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Introduction
There has been a great volume of research on the pulmonary delivery of peptides and
proteins[1]. However, bioavaila-bility remains problematic and must be improved as
subsequent injury to the lungs still occurs. Natural pulmonary
surfactant (PS) is able to disperse on the surface of alveolar
cells as a thin film, consisting mainly of phospholipids and
surfactant proteins. PS therapy has already been applied to
pulmonary diseases such as respiratory distress syndrome
(RDS)[2] and asthma[3] with some success. Because of its
more homogenous and peripheral lung distribution than other
liquids and its inherent therapeutic potential, PS has been
proposed to be used as a carrier for
antibiotics[4], cortico-steroids, and recombinant adenoviral
vectors[5]. From a formulation standpoint, the lipid-based drug delivery system
should be compatible with lung tissue, and not readily elicit
immunogenic reactions, since they resemble physiological
PS in components. It is expected that PS could also be
employed as a protein and peptide carrier in pulmonary
delivery.
Based on previous studies[6,7], we prepared 4
formulations of artificial pulmonary surfactants (APS) as carriers of
insulin (INS) as a model drug. The in vivo bioavailability of
INS-APS and the correlation between minimal surface
tension and the bioavailability of INS-APS were investigated
after intratracheal instillation (IT) in normal rats. Injury to
the lungs of normal rats after 7 d of consecutive
administration was primarily investigated.
Materials and methods
Materials 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC, Sigma, St Louis, MO, USA); L-α-phosphatidyl-DL-
glycerol sodium salt (PG, Sigma, USA); tyloxapol (Tyl, Sigma,
USA); 1-hexadecanol (Hex, Acros Organics, Pittsburgh, PA,
USA); lecithin (Shanghai Boao Bioscience Co, Shanghai,
China); palmitic acid (PA, Sinopharm Group Chemical
Reagent Co, Shanghai, China); INS powder (Jiangsu
Wan-bang Biochemical & Pharmaceutical Co, Xuzhou, China);
enzymatic glucose reagent kit (Shanghai Shenfeng
Biochemistry Reagent Factory, Shanghai, China);
Radioimmunoassays (RIA) kit (Beijing Furui Bioengineering Co, Beijing,
China). Other chemicals were of Analytical reagents
(AR). ZX-98 rotavapor (Shanghai Organic Chemical Institute,
Shanghai, China); KPS-2 supersonic microniser (Shanghai
Kaibo Supersonic Apparatus Factory, Shanghai, China);
particle size system (Nicomp 380/ZLS, Santa Barbara, CA,
USA); pulsating bubble surface tensiometer (modified based
on Enhorning's method[6,8]); UV-754 ultraviolet
spectrophotometer (Shanghai Analytical Instrument General Factory,
Shanghai, China); photomicroscope (Olympus BX-50, Tokyo,
Japan); and Wistar rats (bred by the laboratory animal
department of Fudan University, Shanghai,
China). INS-APS and blank DPPC dispersions employed in this experiment
were produced within our laboratory.
Preparation of INS-APS The composition of 4 APS
dispersions were as follows: DPPC/lecithin/PA (6:3:1,
w/w/w), DPPC/Hex/Tyl (13.5:1.5:1,
w/w/w), DPPC/PG (3:1, w/w), DPPC/Tyl (13.5:1,
w/w). DPPC/lecithin/PA, DPPC/Hex/Tyl and DPPC/PG were prepared by a thin-film sonication method.
Various lipids and additives dissolved in ethanol/chloroform
were mixed; the organic phase was evaporated by rotavapor
and the residual solvent was removed in a vacuum overnight.
Lipids and additives formed a thin film on the flask wall and
were hydrated in sterile saline at 50 °C for 2 h, then sonicated
for 5 min for further dispersing. DPPC/Tyl was prepared by
a direct sonication method. DPPC and Tyl were mixed in
sterile saline and sonicated for 5
min[9]. The resulting APS dispersions were filtrated through a 0.45 µm filter. INS
powder (28 IU/mg) was solubilized with 1 mL of 0.1 mol/L HCl; a
sterile saline solution was added to make the final INS
concentration of 4 IU/mL.
