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Long Circulating Self-Assembled Nanoparticles from Cholesterol-Containing Brush-Like Block Copolymers for Improved Drug Delivery to Tumors Thanh-Huyen Tran, Chi Thanh Nguyen, Laura Gonzalez-Fajardo, Derek Hargrove, Donghui Song, Prashant Deshmukh, Lalit Mahajan, Dennis Ndaya, Laijun Lai, Rajeswari M Kasi, and Xiuling Lu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5013822 • Publication Date (Web): 13 Oct 2014 Downloaded from http://pubs.acs.org on October 19, 2014

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Biomacromolecules

Long Circulating Self-Assembled Nanoparticles from Cholesterol-Containing Brush-Like Block Copolymers for Improved Drug Delivery to Tumors

Thanh-Huyen Tran1, Chi Thanh Nguyen2, Laura Gonzalez-Fajardo1, Derek Hargrove1, Donghui Song,1 Prashant Deshmukh3, Lalit Mahajan2, Dennis Ndaya3, Laijun Lai4, Rajeswari M. Kasi2,3, Xiuling Lu1*

1

Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269 2

Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, Connecticut 06269 3

4

Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269

Department of Allied Health Sciences, University of Connecticut, Storrs, Connecticut 06269

*Corresponding author: [email protected], Phone: (860) 486-0517, Fax: (860) 486-2076 1 ACS Paragon Plus Environment

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ABSTRACT

Amphiphilic brush-like block copolymers comprised of polynorbonene-cholesterol/polyethylene glycol (P(NBCh9-b-NBPEG)) self-assembled to form a long circulating nanostructure capable of encapsulating the anticancer drug doxorubicin (DOX) with high drug loading (22.1% w/w). The release of DOX from the DOX-loaded P(NBCh9-b-NBPEG) nanoparticles (DOX-NPs) was steady with less than 2% per day in PBS. DOX-NPs were effectively internalized by human cervical cancer cells (HeLa) and showed dose-dependent cytotoxicity, while blank nanoparticles were non-cytotoxic. The DOX-NPs demonstrated a superior in vivo circulation time relative to free DOX. Tissue distribution and in vivo imaging studies showed that DOX-NPs preferentially accumulated in tumor tissue with markedly reduced accumulation in the heart and other vital organs. The DOX-NPs greatly improved survival and significantly inhibited tumor growth in tumor-bearing SCID mice compared to untreated and free DOX-treated groups. The results indicated that the self-assembled P(NBCh9-b-NBPEG) may be a useful carrier for improving tumor delivery of hydrophobic anticancer drugs.

KEYWORD: brush-like block copolymer, self-assembled nanoparticles, long circulating, drug delivery

2 ACS Paragon Plus Environment

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INTRODUCTION

In recent years, self-assembled nanoparticles of amphiphilic block copolymers have emerged as one of the most promising nanocarriers for various hydrophobic anticancer drugs. The well-defined core-shell structures allow encapsulation of the drugs in the core while the hydrophilic shell allows for increased water solubility and stability

1, 2

. Furthermore,

nanoparticles of appropriate size and surface properties may accumulate in tumor sites through the enhanced permeability and retention (EPR) effect resulting from abnormalities in tumor blood and lymphatic vasculature

3, 4

. Various self-assembled nanoparticles have been developed

for delivery of anticancer drugs 5-7. However, most of these have not shown beneficial effects in clinical trials 8. This might be attributed to the lack of stability of polymer micelles after dilution in vivo and low drug loading levels. Furthermore, many synthetic biodegradable copolymers upon erosion in vivo yield non-endogenous oligomers and monomers that might adversely interact with the surrounding tissue 9. To increase drug loading capacity and minimize the toxicity of the polymer carrier and its degradation products, appropriate architecture and composition of the block copolymers should be designed using biologically compatible components. Cholesterol is an important component of cell membranes involving in lipid organization, signal transduction, cell adhesion, and cell migration 10. Copolymers with cholesterol end-groups are interesting materials for various biomedical applications including serving as membranes for cell attachment and proliferation, forming the basis of polymeric scaffolds, and as materials with improved blood compatibility

11, 12

. However, most current amphiphilic polymer architectures

that contain cholesterol are conjugates or linear copolymers with only one or a few cholesterol


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molecules which resulted in formation of micelles which have low stability after dilution in vivo 6, 13, 14

. In addition, most of these cholesterol-containing delivery systems had a limited drug

loading capacity and fast drug release with premature burst release. This may result in the loss of a significant amount of drug from the nanocarriers in the circulation before they can reach tumor tissues 6, 13, 15. Recently, brush polymers have gained increasing interest in nanoscience due to their characteristic topological and large domain structures

16, 17

. These polymers have been utilized

for applications in drug delivery such as controlled drug release and targeted drug delivery in vitro

18-25

. For example, Grubbs and co-workers synthesized brush polymer drug conjugates

containing a polynorbornene backbone and polyethylene glycol (PEG) chains with UV-triggered drug release behavior

23

. Likewise, Zou et al. synthesized PEG-based pH-sensitive brush

polymer drug conjugates which showed well-shielded drug moieties and acid-triggered drug release 24. Brush polymers with numerous end-hydrophobic groups may allow high drug loading and strong shielding effects for anticancer drugs 22, 25. However, limited information is available on the in vivo behavior of nanoparticles prepared from the brush polymers and the interaction between the nanoparticles and tumor tissue. In this study, self-assembled nanoparticles were prepared from new amphiphilic cholesterolbased brush-like block copolymers comprised of polynorbonene bearing a cholesterol block and a polyethylene glycol (PEG) block (P(NBCh9-b-NBPEG)) (Scheme 1). The potential of the P(NBCh9-b-NBPEG) nanoparticles as a delivery system for doxorubicin (DOX), an anticancer drugs, whose clinical use is limited by its severe cardiotoxicity 26, 27, was evaluated in vitro and in vivo by measuring drug loading capacity, drug release, cellular uptake behavior, cytotoxicity, in vivo circulation time, biodistribution, and antitumor efficacy in tumor-bearing mice.


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Scheme 1. Self-assembly of cholesterol-based brush block copolymers in aqueous media.

EXPERIMENTAL SECTION

Materials Doxorubicin hydrochloride (DOX.HCl) and idarubicin hydrocholoride were purchased from Biotang Inc (Waltham, MA, USA). Pyrene was obtained from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Triethylamine (TEA) and dimethyl formamide (DMF) were purchased from Fisher Scientific (Boston, MA, USA). Penicillin-streptomycin, 0.25% (w/v) trypsine-0.03% (w/v) EDTA solution, RPMI 1640, and DMEM medium were purchased from American Type Culture Collection (Rockville, MD, USA). Human cervical cancer cells (Hela) and human lung


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cancer cell lines (A549) were purchased from the National Cancer Institute (Frederick, MD, USA). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Norcross, GA, USA). Draq5, 1,1′-Dioctaecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) and in vitro toxicology assay kits (MTT based) were obtained from Invitrogen (Carlsbad, CA, USA). Spectra/Pro membranes were purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA). All chemicals were analytical grade and used without purification.

