- Email: [email protected]
Equilibrium constants for the extraction of Fe(III) from 6M hydrochloric acid solutions by tri-n-octylammonium chloride in chloroform
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LETTERS
Transdermal protein delivery by a coadministered peptide identified via phage display Yongping Chen1,2, Yuanyuan Shen1,2, Xin Guo1,2, Caoshou Zhang1, Wenjuan Yang1, Minglu Ma1, Shu Liu1, Maobin Zhang1 & Long-Ping Wen1 Efficient transdermal drug delivery of large hydrophilic drugs is challenging. Here we report that the short synthetic peptide, ACSSSPSKHCG, identified by in vivo phage display, facilitated efficient transdermal protein drug delivery through intact skin. Coadministration of the peptide and insulin to the abdominal skin of diabetic rats resulted in elevated systemic levels of insulin and suppressed serum glucose levels for at least 11 h. Significant systemic bioavailability of human growth hormone was also achieved when topically coadministered with the peptide. The transdermal-enhancing activity of the peptide was sequence specific and dose dependent, did not involve direct interaction with insulin and enabled penetration of insulin into hair follicles beyond a depth of 600 lm. Timelapse studies suggested that the peptide creates a transient opening in the skin barrier to enable macromolecular drugs to reach systemic circulation. As the largest organ of the human body, skin provides a painless interface for systemic drug delivery1,2. However, the permeability of foreign molecules, especially large hydrophilic molecules, across the stratum corneum in the outer skin surface is extremely low3. In the last 50 years a large number of chemical penetration enhancers (CPEs), belonging to several different classes such as surfactants, fatty acids, fatty esters and Azone-like compounds, have enabled limited skin permeation enhancement4,5. CPEs have two major limitations: skin irritation and inability to deliver large molecules. Without physical enhancement, mediated by iontophoresis6, ultrasound7 or microneedles8, CPEs are generally unable to deliver therapeutic levels of relatively large drugs (molecular mass 4500 Da) through intact skin to the systemic circulation9. Recent progress in high-throughput screening to identify enhancer combinations10 and application of CPE design principles11 may help overcome these two limitations, but transdermal delivery of large hydrophilic proteins remains a formidable task. Phage display peptide libraries are commonly used to obtain defined peptide sequences that interact with a particular molecule. As many as 109 different peptides are expressed on the phage surface by fusion to one of the phage surface proteins, and the desired peptides are selected on the basis of binding to the target molecule12.
1Hefei 2These
Phage libraries have been used to select for peptides that bind to immobilized proteins, carbohydrates, cultured cells and even inorganic material. An extension of this technique is in vivo phage display, which entails the injection of a peptide display phage library into the bloodstream of an animal and subsequent isolation and identification of phage that have the ability to home to a particular organ or tissue13–15. Filamentous phage, known to reach the brain after intranasal administration, have been used as a carrier to deliver antibodies against amyloid plaques16. Furthermore, a recent study used in vivo phage display to identify peptide sequences that induced the transport of phage across the gastrointestinal mucosal barrier17. These studies prompted us to study whether in vivo phage display could identify peptides with transdermal capability. We applied Ph.D.-C7C, a disulfide-constrained, phage-display, peptide library, onto the abdominal skin of BALB/cA nude mice and recovered phage particles from the blood circulation. Recovered phage were amplified and used for the next round of in vivo selection. Amplified phage from the first round exhibited a transdermal efficiency two orders of magnitude higher than that of the library phage. After the second round, twelve clones were randomly picked and sequenced. Eight of them contained an identical nucleotide sequence insert (termed PH-1), which coded for the displayed peptide CSSSPSKHC. When tested in Wistar rats, a representative phage carrying this sequence consistently crossed the skin barrier and reached the bloodstream, achieving an average titer of 4,556 infective phage particles per milliliter of blood 1 h after topical administration. In contrast, a control phage randomly selected from the library exhibited an average titer of 6.8 infective phage particles per milliliter of blood, with zero infective phage particles detected in the majority of the experiments (n ¼16). As PH-1 differs from the random phage only in the displayed peptide sequence, the phage-displayed peptide most likely interacts with the skin to enable skin barrier penetration of PH-1. If the displayed peptide interacts with the skin in a specific manner that can be saturated, an excess of the displayed peptide would inhibit PH-1’s transdermal activity . To test this model, we synthesized a cyclic peptide named TD-1 (ACSSSPSKHCG; the flanking A and G were derived from the M13 coat protein and included to make the peptide more stable). As expected, coadministered TD-1 inhibited the
National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, Hefei, Anhui 230027, China. authors contributed equally to this work. Correspondence should be addressed to L.-P. Wen ([email protected]).
