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ARTICLE IN PRESSG ModelOLSUB-6575; No. of Pages 8

Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

j o ur nal ho me pa ge: www.elsev ier .com/ locate /co lsur fb

olymeric micelles for acyclovir drug delivery

licia J. Sawdon, Ching-An Peng ∗

epartment of Chemical Engineering, Michigan Technological University, Houghton, MI 49931, United States

r t i c l e i n f o

rticle history:eceived 18 June 2014eceived in revised form 31 July 2014ccepted 12 August 2014vailable online xxx

eywords:

a b s t r a c t

Polymeric prodrug micelles for delivery of acyclovir (ACV) were synthesized. First, ACV was used directlyto initiate ring-opening polymerization of �-caprolactone to form ACV-polycaprolactone (ACV-PCL).Through conjugation of hydrophobic ACV-PCL with hydrophilic methoxy poly(ethylene glycol) (MPEG)or chitosan, polymeric micelles for drug delivery were formed. 1H NMR, FTIR, and gel permeationchromatography were employed to show successful conjugation of MPEG or chitosan to hydrophobicACV-PCL. Through dynamic light scattering, zeta potential analysis, transmission electron microscopy,

cycloviring-opening polymerizationicellar drug deliveryethoxypolyethylene glycol

hitosan

and critical micelle concentration (CMC), the synthesized ACV-tagged polymeric micelles were charac-terized. It was found that the average size of the polymeric micelles was under 200 nm and the CMCsof ACV-PCL-MPEG and ACV-PCL-chitosan were 2.0 mg L−1 and 6.6 mg L−1, respectively. The drug releasekinetics of ACV was investigated and cytotoxicity assay demonstrates that ACV-tagged polymeric micelleswere non-toxic.

© 2014 Published by Elsevier B.V.

. Introduction

Due to the fact that nanoparticles can be prepared using a varietyf polymers, biodegradable polymers have been extensively stud-ed for the field of polymer therapeutics [1–3]. While there haveeen many biodegradable polymeric nanoparticles synthesized forrug delivery, polymeric micelles have numerous advantages overther proposed colloidal delivery systems [4–7]. Many studies havehown the promise micelles have for drug delivery because they cane tailored for prolonged blood circulation time, cellular selectiv-

ty, and for their controlled release capabilities [8–10]. In addition,icelles can be specifically synthesized to increase a drug’s solu-

ility and bioavailability [11–14].Acyclovir (ACV) is a guanosine-based prodrug most commonly

sed for the treatment of infections caused by herpes simplexirus (HSV) types 1 and 2, varicella zoster virus and, to a lesserxtent, cytomegalovirus and Epstein–Barr virus [15]. Moreover,rodrug ACV can be converted to its cytotoxic phosphorylatedorm by herpes simplex virus thymidine kinase (HSV-TK) gene for

Please cite this article in press as: A.J. Sawdon, C.-A. Peng, Polymeric m(2014), http://dx.doi.org/10.1016/j.colsurfb.2014.08.011

ancer therapy [16]. That is, if the HSV-TK gene is delivered toctively dividing cancer cells, and ACV is subsequently adminis-ered to the cells, the TK enzyme phosphorylates ACV, yielding toxic

∗ Corresponding author at: Department of Chemical Engineering, Michigan Tech-ological University, 1400 Townsend Drive, Houghton, MI 49931, United States.el.: +1 906 487 2569.

E-mail address: [email protected] (C.-A. Peng).

ttp://dx.doi.org/10.1016/j.colsurfb.2014.08.011927-7765/© 2014 Published by Elsevier B.V.

metabolites which cause death in prodrug treated HSV-TK express-ing cells [17–19]. However, due to ACV’s poor water solubilityand ensuing low bioavailability, alternative delivery approachesare required to increase the therapeutic potential of ACV. Severalmethods reported are to couple ACV to biocompatible hydrophilicpolymers [20–22] or encapsulation into drug carriers [23–25].Although these processes increase the bioavailability of ACV as wellas offer a practical treatment for patients, they are labor-intensiveand cost-ineffective. Recently, we have shown that ACV can beused as an initiator to proceed ring-opening polymerization of �-caprolactone (�-CL) to form hydrophobic ACV-polycaprolactone(ACV-PCL) [26], which makes this an economically attractiveapproach compared with the aforementioned conjugation andencapsulation methods.

