Physiologically Stable Hydrophilic C60 Nanoparticles for

Subscriber access provided by Iowa State University | Library

Article

Physiologically Stable Hydrophilic C60 Nanoparticles for Photodynamic Therapy Daisuke Iohara, Fumitoshi Hirayama, Makoto Anraku, and Kaneto Uekama ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01862 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32 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

ACS Applied Nano Materials

Physiologically Stable Hydrophilic C60 Nanoparticles for Photodynamic Therapy Daisuke Iohara✻,†,‡ , Fumitoshi Hirayama†,‡, Makoto Anraku†,‡ and Kaneto Uekama† †Faculty

‡DDS

of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan

Research Institute, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan

KEYWORDS: fullerene, nanoparticle, in vivo aggregation, biodistribution, photodynamic therapy

ABSTRACT Hydrophilic C60 nanoparticles that are highly stable in living systems were prepared with sugammadex, an anionic γ-cyclodextrin derivative, via a simple procedure for use in biological applications. The prepared C60/sugammadex nanoparticles showed outstanding stability under physiological conditions and even in much harsher conditions. The sugammadex interacted with C60 nanoparticles through strong host-guest interactions on the particle surface, producing a negatively charged layer on the surface of nanoparticles, which contributed to the high stability of the nanoparticles. In addition, the nanoparticles were highly stable in the presence of singly

ACS Paragon Plus Environment

1

ACS Applied Nano Materials 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

Page 2 of 32

charged cations which are present in abundance in living systems. The stable C60/sugammadex nanoparticles showed a significantly different biological behavior compared to less stable C60 nanoparticles after intravenous administration. Most of the C60 particles accumulated and remained in organs of the reticuloendothelial system (RES) after administration, which are susceptible to forming aggregates in physiological conditions. On the other hand, the C60/sugammadex nanoparticles showed a completely different biological behavior, i.e. longer blood circulation, low RES uptake and elimination with time from organs. The photodynamic activity of C60/sugammadex nanoparticles was evaluated both in vitro and in vivo, and an outstanding antitumor effect was achieved based on the generation of reactive oxygen species under light irradiation. We envision that such stable C60 nanoparticles would be a desirable approach for extending the biological applications of these materials and the precise evaluation of C60 activity in living systems.

1. INTRODUCTION Rapid advances in nanotechnology have stimulated an increased interest in carbon-based nanomaterials including carbon nanotubes, graphenes and fullerenes for use in medical applications because of their multifunctional characteristics such as high mechanical strength, electrical and thermal conductivity, unique optical properties and high surface area that permits them to deliver large amounts of drugs.1, 2 Among these materials, fullerenes have gathered much attention for practical applications that take advantage of their unique spherical structure, physical properties and biological activities.3 For example, fullerene C60 can function as an antioxidant,4, 5 a bioimaging agent6,

7

and as a gene8 or drug carrier.9 C60 can function as an efficient


2

Page 3 of 32 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


photosensitizer in photodynamic therapy (PDT) due to light absorption at relatively long wavelengths and the high quantum yield in photoexcitation reactions.10, 11 PDT, which involves the production of reactive oxygen species (ROS), is an effective method for destroying tumor tissues without damaging healthy, surrounding tissues.12-14 However, the practical use of these potential biomedical applications of C60 have been hampered by the fact that it is only sparingly soluble in water.15, 16 Although numerous water soluble C60 derivatives have synthesized so far for biomedical applications,17 C60 readily forms aggregates in polar solvents, resulting in large particles or insoluble aggregates that are formed during storage, especially in biological media which contains relatively high levels of salts. In PDT, a photosensitizer is systemically administered and tumor sites are then irradiated by a light source to site-selectively generate ROS, leading to cell death and tissue destruction. When such nanomaterials are administered, they encounter biological media including blood plasma, lymph fluid and the cytosol, where they would undergo multifold interactions with proteins, lipids, cells and mineral salts through hydrophobic or electrostatic interactions, which can easily lead to aggregation and sedimentation.18,

19

Such unexpected

changes in nanostructure clearly would affect the biological activity and biodistribution of the nanomaterials, result in a lowered efficacy and undesirable side effects.20,

