.4. Effect Nanoparticle Property Release Different charge onofdrug release in a weakon orDrug strong acid with release media.pH Figure 10 shows the drug release of surface charge on drug release in a weak or strong acid release media. 10 shows the CHCP and CHP −3.11 nanoparticles in the release media with pH 6.8 and 4.0. InFigure the release media withdrug Since nanoparticle hydrophobicity regularly affected the drug release as described in above release of the CHCP and CHP nanoparticles in for theCHP release media with pH 6.8 and 4.0. In the ´3.11and pH 6.8, drug was 63.8%−3.11 and 66.2% and CHCP nanoparticles, respectively, experiments, werelease choseamount CHCP CHP nanoparticles to−3.11 examine the effect of nanoparticle surface release media with pH 6.8, the drug release amount was 63.8% and 66.2% for CHP and CHCP ´3.11 at 48 h. on In the release media the acid drugrelease release media. amountFigure was 60.5% and 64.8%, respectively, so charge drug release in a with weakpH or 7.4, strong 10 shows the drug release of nanoparticles, respectively, at 48 h.in In release. the release media with pH 7.4, amount aCHCP weak-acid release favored drug The drug release was fast and substantially increased and CHP −3.11media nanoparticles the release media with pH 6.8 and 4.0. Inthe the drug releaserelease media with wasfor 60.5% and 64.8%, respectively, soand a and weak-acid release media favored drug release. The drug release media with pH 4.0:was 94.2% 81.9% for −3.11 nanoparticles, respectively, pH 6.8, the drug release amount 63.8% 66.2% forCHP CHP −3.11and andCHCP CHCP nanoparticles, respectively, at 48 h. We inferred that strong acid release media (pH 4.0) may lead to degradation of the selfrelease was fast and substantially increased for release media with pH 4.0: 94.2% and 81.9% at 48 h. In the release media with pH 7.4, the drug release amount was 60.5% and 64.8%, respectively, so for aggregated nanoparticles [18,22,42]. It was because that CHP or CHCP molecules were hydrolyzed CHPa´weak-acid respectively, at 48 h. release We inferred acid release media releasenanoparticles, media favored drug release. The drug was fastthat andstrong substantially increased 3.11 and CHCP ester bond with strong acid94.2% interaction and for release time prolonging, which led the spherical that for release media pH 4.0: 81.9% CHPnanoparticles −3.11 and CHCP[18,22,42]. nanoparticles, respectively, (pHat 4.0) may lead to with degradation of theand self-aggregated It was because of pullulan nanoparticles to become segment degradation. As a result of selfthis ator 48CHCP h. We inferred thatwere strong acid release media (pHthe 4.0) may lead to degradation of and the CHPstructure molecules hydrolyzed atloose esteruntil bond with strong acid interaction release change, the generous release of the nanoparticle core loading drug happened, which was observed aggregated nanoparticles [18,22,42]. It was because that CHP or CHCP molecules were hydrolyzed time prolonging, which led the spherical structure of pullulan nanoparticles to become loose until as markedly increased drug release amount,and showed in Figure 10. The drugwhich release rates CHCP at aester bond with strong interaction release prolonging, led thefor spherical the segment degradation. Asacid a result of this change, thetime generous release of the nanoparticle core nanoparticles were 64.8%, 66.2% andto81.9% in the release with pH 7.4, 6.8 andAs 4.0,a respectively, structure of pullulan nanoparticles become loose untilmedia the segment degradation. result of this loading drug happened, which was observed as a markedly increased drug release amount, showed as compared with CHPrelease −3.11 nanoparticles, with 60.5%, 63.8% and 94.2% release, respectively. media change, the generous of the nanoparticle core loading drug happened, which wasThe observed in Figure 10. affected The drug ratesfor forCHP CHCP nanoparticles were 64.8%,which 66.2% meaningful and 81.9% for in the acid value therelease drug release more thanin CHCP nanoparticles, as a markedly increased drug release amount, showed Figure 10. The drug releaseisrates for CHCP release media with pH 7.4, 6.8 and 4.0, respectively, as compared with CHP nanoparticles, 3.11respectively, rationally designing nano-drug slow-release preparations in vivo application. nanoparticles were 64.8%, 66.2% and 81.9% in the releasefor media with pH 7.4, 6.8 and´4.0,

withas60.5%, 63.8% and 94.2% release, respectively. The media acid value affected the drug release compared with CHP −3.11 nanoparticles, with 60.5%, 63.8% and 94.2% release, respectively. The media for CHP more than CHCP nanoparticles, which meaningful for rationally designing nano-drug acid value affected the drug release for CHP moreisthan CHCP nanoparticles, which is meaningful for slow-release preparations for in vivo application. rationally designing nano-drug slow-release preparations for in vivo application.

