Gold Nanorod Mediated Chlorhexidine Microparticle Formation and

Subscriber access provided by UNIV OF NEWCASTLE

Article

Gold Nanorod Mediated Chlorhexidine Microparticle Formation and Near-infrared Light Induced Release Dong Luo, Md. Samiul Hasan, Saroash Shahid, Boris khlebtsov, Michael J. Cattell, and Gleb B. Sukhorukov Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01656 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017

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 free 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 accessible to all readers and 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.

Langmuir 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 36

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

Langmuir

1

Gold Nanorod Mediated Chlorhexidine

2

Microparticle Formation and Near-infrared Light

3

Induced Release

4

Dong Luoa*, Md. Samiul Hasan b, Saroash Shahidb, B. N. Khlebtsov cd, Michael J. Cattellb, Gleb

5

B.Sukhorukova*

6

a

7

4NS, United Kingdom

8

b

9

London E1 2AD, United Kingdom

School of Engineering and Materials Science, Queen Mary University of London, London E1

Barts and The London School of Medicine and Dentistry, Queen Mary University of London,

10

c

11

d

12

Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia

13

Key words: chlorhexidine, gold nanorods, near-infrared light, capsules, controlled release

14

ABSTRACT

15

Gold nanorods (GNR) are good light harvesting species for elaboration of near-infrared (NIR)

16

responsive drug delivery systems. Herein, chlorhexidine microparticles are grown directly on the

17

surface of gold nanorods and then stabilized with polyelectrolyte multilayer encapsulation,

Saratov State University, Astrakhanskaya Street 83, Saratov 410012, Russia Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of

1 Environment ACS Paragon Plus

Langmuir

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

1

producing novel composite drug-GNR particles with high drug loading and NIR light sensitivity.

2

Crystallization of chlorhexidine is caused by the ionic strength of the chloride solution that has

3

been demonstrated via formation of a homogeneous porous spherical structure at 0.33 M CaCl2.

4

By introducing GNRs into the CaCl2 solution, the nucleation of chlorhexidine molecules as well

5

as size of produced spheres are affected, since GNRs act as sites for chlorhexidine nucleation.

6

Similarly, when GNRs are replaced by chlorhexidine seeds (5.2±1.7 µm), a core-shell crystal

7

structure is observed. The encapsulated GNR/chlorhexidine composites are responsive to NIR

8

light (840 nm) that increases the temperature at the chlorhexidine crystals, followed by

9

microparticle dissolution and rupture of capsules which is illustrated with confocal microscopy

10

and SEM. Furthermore, a stepwise burst release of chlorhexidine can be induced by multiple

11

cycles of NIR light exposure. The GNR/chlorhexidine composites show good biocompatibility

12

and antimicrobial activity. The proposed method of antibacterial drug release may therefore

13

indicate that this NIR responsive chlorhexidine composite may be useful for future clinical

14

applications.

15 16

INTRODUCTION

17

Gold nanoparticles and nanorods based carriers are extensively used for drug delivery and

18

photothermol therapy due to their facile surface modification and intrinsic plasmonic properties.1,

19

2, 3

20

energy into heat. The general approach is to attach drug molecules or therapeutic agents, such as

21

DNA and RNA, to their surface and then selective release can be achieved via photothermal

22

treatment.4,

23

polyelectrolyte microcapsules,8 and nanogels9 to facilitate stimuli responsive drug release. These

Upon near infrared (NIR) light irradiation, the gold particles can efficiently convert the

5

They are also incorporated into carriers such as micelles,6 liposomes,7


Page 2 of 36

Page 3 of 36

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

Langmuir

1

NIR light responsive carriers can also demonstrate a step wise in vitro drug release pattern,10

2

with specific intracellular release of cargo triggered by light irradiation.11 Previously a NIR

3

mediated tumor cell apoptosis was achieved with gold nanorod based carriers in vivo.5 Hollow

4

vesicles consisting of gold nanoparticles and gold nanocages were also reported, which were

5

supposed to be more sensitive to the NIR light because of the high gold content.12, 13 Moreover,

6

their hollow structure was also advantageous over the other carriers due to the drug loading

7

capability.

8

Another approach to make the most of the photothermal properties of gold nanoparticles and

9

nanorods for drug delivery is achieved by directly constructing drug on top of the particles. For

10

instance mesoporous silica shells were deposited on the surface of gold particles to produce core-

11

shell nanocomposites.14, 15 Since this was first reported by Liz-Marzan et al., (1996),16 these

12

gold/silica composites have attracted great attention for usage in controlled drug delivery. This is

13

due to the porous silica shells acting as a good drug container and the gold core serving as an

14

efficient photothermal convertor. The release kinetics of the payload from such composites can

15

be easily tuned by NIR light illumination and with targeted delivery realised via modification of

16

the silica surface.1, 5 The mechanism of formation of these core/shell structures consists of two

17

stages: hydrolysis to form silica oligomers and mesoporous silica growth on the surface of the

18

gold seeds via Ostwald ripening.17 Because gold has little affinity for silica, silane coupling

19

agents are usually used as surface primers.14, 16 However, since most of the payload molecules

20

are just physically entrapped in silica cavities, gating moieties on the composite particle surface

21

are always needed to regulate the release behavior, and the drug loading rate is also limited.18

22

Therefore, if the drug crystals could be grown directly onto the surface of the gold nanoparticles

23

or gold nanorods, it would be more straightforward to trigger the release with an NIR light. The


Langmuir

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 4 of 36

1

release behavior could also be easily tuned by further encapsulating with multilayers using the

2

layer-by-layer (LbL) assembly technique.19, 20

3

Chlorhexidine is a broad spectrum antibacterial agent commonly used in dentistry.21,

4

incorporated into mouth rinses for the treatment of gingivitis

5

into a cross-linked gelatin chip (PerioChip®) for the treatment of periodontal pockets.24 However,

6

a lack of controllable release of chlorhexidine and long term effect may lead to repetitive

7

infections. The biguanidine groups of chlorhexidine molecules have a strong coordinating ability,

8

which was utilized to produce other chlorhexidine formulations to improve its antibacterial

9

performance.25,

26, 27

23

22

It is

, reduction of dental plaque or

In our previous study, by tuning the ion type and concentration,

10

chlorhexidine spheres with homogeneous size and morphology were produced, and the crystal

11

growth could be further modulated by the temperature.28 The chlorhexidine spheres could be

12

encapsulated with layer-by-layer assembly to achieve a sustained release.28, 29 Therefore, if gold

13

nanorods could be incorporated into the porous chlorhexidine crystal structure, NIR light stimuli

14

responsive properties could be enabled, which would be very attractive in clinical applications.

15

In the present study, gold nanorods were used to functionalize chlorhexidine spheres to produce

16

a NIR light responsive formulation. The effect of gold nanorods on chlorhexidine crystal

17

formation was investigated. The formation of functionalized chlorhexidine was achieved in the

18

current work and the presence of gold nanorods was effective in the controlled crystallization of

19

chlorhexidine particles. The functionalized chlorhexidine spheres could be stabilized by LbL

20

encapsulation and the resultant capsules could be burst using a NIR light to provide snap

21

chlorhexidine release. The NIR light responsive chlorhexidine formulation has promising

22

applications in medicine and dentistry.

23


Page 5 of 36

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

Langmuir

1

EXPERIMENTAL SECTION

2

Materials

3

Chlorhexidine diacetate (99%), Poly(allylamine hydrochloride) (PAH, 56 kDa), Poly(sodium 4-

4

styrenesulfonate) (PSS, 70 kDa), Rhodamine B Isothiocyanate dye (TRITC, 99%), Fluorescein

5

isothiocyanate isomer I (FITC, >90%), Calcium Chloride (99.9%), Gold(III) chloride (HAuCl4,

6

>99%), Sodium borohydride (96%), Silver nitrate (AgNO3, >99%), Dulbecco's Modified Eagle's

7

Medium (DMEM), fetal bovine serum (FBS), MTT, streptomycin, glutamine, broth were all

8

purchased from Sigma-Aldrich. Hexadecyltrimethylammonium bromide (CTAB, 96%) and

9

Ascorbic acid (>99%) were purchased from Fluka. All the chemicals were used directly without

10

further purification.