Received 22 August 2005; accepted 3 January 2006; published online 26 March 2006; doi:10.1038/nbt1193
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b 4.5
TD-1 (µg) 0 4 8 16
Serum insulin (ng/ml)
4.0
*
3.5
***
** **
3.0 2.5 2.0 1.5 1.0 0.5 0.0
5
0
10
15
20
25
0
2
4
Time (h)
6
8
10
12
Blood glucose (% initial)
Pretreatment 5h
3 2 1
20
100 TD-1 (µg)
*** ***
***
500
1,000
***
***
*** 2
4
6
8
10
12
Time (h)
f
100 80 60 40 20
AP-1 100 µg TD-1 500 µg TD-1 100 µg Saline
1.8 1.6
*
1.4 1.2 1.0 0.8
*
0.6 0.4 0.2
0
0 0
TD-1 + insulin AP-1 + insulin SLA/PP + insulin Insulin TD-1 Insulin subcutaneous
**
0
e 120
5 4
120 110 100 90 80 70 60 50 40 30 20 10 0
Time (h)
Serum growth hormone (ng/ml)
d
c TD-1 + insulin AP-1 + insulin SLA/PP + insulin Insulin TD-1 Insulin subcutaneous
Blood glucose (% initial)
2,400 2,200 2,000 1,800 1,600 1,400 1,200 1,000 800 600 400 200 0
Serum insulin (ng/ml)
© 2006 Nature Publishing Group http://www.nature.com/naturebiotechnology
Blood radioactivity (cpm/ml)
a
0.0 0
20
100 TD-1 (µg)
500
1,000
0
2
4 Time (h)
6
8
Figure 1 Systemic protein drug delivery mediated by TD-1. (a) 125I-Insulin delivery. 125I -insulin (5,000,000 c.p.m.) was topically coadministered to normal rats with various amounts of TD-1. Radioactivity in the whole blood samples was measured at various time points after administration. (b,c) Delivery of therapeutic levels of insulin. Streptozotocin-induced diabetic rats were topically administered as shown, with the following dosing: TD-1, 500 mg; AP-1, 500 mg ; SLA/PP, 0.5% (wt/vol); porcine insulin, 70 mg, with the exception of subcutaneous treatment (14 mg). At various time points after administration, serum insulin concentration (b) and blood glucose level (c) were measured. Glucose levels were normalized against the initial (0 h) value. Mean ± s.e.m. (n ¼ 6 for glucose and n Z 3 for insulin). *P o 0.05, **P o 0.01, ***P o 0.001. (d,e) Dose-response of TD-1. Porcine insulin (70 mg) and various doses of TD-1 were topically coadministered to streptozotocin-induced diabetic rats. Serum insulin (d) and blood glucose (e) levels were measured before and 5 h after administration. Mean ± s.e.m. (n ¼ 6 for glucose and n Z 3 for insulin). (f) Transdermal delivery of human growth hormone. 500 mg of recombinant human growth hormone (495% pure) was topically coadministered to dexamethasone-treated rats with AP-1, two different doses of TD-1, or saline as indicated. At various time points after administration, serum growth hormone levels were measured. Mean ± s.e.m. (n Z 3) *P o 0.05.
