J. MICROENCAPSULATION,
1991, VOL. 8,
NO,
3, 369-374
Effect of nanoparticles on transdermal drug delivery M A R K U S J. CAPPEL? and J O R G KREUTERS Institut fur Pharmazeutische Technologie, J. W. Goethe-Universitat, GeorgVoigt-Strasse 16, D-6000 Frankfurt, Germany
Journal of Microencapsulation Downloaded from informahealthcare.com by Ryerson University on 11/06/12 For personal use only.
(Received 3 December 1990; accepted 14 December 1990)
The purpose of the present study was to assess by in vitro means the effect of poly (methylmethacrylate) nanoparticles and poly (butylcyanoacrylate) nanoparticles on transdermal drug delivery. Methanol and octanol were chosen as test permeants. In order to distinguish between thermodynamic effect and those due to biological consequences, two different membranes were employed, i.e., full thickness hairless mouse skin and silicone elastomer sheeting (175 pm). It is evident that poly (methylmethacrylate) nanoparticles and poly (butylcyanoacrylate) nanoparticles increase the permeability of methanol through hairless mouse skin by a factor of 1.2-2. The permeability of lipophilic octanol is either unaffected by nanoparticles or decreases as a function of nanoparticle concentration depending on the lipophilicity of the polymer material.
Introduction T h e use of nanoparticles as drug carrier systems has been extensively reviewed by Kreuter (1988). Just to name a few applications: they have been employed to enhance the myotic response of rabbits (Harmia et al. 1986), as well as to achieve a better adjuvant effect for vaccines (Kreuter and Speiser 1976 a, b, Kreuter and Liehl 1981). Nanoparticles and their physico-chemical properties have been very well characterized in the past years. T h e concept of the skin being an important permeability barrier which enables the human being to survive in a dry environment has always been recognized. However, it is a conditio sine qua non that topically applied drugs have to circumvent this unique permeation barrier in order to trigger a pharmacodynamic response in the skin or to permeate through the skin in amounts necessary to initiate systemic therapeutic effects. Transport can be facilitated by a number of means, e.g., by using chemicals such as alcohols, oleic acid, AzoneTM,dimethylsulfoxide, or by iontophoresis or by phonophoresis. Nanoparticles were shown to be able to enhance the permeability of some substances through membranes such as cellotape membranes (Kreuter et al. 1983). T h e mechanism of this effect is so far not understood. T h e aim of the present study was to assess by in vitro means the effect of poly(rnethylmethacry1ate) nanoparticles and poly(butylcyanoacry1ate) nanoparticles on the permeation of methanol and octanol through hairless mouse skin. These two alkanols were chosen as test permeants since their permeation through hairless mouse skin with respect to all variables such as sex, age, anatomical site, and hydration are very well documented in the literature. In order to distinguish between thermodynamic effects and those due
t Present affiliation:Cygnus Research Corporation, 400 Penobscot Drive, Redwood City, CA 94063, U.S.A. $ To whom correspondence should be addressed. 0265-2048/91 $3.00 0 1991 Taylor & Francis Ltd
3 70
M . J . Cappel and J . Kreuter
to biological consequences, silicone elastomer membrane was employed as a comparison to hairless mouse skin.
Journal of Microencapsulation Downloaded from informahealthcare.com by Ryerson University on 11/06/12 For personal use only.