11

Synthesis of gold nanorods

12

Gold nanorods were synthesized according to a reported seed mediated growth protocol.30

13

Briefly, the seed solution was prepared by mixing 1 mL of 0.1 M CATB and 0.025 mL of 10

14

mM of HAuCl4. While stirring, 0.1 mL of ice-cold 10 mM NaBH4 was added. The mixture was

15

kept at 25 ºC. Then 50 mL of 0.1 M CTAB, 1 mL of 4 mM AgNO3, and 2.5 mL of 10 mM

16

HAuCl4 were mixed to produce the growth solution. Then 0.5 mL of 0.1 M ascorbic acid was

17

added to the growth solution. Finally, 0.5 mL of the seed solution was added to the growth

18

solution at 27 ºC and the reaction was kept constant at this temperature for 6 hours. The CTAB

19

stabilized GNRs were centrifuged (13000 g, 30 min) and ultrasonicaly redispersed in PSS

20

solution (1 mg/mL) followed by incubation for 1 h. Then PSS coated nanorods were centrifuged

21

(13000 g, 30 min) again and redispersed in water.

22

Fabrication of chlorhexidine spheres


Langmuir

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

1

Chlorhexidine particles were fabricated by precipitation of chlorhexidine diacetate with CaCl2

2

which was reported in our previous work.28 Briefly, 15mg/mL chlorhexidine diacetate solution

3

and 0.33 M CaCl2 solution were mixed together at ratio of 1:1 by volume at room temperature.

4

The mixtures were shaken for 1 min, then centrifuged at 2000 rpm for 1 min (Eppendorf

5

centrifuge 5417C, Germany). Then the precipitates were washed three times with CaCl2 solution

6

to reduce the dissolution of the chlorhexidine compounds. The resulting chlorhexidine

7

compounds were coated with gold and characterized using a scanning electron microscope

8

(SEM, FEI inspect-F, USA) with voltage of 10 kV, working distance of 10 mm and spot of 3.5.

9

In addition, EDX mapping was used to analyze the elemental distribution in the chlorhexidine

10

spheres. The produced chlorhexidine spheres were freeze dried at -107 ºC, 0.009 mBar for 1 day

11

(ScanVac Cool Safe Freeze Drying, Denmark), and the powder was analyzed using Fourier

12

Transform Infrared Spectroscopy (FTIR-Bruker, USA).

13

For visualization of the chlorhexidine spheres using confocal microscopy, the spheres were

14

labelled using Rhodamine B (RhB). Spherical chlorhexidine particle fabrication was carried out

15

using the previous procedure, but before mixing the chlorhexidine diacetate solution with the

16

CaCl2 solution, RhB was added to the chlorhexidine diacetate solution. The synthesized particles

17

were centrifuged and washed as described previously. Particles were characterized using

18

confocal microscopy (Leica TS confocal scanning system, Germany).

19

Gold nanorod functionalization of the chlorhexidine spheres

20

To functionalize the chlorhexidine spheres with gold, the gold nanorod suspension was pre-

21

mixed with 0.8 mL of 0.33 M CaCl2, and then the mixture was introduced to 0.8 mL of 15

22

mg/mL chlorhexidine diacetate solution. Specifically, a series of gold suspensions, 5, 10, 50,

23

100, 200, and 400 µl (0.45 mg/mL), were premixed with CaCl2 solution to determine the


Page 6 of 36

Page 7 of 36

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

Langmuir

1

influence of nanoparticles on chlorhexidine growth. All the procedures were the same as

2

previous described and the gold nanorod functionalized chlorhexidine spheres were also freeze

3

dried. The number of particles produced from all the mixtures was counted using a

4

hemocytometer. Both field emission and back scattered SEM (FEI Inspect F, Eindhoven, The

5

Netherlands) were used to characterize the synthesized particles, and the size of the gold-

6

chlorhexidine composites were measured using image analysis software (Nano Measure, version

7

1.2). The gold nanorod functionalized chlorhexidine spheres were also characterized using

8

Thermo-gravimetric analysis (TGA, Q50, USA) at 10°C/min under a nitrogen atmosphere and

9

over a temperature range of 100-1000 °C.

10

Fabrication of core-shell chlorhexidine spheres

11

Growth of chlorhexidine spheres was also tuned and separated into two stages. The first stage

12

involved the slow growth of small chlorhexidine crystals at low temperature and second the fast

13

growth of chlorhexidine shells on top of the initial primary crystals. To visualize the two stages,

14

chlorhexidine diacetate solutions were mixed with FITC and RhB accordingly. To produce small

15

chlorhexidine primary crystals, both the chlorhexidine diacetate (15mg/ml) and CaCl2 solutions

16

(0.33 M) were kept in an ice bath for one hour. The mixing of these solutions as described

17

previous resulted in immediate precipitation of small chlorhexidine crystals. These pre-produced

18

chlorhexidine crystals at 5, 50, 100, 200 and 400 µl (1.63×107 crystallites/mL) were separately

19

added to 0.33 M room temperature CaCl2, and 15 mg/ml chlorhexidine diacetate solutions. After

20

1 minute, the mixtures were washed with CaCl2 and characterized using confocal microscopy.

21

The size effect induced by the chlorhexidine primary crystals was determined by analyzing the

22

size distribution of produced chlorhexidine spheres using image analysis software (Nano

23

Measure, version 1.2).


Langmuir

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

1

LbL assembly on gold-chlorhexidine composites

2

Stabilization of chlorhexidine spheres was achieved by using LbL self-assembly. PAH (2

3

mg/mL) and PSS (2 mg/mL) were used as polyelectrolytes to be deposited on the gold-

4

chlorhexidine composite surface. The LbL assembly procedure is described in a previous study28

5

and to reduce the dissolution of gold-chlorhexidine spheres, 0.33 M CaCl2 was added in both the

6

PAH and PSS solutions. The PAH was added to the gold-chlorhexidine spheres as the first layer

7

and mixture was shaken (Vortex-Genie 2, Germany) for 10 min. Then the mixture was

8

centrifuged (Eppendorf centrifuge 5417C, Germany) at 2000 rpm and washed with 0.33 M CaCl2

9

solution (3 times) to remove the excess PAH. The second PSS layer was assembled using the

10

same procedure. After assembling six layers, the encapsulated chlorhexidine particles with the

11

structure Chlorhexidine/(PAH/PSS)3 were produced. The supernatant during each layer assembly

12

and after washing was collected for UV measurement to determine chlorhexidine loss during the

13

LbL process (Lambda 35, Perkin Elmer, USA). For fluorescence imaging, one of the PAH layers

14

was labelled with FITC and the chlorhexidine core was labelled with RhB. SEM and confocal

15

microscopy were used to characterize the synthesized gold-chlorhexidine composite capsules.

16

Rupture of gold functionalized chlorhexidine capsules by NIR light irradiation

17

To irradiate the chlorhexidine capsules a customised laser setup was used.31 A 100 mW laser

18

diode (840 nm) was coupled with a simple optical microscope (100 × objective, Edmund

19

Scientific, USA), and the focused laser spot was tuned by adjusting the operating laser voltage.

20

In addition, the white light source and XYZ stages allowed samples to be easily located and

21

focused. The laser beam passed through the objective in the Z direction and was focused at the

22

sample to irradiate the specific site. A CCD camera was connected to a computer to capture this

23

event. Thus, once aligned and focused, an image of sample and a laser spot could be observed on


Page 8 of 36

Page 9 of 36

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

Langmuir

1

the screen. In the current work, remote triggering of the gold functionalized chlorhexidine

2

capsules was carried out using this laser setup.