transdermal activity of PH-1 in a dose-dependent manner and at amounts over 5 mg, completely abolished PH-1’s transdermal activity (Supplementary Fig. 1 online). A control peptide named AP-1 (ACNATLPHQCG; a cyclic 11-mer with the same flanking amino acids and an unrelated internal sequence) had no effect on PH-1’s ability to traverse the skin. This result strongly suggested that PH-1’s transdermal activity involved a specific interaction between the displayed PH-1 peptide and an unknown skin component. In contrast to the effect on PH-1 phage, coadministered TD-1 enhanced the transdermal penetration of the control phage, but the activity was relatively low and somewhat inconsistent, as we observed this enhancing activity on only 20% of the tested rats (data not shown). This finding led us to investigate whether TD-1 could deliver protein drugs transdermally. Insulin was selected as the first test candidate owing to the high market demand to deliver this drug noninvasively. We coadministered 125I-insulin and three different doses of TD-1 to the abdominal skin (an area B4 cm2) of rats and measured radioactivity in the systemic circulation. Significant levels of 125I were detectable in the whole blood 2 h after administration and continued to increase for 24 h (Fig. 1a). All three doses of TD-1 were effective. In the absence of TD-1, a low 125I level was detectable above background in the bloodstream, especially 24 h after administration. In principle, 125I detected in the blood could be a result of any of the following: (i) 125NaI contamination (125NaI, a small molecule present as a contaminant (o5%) in the 125I-insulin preparation, may penetrate the skin barrier without the aid of TD-1); (ii) skin degradation of 125I-insulin and subsequent delivery of the breakdown products to the blood; (iii) systemically delivered 125I-insulin without degradation in the skin and with degradation in the blood; (iv) systemically delivered 125I-insulin without any degradation
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in the skin and blood. As insulin cannot traverse the skin barrier, the low level of 125I detected in the blood of the rat treated without TD-1 was most likely due to the delivery of either 125NaI or degraded 125I-insulin. To confirm insulin delivery and determine whether TD-1–delivered insulin can elicit therapeutic effects, we used streptozotocin-induced diabetic rats. In untreated diabetic rats the measured levels of insulin were relatively constant, ranging between 0.2 and 0.7 ng/ml, and were most likely due to low levels of endogenous insulin or a cross-reacting component in the radioimmunoassay. Upon topical coadministration of a saline solution of TD-1/porcine insulin (500:70 mg), we detected serum insulin at an elevated level at 2 h (0.66–1.18 ng/ml, P ¼ 0.100, compared to TD-1 alone), peaked at 5 h (2.85–4.76 ng/ml, P ¼ 0.002) and returned to basal level at 11 h (0.37–0.81 ng/ml, P ¼ 0.169) after administration (Fig. 1b). Correspondingly, blood glucose was decreased at 2 h (65.7–84.4%, P ¼ 0.004, compared to TD-1 alone) after administration, reached its lowest level at 8 h (16.2–38.4%, P ¼ 6.7710e-005) and was sustained at a significantly reduced level for at least 11 h (25.4–52.9%, P ¼ 0.000149) (Fig. 1c). The control peptide AP-1 was inactive. The CPE combination sodium laureth sulfate and phenyl piperazine (SLA/PP), shown to enhance transdermal delivery of a variety of large molecular weight drugs11, was also found to be ineffective in our system. This discrepancy could be due to the different model systems used to assess transdermal delivery: in vitro studies with isolated porcine skin and an in vivo study with hairless rats11 versus our in vivo studies on shaved rats. Alternatively, SLA/PP may show drug selectivity and may work for some protein drugs but not for insulin. TD-1 without insulin had no effect on either blood glucose or serum insulin level, indicating that the glucoselowering effect observed with TD-1 and insulin coadministration was
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LETTERS Table 1 Transdermal activity of TD-1 peptide variants Blood glucosea Sequence
Meanc (%)
Range (%)
S.e.m. (%)
P valued
Meanc (ng/ml)
Range (ng/ml)
S.e.m. (ng/ml)
P valued
TD-1 TD-2
ACSSSPSKHCG CSSSPSKHC
38.68 68.44
38.05–44.48 58.76–70.31
2.04 6.64
1 0.0017
3.9224 1.7480
2.874–4.451 1.654–1.881
0.45633 0.0685
1 0.0092
TD-11 TD-24
SSSPSKH ACSASPSKHCG
92.93 82.17
86.28–102.6 76.63–85.50
3.07 2.79
0.0001 0.0002
0.1938 0.7442
0.162–0.252 0.607–0.873
0.0291 0.077
0.0012 0.0024
TD-3 TD-6
ACSSSASKHCG ACSSSPAKHCG
89.80 66.65
80.60–100.0 58.55–76.87
5.62 5.58
0.0012 0.0158
0.4017 1.6198
0.311–0.429 1.416–1.899
0.0677 0.1446
0.0014 0.0086
TD-22 TD-23
ACSSSPSAHCG ACSSSPSKACG
85.90 81.36
82.05–90.38 74.17–85.53
2.42 3.61
0.0001 0.0006
0.7939 1.0422
0.761–0.841 0.813–1.265
0.0241 0.1305
0.0024 0.0037
TD-10 TD-4
ACSSSSSKHCG ACSSSPSDHCG
53.79 89.68
28.10–90.36 87.46–93.50
6.86 1.92
0.6654 5.58E-5
3.4505 0.7765
3.221–3.604 0.718–0.850
0.1165 0.0389
0.3730 0.0024
Peptide
h value, expressed as % of the 0 h value. b5 h value minus 0 h value. cn ¼ 3, with the exception for the blood glucose of TD-1,TD-11,TD-10 and TD-4 (n ¼ 6). dCompared to TD-1.