Polymeric micelles consist of an inner core made of a hydropho-bic block copolymer and an outer corona made of the hydrophilicblock of the copolymer. PCL, having been widely used as the core-forming hydrophobic segment of nanoparticles, was selected asthe model polymer for this study. PCL is a semi-crystalline, lin-ear resorbable aliphatic polyester. It has been commonly used indrug delivery systems because it is biodegradable and biocom-patible [27–29]. PCL is commonly synthesized by ring-openingpolymerization of �-CL using an alcohol as an initiator and stan-nous (II) octoate (Sn(Oct)2) as a catalyst [30,31]. In addition to

icelles for acyclovir drug delivery, Colloids Surf. B: Biointerfaces

using alcohol as the initiator, methoxy-poly(ethylene oxide) andstarch have been employed as macroinitiators to form amphiphilicpolymers. [32,33]. In this study, prodrug ACV possessing hydroxylgroups was used as the initiator to obtain prodrug-PCL. Then,

dx.doi.org/10.1016/j.colsurfb.2014.08.011


http://www.sciencedirect.com/science/journal/09277765

http://www.elsevier.com/locate/colsurfb

mailto:[email protected]


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2.4. Preparation of polymeric prodrug micelles

ACV-PCL-MPEG and ACV-PCL-chitosan micelles were formedsimilarly. Briefly, 10 mg of ACV-tagged amphiphilic polymer was

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hydrophilic compound (MPEG or chitosan) was grafted onhe hydrophobic prodrug-PCL to form the amphiphilic blockopolymer which already has the drug attached started fromhe ring-opening polymerization. These synthesized ACV-taggedmphiphilic polymers can self-assemble in aqueous medium toorm polymeric prodrug micelles for use as nanocarriers in drugelivery.

Individual conjugation of ACV-PCL to a wide array of biocompat-ble hydrophilic polymers to form polymeric micelles each has theirwn advantages for drug delivery. With this in mind, we choose tossess the successful conjugation of two model hydrophilic poly-ers, MPEG and chitosan, to hydrophobic ACV-PCL. Chitosan is a

atural polysaccharide derived from deacetylation of chitin. Chi-osan’s biocompatible and biodegradable features have attracted

uch attention in biomedical and pharmaceutical research [28,34].imilarly, MPEG is a biocompatible hydrophilic polymer com-only used in polymeric micelle formation. MPEG is inexpensive,

on-toxic and is widely used to covalently modify biologicalacromolecules and surfaces [10,35,36]. Hence, ACV-PCL-MPEG

nd ACV-PCL-chitosan copolymers were synthesized. The chemicaltructure and physical properties of the copolymers were char-cterized and micelle formation investigated. The drug releaserofiles of ACV from polymeric prodrug micelles and the biocom-atibility of polymeric prodrug micelles were investigated in thistudy.

. Experimental

.1. Materials

ACV was purchased from TCI (Tokyo, Japan). N,N′-dicyclohexylarbodiimide (DCC), �-CL, pyrene, and succinic anhydride wereurchased from Acros Organics (Geel, Belgium). Sn(Oct)2, CDCl3ith 1% tetramethylsilane (TMS), deuterated dimethyl sulfoxide

DMSO-d6), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF),ichloromethane (DCM), methanol, 2-propanol, hexane, toluene,ethoxypolyethylene glycol amine (MPEG-NH2; MW = 5000), and

hitosan oligosaccharide lactate (MW = 5000) were all purchasedrom Sigma–Aldrich (St. Louis, MO). Ethyl ether was purchasedrom J.T. Baker (Austin, TX). N-hydroxysuccinimide (NHS) was pur-hased from Alfa Aesar (Ward Hill, MA). Acetone was purchasedrom Pharmco-AAPER (Shelbyville, KY). Pyridine and hydrochloriccid (HCl) were purchased from EMD (Philadelphia, PA). Sodiumhloride (NaCl) and magnesium sulfate were purchased fromhowa (Tokyo, Japan). All reagents were used as received withouturther purification.

