2012 12th IEEE International Conference on Nanotechnology (IEEE-NANO)

The International Conference Centre Birmingham

20-23 August 20112, Birmingham, United Kingdom

Abstract— The disinfectant loaded micelle fabrication were

investigated as a carrier for 2-benzyl-4-chlorophenol (OBPCP),

a disinfectant agent. Micelles were assessed in terms of the drug

entrapment efficiency by suitable fabrication process. Results

showed that a film sonication method provided the highest drug

loading density at 16.35 ± 0.52%. To utilize numerous

advantages of these polymer micelles, micelles containing

OBPCP were coated on the surface of catheters using Layer-by-

layer (LbL) dip coating technique, highlighting the protocol as

an improvement in the prolong release of the drug from a

period of several hours to several weeks.

I. INTRODUCTION

Over the past few decades, there has been a lot of

research focus on the drug delivery systems using polymeric

micelles as drug carriers. Polymeric micelles are nano-size

particles that have a size between 10 to 100 nanometer and

fabricated by self-assembly of block copolymers or graft

copolymers. Hydrophobic segment assemble at core of

particles while hydrophilic segment covers the core of the

particle (brush-like protective corona). These particles are

able to collect hydrophobic drugs inside. They have shown

the ability to solubilize hydrophobic drugs where the

solubility of paclitaxel, an anticancer drug, is increased from

0.0015 to 2 mg/ml [1] and micelles also enhance drug

pharmacokinetics [2]. Moreover, an entrapment of the drug

using these micelles can enhance delivery efficiency and

reduces the side effects [3]. A 2-benzyl-4-chlorophenol

(OBPCP) is usually coated on medical surface which is used

as chemicals disinfectant for protection and decreasing the

risk of infection due to rapidly inactivate microorganisms

and chemical structure as shown in Fig. 1(c). The

conventional method for coating antibacterial substances is

the direct coating on medical surface providing a high dose at

a local site without exceeding drug concentration in the

systemic toxicity level. However, the problem arose from the

limited release time of the antibacterial substances where the

effectiveness declines and vanishes within a few hours or

days. This resulted in the cause of bacterial related infection

which can lead to death in some cases.

*This research project is supported by Mahidol University.

H. Pheungkham, C. Nagambenjawong, and M. Theerasilp are with the Biomedical Engineering Department, Mahidol University, Nakorn Pathom,

73170 Thailand.

N. Nasongkla is with the Biomedical Engineering Department, Mahidol University, Nakorn Pathom, 73170, Thailand (Corresponding author to

provide phone: 662-889-2138 ext 6357; fax: 662-899-2138 ext 6367; e-mail:

norased.nas@mahidol.ac.th).

Figure 1. Chemical structure of (a) Poly(ethylene glycol)-b-poly( -caprolactone) (PEG-b-PCL), (b) Poly(acrylic acid) (PAA), (c) 2-benzyl-4-

chlorophenol (OBPCP).

Layer-by-Layer (LbL) deposition is one of the most used

thin film fabrication techniques. The films can be formed by

deposition of alternating layers of oppositely materials

properties. It can be used in many applications including

controlled drug release, biosensors, and cell patterning [4].

The films can be coated on any geometry of substrates such

as orthopedic implants, stents, and catheter. This technique

can control inter-diffusion and increase drug loading. Films

can be created through electrostatic interactions, covalent

interactions, and hydrogen bonding generating coatings with

various thicknesses ranging from several nanometers to

micrometers. Therapeutics agents can be incorporated into

these multilayer films either directly or within a carrier such

as micelles.