21

Although the

functionalization of C60 with polar or ionic groups such as hydroxyl22 or carboxyl23 moieties is a widely used approach for preparing water soluble forms of C60, most of these hydrophilic derivatives have yet to be evaluated for their efficacy in vivo. The chemical modification of C60 generally decreases its photosensitizing ability due to the collapsed π-conjugated system, thus, the development of hydrophilic types of C60 that are stable under physiological conditions without any


3


Page 4 of 32

chemical modifications would be a desirable approach for extending the biological applications of these materials and for a precise evaluation of biological activity of C60 itself. In previous studies, we reported on the preparation of hydrophilic C60 nanoparticles that have not been chemically modified by using 2-hydroxypropyl-β-cyclodextrin (HP-β-CD).24 These C60 nanoparticles were found to be stable in water and could be used to readily generate high levels of ROS compared to the aggregated form.25,

26

On the other hand, polyhydroxylated fullerenes

(fullerenol, C60(OH)10) nanoparticles prepared with HP-β-CD were found to show a hepatoprotective effect which is attributed to their ability to scavenge ROS such as O2•－, NO and peroxynitrite.27, 28 In this paper, we report on the development of C60 nanoparticles that are highly stable under physiological conditions. This was achieved by using sugammadex, an anionic -cyclodextrin (CD) derivative, for biological applications (Figure 1). Sugammadex (tradename：Bridion®), is produced from γ-CD by modification with eight carboxyl thio ether groups at the primary hydroxyl side of the host molecule. Negatively charged substituents extend the cavity allowing greater encapsulation of guest molecules as well as contributing to the aqueous nature of the CD.29 Sugammadex is clinically used as an agent for reversing neuromuscular blockage caused by the administration of rocuronium or vecuronium during general anesthesia.30 The findings reported here indicate that the use of sugammadex results in dramatically more stabilized C60 nanoparticles under physiological conditions and harsher conditions by virtue of an electric layer that is formed on the nanoparticles thorough strong host-guest interactions on the surface of nanoparticles. In addition, we report here that these stable C60 nanoparticles have different biological characteristics after an intravenous administration compared with other C60/CD


4

Page 5 of 32 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


nanoparticles which are susceptible to forming aggregates in physiological conditions. In addition, these highly stable C60 nanoparticles show superior in vivo photodynamic activity.

(CH2SCH2CH2COO－)8

+ Sugammadex

C60

＝

(CH2SCH2CH2COO－)8

・physiologically stable ・longer blood circulation ・low RES uptake ・elimination from organs

Scab

Strong host-guest interactions on the surface of nanoparticles

Outstanding antitumor effect

Intravenous administration C60/sugammadex nanoparticle

Photoirradiation Tumor-bearing mouse

Photodynamic therapy

Figure 1. Image of physiologically stable C60/sugammadex nanoparticles for photodynamic therapy.

2. EXPERIMENTAL SECTION Materials C60 (nanom purple SUH) and C70 (nanom orange) were purchased from Frontier Carbon Co. (Tokyo, Japan). Sugammadex (Bridion®) was purchased from MSD K.K. (Tokyo, Japan) and freeze dried for use in the study. -CD was a gift from the Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). All other materials and solvents were of analytical reagent grade and Milli-Q water was used throughout the study.