.2. Characterization methods

Gel permeation chromatography (GPC) analyses were per-ormed on a Waters 1525 binary HPLC pump equipped with

Waters 2414 refractive index detector (Milford, MA). Waterstyragel HR 3 (MW = 500–30,000) and HR 4E (MW = 50–100,000)olumns were equipped. Molecular weight calibration was per-ormed with polystyrene standards that covered a MW range of00–4.3 × 104. GPC analyses were performed in THF at a flowate of 1 mL min−1 with an injected volume of 50 �L. 1H NMRpectra were obtained from a Varian Unity/Inova 400 MHz instru-ent (Sparta, NJ). To obtain FTIR spectra by a Jasco FTIR-4200

pectrometer (Tokyo, Japan), a small amount of polymeric sam-


le was loaded onto a silicon wafer and THF was added dropwiseo dissolve the sample and evaporated afterwards. This wasepeated until the entire sample was dissolved and a film hadormed.

PRESSs B: Biointerfaces xxx (2014) xxx–xxx

2.3. Synthesis of ACV-tagged amphiphilic polymers

2.3.1. Synthesis of ACV-PCLACV (50 mg) was weighed and mixed with �-CL (2.25 mL) under

a sonication bath for 5 min at room temperature. Sn(Oct)2 (0.5 wt%of �-CL) was then added into the mixture. The entire solution wasplaced into a 3-necked round-bottom flask. The system was purgedwith nitrogen and immersed in an oil bath at 140 ◦C for 24 h. Thecrude product was cooled to room temperature, dissolved in DCM,and precipitated by cold methanol. The product was then vacuumdried by a rotary evaporator at 40 ◦C.

2.3.2. Synthesis of ACV-PCL-COOHACV-PCL (0.5 mmol) and succinic anhydride (1 mmol) were

weighed and dissolved in toluene in a 3-necked round-bottom flask.One mmol pyridine was added and the solution was reacted undernitrogen at 70 ◦C for 48 h. The product was then precipitated by coldhexane, and spun down. The pellet was re-dissolved in DCM andwashed twice each with 10% (v/v) HCl and saturated NaCl solution.The organic phase was isolated and dried with magnesium sulfatethen filtered. The carboxylated ACV-PCL was recovered by precipi-tation in cold hexane and then vacuum dried by rotary evaporationat 40 ◦C.

2.3.3. Synthesis of ACV-PCL-NHSACV-PCL-COOH (0.54 mmol) and NHS (2.7 mmol) were weighed

and mixed in 15 mL DCM, and then DCC (2.7 mmol) was added. Thereaction was run under a nitrogen environment at room tempera-ture for 24 h. The precipitated byproduct 1,3-dicyclohexylurea wasremoved by vacuum filtration. The filtrate was added into 35 mLdiethyl ether and cooled to 4 ◦C for 4 h to precipitate ACV-PCL-NHS. The precipitate was collected by centrifugation at 3500 rpmfor 5 min, washed with 2-propanol and solvent removed by rotaryevaporation at 40 ◦C.

2.3.4. Synthesis of ACV-PCL-MPEGACV-PCL-NHS (10 mg) and MPEG-NH2 (10 mg) were weighed

and dissolved by 20 mL DCM in a round-bottom flask. The flaskwas purged with nitrogen and the solution was stirred for 24 h. Thesolution was then dialyzed (MWCO = 6–8 kDa, Spectra/Por, RanchoDominguez, CA) against pure DCM to remove remaining MPEG-NH2. ACV-PCL-MPEG was recovered by rotary evaporation at 40 ◦C.

2.3.5. Synthesis of ACV-PCL-chitosanACV-PCL-NHS (10 mg) was dissolved in 5 mL acetone and slowly

added to chitosan solution (20 mg chitosan oligosaccharide lac-tate dissolved in 25 mL deionized water). The mixture, purgedwith nitrogen, was stirred in a round-bottom flask for 24 h. Thereacted solution was vacuum dried to remove acetone and thenlyophilized. The amphiphilic polymer was then dissolved in DCMand dialyzed (MWCO = 6–8 kDa, Spectra/Por) against pure DCM toremove unreacted chitosan. ACV-PCL-chitosan was recovered byrotary evaporation at 40 ◦C.


dissolved in 2 mL acetone. The solution was then added dropwise to10 mL deionized water under sonication. Acetone was removed byrotary evaporation and the final solution was collected by filteringthrough a 0.45 �m filter.