The objective of this study is to improve loading

efficiency of OBPCP through encapsulation inside polymeric

micelles for the control release from the medical device

surface. The amphiphilic block copolymers between

poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL)

were used in this study because of their excellent properties,

including biocompatibility, low toxicity, as well as an

absence of antigenicity and immuno-genicity. OBPCP

containing micelles were prepared by 3 different methods,

namely the dialysis, solvent evaporation and film sonication

technique [5]. Then, OBPCP-micelles were on surface of

substrate by LbL technique base on hydrogen bonding

interaction between poly(acrylic acid) (PAA) as hydrogen-

bond donor and hydrophilic segment of micelles as

hydrogen-bond acceptor. Advantage of using hydrogen bond

interaction is suitable for the surface delivery of hydrophobic

and neutral drugs, which cannot undergoes electrostatic

interaction to form polyelectrolyte multilayers. Films could

be prepared to have high drug loading efficiency as high as

90 wt% as previously studied by Hammond et al [6]. The

surface composition of the PEG-b-PCL/PAA film was

confirmed by ATR-FTIR spectroscopy and demonstrated the

extended release behavior of drug from the prepared film

over a period of several weeks.

Preparation and Characterization of Polymeric Micelles for

Disinfectant Coating of Catheters.

Hathaichanok Pheungkham, Chayanon Ngambenjawong, Man Theerasilp, and Norased Nasongkla

2012 12th IEEE International Conference on Nanotechnology (IEEE-NANO)

The International Conference Centre Birmingham

20-23 August 20112, Birmingham, United Kingdom

II. MATERIALS AND METHODS

A. Materials

PEG-b-PCL (PEG and PCL, Mw = 5 and 5 kDa) was synthesized according to the method developed by Nasongkla et al [7] and chemical structure as shown in Fig. 1(a). Silicone substrates (0.5 in. diameter and 4 in. length) were purchased from supplier. PAA (Mw = 100 kDa) (chemical structure as shown in Fig. 1 (b)) and other reagents were purchased from Acros Organics (NJ, USA).

B. Preparation and characterization of OBPCP-micelles

with difference methods.

Three different methods (dialysis (DS), solvent

evaporation (SE), film sonication (FS)) were used to prepare

OBPCP-micelles. For all fabrication methods, a 10% w/w

theoretical OBPCP loading were used unless otherwise

stated.

1) Dialysis method, this procedure was carried out by

dissolving 1 mg of OBPCP and 9 mg of diblock copolymer

in THF. The mixture solution was transferred to a dialysis

tubing (MWCO = 50 kDa) and dialyzed against water

overnight at 4 C. Then, the micelle solution was filtered

through centrifugal filter devices with a MWCO of 50 kDa to

remove OBPCP sequential centrifugation until no observable

changes in the retained volume.

2) Solvent evaporation method, this procedure was carried

out by dissolving 1 mg of OBPCP and 9 mg of diblock

copolymer in THF and transferred to an aqueous solution.

The mixture was sonicated at the power level of 60% for 1

min. Next, the solution was gently stirred at room

temperature until the organic phase completely evaporates.

Free OBPCP was then removed from the micelle solution

using the same method as described above.

3) Film sonication method, this procedure was carried out

by dissolving 1 mg of OBPCP and 9 mg of diblock copolymer in THF. Solvent was evaporated to yield a solid

film, to which 10 mL of water was subsequently added and

then vigorously sonicated at a power level of 60% for 1 min.

Free OBPCP was then removed from the micelle solution

using the same method as described above.

Then the particle size of OBPCP-micelles, drug loading,

and in vitro release of OBPCP from micelles were

determined by previously study by Nasongkla et al [7].

Loading density, yield and loading efficiency of OBPCP

were then determined utilizing the following set of equations

(1), (2), and (3), respectively:

C. Preparation of LbL film by dip coating technique

Two distinct substrates with (Section D) and without

coating the substrate surface by diblock copolymers were dip

coated. Firstly, the substrates were dipped into PEG-b-PCL

micelle solution (400 mg/L, pH 2.5) for 10 min and rinsed

with water (pH 2.5) to remove weakly bond for 1 min. Then,

the substrates were immerged into PAA solution (1.44 mg/L,

pH 2.5) for 10 min and rinsed again with water (pH2.5) for 1

min. This cycle provides a bilayer of PAA and OBPCP-

micelles. The dipping process was repeated until the desired

number of bilayers was obtained as shown in Fig. 2. Control

experiment was carried out by pure-OBPCP dip coating

substrate at the same number of layers.