Synthesis and purification of P(NBCh9-b-NBPEG) Synthesis of norbornene functionalized monomers, 5-{9-(cholesteryloxycarbonyl)nonyloxycarbonyl}-bicyclo[2.2.1]hept-2-ene (NBCh9) and methoxy polyethylene glycol (MPEG) (Mn = 2 Kg/mol) functionalized norbornene (NBPEG) are reported in our previous publications 28, 29

. All block copolymers were synthesized using ring opening metathesis polymerization

(ROMP), the synthesis details are described in previous publication 29. Preparation and characterization of self-assembled nanoparticles and DOX-NPs Blank self-assembled nanoparticles were prepared by a dialysis method. Briefly, the P(NBCh9-b-NBPEG) was dissolved in DMF with the aid of sonication. The solution was then transferred to a dialysis bag (MWCO: 10,000 Da) and dialyzed against distilled water for 48 h. To prepare DOX-loaded P(NBCh9-b-NBPEG) nanoparticles (DOX-NPs), DOX.HCl was first dissolved in DMF containing 2 equivalents of TEA and stirred overnight in the dark to form hydrophobic DOX and TEA.HCl. Each copolymer was added, and then the solution was stirred for another hour in the dark. The solution was then dialyzed against distilled water for 48 h to remove free DOX and solvents. The precipitated DOX was removed by centrifugation at 8000


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rpm for 10 min, followed by filtration through 0.45 µm syringe. The final products were collected after lyophilization. The average particle size, size distribution and zeta-potential of the DOX-NPs (1 mg/mL) were measured using a dynamic light scattering (DLS) instrument (Malvern Zetasizer). The morphologies of DOX-NPs were imaged by TEM (FEI TecnaiBiotwin, Eindhoven, Netherlands). Specimens were prepared by adding a suspension of the nanoparticles dropwise to a Formvar/carbon film grid followed by air-drying. The Critical Aggregation Concentration (CAC) of the P(NBCh9-b-NBPEG) copolymers was determined by fluorescence measurements using pyrene as a hydrophobic probe. Pyrene solutions (3x10-4 M) in acetone were added to glass tubes and were subsequently evaporated to remove the organic solvent. Various concentrations of copolymer solutions (10 mL) were added to the tubes and sonicated for 3 h at 60 °C to equilibrate the pyrene and the nanoparticles. The copolymer concentrations ranged from 0.005 to 0.5 mg/mL and the final concentration of pyrene was 6.0x10-7 M. The emission spectra of pyrene were recorded from 350-450 nm using a fluorescence spectrophotometer (Perkin Elmer LS-55B, USA) at an excitation wavelength of 336 nm. For the measurement of the intensity ratio of the first (374.5 nm) and the third highest energy bands (386 nm) in the pyrene emission spectra, the slit opening for the excitation and emission spectra was set at 5 nm. The amount of DOX-loaded into nanoparticles was determined by a colorimetric method. The lyophilized DOX-NPs (0.5 mg) were dissolved in DMF (2 mL) to obtain clear solutions. The absorbance at 480 nm was detected with a UV-VIS spectrophotometer (Shimadzu, Japan). DOX solutions were prepared at various concentrations and the absorbance at 480 nm was measured to


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generate a calibration curve for calculating drug-loading content. The drug-loading content (DLC) and encapsulation efficiency (EE) were calculated using the following equations: DLC =

EE =

Amount of DOX in nanopartic les × 100 Amount of DOX - loaded nanopartic les

Amount of DOX in nanopartic les × 100 Amount of DOX used for nanopartic le preparatio n

Stability and in vitro release of DOX from the P(NBCh9-b-NBMPEG) nanoparticles Lyophilized DOX-NPs (1 mg/mL) were suspended in the serum-containing phosphatebuffered saline (PBS) solution (50% FBS), followed by sonication for 10 min and filtration through a 0.45 µm syringe filter membrane. The particle size of the nanoparticles stored at 4 ºC was monitored over the storage time using a Malvern Zetasizer. In vitro release of DOX from the nanoparticles was studied using a dialysis method. Briefly, lyophilized DOX-NPs (6 mg) were suspended in 3 mL of PBS (0.01 M, pH 7.4), followed by sonication for 10 min to yield an optically clear suspension. The suspensions were introduced into 5 mL-dialyzers (MWCO: 10,000 Da) and immersed in 20 mL of PBS containing Tween 80 (0.1% w/v) at 37 °C in a shaking bath at 100 rpm. At selected time intervals, aliquots (10 mL) were removed from the dissolution medium and an equivalent volume of fresh medium was compensated. The concentration of DOX was immediately measured by UV at 480 nm. The percentage of DOX released was calculated based on a standard curve of known DOX concentrations.


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Cytotoxicity of DOX-NPs HeLa cells (7500 cells/well) were seeded on 96-well plates and cultured in 200 µL of DMEM supplemented with 10% FBS, 1% antibiotics, and 1% L-glutamine for 24 h at 37 °C and 5% CO2. After incubation, various concentrations of the blank nanoparticles (0.2-1 mg/mL), DOXNPs, and free DOX (1-50 µg/mL of DOX equivalents) dissolved in DMEM without supplements were added. After 24 h of incubation with free DOX and DOX-NPs, and 48 h with blank nanoparticles,

cytotoxicity

was

determined

using

3-[4,5-dimethylthiazol-2-yl]-3,5-

diphenyltetrazolium bromide dye (MTT dye, final concentration of 0.5 mg/mL) uptake at 540 nm on a microplate reader (Tecan group Ltd., Männedorf, Switzerland).

Intracellular uptake of DOX-NPs To observe the cellular uptake, HeLa cells were seeded at a density of 1.0 × 105 cells/well in an 8-well chamber of a Lab-Tek II chamber slide and pre-incubated for 24 h at 37 °C and 5 % CO2. Serum-free DMEM containing free DOX and DOX-NPs at equivalent doses (25 µg/mL) was added to each well, followed by incubation for 2 h at 37 °C. To investigate time-dependent uptake behavior of DOX-NPs from 2 h to 24 h, A549 cells were seeded at a density of 1.0 × 105 cells/well and treated with DOX-NPs at an equivalent DOX dose of 25 µg/mL. The cells were then rinsed with PBS, stained with 10 µM Draq5 and fixed with 4% formaldehyde solution for 10 min. Cover glasses were then placed on glass slides. The cellular uptake of free DOX and DOX-NPs was imaged by confocal laser scanning microscopy (Leica, England) at an excitation wavelength of 488 nm for DOX and 633 nm for Draq5. To quantify cellular uptake, HeLa cells (5 x 105 cells/well) in 0.5 mL were grown on a 24-well plate at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. Serum-free DMEM containing


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free DOX and DOX-NPs at equivalent doses (25 µg/mL) was added to the cells which were subsequently incubated for 2 h. The cells were then washed three times with PBS, harvested by trypsinization and transferred into Fluorescence Activated Cell Sorter (FACS) tubes. All samples were analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA) to determine cellular internalization. Fluorescence measurements of intracellular DOX were performed in the FL2 channel.

In vivo circulation time and tissue distribution of DOX-NPs All animal studies were performed according to an approved protocol by Institutional Animal Care and Use Committee (IACUC) at the University of Connecticut. For in vivo determination of the circulation time, in-house bred Balb/c mice at 6-8 weeks old were randomly divided into two groups of 5 mice per group and were injected intravenously (i.v) with free DOX and DOX-NPs at a single dose of 5 mg/kg of equivalent DOX. At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h post injection, blood (150 µL) was collected via facial vein into heparinized tubes for two time points and then mice were sacrificed for blood collection of next time point by cardiac puncture. Each group of mice had three time points for blood collection. Plasma was separated by centrifugation (3000 rpm, 10 min, 4 ºC) and stored at -80 ºC until analyzed. For the tissue distribution study, tumor-bearing severe combined immunodeficient (SCID) between 6 and 8 weeks-old mice were inoculated with 0.1 mL PBS containing 2 x 106 human lung cancer cells (A549) into the right flank. Four weeks after tumor implantation, free DOX and DOX-NPs (100 µL) at 2.5 mg/kg of equivalent DOX were injected into the tail-vein. Twenty four hours after injection, mice were sacrificed for blood and organ collection including tumor,


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liver, spleen, kidney, lung, and heart. Tissues were then weighed and homogenized in the lysis buffer (0.1 M Tris-HCl, 2 mM EDTA, 0.1% Triton X-100) using a probe sonicator (Qsonica LLC, CT). The lysate of each tissue was centrifuged at 14,000 rpm for 20 min at 4 ºC. The blood was centrifuged at 3000 rpm for 10 min at 4 ºC. Sample extraction was performed using a reported procedure with some modification

30

.