An aliquot of the INS solution was added to each of the
APS dispersions and sonicated for 2 min. Final preparations
of INS-APS (INS/DPPC/lecithin/PA, INS/DPPC/Hex/Tyl,
INS/DPPC/PG, INS/DPPC/Tyl) dispersed in saline contained
13.5 mg/mL DPPC and 4 IU/mL INS.
Measurement of particle size The particle sizes of all
INS-APS were determined with a laser diffraction particle
size analysis system.
Measurement of minimal surface tension
(γmin) The minimal surface tension of APS and INS-APS was determined by
a pulsating bubble surface tensiometer (modified based on
Enhorning's method[6,8]). Each dispersion (5 µL) was added
to the gas-liquid interface of the air bubble.
According to the shape of the bubble during pulsating cycles at the
temperature of 37 °C, the γmin was calculated according to the
Bashforth-Adams formulation[10].
Pharmacodynamic and pharmacokinetic experiments
INS preparations administration In all of the
in vivo experiments, healthy Wistar rats [body weight (BW) 200±30
g] had free access to food and water. Before the experiment,
the animals fasted overnight (14 h). Forty-two male rats
were divided into 7 groups randomly as follows: (1)
subcutaneous injection (sc) INS solution (1 IU/kg); (2) IT INS
solution (4 IU/kg); (3) IT INS/DPPC/lecithin/PA (4 IU/kg); (4) IT
INS/DPPC/Hex/Tyl (4 IU/kg); (5) IT INS/DPPC/PG (4 IU/kg);
(6) IT INS/DPPC/Tyl (4 IU/kg); and (7) IT blank DPPC
dispersion. The dose of PA was about 0.2 mL INS-APS per
rat and the dose of sc was about 0.2 mL INS solution per rat.
All of the rats were anaesthetized with aether and
attached to a board with an elevation of 80° to the horizontal
plane. Rat tongues were drawn out and each INS
formulation was instilled intratracheally into the lungs through a
syringe while the rats were inspiring. The rats were
maintained for 30 s on the board after drug delivery and then the
board was rotated to a 30° horizontal elevation for 1 min.
0.4 mL of blood was taken from the tail vein shortly before
drug administration for a baseline sample and subsequently
15, 30, 60, 90, 120, 180, 240, 300, and 360 min after dosing to
determine respective serum glucose and INS
levels. The experimental design of the animal study in this paper was
approved by the appropriate ethical committee on animal
studies at Fudan University (China).
Calculation of the area above the curve (AAC) and
the area under the curve (AUC) of INS formulations
Blood samples were centrifuged at 3000 r/min for 10 min at 4 °C, and
the serum glucose level was determined immediately
according to the glucose oxidase method. The serum INS level was
quantitated by a double-antibody radioimmunoassay using
a commercial RIA kit. The percentage of serum glucose
change and the serum INS level were plotted as a function of
time. The area above the percentage serum glucose change
versus time curve (AAC) and the area under the serum INS
level versus time curve (AUC) were calculated by the linear
trapezoidal method. The relative pharmacological bioavailability
(f)[11] and relative bioavailability
(F)[12] were calculated by the following formulations:
Pilot study on lung injury in rats In lung injury
experi-ments, healthy Wistar rats (BW 200±30 g) had free access to
food and water. Thirty-six male rats were divided into 6
groups randomly as follows: (1) IT INS solution (4 IU/kg);
(2) IT INS/DPPC/lecithin/PA (4 IU/kg); (3) IT
INS/DPPC/Hex/Tyl (4 IU/kg); (4) IT INS/DPPC/PG (4 IU/kg); (5) IT
INS/DPPC/Tyl (4 IU/kg); and (6) control group (normal rats).
Groups 1, 2, 3, 4, and 5 were consecutively administered
INS preparations for 7 d (about 0.2 mL INS-APS per rat) and
once a day as per the above mentioned method.