Figure 10. The mitoxantrone (MTO) release from pullulan nanoparticles in PBS buffer (pH 6.8) at 37 °C in vitro and acetate buffer (pH 4.0) (▲: CHP−3.11, ▼: CHCP; pH 6.8), (■: CHCP, ●: CHP−3.11; pH 4.0). Figure 10. The mitoxantrone (MTO)release releasefrom from7/14 pullulan nanoparticles inin PBS buffer (pH(pH 6.8)6.8) at 37 Figure 10. The mitoxantrone (MTO) pullulan nanoparticles PBS buffer at°C 37 ˝ C in vitro and acetate buffer (pH 4.0) (▲: CHP−3.11, ▼: CHCP; pH 6.8), (■: CHCP, ●: CHP−3.11; pH 4.0). in vitro and acetate buffer (pH 4.0) (N: CHP´3.11 , İ: CHCP; pH 6.8), (: CHCP, ‚ : CHP´3.11 ; pH 4.0).

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2.5.2.5. The Effect ofofNanoparticle upon HSA HSAComplexation Complexation The Effect NanoparticleProperties Propertieson on Drug Drug Release Release upon WeWeexamined affected the the drug drug release releaseofofnanoparticles nanoparticles with examinedwhether whetherHSA HSA complexation complexation affected with different properties. Figure 11 shows the drug release for CHP , CHP and CHCP different properties. Figure 11 shows the drug release for CHP−6.91, CHP−3.11 and CHCP nanoparticles ´6.91 ´3.11 nanoparticles with HSA complexing andrelease in the media. HSA release media. With HSA complexation, thewas drug with HSA complexing and in the HSA With HSA complexation, the drug release release 28.2%, 34.6%for and 38.2% for CHP , CHPnanoparticles, nanoparticles, respectively, 28.2%,was 34.6% and 38.2% CHP −6.91, CHP −3.11 and CHCP respectively, but 42.6%, 60.5% ´6.91 ´3.11 and CHCP 64.8%,60.5% respectively, in PBS. Even for nanoparticles withfor a lower degree of cholesterol substitution butand 42.6%, and 64.8%, respectively, in PBS. Even nanoparticles with a lower degree of bound to HSA molecules with weaker interaction force and less binding amount [35], the effect of cholesterol substitution bound to HSA molecules with weaker interaction force and less binding HSA complexation on of drug release was significant. In the HSAwas release media, the release of amount [35], the effect HSA complexation on drug release significant. In drug the HSA release CHP−6.91 CHP−3.11 and of CHCP nanoparticles was 50.2%, 66.4% and 70.8%, respectively, but 42.6%, media, the, drug release CHP´ , CHP and CHCP nanoparticles was 50.2%, 66.4% and 70.8%, 6.91 ´3.11 60.5% and but 64.8% in PBS, so and without complexation of HSA,complexation the total amount of drug in respectively, 42.6%, 60.5% 64.8% in PBS, so without of HSA, the released total amount nanoparticles increased. Thus, HSA binding with various nanoparticle properties may be vital for of drug released in nanoparticles increased. Thus, HSA binding with various nanoparticle properties designing highly efficient nano-drugs. may be vital for designing highly efficient nano-drugs.

Figure 11.11.The pullulan nanoparticles nanoparticlesupon uponhuman human serum albumin Figure Themitoxantrone mitoxantrone (MTO) (MTO) release release of pullulan serum albumin ˝ Cin (HSA) complexation invitro vitro(●:(‚CHP : CHP , N: −3.11 CHP , İ : CHCP); and in the HSA (HSA) complexationininPBS PBSat at 37 °C −6.91´6.91 , ▲: CHP , ▼: CHCP); and in the HSA release ´3.11 media (■: CHP −6.91 , ►: CHP −3.11 , ◄: CHCP). release media (: CHP´6.91 , §: CHP´3.11 , đ: CHCP).