5


Page 6 of 32

Preparation and characterization of C60/sugammadex nanoparticles C60 powder was ground with sugammadex or -CD at different mole ratios (generally 1:2 for guest:host), using an automatic magnetic agitating mortar (MNV-01, AS ONE, Tokyo, Japan) for 3 hr at 4ºC under reduced pressure.24 The resulting pulverized C60/CDs were dispersed in water or phosphate buffered saline (PBS) by ultrasonication for 5 min, to give about 1.0 mM of C60. Particle sizes of the C60/CDs nanoparticles were determined by means of dynamic light scattering (DLS8000HL, Otsuka Electronics Co., Ltd., Tokyo, Japan) equipped with a He-Ne laser (10 mW) operating at 632.8 nm. Zeta potentials for the nanoparticles were determined by a Zetasizer (ELSZ, Otsuka Electronics Co., Ltd., Tokyo, Japan). The long term dispersibility of nanoparticles was monitored by the changes in particle size and -potential during storage. The dispersibility of C60 nanoparticles in plasma was evaluated by adding 50 L of C60/sugammadex or /γ-CD nanoparticles (C60 = 5 mM) to 100 L of rabbit’s plasma. To understand the interaction of sugammadex with C60 nanoparticles, 1.0 mL of a C60/-CD colloidal solution (C60 = 0.25 mM) was mixed with 1.0 mL of a NaCl solution (14 mM), and, at 5 min after the mixing, the sugammadex powder (4.3mg, 1 mM) was dissolved in the mixed solution. Changes in particle size and -potential were monitored as a function of elapsed time. In contrast, various concentrations of -CD (1~20 mM) were added to the C60/sugammadex nanoparticles prepared in a 100 mM NaCl solution and, after 1 day, the particle size and -potential of the nanoparticles were measured in the same manner.

Aggregation behavior of C60 nanoparticles in mineral salt solutions.


6

Page 7 of 32 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


The effects of counter anions on the aggregation of C60 nanoparticles was studied by adding solutions of mineral salts to the preparations. A 0.25 mL aliquot of C60/sugammadex and a C60/CD colloidal solution (C60 = 1.0 mM) were mixed with 1.75 mL of various concentrations of NaCl solutions. The solution was placed in the dark at 25ºC for over 3 hours in order to allow changes in particle size to occur. The same experiments were also conducted using KCl, CaCl2 and MgCl2 solutions, and the particle size and -potential were measured for the samples.

Biodistribution of C60 nanoparticles after an intravenous administration to tumor-bearing mice The care and maintenance of animals were conducted in accordance with the institutional guidelines of the Institutional Animal Care and Use Committee of Sojo University. Preparation of a mouse tumor models Mouse sarcoma S-180 cells were acclimatized to the in vivo conditions by intraperitoneal growth in ddY mice (Kyudo Co. Ltd., Saga, Japan). The collected cells were washed with saline and implanted in 6~7 week old ddY mice (2 x 107 cells/mL, 100 L/mouse) by subcutaneous injection in the dorsal skin. Tumor-bearing mice were used for the biodistribution studies and photodynamic therapy when the tumor mass reached a diameter of 8 mm. Biodistribution of C60/CDs nanoparticles in tumor-bearing mice For the intravenous injection, C60/sugammadex nanoparticles were prepared in saline, while C60/γ-CD nanoparticles were prepared in 5% glucose. The nanoparticles (C60 = 30 mg/kg) were intravenously injected into the tumor-bearing mice and then sacrificed at predetermined intervals to collect organs and blood. Extractions of C60 from the organs and blood were conducted using a previously reported method with minor modifications.5 Two hundred mg of organs, except for the


7


Page 8 of 32

liver (100 mg), were weighed and then homogenized with 2.0 mL of mixed solution of 0.1M sodium dodecyl sulfate (SDS) and acetonitrile. The homogenizer was rinsed with 1.0 mL of acetonitrile and the rinsing liquid was transferred to the same homogenized liquid. Eight mL of toluene containing 2.0 μg/mL of C70 as an internal standard (IS) was added and the mixture was shaken for 12hr at 1200 rpm. After centrifugation, 1.0 mL of the supernatant was dried and dissolved in the HPLC mobile phase. For extraction of C60 from the blood, 100 μL of whole blood was mixed with 900 μL of 0.1M SDS and 2.0 mL of acetonitrile was added. After adding 8.0 mL of toluene containing 2.0 μg/mL of IS, the mixture was shaken for 12hr and treated in the same manner for organs. The concentration of C60 in the samples was determined by a Hitachi HPLC System (Tokyo, Japan) consisting of a UV-Vis detector, autoinjector and pump. Separations were carried out with the ODS-A column 15 x 4.6 mm, 5 μm (YMC Co., Ltd., Tokyo, Japan) protected by a 23 x 4.0 mm, 5 μm ODS-A pre-column. The mobile phase was toluene-acetonitrile (55 : 45) at a flow rate of 1.0 mL. Eighty μL of sample solutions were injected and detected at a wavelength of 330 nm. The concentration of C60 was calculated from calibration curves which were prepared for each organ and blood. C60/CDs nanoparticles (C60 = 0.1 ~ 2 μg/mL) were added to the homogenate of organs and whole blood, and the same procedure for extraction was carried out for the quantitative determination of C60.