The control group (normal rats) was fed with lab chow and water without
any administration for 7 d. All of the rats were sacrificed on
d 7. The changes of the pulmonary edema index and
histopathology of lungs were investigated to evaluate the
severity of injury to the lungs.
Pulmonary edema index All of the rats were sacrificed
on d 7. The whole lungs were removed and dissected. The
right-sided lungs were weighed both before drying
(WW) and after drying (WD) at 60 °C. The pulmonary edema index
was calculated by the following equation:
Index=WW/WD [12].
Histopathology observation The left-sided lungs were
fixed in 10% formalin, dehydrated, embedded in paraffin,
sectioned using a microtome, and stained using
hematoxylin-eosin. The lung slices were observed under a
photomicroscope and photographic records were taken.
Statistical analysis Data were expressed as mean±SD.
Statistical significant differences were evaluated with an
analysis of ANOVA in the groups and Bonferroni (Dunnett)
t-test between 2 groups.
Results
Appearance and particle size of INS-APS All APS
dispersions and INS-APS were dispersed in sterile saline
homogeneously and had emulsive lights. The appearance
of APS and INS-APS particles were circular and smooth. The
particle sizes of INS-APS (INS/DPPC/lecithin/PA,
INS/DPPC/Hex/Tyl, and INS/DPPC/PG, INS/DPPC/Tyl) were 395.55±
17.14, 439.78±46.84, 460.63±45.18, and 390.73±35.53 nm,
respectively. There was no significant difference
(P>0.05) between them.
γmin of various dispersions The
γmin of APS and INS-APS is shown in Table 1. The
γmin value of APS and INS-APS
decreased to 10 mN/m compared to the surface tension of
H2O (72 mN/m) and the γmin of DPPC (32 mN/m) and INS
solution (64.48 mN/m). There were significant differences
(P<0.05) in the γmin of 4 APS dispersions and in the
γmin of 4 INS-APS, but no significant difference
(P>0.05) in the
γmin between all APS and corresponding INS-APS. The
lowest γmin of APS and INS-APS were obtained by DPPC/
Tyl and INS/DPPC/Tyl. The relative potency of the
γmin
of 4 INS-APS was
γINS/DPPC/Tyl<γINS/DPPC/PG
<γINS/DPPC/Hex/Tyl<
γINS/DPPC/lecithin/PA. The same trend of the
γmin was observed in APS dispersions.
Hypoglycemic effects and plasma INS levels after
pulmonary delivery of INS preparations As illustrated in
Figure 1, the maximum blood glucose reduction after IT of INS
preparations at a dose of 4 IU/kg was more than 60%. The
values of the AAC of the 4 INS-APS (14147.16~19876.04)
were significantly higher (P<0.05) than the value of the INS
solution (13351.13). The optimal hypoglycemic effect of 4
INS-APS was obtained with INS/DPPC/Tyl, which generated
a maximum glucose reduction of 80% and a maximum AAC of
19876.04. The maximum glucose reduction of the remaining
3 INS-APS (INS/DPPC/lecithin/PA, INS/DPPC/Hex/Tyl, and
INS/DPPC/PG) were 63%, 76%, and 74%, respectively. The
AAC and relative pharmacological bioavailability (f) of
the INS formulations are listed in Table 2. The AAC of
INS/DPPC/Hex/Tyl, INS/DPPC/PG, and INS/DPPC/Tyl were
significantly higher (P<0.05) than that of INS/DPPC/
lecithin/PA. The relative pharmacological bioavailability
followed the sequence:
fINS/DPPC/Tyl>fINS/DPPC/Hex/Tyl
>fINS/DPPC/PG>f
INS/DPPC/lecithin/PA. The control group showed that DPPC
dispersion alone did not induce any decrease in the glucose
level.