Materials and methods Methyl methacrylate (Fluka AG, Buchs, Switzerland) was purified from polymerization inhibitors by washing 100 ml of the monomer three times with 20 ml of a solution of 5 g sodium hydroxide and 20 g sodium chloride in 100 ml distilled water. After this, the monomer was washed three times with 20ml distilled water. Poly (methylmethacrylate) nanoparticles were produced as follows: 10.00ml of the purified monomer was dissolved in 10000 ml water, and this solution was irradiated with 500 krad y-rays at a rate of about 2 krad min- using a 60Co-source (Kreuter and Zehnder 1978). T h e resulting nanoparticles were then dried by lyophilization and stored in dry form. Poly (butyl-2-cyanoacrylate) nanoparticles were prepared according to the method described by Douglas et ad. (1984). Briefly, butyl-2-cyanoacrylat (SichelWerke, Hannover, Germany, stored at 4°C) and dextran 70 (molecular weight 70 000) were used as received. All other chemicals were reagent grade. I n order to prepare poly (butylcyanocrylate) nanoparticles, 0.25 ml of the monomer was added dropwise to 24.75 ml of a stirred aqueous solution of 0 5 per cent (w/v) dextran 70 in 0.01 N hydrochloric acid at room temperature. In order to fully disperse the monomer, the dispersion was agitated with a glass coated magnetic stirring bar at 1000 rpm. After 2 hours, the resulting suspension was filtered through a sintered glass funnel (grade 4, pore size 11-16pm) and lyophilized. Prior to use, the appropriate amount of lyophilized nanoparticles was dispersed in saline. Stock solutions of 3H methanol and I4C octanol were prepared in sodium solution for irrigation. T h e actual alcohol concentration was less than lop4mole so that any penetration enhancing effect by the alcohol itself could be eliminated. Also, the stability and integrity of the nanoparticles were not affected by this low concentration of each alkanol. T h e set-up and the design of the diffusion experiment has been described in detail (Cappel and Kreuter, 1991a). Briefly, the infinite dose technique was chosen in these experiments. Side by side diffusion cells made of glass had a half cell volume of 1.5 cm3 and a surface area of 0-78cm'. Both compartments were equipped with two ports, one port to accommodate the shaft of the teflon stirrer and one sampling port. In order to distinguish between changes in thermodynamic parameters and effects due to biological consequences, two different membranes were employed in this study: (i) silicone elastomer sheeting (SilasticTMsheeting Q7-4840,175 pm thick, Dow Corning, Corporation, Midland, MI, USA), and (ii) full thickness hairless mouse skin (strain hr/hr-C3H/TifBom, Bonmice, BomholgPrd Breeding and Research Centre Ltd, Ry, Denmark). The female hairless mice were in excess of 120 days and were sacrificed with CO,. Both, abdominal and dorsal skin was used in the experiments. Two sequential diffusion experiments were carried out. T h e first sequential run was initiated to assess the effect of the nanoparticles on the permeation of 3H methanol and I4C octanol. The medium in the donor compartment constituted of nanoparticles in saline at four different concentrations, i.e. 0-05,0-1,0-5 and 1.0 per cent, neat saline constituted the medium in the receiver chamber. A constant
'
371
Journal of Microencapsulation Downloaded from informahealthcare.com by Ryerson University on 11/06/12 For personal use only.
Eflect of nanoparticles on transdermal drug delivery
temperature of 37°C was maintained throughout the entire course of the permeation experiment. T h e contents of each half-cell were stirred at 150 rpm. Samples of the receiver chamber were taken after predetermined time intervals and replaced with neat saline. T h e samples were processed for liquid scintillation counting and counted in a Beckman LS 1501 (Beckman Instruments, Munchen, Germany) scintillation counter. After finishing the first set of experiments, care was taken to remove the nanoparticle suspension quantitatively. T h e second sequential run was carried out in order to determine the reversibility of the previously observed nanoparticle effects. With the same membrane still mounted in the diffusion cell, saline constituted the medium in both the donor and the receiver chamber. T h e diffusion experiment was carried out as described previously.
Results and discussion Generally, poly (methylmethacrylate) nanoparticles slightly enhance the permeation of hydrophilic methanol through full thickness hairless mouse skin by a factor
T~~Silicone
60 7
Methanol Elastomer Membrane
7 I
.
"
"
0.05
0.1
0.5
1.0
x Concentration of PMMA Nanoparticles NaCl 0.9%
7:
-
Nanopart.
0.05
0.1
0.5
1.0
Concentration o f PMMA Nanoparticles
NaCl 0 . 9 ~
@@Nanopart. Octanol
Octanol Silicone Elastomer Membrane
-
Hairless Mouse Skin
_-.______
70
60
\
5
E
~1000
7
0
0 800 t 600 a
50
0
40
Z
30
0 W
0 0
400
*:
E a 200
a
n
005 3:
0.1
0.5
1.0
Concentration of PMMA Nanoparticles
- NaCl 0 9 z
@#
Nanopart.
20
10 0 j!
0.05
0.1
0.5
1.0
Concentration of PMMA Nanoparticles
- NaCl 0.97
Nanopart.
Figure 1. Effect of different concentrations of poly(methylmethacry1ate) nanoparticles on the permeation of methanol and octanol through either silicone elastomer membrane or full thickness hairless mouse skin. All data represent the mean of three experiments fSD. The straight line depicts the permeability coefficient of the tests permeants when neat saline constitutes the medium in the donor compartment.
M . J . Cappel and J . Kreuter
Journal of Microencapsulation Downloaded from informahealthcare.com by Ryerson University on 11/06/12 For personal use only.