In vivo photodynamic activity of C60/sugammadex nanoparticles after intravenous administrations to tumor-bearing mice Tumor-bearing mice were divided into four groups of 7~10 mice: no treatment, light irradiation (90 J/cm2), i.v. administrtion of C60/sugammadex (50 mg/kg) and i.v. administration of C60/sugammadex plus light irradiation. The light were supplied from a xenon light source (MAX-


8

Page 9 of 32 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


303, Asahi spectra Co., Ltd., Tokyo, Japan) with power of 150 mW/cm2, 400 ~ 700 nm. The treatments

were carried out according to the schedule as shown in the PDT section. Tumor volume (mm3) was calculated as (W2×L)/2 by measuring the length (L) and width (W) of the tumor on the dorsal skin.

Statistical analysis Data are presented as the median values from n samples and the results are reported as the mean ± S.E. Significant differences between the data were calculated by using student’s t-tests. For all analyses, values of p < 0.05 were regarded as statistically significant.

3. RESULTS AND DISCUSSION Preparation and characterization of highly stable C60 nanoparticles under physiological conditions C60 nanoparticles were prepared by simply grinding C60 with powdered sugammadex or γ-CD. γ-CD was selected as a reference because of their similar cavity size of CD ring. The resulting powdered C60 that was ground with γ-CD could be easily dispersed in water, giving ca. 56 nm sized nanoparticles, but large aggregates were produced that rapidly precipitated in phosphate buffered saline (PBS) (Figure 2(A)). In sharp contrast, the C60/sugammadex powder could be easily dispersed both in water as well as in PBS. It is noteworthy that sugammadex produced nanoparticles with particle sizes of ca. 40 nm, even in PBS, almost the same size as produced in water (ca. 30 nm). The colloidal solutions were more transparent than those of -CD, probably due to the smaller particle size of C60 in solution. In comparing the -potentials of those C60/CDs particles, C60 nanoparticles prepared using -CD were -21 mV in water, while the value shifted to


9


Page 10 of 32

positive values in PBS. C60/sugammadex was highly negatively charged in water and even in PBS, -51 mV and -37 mV, respectively. To examine the issue of how sugammadex contributes to nanoparticle formation and dispersibility, C60 nanoparticles were prepared with various amounts of sugammadex. The mean particle diameter of C60 decreased with increasing amounts of sugammadex added in the grinding process (Figure 2(B)). C60/sugammadex formed nanoparticles with diameters less than 100 nm both in water and in PBS over the molar ratio of 1 : 0.5 (C60 : sugammadex). Zeta-potentials were also monitored for the C60 nanoparticles prepared with the various molar ratio (Figure 2(C)). The values decreased to negative values by the addition of sugammadex, reaching -54 mV in water and -37 mV in PBS when a molar ratio of 1 : 1 (C60 : sugammadex) was used. The high negative value of -46 mV at molar ratio of 1 : 0.5 in water was sufficient to ensure the dispersibility of these nanoparticles; thus, the particle size was nearly the same between 0.5 : 1 and 1 : 1 molar ratios. In the case of a molar ratio of 1 : 1, additional sugammadex can gain access to the surface of nanoparticles leading to the changes in -potential. Powder x-ray diffraction data were collected for the powders ground with sugammadex (Figure S1). The characteristic peaks of C60 broadened significantly and eventually nearly disappeared with increasing amounts of sugammadex, indicating that C60 clearly interacts with sugammadex during the formation of stable C60 nanoparticles. The long term dispersibility of C60/sugammadex nanoparticles in water and in PBS was examined (Figure 2(D) and (E)). C60 nanoparticles prepared at molar ratios of 1: 0.5 ~ 3 (C60 : sugammadex) were dispersed in water or PBS, and then stored in the dark at 25˚C. The C60 nanoparticles that were dispersed in water maintained their initial particle size during storage and no aggregation and sedimentation was observed for all samples over a 1 month period, as shown in Figure 2(D). On the other hand, obvious aggregation and sedimentation of C60 particles was