Figure 2 shows the serum INS level after pulmonary
administration. The serum INS level was enhanced after
administration of INS-APS. The highest serum INS level
was obtained by 172 µIU/mL of INS/DPPC/Tyl at 15 min,
which was slightly higher than the level of INS/DPPC/PG
(161 µIU/mL), 1.3 times that of INS/DPPC/Hex/Tyl (131
µIU/mL), 1.8 times that of INS/DPPC/lecithin/PA (93
µIU/mL), and 2.7 times that of the INS solution (64 µIU/mL) after
IT. The difference in the AUC of the INS formulations was
similar to that of pharmacological bioavailability (Table 2).
The value of the AUC of INS/DPPC/Tyl (27243.84±5628.883)
was significantly higher (P<0.05) than the values of the
other 3 INS-APS (15947.78~23097.94). The AUC and the
relative bioavailability (F) of INS/DPPC/lecithin/PA were
lowest of the 4 INS-APS. The relative bioavailability
followed the sequence:
FINS/DPPC/Tyl>FINS/DPPC/PG
>FINS/DPPC/Hex/Tyl>FINS/DPPC/lecithin/PA. DPPC dispersion alone seemed to have no
effect on the serum INS level.
The duration of glucose levels maintained at a level
below 80% after pulmonary delivery of INS were
summarized. This was provided as a means of evaluating the prolonged
hypoglycemic effects of the INS
preparations[13]. As displayed in Figure 1, the duration of IT of INS-APS were all
above 6 h, which was longer than the hypoglycemic effect of
an INS solution sc injection (4 h) and IT (5 h).
Correlation between γmin and bioavailability of INS-APS
The correlation coefficients between the
f, F, and γmin values of the INS preparation were -0.7421 and -0.7245, whereas the
correlation coefficients between the f,
F, and γmin of INS-APS were -0.9688 and -0.8632, respectively (Figure 3).
Pulmonary edema index After 7 d of consecutive
administration, a significant difference (P<0.05) in the
pulmonary edema index of the 6 groups was observed (Table 3).
The index for the INS solution group was significantly higher
(P<0.05) than that of the control group, and the indices of
the INS-APS groups were significantly lower than that of
the INS solution group. This indicated that the INS-APS
groups could alleviate lung injury while the INS solution
conferred marked injury to the lung tissue following 7 d of
consecutive administration. The pulmonary edema indices
of INS/DPPC/Tyl and INS/DPPC/PG showed no significant
difference compared with the control group
(P>0.05).
Histopathological examination of lungs As shown in
Figure 3, following IT for 7 consecutive days, thickened
alveolar septa were clearly observed in the lung slices in rats
administrated with INS solution (Figure 4B), as compared to
the control group (Figure 4A). Diffused disruption of
alveolar capillaries with leakage of red blood cells into the alveolar
lumina and lung interstitium was generally noted in the INS
solution group. Prominent inflammatory cell infiltration and
lung vascular epithelial degeneration were also observed in
this group. The pathological changes became less severe in
the slices after INS-APS administration (Figure 4C_4F). The
width of alveolar septa was slighter broader than the control
group. Disruption of the alveolar wall and leakage of red
blood cells were not evident. There was only very slight
visible epithelial degeneration and no other alveolar
structural changes observed in the INS-APS groups, as compared
with the control groups. Inflammatory cell infiltration was
still found in some samples although this was only negligible.
Discussion
The natural PS is a complex mixture produced by alveolar
type II cells in lung. It can stabilize lungs for efficient
respiration and prevent or treat RDS by reducing alveolar surface
tension and increasing lung compliance. Due to the small
diameter of peripheral airways, fluid with a high surface
tension requires high pressure delivery for distribution.
Studies have shown that PS is superior to saline in distributing a
radioactive colloid within healthy lungs, and is more
homogenous and peripheral in lungs than
saline[14]. PS carry out this function by forming a film at the air-liquid interface. This
film mainly consists of lipids and surfactant proteins. The
hydrophilic polar heads of amphiphilic phospholipid
molecules remain in water while the fatty acids turn towards air.
The phospholipids molecules interluding in water affect the
affinity in molecules. Due to this film, surface tension is
lowered and undergoes considerable changes during the
respiratory cycle[15].