3

The chlorhexidine capsule suspension was placed on a thin glass slide and the glass slide was

4

marked to locate the particles. After application of the laser beam, the chlorhexidine capsules

5

were broken which led to the dissolution of the chlorhexidine crystals and remaining

6

polyelectrolytes shells. The sample was then dried in air and laser triggered site was

7

characterized using SEM and confocal microscopy.

8

NIR light controlled release

9

Chlorhexidine release was performed in deionized H2O (n=3). 50 µl of gold-chlorhexidine

10

capsules were diluted into 400 µl for each sample in NMR tube (5 cm in length), and was

11

irradiated by the laser beam from to top (100 mW) for 30 min (laser on) at each time point. Then

12

the capsule suspensions were incubated at room temperature for 24 h (laser off). Supernatants

13

(200 µl) from each sample at each time points (24, 24.5, 48, 48.5, 72, 72.5, 96, 96.5, 120, 120.5,

14

144, 168 h) were collected and replaced with equivalent fresh deionized H2O. Chlorhexidine

15

release was determined by UV-vis absorption (Lambda 35, Perkin Elmer, USA) at 254 nm

16

according to the established calibration curve. Chlorhexidine release from capsules without laser

17

treatment was normalized as the experimental control.

18

Cytotoxicity assay

19

Cytotoxicity of chlorhexidine particles was evaluated with a standard MTT assay with L929 cells

20

(ECACC 85011425). Briefly, cells were cultured in DMEM supplemented with 10% fetal bovine

21

serum (FBS) with 100 IU/mL penicillin, 100 µg/mL streptomycin and 2 mmol/L glutamine in an

22

humidified incubator with 10% CO2 and 95% air at 37°C, and seeded in 96-well microtiter plates

23

at 10000 cells per well. Following overnight incubation, medium was removed and cells were


Langmuir

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 10 of 36

1

washed twice with PBS. The cells were then treated with different concentrations (ranging from

2

0.0000625 to 0.008%) of chlorhexidine diacetate, uncoated and coated Au/CHX particles for 24

3

and 48 hours. Following treatment, culture medium containing the treatments were removed and

4

50 µL of 5 mg/mL tetrazolium salt MTT was added to each well and incubated in 37 °C for 2 h.

5

Then 100 µl of isopropanol was added to each well and measured with a plate reader at 570 nm.

6

Antimicrobial assay

7

Porphyromonas gingivalis (strain-381) and Escherichia coli (NCTC 9003) were grown in brain

8

heart infusion (BHI) broth in an anaerobic environment with an atmosphere of 5% H2, 10% CO2,

9

and 85% N2 at 37°C. To determine the Minimum Inhibitory Concentration (MIC) of NIR

10

induced chlorhexidine containing solution against P. gingivalis and E.coli, 96-well flat-bottomed

11

microtiter plates were used with a final assay volume of 250 µl/well (225 µl/well of bacterial

12

solution and 25 µl/well of chlorhexidine suspensions from 30 min NIR light illumination, with

13

concentration ranging from 0.00008125-0.0013%). Negative control (bacterial inoculum only but

14

no chlorhexidine) and blank (medium only) wells were also included. The microtiter plates were

15

incubated for 0, 24 and 48 hours in anaerobic conditions as above and the optical density (OD)

16

was determined at 595 nm to quantify bacterial growth. The MIC was defined as the lowest

17

concentration of chlorhexidine that inhibited the growth of microorganism at each time point.

18 19

RESULTS AND DISCUSSION

20

Chlorhexidine spheres with gold nanorods


Page 11 of 36

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

Langmuir

1

Based on the seed mediated growth method, the homogeneous gold nanorods used are shown in

2

Figure 1a. According to the TEM image, the gold nanorods have an average length of 85 nm and

3

width of 20 nm. UV-vis spectroscopy of the gold nanorods (Figure 1b) displayed their

4

absorbance peak at 840 nm. The surface plasmon resonance band was largely dependent on the

5

shape and ratio of length to width of the gold nanorods, their surface properties, as well as the

6

mediums surrounding the particles.32, 33 The plasmonic properties of gold nanorods make them

7

promising candidates in light responsive drug delivery, since they have great photothermal

8

conversion ability and their absorption band in the near infrared range is especially preferred for

9

in vivo applications.34

a

b

10 11

Figure 1. (a) TEM image of gold nanorods; (b) UV-vis spectrum of gold nanorods.

12

In a previous study the authors reported a new method to produce chlorhexidine spheres, with

13

homogeneous size and morphology, and proved that the crystal growth of the drug spheres could

14

be tuned by temperature and ion concentration.28 In medicine and dentistry24 a more sustained

15

and stimuli responsive chlorhexidine delivery system against bacteria is also desirable. This


Langmuir

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 36

1

could be achieved by combining the chlorhexidine spheres with the gold nanorods. As

2

demonstrated in Figure 2 gold nanorod functionalized chlorhexidine spheres were synthesized by

3

introducing the gold nanorods into the CaCl2 solution. Without gold nanorods, the chlorhexidine

4

spheres had a porous surface morphology, which were comprised of small dendrites (Figure 2b).

5

In contrast, for the gold nanorods functionalized spheres (with 400 µl nanorods), gold was

6

clearly observed in the backscattered images (Figure 2c), and at high magnification small gold

7

nanorod

8

GNRs/chlorhexidine composites also demonstrated a absorption peak at 840 nm (Figure 1b). The

9

incorporation of gold particles into drug delivery carriers has been extensively studied. For

10

instance, hollow gold nanospheres were loaded into PLGA microspheres together with

11

paclitaxel, via a double-emulsion solvent evaporation method, and the paclitaxel release was

12

modulated by using a NIR light.35 Gold nanorods and doxorubicin were incorporated into

13

crosslinked poly(β-amino ester) particles, with the glass transition temperature (Tg) close to

14

body temperature. Therefore, transformation of the polymer from a glassy to a rubbery state

15

induced by an NIR light produced an instant burst release of doxorubicin.36 For both examples

16

drug release benefited from enhanced diffusion through the polymer networks. The choice of

17

polymers with either a lower Tg

18

isopropylacrylamide) (PNIPAm),37 is essential for the design of NIR responsive drug carriers.

19

The incorporated gold nanorods in the chlorhexidine drug crystals have the potential to generate

20

heat upon NIR light irradiation, and thus deconstruct the porous drug crystals and induce

21

chlorhexidine release.

clusters

were

present

on

the

chlorhexidine

dendrites

(Figure

2d).

The

or high thermal sensitivity, such as poly(N-


Page 13 of 36

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

Langmuir

a

b

c

d

1 2

Figure 2. BSEM images of (a)chlorhexidine sphere and (c)gold-chlorhexidine composites; SEM

3

images show the surface morphology of (b)chlorhexidine sphere and (d) gold-chlorhexidine

4

composites.