due to the delivered exogenous insulin and not a physiological response elicited by TD-1 (Fig. 1b,c). In comparison, subcutaneous injection of insulin (14 mg) resulted in a rapid and short-lasting insulin-elevating and glucose-lowering effect, with a peak effect between 2 h (insulin 2.44–4.40 ng/ml, P ¼ 0.031, glucose 6.6– 44.0%, P ¼ 1.85e-007, compared to 0 hour value) and 3 h (insulin 3.62–4.05 ng/ml, P ¼ 0.001, glucose 3.9–42.4%, P ¼ 7.06e-008) after administration, and a duration of less than 5 h (Fig. 1b,c). The ability of TD-1 to transdermally deliver insulin is dose dependent. 20 mg of TD-1 (topically coadministered with 70 mg of insulin) significantly elevated insulin levels (0.92–1.17 ng/ml, P ¼ 0.016 compared to 0 mg TD-1 group) without affecting glucose levels (89.726–125.379%, P ¼ 0.569 compared to 0 mg TD-1 group) (Fig. 1d,e). However, 100 mg of TD-1 had both insulin-elevating (2.33–2.65 ng/ml, P ¼ 0.002) and glucose-lowering (30.8–91.0%, P ¼ 0.006) effects. Maximum insulin-elevating (3.29–4.76 ng/ml, P ¼ 0.001) and glucose-lowering (27.5–44.5%, P ¼ 3.41e-005) effects were observed at 500 mg. A dose-dependent effect was also observed with insulin. With coadministered TD-1 fixed at 500 mg, 35 mg of insulin was sufficient to significantly enhance serum insulin (1.86–2.03 ng/ml, P ¼ 7.10e-005 compared to 0-mg insulin group) and lower blood glucose (49.5–61.6%, P ¼ 0.00026 compared to 0-mg insulin group), whereas 70 mg of insulin was needed to achieve the maximum
a
b
Pretreatment
4.0
response (insulin 3.29–4.76 ng/ml, P ¼ 0.013, glucose 27.5–44.5%, P ¼ 0.00039) (Supplementary Fig. 2 online). To assess whether TD-1 can deliver other protein drugs, we coadministered recombinant human growth hormone (hGH, 500 mg) and TD-1 at two different doses to Wistar rats pretreated with dexamethasone (12.5 mg/kg) for 2 d. Dexamethasone pretreatment effectively lowered the endogenous growth hormone of the rats to undetectable levels (data not shown), enabling easy detection of the delivered hGH. TD-1 (100 mg) enabled hGH to reach systemic circulation, with significant amounts at 2 h after administration (0.57–1.68 ng/ml, P ¼ 0.016 compared to hGH alone) (Fig. 1f). The effect was similar with 500 mg of TD-1 (0.33–0.91 ng/ml, P ¼ 0.025) but was somewhat less effective than 100 mg. Although the difference between 100 mg and 500 mg was not statistically significant (P ¼ 0.189 for the 2-h values), it nevertheless indicates that the dose-response profile of TD-1 may reach a plateau beyond which TD-1’s effect could diminish. This suggests that the optimum amount of TD-1 for transdermal use may depend on the particular drug to be delivered. TD-1’s effect on hGH delivery was specific, as the control peptide AP-1 was inactive. To assess the sequence specificity of TD-1, we chemically synthesized a series of peptide variants of TD-1 (Table 1). Removing the terminal A and G residues (TD-2) led to a significant loss of activity.