D. Coating the substrate surface by block copolymers

This procedure was carried out by dissolving diblock copolymer (PEG (5 kDa)-b-PCL (12.6 kDa)) in organic solvent and transfer to water. It should be noted that the concentration of diblock copolymer used in this step is lower than the known critical micelle concentration (55.69 mg/L). This is to ensure that polymers are predominantly in the dissociated free polymer chains rather than micellar architecture. Then, the substrates were immerged into mixture solution and sonicated at a power level of 60% for 1 min. The process was repeated until the desired number of layers was obtained.

Figure 2. Schematic of layer-by-layer deposition used to assemble from

PEG-b-PCL/PAA films on support substrates.

E. Characterization of the qualitative of LbL film coating

Each layer of films was analyzed by ATR-FTIR

spectroscopy. PAA and the PEG-b-PCL polymer in the film

were recorded by Nicolet 6700 for a total of 64 scans at a

resolution of 4 cm-1

.

F. OBPCP-loading content on substrate determination

After surface coating, the substrates were rinsed three

times with acetone for 3 min. Then, the solvent was

evaporated to yield solid samples and dissolved in a mixture

solvent of DMSO and chloroform (1:1 v/v) before subjected to the UV-Vis analysis. The absorbance of the micelle

solution at 288 nm was measured to determine the OBPCP

content using the previously established calibration curve.

2012 12th IEEE International Conference on Nanotechnology (IEEE-NANO)

The International Conference Centre Birmingham

20-23 August 20112, Birmingham, United Kingdom

G. In vitro release of OBPCP from the substrates

After substrate was coated, enclose substrate in dialysis

bag before immersing in phosphate buffer saline solution

(PBS) in order to measure only the free drug released from

micelles into the solution. OBPCP-micelles coatings

substrates will be immersed into 15 mL of PBS (pH 7.4) in

dialysis tubing with a MWCO of 50 kDa and transferred into

80 mL of PBS. Release studies were performed at 37°C in a

shaker-incubator. At selected time intervals, the buffer

solution outside the dialysis tubing was removed and superseded with fresh buffer solution. OBPCP

concentrations were determined based on absorbance of the

UV-Vis analysis at wavelength 282 nm. Experiments were

performed in triplicate for the both conditions of film

coating.

III. RESULTS AND DISCUSSION

A. Characterization of OBPCP-micelles with difference

micelles fabrication methods.

OBPCP-loaded micelle size of each the fabrication

methods was measured by dynamic light scattering (Table 1).

At 10% theoretical loading, these three distinct methods

provided micelles with suitable size. The dialysis and solvent

evaporation method produced micelles at 28.28 ± 0.56 nm

and 34.05 ± 0.88 nm, respectively where the film sonication

method produced micelles with a slightly greater mean

diameter (43.16 ± 2.08%). This is due to the increased

OBPCP-loaded within the micelle core [8].

Moreover, micelle preparation method was found to affect

the drug loading (Table 1). At 10% theoretical loading, the

dialysis method provided the lowest drug loading (7.07±

1.49%), loading efficiency (57.91± 11.44%) and micelle

yield (64.62 ± 1.88%). This is due to the fact that OBPCP

lost into the surrounding aqueous medium during dialysis

process. The solvent evaporation method is another widely

used method for micelle fabrication, and we successfully

fabricated polymeric micelles with high OBPCP loading

(9.21± 0.07%). However, both the dialysis and solvent

evaporation methods have slow processes of micellar

formation. They require the time for remove organic solvent.

Conversely, the film sonication method had the shortest

processes than the other micelle preparations. Among all

three fabrication methods, this method produced the highest

OBPCP loading with 9.35 ± 0.075% drug loading, loading

efficiency of 87.11 ± 8.33%, and micelle yield of 43.16 ±

2.08%. With a subsequent increase in theoretical drug

loading to 20%, OBPCP loading in micelles increased to

16.5 ± 1.0%. Taken together, all the data indicated that the

film sonication method created the highest OBPCP loading

in micelles compared to all the fabrication methods.