5 6

Tuneable chlorhexidine crystal growth

7

To further understand the role of gold nanorods in chlorhexidine particles formation, different

8

amounts of gold nanorods was dispersed into CaCl2 solutions. Figure 3 illustrates the influence

9

of gold nanorods on the size of chlorhexidine spheres. Without the addition of gold nanorods the

10

chlorhexidine spheres had a mean (SD) diameter of 24.0 (5.0) µm. Adding 5 µl of gold nanorods

11

produced a slight size reduction of 22.9 (4.6) µm. The average chlorhexidine sphere diameters

12

gradually decreased on increasing addition of gold nanorods, with a 400 µl gold nanorod

13

addition giving a mean (SD) diameter of 14.5 (1.6) µm. There was a correlation between the

14

amount of gold nanorods added and the mean chlorhexidine particle diameter (r2=0.98, Figure


Langmuir

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 36

1

3g). SEM images indicated there was no distinct difference in the morphology of chlorhexidine

2

spheres at different gold nanorods addition. In terms of the particles size distribution, with less

3

gold nanorods added, the size distribution was broader (Supporting information, Figure S1). The

4

chlorhexidine precipitation efficiency (>98%) was also similar for all the samples (Supporting

5

information, Table S1). When increasing the amount gold nanorods however, the chlorhexidine

6

spheres were more homogeneous. The crystallisation of the chlorhexidine spheres involves a

7

rapid nucleation and crystal growth process, so it is likely that the gold nanorods in the solution

8

may act as sites for crystallization. This is supported by the data in Figure 3g, h where

9

chlorhexidine particle number and size was correlated with the amount of gold nanorods added

10

to solution (r2=0.98, Figure 3g; r2=0.98, x from 0 to 200, Figure 3h). The remaining weight of

11

functionalized chlorhexidine spheres also increased as a function of increased amount of gold

12

nanorods as indicated by the TGA result (Supporting information, Figure S2).

13

Gold nanoparticles were previously used as seeds to produce hybrid silica particles,16 and

14

CdSe/ZnS quantum dots were also used to grow mesoporous silica shells on their surface.38

15

Therefore, we assume that the amount of gold nanorods in the solution was a key factor to

16

determine the chlorhexidine sphere size and number. It was however noted that any excess gold

17

nanorods could cluster and be trapped in the crystals (Supporting information, Figure S3). It is a

18

common approach to control crystals size by adjusting the seed amount in nanoparticle synthesis.

19

Similarly for gold nanoparticle synthesis, the mean particle size can also be tuned, with the mean

20

particle size decreased with increased seed concentration, and at low seed and gold precursor

21

ratio, large particles and secondary nucleation were observed.39 Interestingly, gold nanorods can

22

also be replaced by other nanoparticles (e.g. Fe3O4 nanoparticles), and the same effect on

23

chlorhexidine crystallization has been demonstrated by the authors. The current functionalization


Page 15 of 36

1

of chlorhexidine crystals with Gold or Fe3O4 nanoparticles also opens up a door for its stimuli

2

responsive release.

a

b

c

d

e

f

g

h

450

4

Mean CHX particle number per ml (×10 )

30 28

Mean CHX particle diameter (µm)

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

Langmuir

26 24 22 20 18 16 14

400

350

300

250

200

150

12

-50

10 -50

0

50

100

150

200

250

300

350

400

0

50

450

100

150

200

250

300

350

400

450

Amount of Au NR added (µL)

Amount of Au NR added (µL)

3

Figure 3. Effect of gold nanorods on chlorhexidine crystallization. SEM images of gold-

4

chlorhexidine composites with different amount of gold nanorods: (a) no gold nanorods; (b) 5 µl;

5

(c) 50 µl; (d) 100 µl; (e) 200 µl; (f) 400 µl gold nanorods (0.45 mg/mL), and (g) mean

6

chlorhexidine particle diameter and (h) chlorhexidine particle numbers as a function of GNRs

7

amount. Inset images are individual particles at high magnification (×8000).


Langmuir

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 16 of 36

1

In a parallel experiment, small chlorhexidine spheres were used as primary particles instead of

2

gold nanorods to further reveal the mechanism of chlorhexidine particles formation. This was

3

achieved by keeping the original chlorhexidine diacetate and CaCl2 solutions in an ice bath and

4

carrying out the synthesis to produce primary chlorhexidine particles with a mean (SD) diameter

5

of 5.2 (1.7) µm (Figure 4a and Supporting information, Figure S4), while chlorhexidine spheres

6

synthesized at room temperature had a mean (SD) diameter of 17.2 (1.9) µm (Figure 4b and

7

Supporting information, Figure S4). By introducing the chlorhexidine primary particles into the

8

CaCl2 solution, growth of a second shell of chlorhexidine crystals were successfully achieved.

9

FITC labelled chlorhexidine primary particles were presented as the core (green label, Figure 4a,

10

c) and the new shells grown from the interface around the primary particles were visible when

11

labelled with RhB (red label, Figure 4d, e). At the transmitted channel, a clear boundary between

12

the chlorhexidine primary particles and outside shell was identified (Figure 4f). These

13

observations suggest a seed mediated crystallisation of chlorhexidine crystals, which supports the

14

previous hypothesis. Similarly, the mean particle diameter of the chlorhexidine spheres

15

decreased as the amount of primary chlorhexidine particles increased (Supporting information,

16

Figure S4 and S5). The chlorhexidine particle size distribution also narrowed at increasing

17

primary particle addition (Supporting information, Figure S5 and S6). An epitaxial growth on the

18

surface of the primary seed crystal may also be likely and is present in other systems where the

19

chemistry and crystal lattice constants are favourable.40

20 21 22


Page 17 of 36

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

Langmuir

a

b

d

e

c

f

1 2

Figure 4. Confocal images of chlorhexidine spheres. (a) chlorhexidine primary particles

3

produced in an ice bath (labelled with FITC); (b) large chlorhexidine spheres produced at room

4

temperature (labelled with RhB); core-shell chlorhexidine particles produced based on the small

5

chlorhexidine primary particles at (c) green channel, (d) red channel and (e) merged image; (f) at

6

transmitted channel.

7 8

Mechanism of chlorhexidine crystal growth

9

Theoretically, the growth of nano/micro particles involved two main stages: rapid nucleation to

10

form primary seeds and sustained growth of precursors on the primary seeds.41 The formation of

11

homogenous porous chlorhexidine spheres was a very rapid process, which was dependent on the

12

type and concentration of ions and temperature of the bulk solutions, as proposed in our previous

13

study.28

Herein, with gold nanorods or chlorhexidine primary particles, the chlorhexidine


Langmuir

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 18 of 36

1

particle forming process is presented schematically in Figure 5. At the first stage of synthesis,

2

when chlorhexidine diacetate was mixed with CaCl2, monomers were formed immediately

3

following basic coordination chemistry of guanidines.42 The biguanidines of chlorhexidine have

4

strong coordination capability with ions, such as Cu2+, Zn2+ or Ag+.43, 44 According to the inset

5

FTIR spectrum (Figure 5), the typical chlorhexidine band of C=N was shifted from 1610 cm-1 to

6

1621cm-1 and N-H stretching vibration of the Alkyl-NH-Aryl, (Alkyl)2NH and =NH at 3118,

7

3303 and 3190 cm-1 was distinctly increased.44 This is evidence that Ca2+ had coordinated with

8

chlorhexidine. The presence of Cl- may also help to accelerate the precipitation of these

9

complexes since the Cl- ion is thought to reduce chlorhexidine solubility.45 Interestingly there

10

was a homogenous and copious distribution of Ca2+ and Cl- in the chlorhexidine spheres via EDX

11

mapping (Supporting information, Figure S7). Therefore, the addition of CaCl2 to the

12

chlorhexidine diacetate solution facilitated the formation of chlorhexidine nucleation and

13

monomers for crystal growth. The addition of gold nanorods or chlorhexidine primary particles

14

in the solution, appears to have encouraged surface crystallization to produce more primary

15

crystallites. Research on the growth of mesoporous silica shells on gold nanoparticles, indicates

16

the ratio of gold nanoparticles to silica precursors was a key factor to determine the gold

17

occupancy and cluster sizes for the synthesized composites.17 This could explain why gold was

18

only detected in the backscattered SEM images for samples with excess addition of gold

19

nanorods. At more reduced additions of gold particles crystallization sites were favoured, while

20

excess gold particles could lead to flocculation and clusters observed on the chlorhexidine sphere

21

surfaces (Figure 2d). In addition, the negatively charged gold nanorods may have a high affinity

22

to positively charged chlorhexidine molecules, which encouraged crystallization of chlorhexidine

23

on their surfaces. This is advantageous as coating of gold colloids with silica requires a surface