Briefly, plasma (50 µL) or tissue (100 µL) was spiked with 100 µL of idarubicin (1 µg/mL) as an internal standard (IS). After adding 100 µL of 1.0 M Tris buffer solution, the extraction of DOX and IS was performed twice by adding 2.5 mL of a choloroform/methanol (75:25, v/v) mixture and vortexing for 5 min. After centrifugation at 8000 rpm for 10 min, the samples in the organic phase were collected evaporated to dryness under a flow of nitrogen at ambient temperature. Dry residues from plasma and tissues were dissolved in 100 µL methanol followed by centrifugation to remove any precipitate. The resulting supernatant was analyzed using an HPLC apparatus equipped with an autosampler and a fluorescence detector (Shimadzu, Kyoto, Japan). A 20-µl sample was injected onto a C18 column (Kinetex 5 µm, 150 x 4.6 mm, Phenomenex, CA) using a mixture of 0.05 M sodium acetate (pH 4.0) and acetonitrile (73.5:26.5) as the mobile phase. The flow rate was 1 mL/min and the signals were monitored by fluorescence detection at excitation and emission wavelength (Ex/Em) of 480/558 nm.

In vivo imaging of the P(NBCh9-b-NBPEG) nanoparticles The biodistribution of P(NBCh9-b-NBPEG) self-assembled nanoparticles was assessed by in-vivo near-infrared (NIR) imaging. A NIR fluorophore, DiR, was loaded into the nanoparticles by a dialysis method as described in Section 2.5. Briefly, the P(NBCh9-b-NBPEG) (10 mg) and DiR (0.6 mg) were dissolved in DMF (3 mL). The resulting solution was dialyzed against 11 ACS Paragon Plus Environment

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distilled water for 48 h, and then filtered through a 0.45 µm membrane before lyophilization. The loading content of DiR was determined spectrophotometrically at a wavelength of 750 nm. The tumor models were established by subcutaneous injection of A549 cells (2 × 106 cells in 100 µL of PBS) into the flank of male SCID mice. When the tumor reached an acceptable size, the mice were treated with the DiR-loaded self-assembled nanoparticles (5 µg/kg of equivalent DiR) via tail-vein injection. Whole body images were obtained at 1, 5, and 24 h after injection using the Maestro in vivo imaging system (Cambridge Research & Instrumentation, Inc., Woburn, MA, USA). Images of various organs, including heart, kidney, liver, spleen, lung, and tumor, were also obtained after sacrifice of the mice 24 h after injection.

Evaluation of antitumor activity and toxicity The antitumor efficacy of DOX and DOX-NPs was evaluated in SCID mice inoculated subcutaneously in the right flank with 0.1 mL PBS containing 2 × 106 A549 cells. When the tumors grew to approximately 20-30 mm3 (9 days after tumor implantation), the mice were randomly divided into three groups (n=5-6), and this day was designated as day 0. The mice were injected intravenously twice a week via tail vein with saline (control), free DOX and DOXNPs (100 µL) at 2.5 mg/kg of equivalent DOX. Antitumor activity was evaluated in terms of tumor volume which was calculated using the following equation: Tumor volume (mm3) = width2 × length/2. The body weight was measured simultaneously as an indicator of the systemic toxicity. A separate study was conducted to assess the liver toxicity of the treatments. SCID mice bearing A549 tumors were administered free DOX, DOX-NPs or blank NPs twice a week via tail vein injection at 1 mg/kg of equivalent DOX. The reduced dose was to ensure the survival of the free 12 ACS Paragon Plus Environment

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DOX group (n=3-5). Blood, liver and spleen were collected 45 days after the treatments. Liver function was analyzed based on the alanine transaminase (ALT) plasma test. Liver tissues were fixed by 10% formalin and embedded in paraffin. Tissue slices were subjected to hematoxylin and eosin (H&E) staining. Statistical analysis Data were expressed as mean ± standard deviation. The statistical significance of difference between experimental and control groups was determined using a student’s t-test. A probability (p) of less than 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Preparation and characterization of the P(NBCh9-b-NBPEG) nanoparticles and DOX-NPs Three amphiphilic P(NBCh9-b-NBPEG) brush-like copolymers of MW 400 kDa (400 kDa-50) and 600 kDa (600 kDa-75 and 600 kDa-180) with varied amounts of cholesterol monomer (6-18% w/w) were synthesized 29. Table 1 summarizes the molecular characterization of the brush-like block copolymers. Table 1. Molecular characterization of P(NBCh9)x-b-(NBPEG)y Mn[b] kg/mol Polymer[a]

Weight %[c] PDI

theoretical

GPC

NBCh9

NBMPEG

400 kDa-50

P(NBCh9)50-b-(NBMPEG)170

400

126

1.24

7.7

92.3

600 kDa-75


600

216

1.16

5.7

94.3

600

118

1.16

18

82

600 kDa-180



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[a] number in subscript represents theoretical degree of polymerization, 400 kDa-50 contained ~8% w/w of cholesterol content while 600 kDa-75 and 600 kDa-180 contained ~6% and 18% w/w of cholesterol content, respectively; [b] molecular weight determined using GPC, note: observed molecular weights are lower than theoretically calculated due to the brush architecture; [c] weight percent of monomer is determined by 1H-NMR integration of peaks for cholesterol and PEG side chains, respectively.

These copolymers readily self-assembled in aqueous solution to form nanoparticles due to the hydrophobic interaction between the cholesterol moieties. The self-assembly behavior of the P(NBCh9-b-NBPEG) brush-like copolymers in aqueous solution was characterized by measuring the CAC using pyrene as a hydrophobic fluorescence probe 31. The CAC, which is the threshold concentration of self-aggregation formation, can be determined by the intensity ratio for the two fluorescence emission peaks (I374/I385) of pyrene (Fig. 1A). A linear decrease was observed with an increase in the P(NBCh9-b-NBPEG) concentration. The CAC value of the 400 kDa-50 copolymer was lower than that either of the 600 kDa copolymers containing cholesterol (Table 2) indicating greater stability of 400 kDa-50 copolymer assembled nanoparticles. At the same molecular weight, the 600 kDa copolymer with the higher cholesterol content (600 kDa180) had a greater CAC value than the 600 kDa copolymer with the lower cholesterol content (600 kDa-75), indicating a stronger hydrophobic interaction in the inner core of the P(NBCh9-bNBPEG) nanoparticles with higher cholesterol content. These CAC values were in the typical range of PEG-based block copolymers 6, 32, suggesting that the P(NBCh9-b-NBPEG) copolymers may circulate as self-assembled nanoparticles in vivo for an extended period of time. These