TABLE I

PEG-b-PCL MICELLES PARAMETERS FROM DIFFERENT

FABRICATION PROCEDURES

Micelle

fabrication

method

Theoretical

Loading

(%)

Micelle size

(nm)

Yield (%) Loading

efficiency (%)

Loading

density (%)

DS

SE

FS

10

10

10

15

20

28.28 ± 0.56

34.05 ± 0.88

43.16 ± 2.08

42.50 ± 1.44

44.16 ± 0.78

64.62 ± 1.88

78.16 ± 4.70

84.56 ± 1.29

77.93 ± 0.96

78.44 ± 3.70

57.91 ± 11.44

70.18 ± 4.16

87.11 ± 8.33

75.99 ± 4.81

70.30 ± 4.78

7.07 ± 1.49

9.21 ± 0.07

9.35 ± 0.75

11.59 ± 0.70

16.35 ± 0.52

B. Characterization of the qualitative of LbL film coating

The surface composition of the PEG-b-PCL/PAA film was

confirmed by ATR-FTIR spectroscopy where the peak

corresponding to hydrogen bonding was observed as shown

in Fig. 3. Moreover, the fully protonated PAA peak

(carboxylic acid groups was detected at 1700 cm-1

) without

any peak at 1570 cm-1

(carboxylate anion).

Figure 3. FTIR-ATR spectra of PEG-b-PCL/PAA film on the substrate at

pH 2.5.

C. Micelle-coating content on substrates

The integration of block copolymer micelles into LbL

films by hydrogen bond interaction can increase micelle

content on the surface as shown in Fig. 4. OBPCP containing

micelles increased the OBPCP content on substrate up to 5-

folds, compare to pure-OBPCP dip coating. It should be

noted that there was no difference in the amount of coating

content between non-modified and modified surface with

PEG-b-PCL.

Figure 4. Comparison of OBPCP loading on substrate from different

techniques.

2012 12th IEEE International Conference on Nanotechnology (IEEE-NANO)

The International Conference Centre Birmingham

20-23 August 20112, Birmingham, United Kingdom

D. In vitro release of OBPCP from the substrates

Fig. 5 shows that coating substrates by OBPCP

micelles/PAA LbL could prolong the release and reduce

burst release of OBPCP compared to pure-OBPCP dip

coating substrate. The results showed that OBPCP

completely released from substrate within only one day

where micelles/PAA LbL could prolong the release up to one

month. This is due to hydrogen-bonded LbL films

disintegration upon external pH environment changes. The

carboxylic acid group in PAA used as a hydrogen bond

donor is deprotonated above its critical pH (pKa 4.5).

When OBPCP micelles/PAA-coated substrates were

immerged in physiological conditions, the film deposited on

a substrate gradually disintegration to phosphate buffered

saline at pH 7.4. Moreover, the modification of substrate

surface by block copolymer also play a role in the control of

drug release rate where the substrate that modified with

block copolymer clearly provided slower release than non-

modified surface substrate, possibly due to hydrophilic

segment of block copolymer at the first layer increasing

stability of micelles on the surface.

Figure 5. In vitro release of OBPCP from the substrates into PBS buffer at pH 7.4 (a) accumulative OBPCP release (mg) and (b) accumulative

OBPCP release (%w/w).

IV. CONCLUSION

The results of this study showed that OBPCP was

successfully encapsulated in polymeric micelles with good

yield and loading efficiency. Polymeric micelles loaded with

OBPCP could be coated on the hydrophobic surface and

provided high coating content and prolonged release time.

These coating techniques could potentially be applied as a

film coating to the surface of biomedical devices such as

stents, catheters, and other biomedical implants.

ACKNOWLEDGMENT

This research was supported by National Research

Council of Thailand.

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