Page 19 of 36

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

Langmuir

1

modification using a silane coupling agent to make them vitreophilic.14, 16 For the second stage,

2

the growth of chlorhexidine spheres proceeded via continuous deposition of monomers on the

3

primary particles. This process was monitored by labelling the chlorhexidine primary particles

4

and chlorhexidine monomers with different dyes and a core-shell structure was displayed (Figure

5

4). At low temperature, chlorhexidine crystal growth rate was limited, so small (5.2±1.7 µm)

6

crystals were produced. Once the primary particles were transferred into solutions with

7

monomers, crystal growth continued until the chlorhexidine monomers were depleted. There was

8

also evidence of Ostwald Ripening, as the sacrifice of small particles and bridging between

9

particles was observed in the SEM images of the chlorhexidine precipitates at different time

10

intervals (10, 20, 30, 40, 50 and 60s) (Supporting information, Figure S8).

or

CHX monomers Ca2+

Gold particles Cl

Cl

CHX primary particles Primary particles Crystal growth and Ostwald ripening 3303

3190 C= N 1621

4000

3500

3000

2500

Wa v e l e n g t h / c m

2000

1500

-1

11 12 13

Figure 5. Schematic illustration of chlorhexidine crystallization. Gold particles (red) and small

14

chlorhexidine spheres (green) were used as seeds for chlorhexidine crystals growth; inset is the

15

FTIR spectrum of the chlorhexidine spheres (red) and chlorhexidine diacetate (black).


Langmuir

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 20 of 36

1

Gold-chlorhexidine particle encapsulation and laser triggered release

2

LbL encapsulation of drug crystals is an efficient approach to achieve sustained drug release and

3

improve the solubility for some anticancer drugs.29,

4

encapsulation over other delivery methods is the high drug loading rate since the cores consist of

5

the drug, and their toxicity can be reduced at the same time compared to the equivalent free

6

dose.47,

7

porous structure as the spheres could be easily dissolved in H2O. Successful encapsulation of the

8

chlorhexidine spheres was achieved by carrying out the assembly at moderate conditions and in

9

high salt polyelectrolyte solutions. As presented in Figure 6a, the chlorhexidine sphere cores

10

were still remained intact after encapsulating 6 layers of polyelectrolytes (shells appear in green).

11

The overlapped images indicated that the polyelectrolytes had penetrated into the porous

12

chlorhexidine sphere surface (Figure 6a). Compared to the conventional LbL capsules, the

13

polymer shells here were much thicker, which was due to the deposition of polyelectrolytes at

14

high salt concentration, and also chlorhexidine dissolution during the LbL process.49 The shell

15

thickness of the chlorhexidine capsules was 1.02 µm according to a cross-sectional measurement

16

in our previous study.28 SEM images showed that the chlorhexidine capsules had completely

17

been coated and no dendritic structure could be identified (Supporting information, Figure S9).

48

46

The advantage of drug colloid

LbL encapsulation of the chlorhexidine composites was necessary to stabilize the


Page 21 of 36

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

Langmuir

green

red

transmitted

overlap

a

b

1 2

Figure 6. Confocal images of gold functionalized chlorhexidine capsules before (a) and after

3

laser irradiation (b). One of the PAH layers is labelled with FITC (green) and the chlorhexidine

4

particles were labelled with RhB (red). Arrows indicated the remaining polyelectrolyte shells and

5

inset images are taken at high magnification.

6 7

Gold nanorods have a strong photothermal conversion ability,4 so it was assumed that it would

8

be possible to trigger the release of functionalized chlorhexidine capsules with NIR light

9

irradiation. Using an 840 nm NIR light (up to 100 mW) with our laser setup, the chlorhexidine

10

capsules could be ruptured while the others stayed intact (Figure 6b). Once be focused by the

11

laser beam, the capsule erupted and the exposed chlorhexidine spheres dissolved, as a result,

12

only polyelectrolyte shells remained as indicated by arrows in Figure 6b. There was however still

13

some weak red signal detected within the residual shells, indicating residual chlorhexidine. This


Langmuir

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 22 of 36

1

was due to complexation between the chlorhexidine molecules and the PSS layer via an

2

interaction of its amine group and sulfonic group of PSS. According to the transmitted channel

3

images, debris around the broken polyelectrolyte shells was also observed and confirmed by the

4

SEM images (Figure 7a, b). It was not surprising that the gold nanorod functionalized

5

chlorhexidine capsules were responsive to NIR light since the chlorhexidine crystals were

6

directly growth on top of the nanorods. The energy generated by NIR light irradiation can disrupt

7

the chlorhexidine crystal structures. In a previous study of silica coated gold nanoparticles, the

8

surface temperature of gold particles was suggested to be more than 100 oC upon laser

9

irradiation.50, 51 The local heating by using NIR light irradiation was therefore a more efficient

10

trigger for drug release than simply heating the solution to an equivalent temperature.12 Gold

11

nanoparticles deposited into polyelectrolyte capsules previously revealed rupture of the capsules

12

upon laser irradiation,52 even with a SiO2 layer deposited on the surface of capsules, which

13

allowed snap intracellular cargo release.8 Since the chlorhexidine crystal growth is based on the

14

coordination of Ca2+ and biguanidines, heat generated by gold nanorods could interrupt the

15

binding and even cause a conformational change of chlorhexidine molecules, thus leading to

16

destruction of the crystals. Local heating of gold nanorods may also accelerate dissolution and

17

diffusion of chlorhexidine molecules. According to the in vitro release kinetics (Figure 8),

18

chlorhexidine release occurred in a stepwise fashion after each cycle of laser treatment. A burst

19

release was observed during each laser irradiation cycle. In contrast, the released chlorhexidine

20

content during each cycle for the control was lower, although both groups exhibited a sustained

21

release.

22 23


Page 23 of 36

b

a

1

Figure 7. SEM images of gold functionalized chlorhexidine capsules after laser irradiation.

2

Untreated capsules were still intact but laser triggered ones were broken with polymer shells

3

remaining as indicated by white arrows.

ON

0.6

Cumulative chlorhexidine release (mg)

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

Langmuir

ON

ON

ON

control with NIR light

ON

0.5

0.4

0.3

0.2

0.1

0.0 0

20

40

60

80

100

120

140

160

180

time (h)

4

Figure 8. Cumulative release of chlorhexidine from capsules with (red) and without (black) NIR

5

light irradiation. Five cycles of NIR light on (30 min, 100 mW) were indicated by grey shades;

6

values are the mean of three groups and vertical bars represent the SD.


Langmuir

1

Cytotoxicity and antimicrobial activity

2

The effect of coated Au/CHX spheres on the viability of fibroblast-like cell line (L929) was

3

determined by MTT assay and was compared with uncoated Au/CHX spheres and CHX

4

diacetate. Treatment with these products reduced the viability of the cells in a dose-dependent

5

manner (Figure 9). However, the coated Au/CHX spheres demonstrated reduced cytotoxicity in

6

comparison with uncoated Au/CHX spheres and CHX diacetate at the same concentration.

7

Relative cellular viability was reduced to approximately 50% when treated with 0.0005% of

8

CHX diacetate for 24 hours which was further reduced to about 30% when treated for 48 hours.

9

Treatment with 0.0005% of coated Au/CHX spheres however, showed approximately 90% of

10

cellular viability at 24 hours and was more than 70% at the 48 hour time point. Accordingly,

11

changes in cellular morphology (rounded, swelled and loss of attachment to the wells) were also

12

observed at 0.0005% of CHX diacetate whilst maintaining the normal morphology in coated

13

Au/CHX treated cultures. These suggest that coated Au/CHX spheres are not cytotoxic when

14

compared to CHX diacetate. (Supporting information, Figure S10).