Use of novel nanoparticles as drug carriers has recently attracted broad attention in the drug Use of novel nanoparticles as drug carriers has recently attracted broad attention in the drug developme nt field because of their two potential functions: targeting drug delivery and controlled developme nt field because of their two potential functions: targeting drug delivery and controlled slow release [43–45]. Nano–slow-release drug preparations have been studied widely; for example, slow release [43–45]. Nano–slow-release drug preparations have been studied widely; for example, pullulan nanoparticles with a hydrophobic core loading a hydrophobic drug showed a striking slow pullulan nanoparticles with a hydrophobic core loading a hydrophobic drug showed a striking slow drug release [35–37]. To design a suitable slow-release nano-drug, several crucial factors including drug release [35–37]. To design a suitable slow-release nano-drug, several crucial factors including nanoparticle properties and HSA complexation must be detailed. Before the nano-drug preparation nanoparticle and HSA complexation must be detailed. Before the [46,47]. nano-drug preparation can enter theproperties body to play a function, it cannot get eliminate all the protein coating Protein coating canaffects enter particle the body to play a function, it cannot get eliminate all the protein coating [46,47]. biodistribution, therapeutic efficacy, and mainly drug release amount [48–50].Protein The coating affects and mainly drug release amount [48–50]. amount and particle type ofbiodistribution, protein coatingtherapeutic is decidedefficacy, by nanoparticle properties [51,52]. Therefore, The amount and type of such protein coating is decided by nanoparticle [51,52]. function. Therefore, nanoparticle properties as hydrophobicity and surface control theproperties drug slow-release nanoparticle properties such as hydrophobicity and surface control the drug slow-release function. We found reduced drug release with increased nanoparticle hydrophobicity. However, drug release Wewas found reduced drug release with increased nanoparticle enhanced with a negative-charged nanoparticle surface. hydrophobicity. However, drug release was enhanced with a negative-charged nanoparticle surface. When pullulan nanoparticles enter the body with nano-drug treatment, protein adsorption, especially HSA, may affect the slow-release of the nano-drug preparation. HSA adsorption, has high When pullulan nanoparticles enter the function body with nano-drug treatment, protein affinity for mitoxantrone [53] and can complex with pullulan nanoparticles, depending on thehas especially HSA, may affect the slow-release function of the nano-drug preparation. HSA nanoparticle properties [35]. The drug release of pullulanwith nanoparticle-loaded mitoxantrone showedon high affinity for mitoxantrone [53] and can complex pullulan nanoparticles, depending an intricate process with HSA fast-surface adsorption and slow nanoparticle core embedding. The slow the nanoparticle properties [35]. The drug release of pullulan nanoparticle-loaded mitoxantrone release of pullulan nanoparticle-loaded mitoxantrone on HSA complexation was further enhanced showed an intricate process with HSA fast-surface adsorption and slow nanoparticle core embedding. with an release intricateofmechanism (Figure 12). We investigated nanoparticle hydrophobicity surface The slow pullulan nanoparticle-loaded mitoxantrone on HSA complexationand was further charge to further discuss the HSA complexation effect on drug release. Even if the pullulan enhanced with an intricate mechanism (Figure 12). We investigated nanoparticle hydrophobicity and 8/14