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.5. Determination of critical micelle concentration

The CMC was estimated by using pyrene as a fluorescent probe37]. Briefly, 1 mg mL−1 of polymeric prodrug micelle was formed.arious amounts of deionized water and micellar solution weredded, respectively, to glass vials to obtain micellar concentrationsanging from 5 × 10−7 to 1 mg mL−1. Pyrene in acetone was thendded separately to the prepared vials to get a final concentrationf pyrene in water of 6.0 × 10−7 mg mL−1, slightly lower than theaturation solubility of pyrene in water [38]. The solutions werehen allowed to equilibrate for 8 h. Fluorescent spectra were deter-

ined by a plate reader (Synergy MX, BioTek, Winooski, VT) withn excitation wavelength of 334 nm.

.6. Size and morphology of polymeric prodrug micelles


The average particle size of polymeric prodrug micelles wasetermined by a dynamic light scattering (DLS) instrument (Zeta-izer Nano ZS, Malvern Instruments, United Kingdom) equipped

Scheme 1. Synthetic scheme of (A) ACV-PCL, (B) ACV-PCL-COOH, (C)

PRESSs B: Biointerfaces xxx (2014) xxx–xxx 3

with a red laser at a wavelength of 633 nm and scattering angleof 90◦ at 25 ◦C. The zeta potential of the micelles dispersedin deionized water was determined with a zeta potential ana-lyzer (Zetasizer Nano ZS). Transmission electron microscopy (TEM)image of micelles was performed on a JEM-4000FX (JEOL, Tokyo,Japan) at 80 kV. The TEM samples were prepared by adding 10 �Lof micellar solution (1 mg mL−1) onto a Formvar grid for 5 minand wicking away solution in excess. The samples were negativelystained with 10 �L phosphotungstic acid solution (2 wt%) for 10 sand then 15 s, wicking away excess staining solution each time.

2.7. Drug release kinetics

Polymeric prodrug micelles at a concentration of 1 mg mL−1

were made in phosphate buffered saline (PBS) (1 M, pH 7.4) at


25 ◦C. Two mL of solution was placed in a dialysis tube (Float-A-Lyzer, Spectra/Por) with a MWCO of 3.5–5 kDa. The dialysis bagwas then immersed in 50 mL PBS at room temperature. At specifiedtime intervals, 5 �L of sample was removed and replaced with fresh

ACV-PCL-NHS, (D) ACV-PCL-MPEG, and (E) ACV-PCL-chitosan.


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BS. The amount of ACV released was analyzed by a plate readerBioTek) at 254 nm. All experiments were carried out in triplicate.

.8. Cytotoxicity test

24-well plates were seeded with human colorectal HT29 cellsHTB-38; ATCC, Manassas, VA) suspended in 0.5 mL Dulbecco’s

odified Eagles’ medium (DMEM, Corning Cellgro, Manassas, VA)upplemented with 10% fetal bovine serum (FBS, Atlanta biologi-als, Flowery Branch, GA) and 1% penicillin–streptomycin (Sigma)nd incubated at 37 ◦C in 5% CO2 balanced with humidified air for4 h. In each well, 500 �L of 1 mg mL−1 ACV-PCL-MPEG or ACV-CL-chitosan polymeric micelles (filtered by a 0.22 �m filter) wasdded. After incubation for 48 h, cell viability was assessed usingTT assay. 200 �L of sterile MTT solution (4 mg mL−1) was added

nto the culture wells and incubated for 4 h. The medium contain-ng unreacted MTT was removed and 300 �L DMSO was added toissolve the insoluble purple formazan crystals formed in cellu-

ar mitochondria. The absorbance at 590 nm was measured with alate reader (BioTek) and results were recorded as viability percent-ge calculated against the control group without micellar challenge.ll experiments were carried out in triplicate.