372
of 1.16-13. Figure 1 implies that a concentration of 0.1 per cent poly (methylmethacrylate) nanoparticles seems to be most effective as a penetration enhancer. However, the reversibility experiment clearly demonstrates that the permeability coefficient is still three times that of the saline reference (figure 1). This suggests that there is an irregularity due to biological variation of the hairless mouse skin. T h e diffusion experiments employing silicone elastomer sheeting as a membrane show no significant difference in permeability of methanol. Apparently, the thermodynamic activity of methanol is not affected by nanoparticles. T h e observed effects due to the nanoparticles in this study were generally much less pronounced than those observed previously with other permeants and different membranes (Kreuter et al. 1983 and unpublished results). Regarding the mode of action of nanoparticles, one might hypothesize that they are associated with the skin surface, facilitating drug transport by changing the vehicle/stratum corneum partition coefficient. Kreuter (1990) also demonstrated that nanoparticles are somewhat bioadhesive. T h e permeability of lipophilic octanol through both membranes, i.e., full thickness hairless mouse skin and silicone elastomer sheeting, decreases as a function of poly (methylmethacrylate) nanoparticle concentration (figure 1). Poly (methylmethacrylate) nanoparticles are very lipophilic causing sorption of octanol. Poly (butylcyanoacrylate) nanoparticles show a similar enhancement of the permeability of methanol compared to their poly (methylmethacrylate) counterparts. A detailed listing of enhancement factors is given in table 1. T h e effect of poly (butylcyanoacrylate) nanoparticles is also fully reversible (figure 2). T h e permeability pattern of octanol differs from the previously described phenomena. It is
Table 1. Enhancement Factors (E.F.) of the permeability coefficient for PMMA and PBCA nanoparticles at various concentrations.
a) P M M A nanoparticles Hairless mouse skin Conc.
E.F. MeOH
(%I 0.05 0.1 0.5 1.o
1.168 3.56 1.8 1.09
E.F. OcOH ns.
p < 0.05 p < 0.05 n.s.
1.062 099 0688 0.255
n.s. ns. p < 0.01 p < 0.01
6 ) PBCA nanoparticles Hairless mouse skin
E.F. MeOH
Conc. (%)
0.05 0.1 0.5 1.o
1.291 1.668 1.672 1.518
E.F. OcOH n.s.
p
1991, VOL. 8,
NO,
3, 369-374
Effect of nanoparticles on transdermal drug delivery M A R K U S J. CAPPEL? and J O R G KREUTERS Institut fur Pharmazeutische Technologie, J. W. Goethe-Universitat, GeorgVoigt-Strasse 16, D-6000 Frankfurt, Germany
Journal of Microencapsulation Downloaded from informahealthcare.com by Ryerson University on 11/06/12 For personal use only.
(Received 3 December 1990; accepted 14 December 1990)
The purpose of the present study was to assess by in vitro means the effect of poly (methylmethacrylate) nanoparticles and poly (butylcyanoacrylate) nanoparticles on transdermal drug delivery. Methanol and octanol were chosen as test permeants. In order to distinguish between thermodynamic effect and those due to biological consequences, two different membranes were employed, i.e., full thickness hairless mouse skin and silicone elastomer sheeting (175 pm). It is evident that poly (methylmethacrylate) nanoparticles and poly (butylcyanoacrylate) nanoparticles increase the permeability of methanol through hairless mouse skin by a factor of 1.2-2. The permeability of lipophilic octanol is either unaffected by nanoparticles or decreases as a function of nanoparticle concentration depending on the lipophilicity of the polymer material.
Introduction T h e use of nanoparticles as drug carrier systems has been extensively reviewed by Kreuter (1988). Just to name a few applications: they have been employed to enhance the myotic response of rabbits (Harmia et al. 1986), as well as to achieve a better adjuvant effect for vaccines (Kreuter and Speiser 1976 a, b, Kreuter and Liehl 1981). Nanoparticles and their physico-chemical properties have been very well characterized in the past years. T h e concept of the skin being an important permeability barrier which enables the human being to survive in a dry environment has always been recognized. However, it is a conditio sine qua non that topically applied drugs have to circumvent this unique permeation barrier in order to trigger a pharmacodynamic response in the skin or to permeate through the skin in amounts necessary to initiate systemic therapeutic effects. Transport can be facilitated by a number of means, e.g., by using chemicals such as alcohols, oleic acid, AzoneTM,dimethylsulfoxide, or by iontophoresis or by phonophoresis. Nanoparticles were shown to be able to enhance the permeability of some substances through membranes such as cellotape membranes (Kreuter et al. 1983). T h e mechanism of this effect is so far not understood. T h e aim of the present study was to assess by in vitro means the effect of poly(rnethylmethacry1ate) nanoparticles and poly(butylcyanoacry1ate) nanoparticles on the permeation of methanol and octanol through hairless mouse skin. These two alkanols were chosen as test permeants since their permeation through hairless mouse skin with respect to all variables such as sex, age, anatomical site, and hydration are very well documented in the literature. In order to distinguish between thermodynamic effects and those due
t Present affiliation:Cygnus Research Corporation, 400 Penobscot Drive, Redwood City, CA 94063, U.S.A. $ To whom correspondence should be addressed. 0265-2048/91 $3.00 0 1991 Taylor & Francis Ltd
3 70
M . J . Cappel and J . Kreuter
to biological consequences, silicone elastomer membrane was employed as a comparison to hairless mouse skin.