c
5h
10,000
6,000 4,000 2,000
Serum insulin (ng/ml)
Serum insulin (ng/ml)
8,000
3.0 2.5 2.0 1.5 1.0 0.5
0 25 0, 1: 000 50 ,0 1: 00 10 ,0 1: 00 2, 00 1: 0 1, 00 0
5h
4.0 3.5 3.0 2.5 2.0 1.5 1.0
80 60 40 20
0.0 pH 2.0
pH 3.0
pH 7.0
0 CO
0
5
15
Lapsed time (min)
60
CO
0
5
15
60
Lapsed time (min)
1:
10 0 1, 00 0
1
100
Pretreatment
0.5
0.0 10
d
5.0 4.5
3.5
Blood glucose (% initial)
a5
Bound radioactivity (c.p.m.)
© 2006 Nature Publishing Group http://www.nature.com/naturebiotechnology
Serum insulinb
Coated TD-1 (µg) Coated insulin anti-serum
Figure 2 Exploring TD-1’s mode of action. (a) TD-1 does not bind insulin directly. 125I-insulin was added to ELISA microwell plates precoated with increasing amounts of TD-1 (left panel) or an insulin antibody (right panel), and bound radioactivity was determined after washing. (b) Delivery of different molecular forms of insulin by TD-1. 500 mg of TD-1 and 200 mg of insulin were administered to the abdominal skin of streptozotocin-induced diabetic rats in 100 ml saline (after adjusting the pH to 2.0, 3.0 and 7.0, respectively), and serum insulin was measured before and 5 h after administration. (c,d) Timelapse effect of TD-1. TD-1 (500 mg) in 100 ml of saline was administered to the abdominal skin of streptozotocin-induced diabetic rats and left for 5 min. The skin area was then carefully washed with an excess of saline. After various waiting periods, porcine insulin (70 mg in 100 ml saline) was administered to the same skin site. Serum insulin (c) and blood glucose (d) levels were measured before TD-1 treatment and 5 h after insulin administration. Coadministration (CO) of TD-1 (500 mg) and insulin (70 mg) served as the control. Mean ± s.e.m., (n ¼ 6 for glucose and n Z 3 for insulin).
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a
b
c
d
e
f
g
h
i
j
k
l
TD-11, a peptide consisting of only the internal seven-amino acid sequence of TD-1, was totally inactive for delivering insulin, suggesting that the disulfide constrained nature of TD-1 was important for transdermal activity. A partial alanine scan revealed that nearly every amino acid of the internal sequence is important for TD-1’s transdermal activity, as all alanine scan mutants, including TD-24, TD-2, TD-6, TD-22 and TD-23, resulted in a statistically significant loss of activity when compared to TD-1 (see Table 1), but some amino acids are more important than others. Notably, K-D (TD-4) and P-A (TD-3) substitutions had the most effect among the point mutants and resulted in an almost complete loss of activity. On the other hand, the P-S substitution (TD-10) led to only a slight, insignificant decrease in activity. These results demonstrated that TD-1’s transdermal activity is highly sequence specific. How could a peptide as small as TD-1 achieve transdermal delivery of a protein drug such as insulin? To address this question, we studied the hydrophilic nature of TD-1. As the octanol/water partition coefficient (log Pow) for TD-1 was equal to –3.03, TD-1 is predicted to be highly hydrophilic with poor skin permeability based on various models on log Pow18. Thus, the mechanism of TD-1 might be quite different from that of a typical CPE. One possibility is that TD-1 binds to insulin to facilitate its delivery. To test this, we conducted two types of direct binding assays. In the first assay, 125I-insulin was added to enzyme-linked immunosorbent assay (ELISA) microwell plates precoated with increasing amounts of TD-1 (up to 1 mg per well), and bound radioactivity was determined after washing. No significant radioactivity above the background level was detected at any TD-1 amount (Fig. 2a). In the control, precoating with increasing amounts of an insulin antibody resulted in increasing amounts of 125I-insulin
458
Figure 3 Hair follicle penetration of insulin-FITC. After topical administration to the abdominal skin of rats, vertical and horizontal (taken at B600 mm below the skin surface) skin sections were examined. Light and fluorescent microscope pictures are shown in tandem. (a–h) Follicular penetration of insulin-FITC facilitated by TD-1. 10 mg of insulin-FITC was coadministered with 100 mg TD-1 (a and e), 100 mg AP-1 (b and f), nothing (c and g) or 0.5% SLA/PP (d and h) in 100 ml of saline solution. Microscopy of vertical (a–d) and horizontal (e–h) skin sections 2 h after administration was shown. (i–l) Time-lapse effect of TD-1 on follicular penetration of insulin-FITC. TD-1 (100 mg) was topically administered for 5 min and then carefully washed away with an excess of saline. After a waiting period of 0 (i), 5 (j), 15 (k) or 60 (l) min, insulin-FITC (10 mg) was administered to the same skin site. Microscopy of horizontal skin sections was shown. Magnification 200. Bar ¼ 100 mm.