. Results and discussion

.1. Synthesis and characterization of amphiphilic prodrug


olymers

ACV-PCL was synthesized through ring-opening polymeriza-ion of �-CL, which was initiated exclusively by ACV (Scheme 1A).

Fig. 1. 1H NMR spectra of (iii) MP


The number average molecular weight (Mn) of ACV-PCL polymerafter 24 h reaction time was approximately 1.7 × 104 g mol−1 withpolydispersity index (PDI) of 1.64 as measured from GPC anal-ysis (as shown in Table S1 in the supporting information). The1H NMR spectra of guanosine-based prodrug ACV and ACV-PCL24 h post-synthesis are shown in Fig. S1(i) and (ii), respectively, inthe supporting information. Judging from the ACV-PCL spectrum,chemical shifts at 3.75 (e, f-CH2), 5.44 (d-CH2), 7.68 (b-CH) wereassigned to protons in ACV, while the peaks at ı = 1.36 (3-CH2), 1.64(2-CH2), 2.29 (1-CH2) and 4.04 (4-CH2) correspond with the back-bone chain of PCL polymer. The characteristic resonances of PCLand ACV were both observed in the synthesized polymer. Althoughthe characteristic peaks of ACV in Fig. S1(ii) are much lower thanthose representing PCL, the evidence of coupling of ACV’s protonsin synthesized ACV-PCL is clear.

ACV-PCL was further conjugated with either MPEG or chitosanas shown in Scheme 1B–E. Successful conjugation of MPEG orchitosan was confirmed by GPC, 1H NMR and FTIR analyses. Theincrease in Mn of ACV-PCL to 22.4 kDa from 17.5 kDa correspondswith the addition of MPEG (Table S1 in the supporting informa-tion). Fig. 1 shows the 1H NMR of MPEG-NH2 and ACV-PCL-MPEG.The peak at 3.63 (A-OCH2) attributed to MPEG can be clearly seenin Fig. 1(iv). Due to the conjugation of MPEG to ACV-PCL thoughamide linkage (Scheme 1D), the change of the peak at 1.79 (B-NH2)from a singlet in Fig. 1(iii) to a multiplet in Fig. 1(iv) further con-firms conjugation of MPEG to ACV-PCL. Further characterization


of successful grafting of MPEG to hydrophobic ACV-PCL was exam-ined through FTIR analysis. FTIR spectra of ACV-PCL (A), MPEG-NH2(B) and ACV-PCL-MPEG (C) are shown in Fig. 2. C H stretch-ing vibrations can be seen from 2945–2852 cm−1 for all samples.

EG and (iv) ACV-PCL-MPEG.


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Here, ACV-PCL acts as the hydrophobic segment and MPEG or chi-

Fig. 2. FTIR spectra of (A) ACV-PCL, (B) MPEG-NH2, and (C) ACV-PCL-MPEG.

he typical bending vibration of 1634 cm−1 for the NH2 bendingmide I band can be seen in ACV-PCL and ACV-PCL-MPEG spectra,nd is attributed to ACV. PCL and MPEG blocks were character-zed by prominent absorptions at 1726 cm−1 (C O) and 1112 cm−1

C O C), respectively. These peaks are found in ACV-PCL-MPEG


opolymer ascertaining successful bonding of MPEG to ACV-PCL.GPC analysis revealed that the Mn of ACV-PCL increased from