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Nanomaterials 6, 2 surface charge2016, to further discuss the HSA complexation effect on drug release. Even if the pullulan nanoparticle property changed the HSA binding force and amount [35,36], the drug release for all nanoparticle property changed the HSA binding force and amount [35,36], the drug release for all pullulan nanoparticles was slower. pullulan nanoparticles was slower. In this study, the unique interaction among mitoxantrone, HSA, and pullulan nanoparticles In this study, the unique interaction among mitoxantrone, HSA, and pullulan nanoparticles is is interesting. If HSA did not bind to pullulan nanoparticles, the drug release amount from interesting. If HSA did not bind to pullulan nanoparticles, the drug release amount from pullulan pullulan nanoparticles could be enhanced because the fast-released drug is by HSA nanoparticles could be enhanced because the fast-released drug is absorbed by absorbed HSA as shown in as shown in Figure 12. The subsequent decrease in mitoxantrone concentration near the pullulan Figure 12. The subsequent decrease in mitoxantrone concentration near the pullulan nanoparticles nanoparticles favored further Thisis mechanism confirmed by the presentthat study favored further release. Thisrelease. mechanism confirmed byisthe present study showing the showing drug thatrelease the drug release nanoparticles of pullulan nanoparticles in media. HSA than PBS media. Of course, of pullulan was greater in was HSAgreater than PBS Of course, if nanoparticles cannot bind HSA and bind at same time, theat loaded in the nanoparticles cannot coated by HSA, the be if nanoparticles cannot HSA and samedrug time, loaded drug in be nanoparticles cannot drug release of nanoparticles would be different. Our results have shown that novel nanoparticle coated by HSA, the drug release of nanoparticles would be different. Our results have shown that properties control the in vivo protein and absorption drug slow-release function. Of note,function. we novel nanoparticle properties control the absorption in vivo protein and drug slow-release investigated only the only principal extracellular protein HSA; if weHSA; consider other factors in the Of note, we investigated the principal extracellular protein if wethe consider the other factors human body systems interacting with nanoparticles, the drug release mechanism will more in the human body systems interacting with nanoparticles, the drug release mechanism will more be complicated. be complicated.

Figure 12. 12. Drug released from HSAcomplexation complexationand and HSA release media. Figure Drug released fromnanoparticles nanoparticles upon upon HSA in in thethe HSA release media.

Besides featuring nanoparticleproperties properties and and protein nanoparticles display Besides featuring nanoparticle proteinbinding, binding,pullulan pullulan nanoparticles display an evidently sustained drug release depending on pH value and ion intensity of release media. an evidently sustained drug release depending on pH value and ion intensity of release media. Here, we discuss the drug release from pullulan nanoparticles in acid release media and analyzed Here, we discuss the drug release from pullulan nanoparticles in acid release media and analyzed the effect of nanoparticle negative charge on drug release. Drug release from pullulan nanoparticles the effect of nanoparticle negative charge on drug release. Drug release from pullulan nanoparticles was increased in acid media, and negative surface-charge nanoparticles showed reduced sensitivity wasto increased in acid media, negative surface-charge nanoparticles showed sensitivity this action. Because the and cancer cell environment is acidic and nanoparticles asreduced a drug carrier are to this often action.loaded Because the cancer cell environment is acidic and nanoparticles as a drug carrier are with anticancer drugs to treat cancer, we provide useful information for furtheroften loaded with anticancer drugs to treat cancer, we provide useful information for further investigation investigation of the drug release of nanoparticles with acidic media. of the drug releasea highly of nanoparticles with acidic media. To obtain efficient slow-release drug carrier, the nano-drug release should be examined toTodesign the precise efficient treatmentslow-release requirement drug for in carrier, vivo high-efficacy delivery. Further studybeofexamined other obtain a highly the nano-drug release should factors such as ions and temperature sensitivity of materials is necessary to the in vivo application to design the precise treatment requirement for in vivo high-efficacy delivery. Further study ofof other nano-drug factors such aspreparations. ions and temperature sensitivity of materials is necessary to the in vivo application of

nano-drug preparations.

3. Experimental Section

3. Experimental Section 3.1. Materials

3.1. Materials Cholesterol modified pullulan (CHP) were synthesized as followed [35]: The pullulan power (0.5 g) was dissolved in 30 mL dry DMSO. The cholesterol succinate (CHS) Cholesterol modified pullulan (CHP) were synthesized as followed [35]: in dried DMSO was activated by addition EDC (1.5 equiv. CHS) and DMAP (1 equiv. CHS). The The pullulan power (0.5 g) was dissolved in 30 mL dry DMSO. The cholesterol succinate (CHS) in mixture was reacted by stirring at 45 °C for 48 h. Then, the reactant mixture was dropped into ethanol dried DMSO was activated by addition EDC (1.5 equiv. CHS) and DMAP (1 equiv. CHS). The mixture to obtain CHP conjugate, and it was washed with ethanol, THF and diethyl ether respectively. ˝ C for 48 h. Then, the reactant mixture was dropped into ethanol to was reacted by stirring at 45carboxyethyl Cholesterol modified pullulan (CHCP) were synthesized as followed:

obtain CHP conjugate, and it was washed with ethanol, THF and diethyl ether respectively. Cholesterol modified carboxyethyl pullulan (CHCP) were synthesized as followed: 9/14