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nanoparticles likely have a core/shell structure bearing a hydrophobic cholesterol inner core that could serve as a reservoir for hydrophobic drugs. The hydrophobic DOX was successfully encapsulated into the P(NBCh9-b-NBPEG) selfassembled nanoparticles using a dialysis method. With a DOX feed ratio of 25% (w/w), DLC in the 400 kDa-50 nanoparticles was about 22.1% with EE of 88.4%. However, the drug loading was affected by the molecular weight of the copolymers. Despite having higher cholesterol content than the 400 kDa-50 copolymer, the DLC and EE values were lower in the 600 kDa-180 copolymer (Table 2). This could be attributed to the change in balance between the hydrophilic and hydrophobic segments of the 600 kDa-180 resulting in different self-assembly behavior of the nanoparticles 7. The 600 kDa-180 copolymer with the higher cholesterol content had slightly greater DLC and EE values than the 600 kDa-75. The particle size and morphology of blank P(NBCh9-b-NBMPEG) nanoparticles and DOXNPs were measured by DLS and TEM, respectively. The average particle size of blank P(NBCh9-b-NBPEG) nanoparticles ranged from 124 to 179 nm with a narrow size distribution (PDI less than 0.1) (Table 2). The average size of the nanoparticles increased with increasing molecular size from 400 kDa to 600 kDa. At the same MW of 600 kDa, the nanoparticle size decreased with increasing cholesterol content due to the formation of a more compact hydrophobic inner core. Physical encapsulation of DOX increased particle size and polydispersity of the nanoparticles (Table 2). The TEM images showed that the nanoparticles with and without DOX loading were spherical in shape with diameters of 100-160 nm for blank nanoparticles and 120-170 nm for DOX-NPs (Fig. 1B). The particle sizes determined by TEM were slightly smaller than the size measured by DLS. The 400 kDa-50, 600 kDa-75, and 600 kDa-180 nanoparticles were negatively charged at their surface, as reflected in the zeta-potential


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values of -18.8, -14.9, and -6.9 mV for 400 kDa-50, 600 kDa-75, and 600 kDa-180, respectively (Table 2). This negative charge might be due to the pendant carbonyl group of the cholesterol derivative. The decrease of negative charge when increasing molecular weight of the copolymers might be attributed to the increase in the neutral charged PEG segments that occupied the negative site on the surface of nanoparticles. The physical loading of DOX significantly decreased the negative surface charge of the nanoparticles. The ideal size for self-assembled nanoparticles to accumulate in tumor tissue via the EPR effect is less than 200 nm 33. Therefore, these nanoparticles have sizes and surface charges that are suitable for tumor targeting via the EPR effect. (A)


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(B)

Figure 1. (A) Representative emission spectra of pyrene in 400 kDa-50 copolymer and the plot of intensity ratio as a function of the logarithm of concentration; an arrow from a to m represents a range of concentration from high to low. (B) TEM images of 400 kDa-50 (a), 600 kDa-75 (b), 600 kDa-180 (c), 400 kDa-50-DOX (d), 600 kDa-75-DOX (e), and 600 kDa-180-DOX (f). Nanoparticles were negatively stained with 1% phosphoric tungstic acid.

Table 2. Characterization of P(NBCh9-b-NBMPEG) and DOX-NPs DOX.HCl DLC feed ratio (%) (%)

400 kDa-50

5.4 ± 0.2

124.2 ±5.2

0.09

Zeta potential (mV) -18.8 ± 0.7

600 kDa-75

21.1 ± 0.5

179.1 ± 3.2

0.05

- 14.9 ± 0.1

600 kDa-180

15.9 ± 0.3

144.4 ± 2.1

0.04

- 6.9 ± 0.8

Samples

CAC (mg/L)

EE (%)

Average size ( nm)

PDI

400 kDa-50-DOX

25

22.1

88.4

138.3 ± 4.3

0.20

-3.1 ± 0.2

600 kDa-75-DOX

25

17.2

68.8

197.5 ± 1.5

0.16

-1.6 ± 0.2


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189.4 ± 4.1

0.15

-0.7 ± 0.1

DLC: drug loading content EE: encapsulation efficiency

Stability of DOX-NPs and in vitro DOX release The stability of nanocarriers has been reported to be a key to a successful drug delivery system 34

. Nanocarriers that are administered through the intravenous route encounter interactions with

serum proteins, the major species in the blood components that may alter the stability and the tissue distribution of the carriers

35

. Thus, if the nanocarriers maintain their integrity when

encountering serum proteins, efficient drug delivery to the target tissue can be expected. Taking into account that the P(NBCh9-b-NBPEG) nanoparticles are intended for i.v administration, we evaluated their stability in a medium containing serum. The stability of the DOX-NPs was investigated using DLS to monitor the change in the size of the nanoparticles. The average particle size of DOX-loaded 400 kDa-50, 600 kDa-75, and 600 kDa-180 did not change significantly after 1 week storage at 4 ºC in 50% FBS (Fig. 2A), and no precipitation or aggregation was observed. Two factors may contribute to the excellent stability of the P(NBCh9b-NBPEG) nanoparticles: the hydrophobic interaction among cholesterol ends in the side chain of one copolymer and the strengthened interaction by entanglement of cholesterol segments from different brush copolymers 18. Such excellent stability of the brush copolymer nanoparticles will be beneficial for systemic drug delivery of the nanoparticles with the first in vivo challenge of dilution in plasma. To further assess the potential of the P(NBCh9-b-NBPEG) nanoparticles as drug carriers, a DOX release test was performed in PBS (pH 7.4, 37 ºC) containing 0.1% Tween 80 using a


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dialysis method. The P(NBCh9-b-NBPEG) nanoparticles released DOX in a sustained release pattern without an initial burst release, with approximately 2% release after the first day, and about 25% for 400 kDa-50-DOX and 17% for 600 kDa-180-DOX nanoparticles after 13 days (Fig. 2B). The drug release from 400 kDa-50 nanoparticles was relatively faster than that from 600 kDa nanoparticles. Despite the significant difference in cholesterol content, DOX release from 600 kDa-75 and 600 kDa-180 was similar. Core forming materials have been reported to play an important role in drug release; the strong interaction between hydrophobic drugs and the hydrophobic core often results in the slow release of drug from the nanoparticles 36. It has been reported that the rigid hydrophobic structure and self-associative or liquid crystalline character of cholesterol-containing hydrophobic blocks promote drug encapsulation and stability of paclitaxel-loaded micelles 6. Therefore, the sustained release of DOX from the nanoparticles with the absence of a burst release under the simulated physiological condition might be due to a strong interaction of hydrophobic DOX with numerous hydrophobic cholesterol moieties in the core of the nanoparticles, and the liquid crystalline character of cholesterol-containing blocks. The same trend of drug release was also observed in paclitaxel-loaded PEG-b-PLA stereoblock copolymer assemblies which showed less than 20% drug release within 30 days without significant burst release

37

, and in paclitaxel-loaded linear dendritic amphiphilic polymers with

6% drug released for 48 h and less than 20% drug release within 1 week 38. The sustained release behavior with the absence of an initial burst release effect is useful for delivery of anticancer drugs, in which limited amounts of the drug are released in the blood stream until the nanoparticles reach the tumor tissues where the drug release may be elevated inside cells due to the degradation of the copolymers in the presence of enzymes.


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(A)

(B)


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Figure 2. Stability of DOX-loaded 400 kDa-50, 600 kDa-75 and 600 kDa-180 nanoparticles in PBS/FBS (1:1) stored at 4 °C (A), and the release profiles of DOX-loaded 400 kDa-50, 600 kDa-75, and 600 kDa-180 nanoparticles in PBS (pH 7.4) containing 0.1% Tween 80 at 100 rpm and 37 °C (B). n=3.