10

Page 11 of 32 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


observed in PBS for the samples prepared at low sugammadex concentrations. In the case of samples prepared at a 1 : 0.5 ratio (C60 : sugammadex), the size had increased to over 400 nm at 3 days and then precipitated, and samples prepared at a 1:1 ratio (C60 : sugammadex) gradually aggregated, eventually reaching a size of ca. 280 nm at 14 days, and then precipitated. The aggregation of C60 was probably controlled by the equilibrium among C60 nanoparticles, salts and sugamamdex in the PBS; thus, a rapid aggregation occurred in the case of the sample that was prepared with a low concentration of sugammadex. In contrast, such changes in size were markedly inhibited for the samples prepared at 1:2 and 1:3 ratios (C60 : sugammadex). These nanoparticles maintained the small size of about 100 nm even at 28 days. C60/sugammadex nanoparticles were well dispersed in plasma, while rapid aggregation was observed for C60/γ-CD sample, as shown in Figure 2(F). We attempted to measure the particle size and -potential of C60 nanoparticles in plasma, however, these values of C60 were overlapped with the plasma proteins (particle size : 100 ~ 150 nm, -potential : around -10 mV). These collective results suggest that sugammadex can be useful in ensuring the dispersibility of C60 nanoparticles under physiological conditions, probably due to the highly negatively charged surface conferred by the eight anionic carboxyl groups that were introduced at the rim of the primary ring.


11


(B)

in water Mean particle 30±5 nm diameter

in PBS

in water

40±9 nm

56±10 nm

-potential ‐51±5 mV ‐37±4 mV ‐21±2 mV

in PBS 462±33 nm 1±1 mV

Appearance

(C) 0

500

Molar ratio (sugammadex/C60) 1

2

3

0

● : in water ■ : in PBS

400

 -potential(mV)

-CD

sugammadex

Mean particle diameter(nm)

(A)

300 200 100

-10 -20

● : in water ■ : in PBS

-30 -40 -50

0 0

1

2

3

-60

Molar ratio (sugammadex/C60)

Mean particle diameter (nm)

100

□ : 0.5 (sugammadex/C60) ▲ : 1.0 ● : 2.0 ◇ : 3.0

80 60

(F)

(E)

(D) Mean particle diameter (nm)

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

Page 12 of 32

40 20

□ : 0.5 (sugammadex/C60) ▲ : 1.0 ● : 2.0 ◇ : 3.0

400 300

C60/Sugammadex

C60/-CD

200 100 0

0 0

7

14

21

28

Time (days)

Figure 2.

0

7

14

21

28

Time (days)

Appearances (A), particle size (B) and -potential (C) of C60/sugammadex

nanoparticles. Long term dispersibility of C60/sugammadex nanoparticles in water (D) and in PBS (E). Appearances of C60/CDs nanoparticles after storage in rabbit plasma (F). Each point represents the mean ± S.E. of 3 experiments.

The structure of the C60 nanoparticles was estimated by NMR measurements and monitoring aggregation behavior. NMR measurements were conducted for a colloidal solution to evaluate the interaction of sugammadex with C60 nanoparticles (Figure S2). 13C NMR spectra of a C60 colloidal solution gave two peaks at around 146 ppm, which are assigned to C60 nanoparticles (146.05 ppm) and a C60/sugammadex inclusion complex (145.85 ppm), respectively.24 It is well known that γCD with a large cavity forms a 1:2 (guest : host) inclusion complex with C60,31, 32 thus sugammadex,