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Synthesis of carboxyethyl pullulan (CEP): Pullulan (3.0 g) was mixed with acrylic acid (1.26 mL) at molar ratio 1:1. Then, the mixture was incubated for 4 h at 50 ˝ C with KOH solution as a catalytic agent. The reaction solution was cooled to room temperature and was placed into 500 mL ethanol. Yellowish-brown precipitation was obtained on removing the ethanol solution. It was dissolved with 40 mL distilled water, filtered, and dialyzed against 5000 mL hydrochloric acid solution (pH = 4.5 ˘ 0.2) for 2 days and distilled water to remove hydrochloric acid and other small substance. The CEP dialysate was collected by freezed-dried to obtain a white cotton solid. Synthesis of carboxyethyl pullulan hydrophobically-modified with cholesterol (CHCP): NHS-activated

cholesterol succinate (CSN) were synthesized according to the method which we previously described [54,55]. CEP was dissolved in 10 mL DMSO, and put into round fask in oil bath pan. CSN (CSN/glucose unit = 0.1–0.5 mmol/mmol) and (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC/CSN = 1.0 mmol/mmol) were dissolved in the mixture of DMSO and tetrahydrofuran. CHCP conjugate was prepared by the reaction of CSN with CEP at 45 ˝ C according to the method as the same as CHP synthesis, shown in the above experiment. Pullulan (20 kDa) was substituted with 3.11, 6.03, 6.91 cholesterol moieties per 100 glucose units and named CHP´3.11 , CHP´6.03 and CHP´6.91 . Pullulan (20 kDa) was substituted with 3.46 cholesterol moieties and with 10.1 carboxyethyl moieties per 100 glucose units in one CHCP molecule. HSA (fatty acid-free) was from Sigma-Aldrich (St. Louis, MO, USA). Mitoxantrone was purchased from Beijing Xinze Science and Technology Company (Beijing, China). Dialysis bags (molecular weight cut off 14,000) and other chemical reagent were obtained from Changsha Huicheng Commerce Company (Changsha, China). 3.2. CHP and CHCP Nanoparticle Characterization All types of pullulan nanoparticles were prepared by the dialysis method [35,36]. Briefly, hydrophobically modified pullulan conjugates were dissolved in dimethyl sulfoxide and the mixture solution was dialyzed for 24 h to completely remove dimethyl sulfoxide. Pullulan nanoparticle solutions were passed through a membrane fliter (pore size: 0.45 µm, Millipore). The solutions were collected in bottles to obtain photos of their appearance. The size distribution and zeta potential of the pullulan nanoparticles were determined by dynamic laser light scattering at 25 ˝ C with a BI-90US (HORIBA Ltd., Kyoto, Japan) light-scattering spectrophotometer. To observe morphology, a sample solution (0.5 mg¨ mL´1 ) of pullulan nanoparticles was observed by TEM with a JEM-100C (JEOL Ltd., Kyoto, Japan) electron microscope at 80 ˝ C. 3.3. Mitoxantrone-Loaded Nanoparticle Characterization Drug-loaded pullulan nanoparticles were prepared as described [31]. Briefly, 4 mg mitoxantrone and 40 mg conjugates were dissolved in 20 mL dimethyl sulfoxide, and an amount of triethylamine was added to help the drug load onto nanoparticles. Dimethyl sulfoxide and free mitoxantrone were removed by dialysis (dialysis tube, weight cutoff, 12–14 kDa, Millipore) for 9 h to obtain mitoxantrone-loaded nanoparticles. The sizes of the obtained drug-loaded CHP and CHCP nanoparticles were measured by DLS. Nanoparticle drug encapsulation efficiency and loading capacity were calculated as described [13]. 3.4. In Vitro Drug Release of Pullulan Nanoparticles with Different Properties The solution of mitoxantrone-loaded CHP nanoparticles (a certain amount) was put in Visking dialysis bag and dialyzed against the PBS release media at 37 ˝ C. Then, 2 mL of the release media was collected and replaced with an equal volume of the fresh release media at pre-defined time intervals (Ti). The released amount of drug was determined by UV spectrophotometry (UV-384 plus,