7.5 kDa to 21.7 kDa after grafting with chitosan and had a PDI

Fig. 3. 1H NMR spectra of (v) chitosa

PRESSs B: Biointerfaces xxx (2014) xxx–xxx 5

of 1.42 (Table S1 in the supporting information). Successful con-jugation of chitosan to ACV-PCL was characterized similar toACV-PCL-MPEG. Fig. 3 reveals the 1H NMR of chitosan (v) and ACV-PCL-chitosan (vi). As shown in Scheme 1E, conjugation of chitosanto ACV-PCL is made via amide linkage. The peak at 1.79 (A-NH2)from a singlet to a multiplet in Fig. 3(vi) confirms conjugation of chi-tosan to ACV-PCL. Moreover, the peaks from the aromatic protonson C3–C6 can be seen from ı = 3.28–3.85, slightly shifted down-ward from the peaks shown in the original chitosan sample. Fig. 4depicts the FTIR spectra of ACV-PCL (A), chitosan (B), and ACV-PCL-chitosan (C). OH stretching from 3429–3167 cm−1 as well as peaksat 1634 cm−1 and 1521 cm−1 corresponding to the N–H bendingvibrations of the amide I and II bands are attributed to ACV andchitosan, respectively. The aforementioned peaks as well as the car-bonyl absorption at 1726 cm−1 associated with PCL and a peak at1065 cm−1 (C O C) seen in each spectra are found in ACV-PCL-chitosan. The FTIR results are in line with the results from 1H NMRand demonstrate successful synthesis of ACV-PCL-chitosan.

3.2. Formation and characterization of ACV-tagged polymericmicelles

Due to the amphiphilic nature of the synthesized ACV-PCLcopolymers (i.e., ACV-PCL-MPEG and ACV-PCL-chitosan), they canself-assemble into polymeric prodrug micelles in aqueous medium.


tosan serve as the hydrophilic segments. Polymeric micelles ofACV-PCL copolymer were prepared via the evaporation method[39]. The CMC using pyrene as a hydrophobic fluorescent probe

n and (vi) ACV-PCL-chitosan.


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Fig. 4. FTIR spectra of (A) ACV-PCL, (B) chitosan, and (C) ACV-PCL-chitosan.

as carried out to prove the formation of the micellar structures.yrene can preferentially partition into the interior hydrophobicicrodomains and change the intensities of the first and third


ands in the pyrene fluorescence spectrum [38]. For ACV-PCL-PEG the shift found in the first and third bands was at I335/I330hile for ACV-PCL-chitosan it was at I337/I332. It is believed that this

hange is due to different polymer compositions. Fig. 5A shows the

ig. 5. (A) Plot of the intensity ratio (II/IIII) versus concentration of ACV-tagged polymerichitosan. Insets represent TEM images.


CMC values of ACV-tagged polymeric micelles in aqueous medium.It was determined that the CMCs for ACV-PCL-MPEG and ACV-PCL-chitosan were 2.0 and 6.6 mg L−1, respectively (Fig. 5A(i) and(ii)). Kim et al. reported that the CMC values of block copoly-mers depends upon the block copolymer architecture [40]. Inthis study, the hydrophobic block was kept constant, as ACV-PCL, while the hydrophilic block was changed to either MPEGor chitosan. The low CMC value obtained for ACV-tagged poly-meric micelles could provide evidence for their stability in dilutesolutions.

In order to characterize the morphology of ACV-tagged poly-meric micelles, DLS and TEM analyses were carried out. Fig. 5Bshows the size and morphology of ACV-tagged polymeric micelles.As can be seen from DLS results, sizes of the two types of polymericprodrug micelles were under 200 nm. ACV-PCL-MPEG (Fig. 5B(i))and ACV-PCL-chitosan (Fig. 5B(ii)) micelles showed size distribu-tions with mean diameters of 141.8 and 172.7 nm, respectively. Themorphology of the micelles was further examined by TEM as shownin the insets of Fig. 5B. The size of the micelles in the TEM imageswas slightly lower than the results from DLS with an average parti-cle size of ∼100–150 nm. This size fluctuation is due to the fact thatDLS records the hydrodynamic radius of particles which is oftentimes slightly larger than the actual particle size. Zeta potentialanalysis of ACV-tagged polymeric micelles was also performed. Anaverage zeta potential for ACV-PCL-MPEG micelles was 1.4 mV. Dueto the fact that MPEG has a neutral charge, the results obtained for


ACV-PCL-MPEG micelles are reasonable. In contrast, the detectedzeta potential of ACV-PCL-chitosan micelles was 32.3 mV. This isattributed to the positive charge of the hydrophilic chitosan seg-ment deployed on micellar nanoparticles.

micelles, and (B) particle size distribution of (i) ACV-PCL-MPEG and (ii) ACV-PCL-


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gation. ACV-tagged polymeric micelles were found to have a mean