Cellular uptake and in vitro cytotoxicity The uptake of nanoparticles by cancer cells attributed largely to the therapeutic effects of drug-loaded nanoparticles

39

. Confocal laser scanning microscopy (CLSM) was employed to

investigate the cellular uptake behavior of DOX-NPs in HeLa cells in comparison with that of free DOX. Since DOX itself is fluorescent, it was used directly to investigate cellular uptake without additional markers in the nanoparticles. The nuclei were stained with Draq-5 and changed to a blue color by the confocal software. As shown in Fig. 3A, after 2 h of incubation, free DOX (25 µg/mL)-treated cells presented a strong red color in the nuclei, indicating that free DOX was quickly transported to cytoplasm and diffused to nuclei. Due to its fast cellular uptake, the use of free DOX in cancer therapy may cause severe toxicity since it can diffuse rapidly through the body in both healthy and diseased tissues. On the contrary, the intracellular distribution of DOX in the cells incubated with DOX-NPs was significantly different which is likely through an endocytosis pathway 40. After 2 h of incubation, intense DOX fluorescence was observed in the cytoplasm rather than in cell nuclei, implying that DOX-NPs could be effectively internalized by HeLa cells. To compare the cellular uptake of free DOX and DOX-NPs, flow cytometry analysis was performed. Because the fluorescence intensity is proportional to the amount of DOX internalized by the cells, the mean fluorescence intensity was used to make a quantitative comparison of cellular uptake (Fig. 3B). While the difference in fluorescence intensity of the cells treated with DOX-loaded 400 kDa-50, 600 kDa-75 and 600 kDa-180 nanoparticles was negligible, the cells treated with free DOX showed greater fluorescence 21 ACS Paragon Plus Environment

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intensity than those treated with DOX-NPs at the same equivalent DOX concentration and incubation time. The cellular uptake behavior of DOX-NPs (400 kDA-50) was also investigated in A549 cells at different time points ranging from 2 h to 24 h. At the same setting, the red signal of DOX inside the cells increased with increasing incubation time (Fig. 3C). DOX-NPs mainly localized in the cytoplasm after 2 h and 4 h of incubation. When increasing the incubation time to 8 h, the DOX signal was visible in both cytoplasm and in the cell nuclei. At 24 h after incubation, a strong red signal could be seen in the cell nuclei, indicating that DOX was efficiently released from the nanoparticles and entered the cell nucleus.


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Figure 3. CLSM images of HeLa cells incubated with free DOX and DOX-loaded 400 kDa-50, 600 kDa-75, and 600 kDa-180 nanoparticles for 2 h at 25 µg/mL DOX equivalence (A). Mean fluorescence determined by FACS of HeLa cells incubated with free DOX and DOX-loaded 400 kDa-50, 600 kDa-75, and 600 kDa-180 nanoparticles for 2 h at 25 µg/mL DOX equivalence (B) (n=3). *P < 0.05. Uptake of DOX-loaded 400 kDa-50 (25 µg/mL DOX) in A549 cells at different time points from 2 h to 24 h, scale bars are 10 µm (C).

We next investigated the in vitro cytotoxicity of free DOX, blank P(NBCh9-b-NBPEG) nanoparticles, and DOX-NPs in HeLa cells using the MTT assay. Fig. 4A-B show the viability of HeLa cells treated with blank P(NBCh9-b-NBPEG) nanoparticles, free DOX, and DOX-NPs at different nanoparticle and equivalent DOX concentrations. Blank P(NBCh9-b-NBPEG) nanoparticles showed negligible toxicity to HeLa cells even at a concentration of 1 mg/mL with a cell viability of ≥ 90% 48 h after treatment (Fig. 4A), revealing the low toxicity and good compatibility of the P(NBCh9-b-NBPEG) nanoparticles to the cells. On the other hand, free


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DOX and DOX-NPs dose-dependently (1-50 µg/mL DOX) decreased the cell viability to 5-60% after 24 h of incubation (Fig. 4B). By increasing DOX concentration from 1 µg/mL up to 50 µg/mL, free DOX drastically decreased the cell viability while the DOX-NPs gradually decreased the cell viability. At all DOX concentrations, free DOX showed significantly higher cytotoxicity than DOX-NPs. To induce cytotoxicity, DOX-NPs taken up by cells must release DOX in the free form so that DOX can enter the nucleus and exert its activity. The difference in the cytotoxicity between free DOX and DOX-NPs was possibly due to the difference in the uptake pathway of free DOX and DOX-NPs, and the sustained-release property of DOX-NPs. At similar DOX levels, the cytotoxicity of DOX-loaded 400 kDa-50 was significantly higher than that of DOX-loaded 600 kDa-75, showing the viability value of about 40% for DOX-loaded 400 kDa-50, and 70% for DOX-loaded 600 kDa-75 at 10 µg/mL DOX. However, the cytotoxicity of DOX-loaded 400 kDa-50 and DOX-loaded 600 kDa-180 was not significantly different except for 25 µg/mL DOX. The mechanism of greater cytotoxicty by DOX-loaded 400 kDa-50 was not entirely discerned as there was no significant difference in the drug release and cellular uptake behavior of DOX-NPs from three block copolymers. Collectively, the 400 kDa-50 copolymer provided the best results in terms of particle size, drug loading capacity, drug release, and cytotoxicity; thus, it was subsequently used in in vivo studies.


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(A)

(B)

Figure 4. Viability of HeLa cells incubated with different concentrations of blank 400 kDa-50, 600 kDa-75, and 600 kDa-180 nanoparticles for 48 h (A), and IC50 of free DOX, DOX-loaded 400 kDa-50, 600 kDa-75 and 600 kDa-180 nanoparticles in Hela cells for 24 h (B). n=6.


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In vivo circulation time and biodistribution of DOX-NPs To gain insight into the in vivo behavior of DOX-NPs, the circulation time of these nanoparticles was evaluated in mice and compared with that of free DOX. Fig. 5A shows plasma concentration-time profiles of DOX after i.v injection of free DOX and DOX-NPs at a dose of 5 mg/kg. Plasma concentration of free DOX was 75.6 ± 5 µg/mL at 5 min post-injection but dramatically decreased to 5.3 ± 0.3 µg/mL at 1 h post-injection and to 1.4 ± 0.1 µg/mL at 4 h post-injection, indicating rapid elimination of free DOX from the circulation. DOX was injected intravenously to the mice in its hydrophilic hydrochloride salt form which was quickly distributed out of the blood into tissues

39

, and rapidly eliminated from the blood by renal

filtration due to its limited protein binding 41. In contrast, DOX-NPs displayed markedly delayed blood clearance, showing plasma concentration of DOX of 88.0 ± 5.2 µg/mL at 5 min postinjection, 68.8 ± 7.3 µg/mL at 1 h and 45.4 ± 7.1 µg/mL at 4 h post-injection. Notably, 24 h after administration of the DOX-NPs, the DOX plasma concentration was still 12.6 ± 1.9 µg/mL, whereas it was almost undetectable for the free DOX at this time point. As a result of the rapid clearance of free DOX, the drug found in the serum is believed to be encapsulated in the nanoparticles. The results indicated that the circulation time of DOX was significant increased by encapsulation into the P(NBCh9-b-NBPEG) nanoparticles which might be attributed to “stealth” behavior of the hydrophilic PEG shell and the excellent stability of DOX-NPs 41,42, 43. The DOXNPs with long circulation times are likely to preferentially accumulate in tumor tissues. Motivated by this finding, we next investigated the tissue distribution of DOX-NPs in tumorbearing SCID mice. DOX concentrations were measured in blood, the heart, liver, spleen, lung, kidney and tumor 24 h after administration of free DOX and DOX-NPs at a dose of 2.5 mg/kg. As seen in Fig. 5B, the concentration of DOX following administration of free DOX was greatest