Of the many components of natural PS, DPPC appears to
have the necessary thermodynamic properties to reduce
surface tension effectively. However the high cohesive
energy of compressed monolayers of disaturated long-chain
fatty acids, combined with significant hydration of the
headgroups, inhibits the rapid spreading of DPPC molecules
in an air/liquid interface. Therefore, the synergistic effect of
other lipids are required to reduce the cohesion of DPPC in
lipid mixture, such as PG, cholesterol and free PA, and of
several lung specific proteins[16]. Most research on APS
have documented a potential for antigenicity due to the
presence of a foreign protein[17,18]. In this paper, DPPC was used
as the key ingredient and other components were employed
to prepare APS dispersions. It was shown that APS could
decrease the surface tension at the air/liquid interface
significantly and also enhance INS absorption with resulting
alleviation of lung injury.
This experiment showed that the γmin value of the 4
INS-APS were all lower than DPPC dispersion alone. DPPC and
PG are generally considered as components of PS; in this
study they were employed as 1 formulation of APS. Lecithin
contains unsaturated fatty acid residues which probably
enhance the fluidity of the DPPC
films[16]; furthermore, the addition of PA often makes the dispersion more
homogeneous[19]. DPPC/lecithin/PA was designed and added to INS.
In the DPPC/Hex/Tyl dispersion, Hex acts as a spreading
agent and Tyl as a dispersion agent[20]. So DPPC/Hex/Tyl
and DPPC/Tyl were prepared as formulations of APS. The
difference of the γmin between DPPC/Hex/Tyl and DPPC/Tyl
requires further research. With intratracheally instilled INS
preparation, the serum glucose level of the INS-APS groups
was clearly lower than that of the INS solution group. In
addition, the duration of the hypoglycemic effect of the
INS-APS groups was significantly longer than that of the INS
solution group. The lowest γmin value and the largest
hypoglycemic effect were obtained by the INS/DPPC/Tyl
preparation. The relative pharmacological bioavailabilities
of INS/DPPC/Hex/Tyl, INS/DPPC/PG, and INS/DPPC/Tyl
were higher than INS/DPPC/lecithin/PA, and similar trends
were noted for the relative bioavailability of INS-APS. The
correlation between γmin and f, and
F suggested that the
decrease of the γmin values of APS and INS-APS directly
affected f and F. It was also revealed that the
in vivo
hypoglycemic effect could be predicted with the
γmin values of INS-APS. The mechanism whereby INS absorption was
enhanced is believed to be via an APS-mediated decrease in
the γmin of the gas-liquid interface in alveolar tissue. This
property appears to increase the facility and affect spreading,
which in return increases the absorption area and produces
greater INS absorption with APS.
In comparison to the pulmonary edema index, it was
concluded that INS-APS could decrease lung injury compared
with the INS solution following 7 d of consecutive IT. The
lower index was obtained by INS/DPPC/PG and
INS/DPPC/Tyl, which might contribute to the lower
γmin value of the 2 INS-APS. Combining the results of the pulmonary edema
index and histopathology examination, it was suggested that
the pulmonary delivery of INS-APS might reduce the
severity of any lung injury during administration and would thus
be more appropriate for pulmonary delivery than the INS
solution.
In summary, DPPC/Hex/Tyl, DPPC/PG, and DPPC/Tyl,
with their low γmin, appeared to be effective absorption
enhancers and significantly decreased lung
injury. INS/DPPC/Tyl and INS/DPPC/PG in this study were shown to be
efficient INS pulmonary delivery carriers with several
advantages over drug preparations current utilized clinically.
Acknowledgements
We thank Dr Zhi-qiang JIANG and Prof Yuan-ming MA
for their suggestions.
References
1 Agu RU, Ugwoke MI, Armand M, Kinget R, Verbeke N. The lung
as a route for systemic delivery of therapeutic proteins and
peptides. Respir Res 2001; 2: 198_209.
2 Merrill JD, Ballard RA. Pulmonary surfactant for neonatal
respiratory disorders. Curr Opin Pediatr 2003; 15: 149_54.