bound to the wells, indicating that the coating and detection procedure was working. In the second assay, microwell plates were coated with increasing amounts of TD-1 and then incubated with insulin. Any bound insulin was then detected with a guinea pig anti-insulin antibody coupled with anti-guinea pig IgG conjugated with peroxidase. No significant amount of insulin was found to bind to precoated TD-1 (Supplementary Fig. 3a online), whereas as little as 1 ng of insulin directly coated on the microwell plate could be detected by the same procedure. We also performed the reverse scheme of this experiment, in which we coated the plate with increasing amounts of insulin and then incubated with TD-1-AngII, a fusion peptide composed of TD-1 and angiotensin II sequences. Any bound TD-1AngII was then detected with a rabbit anti-angiotensin II antibody coupled with anti-rabbit IgG conjugated with peroxidase. No significant amount of TD-1-AngII was found to bind to precoated insulin (Supplementary Fig. 3b online), whereas as little as 10 ng of TD-1AngII was detected by the same procedure. We conclude from these results that TD-1 does not bind insulin directly. TD-1 could conceivably mediate skin penetration by altering the molecular form of insulin. Insulin is known to exist as a hexamer under physiological pH and as lower-molecular-weight forms under acidic pH conditions19. To test this possibility, we conducted dynamic light-scattering studies. In the absence of TD-1, insulin at pH 7.0 exhibited an apparent molecular weight of 32,328, consistent with a predominant hexamer form (Supplementary Table 1 online). At pH 2.0, the observed molecular weight of insulin was 14,585, suggesting mostly a dimer configuration, whereas at pH 3.0 (which is close to the pH value for an unadjusted TD-1/insulin mixture in saline) an intermediate molecular weight (20,343) was seen. Importantly, TD-1 did not significantly alter the apparent molecular weights of insulin under any of the three pH conditions. The radius data further supported this conclusion. The transdermal efficiency of insulin (mediated by TD-1) was similar at pH 2.0 (2.95–3.84 ng/ml) and 3.0 (2.85–3.42 ng/ml, P ¼ 0.39 compared to pH 2.0 group) and was about 20% lower at pH 7.0 compared with the pH 3.0 group (2.08– 2.45 ng/ml) (Fig. 2b). However, some insulin precipitated at pH 7.0, which may have affected delivery of the drug at this pH. Overall, the different molecular forms of insulin did not appear to have a major effect on transdermal delivery facilitated by TD-1. To further study the mechanism of TD-1, we performed time-lapse experiments in which we pretreated the skin of diabetic rats with TD-1 for 5 min, washed off the peptide and waited for a period of time before the administration of insulin to the same skin site. As judged by serum insulin levels (Fig. 2c), when there was no waiting period (the washing process took about 2 min), the transdermal efficiency of insulin was about the same as that observed under the TD-1 and insulin coadministration (3.26–4.36 ng/ml, P ¼ 0.805 compared to
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LETTERS coadministered group). A waiting time of 5 min still enabled a significant amount of insulin to reach blood circulation (2.35– 2.61 ng/ml, P ¼ 0.059). But when the waiting period increased to 15 min (1.48–1.86 ng/ml, P ¼ 0.022) or more, the transdermal efficiency of insulin dramatically decreased. Similar results were obtained with blood glucose measurement (Fig. 2d). These results indicated that TD-1 may create a transient opening on the skin barrier that enables insulin to pass through and reach the systemic circulation. To investigate the route of skin penetration for TD-1–mediated protein drug delivery, we used fluorescent microscopy to examine skin sections after topical coadministration of TD-1 and insulin-fluorescein isothiocyanate (insulin-FITC). Vertical sectioning (sectioning perpendicular to the skin surface) revealed green fluorescence, and thus penetration of insulin-FITC deep into hair follicles (Fig. 3a). Little fluorescence was seen outside hair follicles. The control peptide AP-1 or saline did not lead to follicular penetration of insulin-FITC (Fig. 3b,c). In comparison, SLA/PP enabled penetration of insulinFITC into both the hair follicles and the surrounding dermal tissue, but the amount and depth of follicular