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in the liver and lowest in the tumor. In contrast, the concentration of DOX after administration of DOX-NPs was greatest in blood, and the tumor concentration was significantly greater than observed following administration of free DOX. The results suggested that while free DOX was widely distributed in the body, it was mainly captured in the host defense and metabolic organs such as liver, spleen, lung, and kidney and metabolized or rapid excreted by these organs leading to low drug concentration in tumor. Notably, the 6.5-fold lower concentration in liver and 2.3fold higher concentration in tumor of DOX were achieved 24 h after administration of DOX-NPs compared to that of free DOX. The DOX plasma level following administration of DOX-NPs was 11-fold greater than after free DOX administration at the same dose. This high drug concentration in plasma may contribute to further accumulation of the DOX-NPs in tumor tissue with increasing blood circulation time of the nanoparticles. Furthermore, the DOX-NPs decreased the drug concentration in the heart by 3.9-fold compared with free drug. Thus, the P(NBCh9-b-NBPEG) nanoparticles might significantly reduce the cardiotoxicity of DOX since cardiomyopathy is the dose-limiting side effect of free DOX

44

. Meanwhile, the nanoparticles

also decreased the DOX level in the lungs by 10 fold compared with free DOX, which might depress the damage to the lung and increase its biosafety. The results confirmed that DOX-NPs with its PEG shielding effect and excellent stability exhibited longer blood circulation, less uptake by the MPS and higher tumor accumulation than the free drug.


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(A)

(B)

Figure 5. In vivo circulation time of free DOX and DOX-NPs (A) and tissue distribution of DOX and DOXNPs in tumor-bearing SCID mice after 24 h injection (B). Data are presented as mean ±SD (n = 5) *P < 0.05, **P < 0.01.


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In vivo fluorescence images of P(NBCh9-b-NBPEG) nanoparticles in tumor-bearing mice To further assess the biodistribution of the P(NBCh9-b-NBPEG) nanoparticles, in vivo fluorescence images of the P(NBCh9-b-NBPEG) nanoparticles in tumor-bearing SCID mice were obtained using non-invasive near-infrared fluorescence (NIRF) imaging. A hydrophobic NIRF molecule, DiR was used as a model drug for encapsulation into 400 kDa-50 nanoparticles (5% w/w) and emits strong NIR fluorescence that is minimally absorbed by water or hemoglobin. This enables less interference from background fluorescence during non-invasive animal imaging

45

. We investigated the stability of DiR-loaded 400 kDa-50 nanoparticles in

simulated physiological condition, and the results showed less than 2% DiR release within 24 h without any premature release of the dye for 5 h. The DiR-loaded P(NBCh9-b-NBPEG) nanoparticles were administrated to the A549 tumor-bearing SCID mice via tail-vein injection. At 1 h post-injection, fluorescence could be detected throughout the entire animal, and a strong fluorescent signal was visualized in the liver, indicating that the nanoparticles accumulated primarily in the liver (Fig. 6A). The intense whole-body fluorescence was continuously observed at 5 h post-injection, indicating the long circulation time of the DiR-loaded P(NBCh9-b-NBPEG) nanoparticles. Moreover, the contrast of the DiR signal in the tumor compared to the surrounding tissues of the animal was already apparent 5 h post-injection of the P(NBCh9-b-NBPEG) nanoparticles. After 24 h, a strong red signal was observed in tumor tissues and only a low signal was observed in the liver. At 24 h post-injection, the mice were sacrificed and the major organs were isolated to analyze the tissue distribution of the DiR-loaded nanoparticles. As shown in Fig. 6B, the highest NIRF intensity was observed in tumor tissues, while the signal intensities were lower for other tissues, with a particularly low fluorescence signal in the heart. The result showed discrepancy between fluorescence imaging and quantitative biodistribution by HPLC.


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The HPLC results showed that liver and spleen were major accumulation sites while fluorescence imaging indicated that tumor accumulation was predominant. The same trend was also observed by Liu et al. 46 and Hollis et al. 47. This discrepancy was because the fluorescence signals in both liver and spleen are greatly attenuated compared with those in tumor due to intrinsic tissue absorption and light scattering 46. The results suggest that careful attention must be paid while using fluorescence imaging for biodistribution studies of drug delivery systems. (A)

(B)


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Figure 6. In vivo fluorescence images of DiR-loaded nanoparticles in tumor-bearing SCID mice at 1 h, 5 h, 24 h post tail-vein injection (A), and Ex vivo fluorescence images of organs and tumors in SCID mice 24 h after injection (B).

In vivo antitumor efficacy The antitumor efficacy of DOX-NPs was studied in SCID mice bearing lung cancer A549 xenografts. Fig. 7 shows the changes in tumor volume, body weights, and survival rates of the mice treated with free DOX and DOX-NPs at 2.5 mg/kg DOX. The tumor volumes in the control group (saline) and the group treated with DOX-NPs slowly increased to about 230.7 ± 98.2 mm3 and 176.6 ± 44.0 mm3 within 24 days, respectively (Fig. 7A). Although tumor growth was inhibited by free DOX treatment, the body weight of this group of animals dramatically decreased compared to the control and DOX-NPs treated groups (Fig. 7C), suggesting that severe toxicity was induced by free DOX at the given dose. Eventually, all the animals in the DOX group on day 11 had to be terminated for humanitarian reasons. Termination of animals in the free DOX group at day 7 or day 12 post-treatment due to serious systemic toxicity caused by free DOX has also been done by other researchers such as Gou et al.48 and Kiziltepe et al.49. On the contrary, the treatment with DOX-NPs appeared to be well-tolerated; the animals exhibited almost no decrease in body weight and improved survival rate compared to the control and free DOX group. The result demonstrated greatly reduced toxicity of DOX when incorporated into the P(NBCh9-b-NBPEG) nanoparticles. After 24 days, the tumor volume of the control group rapidly increased and reached 883.4 ± 165.7 mm3 at day 52. In contrast, the tumor volume of the group treated with DOX-NPs slightly increased to 224.8 ± 84.7 mm3 and began to decrease after 38 day injection, and was further reduced to 130 ± 102.0 mm3 at day 52, indicating that the treatment group had 31 ACS Paragon Plus Environment

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significantly lower mean tumor volume (p < 0.01) than the control group. In addition, compared to the initial tumor volume (approximately 20 mm3), the tumor volume barely increased in the DOX-NPs treated mice, indicating that DOX-NPs effectively suppressed tumor growth. All mice were sacrificed and tumors were excised after 52 days of treatment. Fig. 7B shows the representative photographs of each group at the end of the experiment. The tumor size of the control group was obviously larger than that of the treatment groups, which was consistent with the results of the relative tumor volume measurements. These results demonstrated that compared to free DOX, DOX-NPs had significantly increased in vivo safety and exerted efficient therapeutic activity in animal models. To evaluate liver toxicity, Alanine Aminotransferase (ALT) level was measured in the tumor bearing SCID mice treated with blank nanoparticles, free DOX or DOX-NPs in comparison with the ALT level of healthy mice and tumor bearing mice injected with saline. Tumor bearing mice treated with free DOX showed increased ALT values in plasma (mean value 146.8 ± 4.946 IU/mL) which is indicative of early signs of liver damage. On the contrary, no alteration of ALT plasma levels was found in the DOX-NPs and blank nanoparticle- treated groups (mean value 17.30 IU/mL) after 45 days of treatment (Figure 8), indicating no liver toxicity. Liver histology revealed moderate glycogen depletion in free DOX group while no tissue damage was identified in DOX-NP and blank NP groups.


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Figure 7. Antitumor efficacy of DOX-NPs in tumor-bearing SCID mice including tumor volume (A), photo of tumor tissues from mice without treatment (a) and treated with DOX-NPs (b)at the end of the study (B), body weight change (C), and survival rate(D). Data are presented as mean ±SD (n = 5), **P < 0.01.