24h

48h

100

80

80

concentration (%)


concentration (%)

0. 00 8

0. 00 2

0. 00 1

0. 00 05

0. 00 02 5

25

0. 00 8

0. 00 4

0 0. 00 2

0 0. 00 1

20

0. 00 05

20

0. 00 02 5

40

0. 00 00 62 5 0. 00 01 25

40

0. 00 01 25

60

0. 00 00 6

viability (%)

100

60

coated Au/CHX uncoated Au/CHX CHX diacetate

120

0. 00 4

coated Au/CHX uncoated Au/CHX CHX diacetate

120

viability (%)

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 24 of 36

Page 25 of 36

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

Langmuir

1 2

Figure 9. Effect of coated and uncoated Au/CHX particles and chlorhexidine diacetate on the

3

relative viability of fibroblast like cell line. The percentages of cellular viability in the presence

4

of the treatment compounds relative to that in the control are shown. The results are the average

5

of six replicates.

6

To demonstrate if the released chlorhexidine induced by NIR light exposure was still functional,

7

antimicrobial assays were performed against anaerobic microorganisms; P. gingivalis and E.coli.

8

50 µl of coated Au/CHX particles (diluted into 400 µl) were exposed to NIR light irradiation for

9

30 min and supernatants were collected for the test. Supernatants from coated Au/CHX particles

10

without exposure were also tested as a control. According to the measurement, the concentration

11

of released chlorhexidine induced by NIR light exposure was about 0.0013% and was only

12

0.00016% without NIR light irradiation.

13

P. gingivalis and E.coli were grown and treated with series of diluted supernatants for 24 and 48

14

hours to determine the MIC. The results showed that NIR induced CHX were able to inhibit

15

(MIC) P. gingivalis at 0.00065%, whereas the growth of E.coli was inhibited at 0.0013% (Figure

16

10), indicating that the released chlorhexidine was still functional. However, supernatant from

17

coated Au/CHX particles without NIR light exposure showed no inhibitory effect against either

18

the P. gingivalis or E.coli, which means the released amount without NIR treatment was too low

19

at these time points to inhibit the bacterial growth (Supporting information, Figure S11).

20


Langmuir

P.gingivalis

E.coli 0h 24h 48h

0.7 0.6

0h 24h 48h

0.7

0.6

0.5 0.5

absorption (a.u.)

0.4

0.3 0.2

0.3

13 0. 00

5 0. 00 06

03 25 0. 00

01 6 0. 00

00 0

01

0. 0

0.

0. 0

00 06

5

32 5 0. 0

00 0.

00

25

01 6

concentration (%)

concentration (%)

5

0.

00 00

81

0

5

0.0 81 2

0.0 3

0.1

25

0.1

25

0.2

00

06 2

Figure 10. Antimicrobial activity of chlorhexidine supernatants from NIR light exposure. 0. 0

1

0.4

0

absorption (a.u.)

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 26 of 36

2 3

The proposed gold nanorod functionalized chlorhexidine capsules with high drug loading rate

4

and NIR light responsive properties have advantages and promising applications in medicine and

5

dentistry. Many current products have limitations due to lack of controlled drug release and

6

duration.24 The gold nanorod functionalized chlorhexidine capsules are useful as they may be

7

injected into sites such as periodontal pockets. As periodontitis is caused by microbial biofilms

8

which are highly resistant to antimicrobial agents, a burst release triggered by NIR light would

9

be essential to eliminate the pathogens, which cannot be achieved otherwise as the minimum

10

inhibitory concentration (MIC) may not be reached without these triggers. Any remaining

11

bacteria may cause a secondary infection, so the non-invasive approach of NIR light induced

12

drug release could help solve this problem. Photodynamic therapy using a diode laser is already

13

used in conjunction with a photosensitizer to decrease or eliminate bacteria53 and it may be

14

possible to combine these therapies to increase its efficacy.

15 16

CONCLUSION


Page 27 of 36

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

Langmuir

1

In the present work, a novel NIR light responsive chlorhexidine composites was proposed, which

2

was based on gold nanorods and chlorhexidine crystallisation. By introducing gold nanorods into

3

chlorhexidine solutions it was possible to control the amount and size of chlorhexidine spheres.

4

Formation of gold functionalized chlorhexidine microparticles was demonstrated in a gold

5

nanorod assisted crystallisation and Ostwald Ripening mechanism. This process was also

6

demonstrated by using chlorhexidine primary particles to control chlorhexidine crystallisation.

7

Encapsulation of the gold nanorod functionalized chlorhexidine crystals using LbL

8

polyelectrolyte multilayers allowed a sustained release and stimuli responsive release when NIR

9

light irradiation was used.

10 11

ASSOCIATED CONTENT

12

Supporting Information

13

SEM images of Au-chlorhexidine particles size distribution, chlorhexidine precipitation

14

efficiency, TGA results of Au-chlorhexidine particles, BSEM images of Au-chlorhexidine

15

particles, SEM images of chlorhexidine particles with different amounts of CHX seeds and their

16

size distribution, EDX mapping of chlorhexidine particle, SEM images of chlorhexidine particles

17

growth as a function of time, encapsulated chlorhexidine particles, cell morphology with CHX

18

treatments, and plates showing the antibacterial activity of CHX treatments are included in the

19

supporting information. These materials are available free of charge on the ACS Publications

20

website athttp://pubs.acs.org.

21

AUTHOR INFORMATION

22

Corresponding Author


Langmuir

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

1

*E-mail: [email protected]; [email protected]

2

Notes

3

The authors declare no competing financial interest.

Page 28 of 36

4 5

ACKNOWLEDGMENT

6

The work was supported by Government of the Russian Federation (grant №14.Z50.31.0004) to

7

support scientific research projects implemented under the supervision of leading scientists. The

8

authors thank Mr. Russell Bailey of Nanovision (QMUL) for help with SEM, Dr Dongsheng Wu

9

for assistance in Confocal laser scanning microscopy. Specially, we have to thank Dr Anton M.

10

Pavlov for the assistance with the assembly of the laser setup. Dong Luo thanks for the financial

11

support from the China Scholarship Council during his PhD study. The authors would like to

12

thank Queen Mary Innovation for providing funding via the Life Sciences Initiative Proof of

13

Concept Fund.

14 15

ABBREVIATIONS

16

GNRs, gold nanorods; CHX, chlorhexidine; LbL, Layer-by-Layer; PAH, poly(allylamine

17

hydrochloride); PSS, poly(sodium 4-styrenesulfonate); RhB, Rhodamine B isothiocyanate; FITC,

18

Fluorescein isothiocyanate isomer I; FTIR, Fourier Transform Infrared Spectroscopy; SEM

19

scanning electron microscopy; CLSM, confocal laser scanning microscopy.

20 21 22


Page 29 of 36

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

Langmuir

1 2

REFERENCES

3

1.

4

Nanorods Coated with Mesoporous Silica Shell as Drug Delivery System for Remote Near

5

Infrared Light-activated Release and Potential Phototherapy. Small 2015, 11 (19), 2323-2332.

6

2.

7

Martinez de la Fuentea, J.; Cui, D. Human Induced Pluripotent Stem Cells for Tumor Targeted

8

Delivery of Gold Nanorods and Enhanced Photothermal Therapy. ACS Nano 2016, 10 (2), 2375-

9

2385.

Liu, J.; Detrembleur, C.; De Pauw-Gillet, M. C.; Mornet, S.; Jerome, C.; Duguet, E. Gold

Liu, Y.; Yang, M.; Zhang, J.; Zhi, X.; Li, C.; Zhang, C.; Pan, F.; Wang, K.; Yang, Y.;

10

3.

Yahia-Ammar, A.; Sierra, D.; Merola, F.; Hildebrandt, N.; Le Guevel, X. Self-Assembled

11

Gold Nanoclusters for Bright Fluorescence Imaging and Enhanced Drug Delivery. ACS Nano

12

2016, 10 (2), 2591-2599.

13

4.