penetration were much less than that observed under TD-1 coadministration (Fig. 3d). These findings were backed up by the results of horizontal sectioning (sectioning parallel to the skin surface) at ~600 mm depth (Fig. 3e–h). We estimated the length of hair follicles on the abdominal skin of rats to be about 800 mm, thus at 600 mm-depth it would be close to the bottom portion, or the bulb region, a region known to be important for providing nutrient supply from the blood circulation to the growing hair follicles. It was also known that there are more blood vessels surrounding the lower half of hair follicles20. Deep follicular penetration of insulin appears to be well correlated with systemic delivery of insulin. Coadministration of AP-1, which did not facilitate transdermal insulin delivery, did not lead to follicular penetration of insulin-FITC (Fig. 3b,f). The time-lapse studies further supported this notion, as the length of the waiting period was inversely proportional to both the extent of follicular penetration of insulin-FITC (Fig. 3i–l) and the amount of insulin delivered to the systemic circulation (Fig. 2c). Time-course studies indicated that follicular penetration of insulin-FITC facilitated by TD-1 was detectable as early as 30 min after administration, reached a maximum at 2 h and was sustained for at least 24 h (Supplementary Fig. 4 online). To assess whether TD-1 itself also enters hair follicles, we conducted fluorescent microscopy on skin sections after topical administration of FITC-labeled TD-1 (TD1-FITC). Follicular penetration of TD-1-FITC, similar to that observed under insulin-FITC and TD-1 coadministration, was clearly seen, but a control peptide (SC-1-FITC; FITC-HPGARPVFPWPG) did not show follicular penetration (Supplementary Fig. 5 online). Various lipophilic molecules21, microspheres22 and liposome formulations23 are known to exhibit follicular penetration, and hair follicles are increasingly being recognized as an important route of entry for transdermal drug delivery24,25. However, definitive proof of transfollicular delivery has been difficult to obtain26. Additional work is needed to ascertain whether the transdermal protein drug delivery facilitated by TD-1 indeed involves the transfollicular route. Furthermore, human skin has lower density of hair follicles than rat skin, so the applicability of TD-1 for transdermal drug delivery in humans remains to be determined. A class of membrane-permeable peptides, called protein transduction domains (PTDs)27, has recently been reported to facilitate epicutaneous delivery of protein and peptide molecules28–30. PTDs, however, deliver cargo only locally and not systemically, and they require physical association (usually achieved through covalent linkage) with the cargo to fulfill the delivery function. In contrast, TD-1
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achieves efficient delivery of a cargo to systemic circulation without linkage or direct association with the cargo. Further research may lead to the development of a new class of peptide-based enhancers with the ability to transdermally deliver hydrophilic macromolecular drugs. METHODS Peptide synthesis. All peptides were synthesized by Shanghai GL Biochemicals, using standard solid-phase FMOC method with automatic peptide synthesizer (CS Bio) and purified to 495% by high-performance liquid chromatography (HPLC). FITC was linked to the N terminus of TD-1 and SC-1 through an aminocaproic (Acp) spacer. The identity of the peptides was independently verified by us, using a mass spectrometer (BIFLEX). Library screening. BALB/cA nude mice (Slaccas) were maintained in the SPF grade animal house under a 12-h light/dark cycles at 24–25 1C with a relative humidity of 50–55%. Institutional guidelines were followed in handling the animals. Mice were anesthetized using 5 ml/kg of 20% solution of urethane. 1011 plaque forming units (pfu) of Ph.D.