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200

150

ALT (IU/L)

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50

0

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Saline

Blank NPs Free DOX DOX-NPs

Figure 8. ALT plasma levels of healthy mice and the tumor bearing mice treated with saline, blank NPs, free DOX or DOX-NPs. ALT is expressed as international units (IU) per liter. Data are reported as mean ± SD.

CONCLUSION

In this study, self-assembled nanoparticles formed from cholesterol-based brush-like block copolymers were successfully prepared for encapsulation of the hydrophobic drug DOX. The DOX-NPs displayed a spherical shape with an average particle diameter below 200 nm, high drug loading capacity, excellent stability in a serum-containing medium, and a slow release profile. In addition, DOX-NPs showed efficient cellular uptake and dose-dependent cytotoxicity in HeLa cells, while blank nanoparticles showed no toxicity. Furthermore, the nanoparticles significantly increased the duration of the drug in the circulation and decreased cardiac accumulation of DOX. The in vivo studies on A549 lung tumor-bearing SCID mice demonstrated 34 ACS Paragon Plus Environment

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that DOX-NPs had greater tumor accumulation with significantly reduced toxicity compared to free DOX. The results suggest that the P(NBCh9-b-NBPEG) nanoparticles are a promising material for delivery of anticancer drugs with minimal toxicity.

ACKNOWLEDGEMENT

The synthesis and characterization work is partly supported by a grant from the National Science Foundation NSF CAREER Award to R.M.K. (DMR-0748398) and ACS PRF 5247200 to R.M.K. managed by the American Chemical Society.

SUPPORTING INFORMATION AVAILABLE

Chemical structures of doxorubicin and DiR dye as well as biodistribution of DiR dye in tumorbearing nude mice are provided as supporting information. This information is available free of charge via the Internet at http://pubs.acs.org/.

REFERENCES

1.

2.

Huang, J.; Zhang, H.; Yu, Y.; Chen, Y.; Wang, D.; Zhang, G.; Zhou, G.; Liu, J.; Sun, Z.; Sun, D.; Lu, Y.; Zhong, Y., Biodegradable self-assembled nanoparticles of poly (D,Llactide-co-glycolide)/hyaluronic acid block copolymers for target delivery of docetaxel to breast cancer. Biomaterials. 2014, 35, (1), 550-66. Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K., Accumulation of sub100 nm polymeric micelles in poorly permeable tumours depends on size. Nat. Nanotechnol. 2011, 6, (12), 815-23. 35 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3. 4.

5. 6.

7. 8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

Page 36 of 40

Phillips, M. A.; Gran, M. L.; Peppas, N. A., Targeted Nanodelivery of Drugs and Diagnostics. Nano today.2010, 5, (2), 143-159. Fang, J.; Nakamura, H.; Maeda, H., The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv. Drug Delivery Rev. 2011, 63, (3), 136-51. Kwon, G. S.; Forrest, M. L., Amphiphilic block copolymer micelles for nanoscale drug delivery. Drug Develop Res. 2006, 67, (1), 15-22. Lee AL, V. S., Gao S, Hedrick JL, Yang YY, The use of cholesterol-containing biodegradable block copolymers to exploit hydrophobic interactions for the delivery of anticancer drugs. Biomaterials. 2012, 33, (6), 1921−28. Wiradharma N, Z. Y., Venkataraman S, Hedrick JL, Yang YY Self-assembled polymer nanostructures for delivery of anticancer therapeutics. Nano Today. 2009, 4, 302−17. Cho, H. J.; Yoon, I. S.; Yoon, H. Y.; Koo, H.; Jin, Y. J.; Ko, S. H.; Shim, J. S.; Kim, K.; Kwon, I. C.; Kim, D. D., Polyethylene glycol-conjugated hyaluronic acid-ceramide selfassembled nanoparticles for targeted delivery of doxorubicin. Biomaterials. 2012, 33, (4), 1190-200. Park J-S, S. M.-H., Positively charged poly(alpha-(omega-aminoalkyl)lycolic acid) for the delivery of a bioactive agent via tissue and cellular uptake. US patent 2003, US 6517869 B1. Heino, S.; Lusa, S.; Somerharju, P.; Ehnholm, C.; Olkkonen, V. M.; Ikonen, E., Dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface. Proc. Natl. Acad. Sci. U S A. 2000, 97, (15), 8375-80. Wan, T.; Zou, T.; Cheng, S. X.; Zhuo, R. X., Synthesis and characterization of biodegradable cholesteryl end-capped polycarbonates. Biomacromolecules. 2005, 6, (1), 524-9. Nagahama, K.; Ueda, Y.; Ouchi, T.; Ohya, Y., Exhibition of soft and tenacious characteristics based on liquid crystal formation by introduction of cholesterol groups on biodegradable lactide copolymer. Biomacromolecules. 2007, 8, (12), 3938-43. Yu, Y.; He, Y.; Xu, B.; He, Z.; Zhang, Y.; Chen, Y.; Yang, Y.; Xie, Y.; Zheng, Y.; He, G.; He, J.; Song, X., Self-assembled methoxy poly(ethylene glycol)-cholesterol micelles for hydrophobic drug delivery. J. Pharm. Sci. 2013, 102, (3), 1054-62. Liu, Y.; Wang, Y.; Zhuang, D.; Yang, J., Bionanoparticles of amphiphilic copolymers polyacrylate bearing cholesterol and ascorbate for drug delivery. J. Colloid Interface Sci. 2012, 377, (1), 197-206. Yinsong Wang, Y. L., Yuanyuan Liu, Yan Wang, Jing Wu, Rongshan Li,; Zhang, J. Y. a. N., pH-sensitive pullulan-based nanoparticles for intracellular drug delivery. Polym. Chem. 2014, 5, 423. Rzayev, J., Synthesis of Polystyrene−Polylactide Bottle brush Block Copolymers and Their Melt Self-Assembly into Large Domain Nanostructures. Macromolecules. 2009, 42, 2135−41. Zhang, Y.; Yin, Q.; Lu, H.; Xia, H.; Lin, Y.; Cheng, J., PEG-Polypeptide Dual Brush Block Copolymers: Synthesis and Application in Nanoparticle Surface PEGylation. ACS Macro Lett. 2013, 2, (9), 809-813. Du, J. Z.; Tang, L. Y.; Song, W. J.; Shi, Y.; Wang, J., Evaluation of polymeric micelles from brush polymer with poly(epsilon-caprolactone)-b-poly(ethylene glycol) side chains as drug carrier. Biomacromolecules. 2009, 10, (8), 2169-74. 36 ACS Paragon Plus Environment