3 Erpenbeck VJ, Hagenberg A, Dulkys Y, Elsner D, Balder R,
Krentel H, et al. Natural porcine surfactant augments airway
inflammation after allergen challenge in patients with asthma.
Am J Respir Crit Care Med 2004; 169: 578_86.
4 Van't Veen A, Gommers D, Mouton JW, Kluytmans JA, Krijt EJ,
Lachmann B. Exogenous pulmonary surfactant as a drug
delivering agent: influence of antibiotics on surfactant activity. Br J
Pharmacol 1996; 118: 593_8.
5 Katkin JP, Husser RC, Langston C, Welty SE. Exogenous
surfactant enhances the delivery of recombinant adenoviral vectors to
the lung. Hum Gene Ther 1997; 8: 171_6.
6 Jiang ZQ, Sun GM, Ma YM. Artificial reconstituted pulmonary
surfactant in prevention and treatment of respiratory distress
syndrome in neonates. Acta Pharmacol Sin 1997; 18:182_4.
7 Mitra R, Pezron I, Li Y, Mitra AK. Enhanced pulmonary
delivery of insulin by lung lavage fluid and phospholipids. Int J Pharm
2001; 217: 25_31.
8 Enhorning G. Pulsating bubble technique for evaluating
pulmonary surfactant. J Appl Physiol 1977; 43: 198_203.
9 Ji Y, Pei YY. Primary study of artificial pulmonary surfactant as
carrier of insulin pulmonary delivery. Chin Pharm J 2006; 41:
766_8.
10 Paddy JF. Surface tension. Part III. Tables relating the size and
shape of liquid drops to the surface tension. In Matijevic E,
editor. Surface and colloid science. New York: Wiley-Interscience;
1969. p151_97.
11 Pan Y, Li YJ, Zhao HY, Zheng JM, Xu H, Wei G,
et al. Bioadhesive polysaccharide in protein delivery system: chitosan
nanoparticles improve the intestinal absorption of insulin
in vivo. Int J Pharm 2002; 249: 139_47.
12 Lu J, Ji Y, Jiang ZQ. Study on the bioavailability and initial
investigation on the rat following pulmonary delivery of insulin
lipid suspension. Chin J Biochem Pharm 2002; 23: 271_4.
13 Zhang Q, Shen ZC, Tsuneji N. Prolonged hypoglycemic effect
of insulin-loaded polybutylcyanoacrylate nanoparticles after
pulmonary administration to normal rats. Int J Pharm 2001; 218:
75_80.
14 Kharasch VS, Sweeney TD, Fredberg J, Lehr J, Damokosh AI,
Avery ME, et al. Pulmonary surfactant as a vehicle for
intratracheal delivery of technetium sulfur colloid and pentamidine in
hamster lungs. Am Rev Respir Dis 1991; 144: 909_13.
15 Enhorning G. Pulmonary surfactant function studied with the
pulsating bubble surfactometer (PBS) and the capillary
surfacto-meter (CS). Comp Biochem Physiol A Mol Integr Physiol 2001;
129: 221_6.
16 Tanaka Y, Takei T, Aiba T, Masuda K, Kiuchi A, Fujiwara T.
Development of synthetic lung surfactants. J Lipid Res 1986;
27: 475_85.
17 Latallo JFK, Chen CL, Eichman J, Bielinska AU, Baker JR.
Enhancement of endrimer-mediated transfection using synthetic
lung surfactant exosurf neonatal in vitro. Biochem Biophys Res
Commun 1999; 264: 253_61.
18 Lewis JF, Brackenbury A. Role of exogenous surfactant in acute
lung damage. Crit Care Med 2003; 31: S324_8.
19 Palmblad M, Gustafsson M, Curstedt T, Johansson J, Schurch S.
Surface activity and film formation from the surface associated
material of artificial surfactant preparations. Biochim Biophys
Acta 2001; 1510: 106_17.
20 Park SY, Hannemann RE, Franses EI. Dynamic tension and
adsorption behavior of aqueous lung surfactants. Colloids Surf B
Biointerfaces 1999; 15: 325_38.
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