14

Nanorods: Their Potential for Photothermal Therapeutics and Drug Delivery, Tempered by the

15

Complexity of Their Biological Interactions. Adv Drug Deliv Rev. 2012, 64 (2), 190-199.

16

5.

17

J.; Chen, H. Y.; Zhu, J. J. Near Infrared-Guided Smart Nanocarriers for MicroRNA-Controlled

18

Release of Doxorubicin/siRNA with Intracellular ATP as Fuel. ACS Nano 2016, 10 (3), 3637-

19

3647.

20

6.

21

Tumor Delivery of Paclitaxel Using Glycolipid-like Polymer Micelles Containing Gold

22

Nanospheres. Biomaterials 2013, 34 (18), 4510-4519.

Alkilany, A. M.; Thompson, L. B.; Boulos, S. P.; Sisco, P. N.; Murphy, C. J. Gold

Zhang, P.; Wang, C.; Zhao, J.; Xiao, A.; Shen, Q.; Li, L.; Li, J.; Zhang, J.; Min, Q.; Chen,

You, J.; Wang, Z.; Du, Y.; Yuan, H.; Zhang, P.; Zhou, J.; Liu, F.; Li, C.; Hu, F. Specific


Langmuir

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

1

7.

2

E.; Viitala, T.; Le Guevel, X.; Yliperttula, M.; Murtomaki, L.; Urtti, A. Light Induced Cytosolic

3

Drug Delivery from Liposomes with Gold Nanoparticles. J. Controlled Release 2015, 203, 85-

4

98.

5

8.

6

Romero, S. Light-Addressable and Degradable Silica Capsules for Delivery of Molecular Cargo

7

to the Cytosol of Cells. Chem. Mater. 2015, 27 (6), 1929-1942.

8

9.

9

Responsive Hybrid Nanogels for Optical Temperature Sensing, Tumor Cell Imaging and

Page 30 of 36

Lajunen, T.; Viitala, L.; Kontturi, L. S.; Laaksonen, T.; Liang, H.; Vuorimaa-Laukkanen,

Ott, A.; Yu, X.; Hartmann, R.; Rejman, J.; Schütz, A.; Ochs, M.; Parak, W. J.; Carregal-

Wang, H.; Yi, J.; Mukherjee, S.; Banerjee, P.; Zhou, S. Magnetic/NIR-thermally

10

Triggered Drug Release. Nanoscale 2014, 6 (21), 13001-11301.

11

10.

12

Nanocarrier with Reversible DNA Valves for Intracellular Controlled Release. Adv. Funct.

13

Mater. 2013, 23 (18), 2255-2262.

14

11.

15

G. B.; Parak, W. J. NIR-light Triggered Delivery of Macromolecules into the Cytosol. J.

16

Controlled Release 2012, 159 (1), 120-127.

17

12.

18

Vesicles as a Drug Delivery Carrier Enabling Rapid Drug Release upon Light Irradiation. ACS

19

Appl. Mater. Interfaces 2013, 5 (9), 3900-3907.

20

13.

21

Logical Intracellular Release Using Gold Nanocage@Smart Polymer Shell. Adv. Funct. Mater.

22

2014, 24 (6), 826-834.

Li, N.; Yu, Z.; Pan, W.; Han, Y.; Zhang, T.; Tang, B. A Near-Infrared Light-Triggered

Carregal-Romero, S.; Ochs, M.; Rivera-Gil, P.; Ganas, C.; Pavlov, A. M.; Sukhorukov,

Niikura, K.; Iyo, N.; Matsuo, Y.; Mitomo, H.; Ijiro, K. Sub-100 nm Gold Nanoparticle

Shi, P.; Ju, E.; Ren, J.; Qu, X. Near-Infrared Light-Encoded Orthogonally Triggered and


Page 31 of 36

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

Langmuir

1

14.

Nooney, R. I.; Dhanasekaran, T.; Chen, Y.; Josephs, R.; Ostafin, A. E. Self-Assembled

2

Highly Ordered Spherical Mesoporous Silica/Gold Nanocomposites. Adv. Mater. 2002, 14 (7),

3

529-532.

4

15.

5

Drug Delivery to Cancer Cells by Aptamer Gated Nanovehicles. Adv. Mater. 2012, 24 (21),

6

2890-2895.

7

16.

8

Shell Particles. Langmuir 1996, 12 (18), 4329-4335.

9

17.

Yang, X.; Liu, X.; Liu, Z.; Pu, F.; Ren, J.; Qu, X. Near-infrared Light-triggered, Targeted

Liz-Marzan, L. M.; Giersig, M.; Mulvaney, P. Synthesis of Nanosized Gold-Silica Core-

Nooney, R. I.; Thirunavukkarasu, D.; Chen, Y.; R., J.; Ostafin, A. E. Self-Assembly of

10

Mesoporous Nanoscale Silica/Gold Composites. Langmuir 2003, 19, 7628-7637.

11

18.

12

Responsive Drug Release System Based on Gold Nanorods and Phase Change Material. J.

13

Mater. Chem. B 2014, 2, 8338–8345.

14

19.

15

Delivery. Chem. Soc. Rev. 2012, 41 (7), 2867-2884.

16

20.

17

Properties of Composite Polyelectrolyte Based Capsules. Adv Drug Deliv Rev. 2011, 63 (9), 716-

18

729.

19

21.

20

Dental Composites with Chlorhexidine and Mesoporous Silica. J. Dent. Res. 2014, 93 (12),

21

1283-1289.

Lee, J.; Jeong, C.; Kim, W. J. Facile Fabrication and Application of Near-IR Light-

De Koker, S.; Hoogenboom, R.; De Geest, B. G. Polymeric Multilayer Capsules for Drug

Antipina, M. N.; Sukhorukov, G. B. Remote Control Over Guidance and Release

Zhang, J. F.; Wu, R.; Fan, Y.; Liao, S.; Wang, Y.; Wen, Z. T.; Xu, X. Antibacterial


Langmuir

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 32 of 36

1

22.

Farrugia, C.; Camilleri, J. Antimicrobial Properties of Conventional Restorative Filling

2

Materials and Advances in Antimicrobial Properties of Composite Resins and Glass Ionomer

3

Cements-A Literature Review. Dent. Mater. 2015, 31 (4), e89-99.

4

23.

5

UDMA-TEGDMA Resin. J. Dent. Res. 2006, 85 (10), 950-954.

6

24.

7

Debruyne, E.; Hildebrand, H. F.; Martel, B. A chlorhexidine-loaded Biodegradable Cellulosic

8

Device for Periodontal Pockets Treatment. Acta Biomater. 2014, 10 (1), 318-329.

9

25.

Anusavice, K. J.; Zhang, N. Z.; Shen, C. Controlled Release of Chlorhexidine from

Tabary, N.; Chai, F.; Blanchemain, N.; Neut, C.; Pauchet, L.; Bertini, S.; Delcourt-

Dupont, N.; Lazar, A. N.; Perret, F.; Danylyuk, O.; Suwinska, K.; Navaza, A.; Coleman,

10

A. W. Solid State Structures of the Complexes Between the Antiseptic Chlorhexidine and Three

11

Anionic Derivatives of Calix[4]arene. CrystEngComm 2008, 10 (8), 975.

12

26.

13

Characterization,

14

Nanoparticles for Applications in Biomedical Materials and Consumer Products. Int

15

J Nanomedicine 2013, 8, 3507-3519.

16

27.

17

Chlorhexidine–Copper(II)

18

Nanocomposites. Mater. Sci. Eng. C 2013, 33 (2), 752-757.

19

28.

20

of Chlorhexidine Spheres and Sustained Release with Multilayered Encapsulation. ACS Appl.

21

Mater. Interfaces 2016, 8 (20), 12652-12660.