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19. Zhao P, L. L., Feng X, Wang C, Shuai X, Chen Y. , Molecular nanoworm with PCL core and PEO shell as a non-spherical carrier for drug delivery. Macromol. Rapid Commun. 2012, 33, 1351−55. 20. Khoee, S.; Rahmatolahzadeh, R., Synthesis and characterization of pH-responsive and folated nanoparticles based on self-assembled brush-like PLGA/PEG/AEMA copolymer with targeted cancer therapy properties: A comprehensive kinetic study. Eur. J. Med. Chem. 2012, 50, 416-27. 21. Yang, Y. Q.; Lin, W. J.; Zhao, B.; Wen, X. F.; Guo, X. D.; Zhang, L. J., Synthesis and physicochemical characterization of amphiphilic triblock copolymer brush containing pHsensitive linkage for oral drug delivery. Langmuir.2012, 28, (21), 8251-9. 22. Yu, Y.; Chen, C. K.; Law, W. C.; Mok, J.; Zou, J.; Prasad, P. N.; Cheng, C., Well-defined degradable brush polymer-drug conjugates for sustained delivery of Paclitaxel. Mol. Pharm. 2013, 10, (3), 867-74. 23. Johnson, J. A.; Lu, Y. Y.; Burts, A. O.; Lim, Y. H.; Finn, M. G.; Koberstein, J. T.; Turro, N. J.; Tirrell, D. A.; Grubbs, R. H., Core-clickable PEG-branch-azide bivalent-bottle-brush polymers by ROMP: grafting-through and clicking-to. J. Am. Chem. Soc. 2011, 133, (3), 559-66. 24. Zou, J.; Jafr, G.; Themistou, E.; Yap, Y.; Wintrob, Z. A.; Alexandridis, P.; Ceacareanu, A. C.; Cheng, C., pH-Sensitive brush polymer-drug conjugates by ring-opening metathesis copolymerization. Chem. Commun. 2011, 47, (15), 4493-5. 25. Nguyen, C. T.; Tran, T. H.; Lu, X. L.; Kasi, R. M., Self-assembled nanoparticles from thiol functionalized liquid crystalline brush block copolymers for dual encapsulation of doxorubicin and gold nanoparticles. Polym. Chem-Uk. 2014, 5, (8), 2774-2783. 26. Sawyer, D. B., Anthracyclines and heart failure. N Engl J Med. 2013, 368, (12), 1154-6. 27. Zhang, S.; Liu, X.; Bawa-Khalfe, T.; Lu, L. S.; Lyu, Y. L.; Liu, L. F.; Yeh, E. T., Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat. Med. 2012, 18, (11), 1639-42. 28. Ahn S-K, L. L., Kasi RM, Synthesis and characterization of side-chain liquid crystalline polymers bearing cholesterol mesogen. J. Polym. Sci. A Polym. Chem. 2009, 47, 2690−1. 29. Deshmukh P, A. S.-k., Merxem LG, Kasi RM, Interplay between liquid crystalline order and microphase segregation on the self-assembly of side-chain liquid crystalline brush block copolymers. Macromolecules. 2013, 46, 8245−52. 30. Alhareth, K.; Vauthier, C.; Gueutin, C.; Ponchel, G.; Moussa, F., HPLC quantification of doxorubicin in plasma and tissues of rats treated with doxorubicin loaded poly(alkylcyanoacrylate) nanoparticles. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2012, 887-888, 128-32. 31. Yinsong W, L. L., Jian W, Zhang Q., Preparation and characterization of self-aggregated nanoparticles of cholesterol-modified O-carboxymethyl chitosan conjugates. Carbohydr Polym. 2007, 69, 597−6. 32. Attia, A. B.; Yang, C.; Tan, J. P.; Gao, S.; Williams, D. F.; Hedrick, J. L.; Yang, Y. Y., The effect of kinetic stability on biodistribution and anti-tumor efficacy of drug-loaded biodegradable polymeric micelles. Biomaterials. 2013, 34, (12), 3132-40. 33. Cho, K.; Wang, X.; Nie, S.; Chen, Z. G.; Shin, D. M., Therapeutic nanoparticles for drug delivery in cancer. Clin. Cancer Res. 2008, 14, (5), 1310-6.


Biomacromolecules

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34. Lv, S.; Li, M.; Tang, Z.; Song, W.; Sun, H.; Liu, H.; Chen, X., Doxorubicin-loaded amphiphilic polypeptide-based nanoparticles as an efficient drug delivery system for cancer therapy. Acta Biomater. 2013, 9, (12), 9330-42. 35. Opanasopit, P.; Yokoyama, M.; Watanabe, M.; Kawano, K.; Maitani, Y.; Okano, T., Influence of serum and albumins from different species on stability of camptothecin-loaded micelles. J. Controlled Release. 2005, 104, (2), 313-21. 36. Sutton, D.; Wang, S.; Nasongkla, N.; Gao, J.; Dormidontova, E. E., Doxorubicin and betalapachone release and interaction with micellar core materials: experiment and modeling. Exp. Biol. Med. 2007, 232, (8), 1090-9. 37. Tan, J. P.; Kim, S. H.; Nederberg, F.; Appel, E. A.; Waymouth, R. M.; Zhang, Y.; Hedrick, J. L.; Yang, Y. Y., Hierarchical supermolecular structures for sustained drug release. Small. 2009, 5, (13), 1504-7. 38. Poon, Z.; Lee, J. A.; Huang, S.; Prevost, R. J.; Hammond, P. T., Highly stable, ligandclustered "patchy" micelle nanocarriers for systemic tumor targeting. Nanomedicine. 2011, 7, (2), 201-9. 39. Park, J.; Fong, P. M.; Lu, J.; Russell, K. S.; Booth, C. J.; Saltzman, W. M.; Fahmy, T. M., PEGylated PLGA nanoparticles for the improved delivery of doxorubicin. Nanomedicine. 2009, 5, (4), 410-8. 40. Guo M, Q. C., Wang C, Liu X, Yan H, Liu K., Multifunctional superparamagnetic nanocarrier with folate-mediated and pH-responsive targeting properties for anticancer drug delivery. Biomaterials. 2011, 32, 185−94. 41. Li, S. D.; Huang, L., Pharmacokinetics and biodistribution of nanoparticles. Mol. Pharm. 2008, 5, (4), 496-504. 42. Sheng, Y.; Liu, C.; Yuan, Y.; Tao, X.; Yang, F.; Shan, X.; Zhou, H.; Xu, F., Longcirculating polymeric nanoparticles bearing a combinatorial coating of PEG and watersoluble chitosan. Biomaterials. 2009, 30, (12), 2340-8. 43. Torchilin, V. P., Micellar nanocarriers: pharmaceutical perspectives. Pharm. Res. 2007, 24, (1), 1-16. 44. Singal, P. K.; Iliskovic, N., Doxorubicin-induced cardiomyopathy. N. Engl. J. Med. 1998, 339, (13), 900-5. 45. Chen X, C. P., Moats RA, In vivo near-infrared fluorescence imaging of intergrin ανβ3 in brain tumor xenografts. Cancer Res. 2004, 64, 8009−14. 46. Liu, Y.; Tseng, Y. C.; Huang, L., Biodistribution studies of nanoparticles using fluorescence imaging: a qualitative or quantitative method? Pharm. Res. 2012, 29, (12), 3273-7. 47. Hollis, C. P.; Weiss, H. L.; Leggas, M.; Evers, B. M.; Gemeinhart, R. A.; Li, T., Biodistribution and bioimaging studies of hybrid paclitaxel nanocrystals: lessons learned of the EPR effect and image-guided drug delivery. J. Controlled Release. 2013, 172, (1), 1221. 48. Pengfei Gou , W. L., Weiwei Mao , Jianbin Tang , Youqing Shen and Meihua Sui, Selfassembling doxorubicin prodrug forming nanoparticles for cancer chemotherapy: synthesis and anticancer study in vitro and in vivo. J. Mater. Chem. B 2013, 1, 284. 49. Kiziltepe, T.; Ashley, J. D.; Stefanick, J. F.; Qi, Y. M.; Alves, N. J.; Handlogten, M. W.; Suckow, M. A.; Navari, R. M.; Bilgicer, B., Rationally engineered nanoparticles target multiple myeloma cells, overcome cell-adhesion-mediated drug resistance, and show enhanced efficacy in vivo. Blood cancer J. 2012, 2, (4), e64.


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Biomacromolecules


Biomacromolecules

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Table of Contents Graphic


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Long Circulating Self-Assembled Nanoparticles ... - ACS Publications

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