Journal of Microencapsulation Downloaded from informahealthcare.com by Ryerson University on 11/06/12 For personal use only.
Materials and methods Methyl methacrylate (Fluka AG, Buchs, Switzerland) was purified from polymerization inhibitors by washing 100 ml of the monomer three times with 20 ml of a solution of 5 g sodium hydroxide and 20 g sodium chloride in 100 ml distilled water. After this, the monomer was washed three times with 20ml distilled water. Poly (methylmethacrylate) nanoparticles were produced as follows: 10.00ml of the purified monomer was dissolved in 10000 ml water, and this solution was irradiated with 500 krad y-rays at a rate of about 2 krad min- using a 60Co-source (Kreuter and Zehnder 1978). T h e resulting nanoparticles were then dried by lyophilization and stored in dry form. Poly (butyl-2-cyanoacrylate) nanoparticles were prepared according to the method described by Douglas et ad. (1984). Briefly, butyl-2-cyanoacrylat (SichelWerke, Hannover, Germany, stored at 4°C) and dextran 70 (molecular weight 70 000) were used as received. All other chemicals were reagent grade. I n order to prepare poly (butylcyanocrylate) nanoparticles, 0.25 ml of the monomer was added dropwise to 24.75 ml of a stirred aqueous solution of 0 5 per cent (w/v) dextran 70 in 0.01 N hydrochloric acid at room temperature. In order to fully disperse the monomer, the dispersion was agitated with a glass coated magnetic stirring bar at 1000 rpm. After 2 hours, the resulting suspension was filtered through a sintered glass funnel (grade 4, pore size 11-16pm) and lyophilized. Prior to use, the appropriate amount of lyophilized nanoparticles was dispersed in saline. Stock solutions of 3H methanol and I4C octanol were prepared in sodium solution for irrigation. T h e actual alcohol concentration was less than lop4mole so that any penetration enhancing effect by the alcohol itself could be eliminated. Also, the stability and integrity of the nanoparticles were not affected by this low concentration of each alkanol. T h e set-up and the design of the diffusion experiment has been described in detail (Cappel and Kreuter, 1991a). Briefly, the infinite dose technique was chosen in these experiments. Side by side diffusion cells made of glass had a half cell volume of 1.5 cm3 and a surface area of 0-78cm'. Both compartments were equipped with two ports, one port to accommodate the shaft of the teflon stirrer and one sampling port. In order to distinguish between changes in thermodynamic parameters and effects due to biological consequences, two different membranes were employed in this study: (i) silicone elastomer sheeting (SilasticTMsheeting Q7-4840,175 pm thick, Dow Corning, Corporation, Midland, MI, USA), and (ii) full thickness hairless mouse skin (strain hr/hr-C3H/TifBom, Bonmice, BomholgPrd Breeding and Research Centre Ltd, Ry, Denmark). The female hairless mice were in excess of 120 days and were sacrificed with CO,. Both, abdominal and dorsal skin was used in the experiments. Two sequential diffusion experiments were carried out. T h e first sequential run was initiated to assess the effect of the nanoparticles on the permeation of 3H methanol and I4C octanol. The medium in the donor compartment constituted of nanoparticles in saline at four different concentrations, i.e. 0-05,0-1,0-5 and 1.0 per cent, neat saline constituted the medium in the receiver chamber. A constant
'
371
Journal of Microencapsulation Downloaded from informahealthcare.com by Ryerson University on 11/06/12 For personal use only.