12

Page 13 of 32 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


a γ-CD derivative, also readily formed an inclusion complex with C60. The content of C60 inclusion complex was determined by UV measurements on the supernatant of the colloidal solution after ultracentrifugation. The resulting value was low (2 ~ 3% w/w), indicating that most of the C60 was dispersed in the form of nanoparticles. The peak for the C60 nanoparticles was accompanied by a small shoulder peak, probably due to interactions of sugammadex on the surface of the C60 nanoparticles. In addition, proton peaks (H3 and H5) corresponding to the inner cavity of sugammadex were split into two peaks, indicating the existence of a strong interaction between the sugammadex and C60 molecules. The interaction of C60 nanoparticles and sugammadex was further studied by monitoring the aggregation behavior in a solution containing both -CD and sugammadex (Figure 3). The C60/-CD colloidal solution was initially mixed with NaCl (14 mM), and, after 5 min, sugamamdex (1 mM) was added to the solution. The C60 particle size increased immediately on the addition of NaCl and the -potential shifted to a positive value as shown in Figure 3(A) and (B). The reduced -potential of particles was accompanied by a weakened interparticle repulsion force, whereby causing the destabilization of the nanoparticles, with the occurrence of aggregation and sedimentation. On the other hand, the aggregation process was completely suppressed by simply adding sugammadex to the solution, and the particle size consequently settled at around 100 nm (Figure 3(A)). The -potential of the C60 nanoparticles first shifted to a positive direction, and the value then came back to – 27 mV immediately after the addition of sugammadex (Figure 3(B)). These results are consistent with competitive host-guest interactions occurring on the particle surface replacing the pre-existing γ-CD with the sugammadex that was added later. In contrast, when the C60/sugammadex preparation was initially dispersed in 100 mM NaCl and γ-CD was then added to the solution, the particle size and potential shifted in parallel with the amount of γ-CD, eventually resulting in precipitation, as


13


shown in Fig. 3(C). This is because the amount of γ-CD added overtook the pre-existing sugammadex covering the nanoparticles and the resulting unstable C60/γ-CD nanoparticles then began to form aggregates, as shown in Figure 3(D). The present results confirm that sugammadex interacted with C60 nanoparticles through host–guest interactions, even though the sugammadex was on the particle surface and covered only the surfaces of the C60 nanoparticles, thus conferring a large negative charge on the surface, a situation that is crucial for stabilizing C60 nanoparticles at physiological conditions.

0

350

 –potential (mV)

250 200 150 100

add sugammadex

50

15

30

45

60

-5 -10 -15

● : C60/γ-CD ○ : add sugammadex

-20 -25 -30

0 0

30

0

● : C60/γ-CD ○ : add sugammadex

300

(C)

Time (min) 15

45

60

-35

add sugammadex

2000

0

1750

-10

1500

-20

1250

Adding -CD

1000

-30

750

-40

500

-50

C60/sugammadex in 100 mM NaCl

250

-60

0 0

Time (min)

5

10

15

20

Molar ratio (-CD/sugammadex)

(D)

Adding sugammadex

Adding NaCl

Adding γ-CD

Stable nanoparticle

Aggregation

Figure 3. Changes in particle size and -potential of C60 nanoparticles in the presence of γ-CD and sugammadex. Particle size (A) and -potential (B) of C60/γ-CD after adding sugammadex (1 mM). Particle size and -potential of C60/sugammadex after adding γ-CD (1 ~ 20 mM) (C). Images of competitive host-guest interactions on the C60 nanoparticle surface (D). Each point represents the mean ± S.E. of 3 experiments.


14

 -potential(mV)

(B)

Mean particle diameter (nm)

(A) Mean particle diameter (nm)

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

Page 14 of 32

Page 15 of 32 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


Surface properties of C60 nanoparticles against aggregation under physiological conditions To better understand the enhanced colloidal stability conferred by sugammadex, the aggregation behavior of particles was monitored in the presence of mineral salts (Na+, K+, Ca2+, Mg2+) as counter anions, which are abundant in living systems (Figure 4). These C60 nanoparticles were prepared at molar ratio of 1:2 (C60 : CD). The particle size of C60/-CD increased with increasing concentration of NaCl and precipitation was observed at concentrations of NaCl above 7 mM (Figure 4(A)). Zeta-potential of the C60/-CD also shifted in a positive direction with increasing NaCl concentrations. On the other hand, such changes in particle size and -potential were not observed in the case of sugammadex nanoparticles at the same NaCl concentrations (

Physiologically Stable Hydrophilic C60 Nanoparticles for

Recommend Documents