Barbour, M. E.; Maddocks, S. E.; Wood, N. J.; Collins, A. M. Synthesis, and

Efficacy

of

Antimicrobial

Chlorhexidine

Hexametaphosphate

Wu, Y.; Zhou, N.; Li, W.; Gu, H.; Fan, Y.; Yuan, J. Long-term and Controlled Release of from

Organically

Modified

Montmorillonite

(OMMT)

Luo, D.; Shahid, S.; Wilson, R. M.; Cattell, M. J.; Sukhorukov, G. B. Novel Formulation


Page 33 of 36

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

Langmuir

1

29.

Santos, A. C.; Pattekari, P.; Jesus, S.; Veiga, F.; Lvov, Y.; Ribeiro, A. J. Sonication-

2

Assisted Layer-by-Layer Assembly for Low Solubility Drug Nanoformulation. ACS Appl. Mater.

3

Interfaces 2015, 7 (22), 11972-11983.

4

30.

5

Resonance of Gold Nanorods by Controlled Etching. Colloid J. 2015, 77 (5), 652-660.

6

31.

7

of Capsules Containing Ag Nanoparticles and IR Dye by Laser Light. Langmuir 2004, 20 (17),

8

6988-6992.

9

32.

Khanadeev, V. A.; Khlebtsov, N. G.; Burov, A. M.; Khlebtsov, B. N. Tuning of Plasmon

Skirtach, A. G.; Antipov, A. A.; Shchukin, D. G.; Sukhorukov, G. B. Remote Activation

Zhang, Q.; Han, L.; Jing, H.; Blom, D. A.; Lin, Y.; Xin, H. L.; Wang, H. Facet Control of

10

Gold Nanorods. ACS Nano 2016, 10 (2), 2960-2974.

11

33.

12

Silver Ions, Surfactants, and Reducing Agents in Gold Nanorod Overgrowth: Pathway Switch

13

between Silver Underpotential Deposition and Gold–Silver Codeposition. Chem. Mater. 2016,

14

28 (8), 2728-2741.

15

34.

16

Adv. Mater. 2010, 22 (44), 4925-4943.

17

35.

18

Paclitaxel from Biodegradable Microspheres: Photothermal Effect and Enhanced Antitumor

19

Activity. Small 2010, 6 (9), 1022-1031.

20

36.

21

from Near-Infrared Light Responsive Polymer-Nanorod Composites. ACS Nano 2011, 5 (4),

22

2948–2956.

Zhang, Q.; Jing, H.; Li, G. G.; Lin, Y.; Blom, D. A.; Wang, H. Intertwining Roles of

Timko, B. P.; Dvir, T.; Kohane, D. S. Remotely Triggerable Drug Delivery Systems.

You, J.; Shao, R.; Wei, X.; Gupta, S.; Li, C. Near-Infrared Light Triggers Release of

Hribar, K. C.; Lee, M. H.; Lee, D.; Burdick, J. A. Enhanced Release of Small Molecules


Langmuir

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

1

37.

2

Lin, D.; Langer, R.; Kohane, D. S. Magnetically Triggered Nanocomposite Membranes: a

3

Versatile Platform for Triggered Drug Release. Nano Lett 2011, 11 (3), 1395-1400.

4

38.

5

Ammonium Bromide-Capped Nanoparticles. Nano lett 2008, 8 (1), 369-373.

6

39.

7

of Citrate-Stabilized Gold Nanoparticles of Up to 200 nm: Size Focusing Versus Ostwald

8

Ripening. Langmuir 2011, 27 (17), 11098-11105.

9

40.

Page 34 of 36

Hoare, T.; Timko, B. P.; Santamaria, J.; Goya, G. F.; Irusta, S.; Lau, S.; Stefanescu, C. F.;

Gorelikov, I.; Matsuura, N. Single-Step Coating of Mesoporous Silica on Cetyltrimethyl

Bastus, N. G.; Comenge, J.; Puntes, V. Kinetically Controlled Seeded Growth Synthesis

Koutsoukos, P. G.; Nancollas, G. H. Crystal Growth of Calcium Phosphates - Epitaxial

10

Considerations. J. Cryst. Growth 1981, 53 (1), 10-19.

11

41.

12

Monodispersed Hydrosols. J. Am. Chem. Soc. 1950, 72 (11), 4847-4854.

13

42.

14

Coord. Chem. Rev. 2001, 214, 91–141.

15

43.

16

Characterization of New Copper(II) Complex Compounds with Chlorhexidine. Part I. Cent Eur.

17

J. Chem. 2010, 8 (3), 543-549.

18

44.

19

and Thermal Decomposition of New Copper (II) Complex Compounds with Chlorhexidine. J

20

Therm Anal Calorim. 2012, 111 (3), 1763-1770.

21

45.

22

Salts. Drug Dev Ind Pharm. 2009, 35 (2), 172-176.

LaMer, V. K.; Dinegar, R. H. Theory, Production and Mechanism of Formation of

Bailey, P. J.; Pace, S. The Coordination Chemistry of Guanidines and Guanidinates.

Călinescu, M.; Negreanu-Pîrjol, T.; Georgescu, R.; Călinescu, O. Synthesis and

Badea, M.; Olar, R.; Iliş, M.; Georgescu, R.; Călinescu, M. Synthesis, Characterization,

Zeng, P.; Rao, A.; Wiedmann, T. S.; Bowles, W. Solubility Properties of Chlorhexidine


Page 35 of 36

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

Langmuir

1

46.

Fuhrmann, K.; Schulz, J. D.; Gauthier, M. A.; Leroux, J. PEG Nanocages as Non-

2

sheddable Stabilizers for Drug Nanocrystals. ACS Nano 2012, 6 (2), 1667–1676.

3

47.

4

Encapsulated Hydrocortisone Crystals by Insulin/Alginate Shells. Langmuir 2011, 27 (4), 1499-

5

1504.

6

48.

7

Electrospun Poly(lactic acid) Fibers Containing Novel Chlorhexidine Particles with Sustained

8

Antibacterial Activity. Biomater Sci. 2017, 5, 111-119.

9

49.

Zhao, J.; Cui, Y.; Wang, A.; Fei, J.; Yang, Y.; Li, J. Side Effect Reduction of

Luo, D.; Zhang, X.; Shahid, S.; Cattell, M. J.; Gould, D. J.; Sukhorukov, G. B.

Ai, H.; Jones, S. A.; de Villiers, M. M.; Lvov, Y. M. Nano-Encapsulation of Furosemide

10

Microcrystals for Controlled Drug Release. J. Controlled Release 2003, 86, 59–68.

11

50.

12

Heating. J. Am. Chem. Soc. 2012, 134 (18), 7628-7631.

13

51.

14

Nanolithography by Plasmonic Heating and Optical Manipulation of Gold Nanoparticles. ACS

15

Nano 2013, 7 (9), 7648-7653.

16

52.

17

Encapsulation and Triggered Release of Small Molecules. Part Part Syst Char. 2013, 30 (11),

18

989-995.

19

53.

20

PDT against Periodontopathogenic Bacteria. Int J Mol Sci. 2015, 16 (11), 27327-27338.

Croissant, J.; Zink, J. I. Nanovalve-Controlled Cargo Release Activated by Plasmonic

Fedoruk, M.; Meixner, M.; Carretero-Palacios, S.; Lohmüller, T.; Feldmann, J.

Yi, Q.; Sukhorukov, G. B. Single-Component Diazo-Resin Microcapsules for

Haag, P. A.; Steiger-Ronay, V.; Schmidlin, P. R. The in Vitro Antimicrobial Efficacy of

21 22


Langmuir

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

1

Page 36 of 36

Insert Table of Contents Graphic and Synopsis Here

or

CHX monomers Ca2+

Gold particles Cl

Cl

CHX primary particles Primary particles Crystal growth and Ostwald ripening 3303

3190 C= N 1621

4000

3500

3000

2500

Wa v e l e n g t h / c m


2000 -1

1500

Gold Nanorod Mediated Chlorhexidine Microparticle Formation and

Recommend Documents