Eflect of nanoparticles on transdermal drug delivery
temperature of 37°C was maintained throughout the entire course of the permeation experiment. T h e contents of each half-cell were stirred at 150 rpm. Samples of the receiver chamber were taken after predetermined time intervals and replaced with neat saline. T h e samples were processed for liquid scintillation counting and counted in a Beckman LS 1501 (Beckman Instruments, Munchen, Germany) scintillation counter. After finishing the first set of experiments, care was taken to remove the nanoparticle suspension quantitatively. T h e second sequential run was carried out in order to determine the reversibility of the previously observed nanoparticle effects. With the same membrane still mounted in the diffusion cell, saline constituted the medium in both the donor and the receiver chamber. T h e diffusion experiment was carried out as described previously.
Results and discussion Generally, poly (methylmethacrylate) nanoparticles slightly enhance the permeation of hydrophilic methanol through full thickness hairless mouse skin by a factor
T~~Silicone
60 7
Methanol Elastomer Membrane
7 I
.
"
"
0.05
0.1
0.5
1.0
x Concentration of PMMA Nanoparticles NaCl 0.9%
7:
-
Nanopart.
0.05
0.1
0.5
1.0
Concentration o f PMMA Nanoparticles
NaCl 0 . 9 ~
@@Nanopart. Octanol
Octanol Silicone Elastomer Membrane
-
Hairless Mouse Skin
_-.______
70
60
\
5
E
~1000
7
0
0 800 t 600 a
50
0
40
Z
30
0 W
0 0
400
*:
E a 200
a
n
005 3:
0.1
0.5
1.0
Concentration of PMMA Nanoparticles
- NaCl 0 9 z
@#
Nanopart.
20
10 0 j!
0.05
0.1
0.5
1.0
Concentration of PMMA Nanoparticles
- NaCl 0.97
Nanopart.
Figure 1. Effect of different concentrations of poly(methylmethacry1ate) nanoparticles on the permeation of methanol and octanol through either silicone elastomer membrane or full thickness hairless mouse skin. All data represent the mean of three experiments fSD. The straight line depicts the permeability coefficient of the tests permeants when neat saline constitutes the medium in the donor compartment.
M . J . Cappel and J . Kreuter
Journal of Microencapsulation Downloaded from informahealthcare.com by Ryerson University on 11/06/12 For personal use only.
372
of 1.16-13. Figure 1 implies that a concentration of 0.1 per cent poly (methylmethacrylate) nanoparticles seems to be most effective as a penetration enhancer. However, the reversibility experiment clearly demonstrates that the permeability coefficient is still three times that of the saline reference (figure 1). This suggests that there is an irregularity due to biological variation of the hairless mouse skin. T h e diffusion experiments employing silicone elastomer sheeting as a membrane show no significant difference in permeability of methanol. Apparently, the thermodynamic activity of methanol is not affected by nanoparticles. T h e observed effects due to the nanoparticles in this study were generally much less pronounced than those observed previously with other permeants and different membranes (Kreuter et al. 1983 and unpublished results). Regarding the mode of action of nanoparticles, one might hypothesize that they are associated with the skin surface, facilitating drug transport by changing the vehicle/stratum corneum partition coefficient. Kreuter (1990) also demonstrated that nanoparticles are somewhat bioadhesive. T h e permeability of lipophilic octanol through both membranes, i.e., full thickness hairless mouse skin and silicone elastomer sheeting, decreases as a function of poly (methylmethacrylate) nanoparticle concentration (figure 1). Poly (methylmethacrylate) nanoparticles are very lipophilic causing sorption of octanol. Poly (butylcyanoacrylate) nanoparticles show a similar enhancement of the permeability of methanol compared to their poly (methylmethacrylate) counterparts. A detailed listing of enhancement factors is given in table 1. T h e effect of poly (butylcyanoacrylate) nanoparticles is also fully reversible (figure 2). T h e permeability pattern of octanol differs from the previously described phenomena. It is
Table 1. Enhancement Factors (E.F.) of the permeability coefficient for PMMA and PBCA nanoparticles at various concentrations.
a) P M M A nanoparticles Hairless mouse skin Conc.
E.F. MeOH
(%I 0.05 0.1 0.5 1.o
1.168 3.56 1.8 1.09
E.F. OcOH ns.
p < 0.05 p < 0.05 n.s.
1.062 099 0688 0.255
n.s. ns. p < 0.01 p < 0.01
6 ) PBCA nanoparticles Hairless mouse skin
E.F. MeOH
Conc. (%)
0.05 0.1 0.5 1.o
1.291 1.668 1.672 1.518
E.F. OcOH n.s.
p