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Molecular Devices, Thermo Fisher Scientific Inc., Waltham, MA, USA), and drug release percentage rate (Q%) was measured as follows [37]. Q% “ pCn ˆ V ` Vn

iÿ “n

Ci q{Wdrug-loading

(1)

i“0

where Cn is the sample concentration at Tn , V is the total volume of release medium, Vi is the sample volume at Ti , Ci is the sample concentration at Ti (both V 0 and C0 were equal to zero), and Tn is the sampling at the Nth time. The drug release amount from CHP and CHCP nanoparticles was collected to compare with the drug release feature. 3.5. In Vitro Drug Release of Pululllan Nanoparticles in Release Media with Different pH Mitoxantrone release was studied in vitro by a dialysis method in phosphate buffered saline (PBS; pH 6.8) and acetate buffer (pH 4.0). Briefly, the solution of mitoxantrone-loaded CHCP nanoparticles was placed into dialysis tubing (dialysis tube, weight cut-off, 12–14 kDa, Millipore) and dialyzed against the release media at 37 ˘ 0.2 ˝ C in an air-bath shaker at 100 rpm. Then, the drug release amount was collected to calculate the release percentage according to the above method. The drug release amount from CHP´3.11 nanoparticles was collected in media at different PH values to compare with the release from CHCP nanoparticles. 3.6. Drug Release of Pulullan Nanoparticles with HSA Complexation CHP nanoparticles can complex with HSA and bind completely and firmly in a 9 h reaction as we previously described [35]. Therefore, we mixed an amount of HSA and drug-loaded nanoparticles to obtain the nanoparticle-drug-HSA complex, then measured the drug release as described previously. We studied the drug release of the three types of pullulan nanoparticles, CHP´6.91 , CHP´3.11 , CHCP, after HSA complexation. We changed the PBS release media to HSA release media at a certain concentration and analyzed the drug release of the three types of pullulan nanoparticles. 4. Conclusions Nanoparticle hydrophobicity and surface charge affected the size formed, drug loading amount, and drug release of pullulan nanoparticles. The size formed and drug release of self-aggregated pullulan nanoparticles decreased with increasing cholesterol substitution. However, the drug-loading amount showed an opposite relationship. Carboxyethyl groups appeared to form a nanoparticle surface-negative charge and disturb the size formed and the drug-loading amount as well as the drug release amount, which were related to nanoparticle hydrophobicity. The amount of drug released from pullulan nanoparticles was increased in the acid media. However, the drug release was weaker from nanoparticles with a negative charge. The drug release from pullulan nanoparticles was slower with HSA complexation than in PBS and HSA media. Nanoparticle hydrophobicity and surface charge affected nano-drug release after nanoparticle-HSA complexation only slightly. Further study of nano-drug slow-release preparations with different properties is crucial to fully understand the potential application of nano-drug carriers in drug delivery. Acknowledgments: This project was supported by the Doctoral Fund (No. 140630) of Hunan Normal University to Xiaojun Tao, Advanced Fund of Hunan Provincial Education Department (No. 15A117) and Xiaoxiang Endowed University Professor Fund of Hunan Normal University (No. 840140-008) to Xiaoping Yang. Author Contributions: Xiaojun Tao and Xiaoping Yang conceived of the idea. Xiaojun Tao, Liming Yuan and Yongchao Xie performed the experiments. Xiaojun Tao, Shu Jin and Dehong Wu analyzed the results. Xiaojun Tao, Kai Ling and Pingfa Lin wrote the paper. Xiaoping Yang guided and supervised the work. Conflicts of Interest: The authors declare no conflict of interest.


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