Fig. 6. In vitro drug release profiles of ACV-PCL-MPEG ( ) and

To determine the drug loading percentage of acyclovir perg of micelle formulation, the absorbance of ACV-tagged poly-eric micelles at t = 0 and t = 48 h was examined and compared

o a standard calibration curve of ACV ranging from 0.002 to.0 mg mL−1. It was found that ACV comprised 8.7% and 3.2% ofCV-PCL-MPEG and ACV-PCL-chitosan micelles, respectively. Theifference in ACV loading percentage is due to different conditionsmployed for the synthesis of ACV-PCL-MPEG and ACV-PCL-hitosan amphiphilic copolymers. While MPEG-NH2 can dissolven organic solvent, chitosan requires an aqueous solvent for it toissolve. Therefore, for ACV-PCL-chitosan synthesis, the ester bondetween ACV and PCL is already weakened, thereby resulting in lessCV coupled with PCL than ACV-PCL-MPEG which is synthesizednder organic solvent environment.

.3. In vitro release of ACV from polymeric micelles

The in vitro release behaviors of ACV from both types of poly-eric prodrug micelles in PBS at 25 ◦C were studied and the results

re shown in Fig. 6. Similar biphasic drug release profile of ACV-PCL-PEG and ACV-PCL-chitosan micelles was observed, with initial

urst release within the first 2 h (∼50% accumulative release) fol-owed by a sustained release pattern up to 48 h. The release of ACVrom both polymeric prodrug micelles was caused by the hydroly-is of ester linkage between ACV and PCL. In the first phase, the ratef drug release from ACV-PCL MPEG micelles was relatively rapidhan that from ACV-PCL-chitosan ones. The reason for this phe-omenon is speculated to be twofold. First, MPEG chains probablyre more hydrophilic and flexible than chitosan chains on micellaranoparticles, leading to a faster hydrolysis and concomitant ACViffusion from hydrophobic PCL cores. Second, the diffusion of ACVolecules might be slowed down by the positively charged chi-

osan on ACV-PCL-chitosan micelles. Such different initial releaserend continued and resulted in higher ACV accumulative releaserom ACV-PCL-MPEG micelles (96%) than from ACV-PCL-chitosan

icelles (82%).

.4. Cytotoxicity study


Biocompatibility is a vital necessity of polymer materials beingsed for drug delivery applications. To evaluate if ACV-tagged poly-eric micelles showed any cytotoxicity toward HT29 colorectal

ells, MTT assay was performed. As shown in Fig. 7, the viability

meric micelles at different dosages. Control (without micelle challenge),ACV-PCL-MPEG micelles, and ACV-PCL-chitosan micelles (mean ± SD, n = 3).

of HT29 cells after treatment for 48 h with either ACV-PCL-MPEGor ACV-PCL-chitosan has little change compared with the untreatedcontrol cells (i.e., cells without any micelle challenge). These resultsdemonstrate that ACV-tagged polymeric micelles do not exhibitapparent toxicity, and are biocompatible.

4. Conclusion

In this study, ACV-PCL-MPEG and ACV-PCL-chitosan polymericmicelles were synthesized and characterized. ACV was used todirectly initiate polymerization of �-CL to form hydrophobicACV-PCL. Compared to conventional methods of incorporatingACV into polymeric carriers by chemical conjugation or physicalencapsulation, our approach is advantageous in terms of elimi-nating drug-loading steps, enhancing drug-carrying capacity, anddecreasing production cost. By grafting ACV-PCL with either MPEGor chitosan, ACV-tagged amphiphilic polymers can self-assembleas micellar nanoparticles in aqueous medium. Structural analysessuch as 1H NMR and FTIR were performed to confirm their conju-


size under 200 nm by both DLS and TEM and have a low CMC. More-over, both ACV-PCL-MPEG and ACV-PCL-chitosan micelles werenon-toxic to HT29 colorectal cells. ACV-tagged polymeric micellesare potential carriers for therapeutic and anticancer drug delivery.


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cknowledgements

This study was partially supported by the Michigan Tech Fundnd the NIH (R15 CA152828-01).

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.014.08.011.

eferences

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http://dx.doi.org/10.1016/j.colsurfb.2014.08.011

http://dx.doi.org/10.1016/j.colsurfb.2014.08.011

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polymeric micelles for acyclovir drug delivery

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