-C7C phage library (New England Biolabs) in 100 ml saline was applied to the abdominal skin of nude mice and spread evenly over an area of B3 cm 3 cm using the side of a pipet tip. One hour after the phage administration, 1 ml of blood was withdrawn from the heart and mixed with 0.5 ml of rapidly growing Escherichia coli ER 2738. After 30 min incubation, the recovered phage were amplified in 20 ml of LuriaBertani (LB) medium for 6 h. Amplified phage were resuspended in PBS and used for the second round of screening, following the same procedure. Phage recovered from the blood sample of the second round were plated out on LB plates containing X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactoside) and IPTG (isopropyl-b-D-thiogalactoside). Twelve blue plaques were randomly picked and subjected to DNA sequencing. Phage delivery. Male Wistar rats (180 g–220 g) were housed at a constant temperature (22 1C) and relative humidity (60%) with a fixed 12-h light/dark cycle, and free access to food and water. The hair in a B3 cm 3 cm area on the abdomen was carefully trimmed with scissors (rats with any visible sign of skin damage were not used). 1012 pfu (phage forming unit) of PH-1 phage or control phage (randomly picked from the library and encoding a displayed peptide of CDQSHPQQC) were applied to the center of the area and spread evenly over the entire area. One hour later, blood was withdrawn from the tail vein and immediately assayed for phage titer. 125I-insulin
delivery. 125I-insulin (specific activity: 170 mCi/mg) was prepared (Beijing Atom High-Tech) using Bolton-Hunter procedure, and purified by HPLC. Five million c.p.m. of 125I-insulin was coadministered with different amounts of TD-1 or 16 mg of AP-1 to the exposed abdominal skin (approximately 2 cm 2 cm) of anesthetized Wistar rats in 100 ml of saline. Blood was withdrawn from the tail vein at the various time points and assayed for 125I radioactivity with Gamma Radioimmunoassay Counter (Chuangxin). Insulin delivery. Male Wistar rats were given an intraperitoneal injection of streptozotocin (90 mg/kg; Sigma). Seven to 10 days after injection blood glucose levels were measured using One-Touch Ultra Glucometer (LifeScan). Rats with a blood glucose level of over 20 mmol/l (normal rats are between 4 and 6 mmol/l) were selected and randomly assigned to treatment groups, with six rats for each group. One treatment group received 70 mg of pharmaceutical-grade (498%, 30 IU/mg, containing 0.35% zinc) porcine insulin (Xuzhou Pharmaceuticals) and 500 mg of TD-1 per rat, coapplied to the exposed abdominal skin (B2 cm 2 cm) in 100 ml saline. The chemical enhancer group received topical administration of 100 ml of 1:1 PBS/ethanol solution containing 70 mg insulin, 0.35% (wt/vol) SLA and 0.15% (wt/vol) PP. Other transdermal treatment groups were all in 100 ml saline. For the subcutaneous treatment group, 14 mg of insulin per rat was injected. At various time points after treatment (0, 2, 5, 8 and 11 h for transdermal and 0, 1, 2, 3 and 5 h for subcutaneous), blood was drawn from the tail vein and assayed for blood glucose as described above and serum insulin by an insulin IRMA kit (Immunotech). The significance of the difference in the serum insulin and blood glucose levels between a test group and the control (TD-1 alone) group at each time point was determined by two-tailed independent samples t-test,
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with the exception of the subcutaneous treatment group (we used two-tailed paired samples t-test to compare each time point after administration with the 0 h data for this group, since the time points for this group were unique). Growth hormone delivery. Male Wistar rats were given a daily subcutaneous injection of dexamethasone (12.5 mg/kg; Sigma). After 2 d treatment they were randomly assigned to treatment groups, with 3 to 4 rats for each group. One treatment group received 500 mg of pharmaceutical-grade recombinant human growth hormone (21.5 kDa full-length protein expressed in E. coli and purified to 495%, with a specific activity of 2.5 IU/mg; Rising Bio-tech) and 500 mg of TD-1 per rat, coapplied to the exposed abdominal skin (B2 cm 2 cm) in 100 ml saline. Other treatment groups were as indicated in the figure legend. At various time points (0, 2, 5 and 8 h) after treatment, blood was drawn from the tail vein and the amount of growth hormone in the serum assayed by ELISA (Diagnostic Systems Laboratories). For statistical analysis, the various groups were compared to the saline+hGH group on each time point, using a two-tailed independent samples t-test. Microscopy of skin sections. Insulin-FITC (Xi-an Huacheng) was dissolved in a small amount of DMF and subsequently diluted in saline for topical administration (final DMF concentration was o0.2%). Insulin-FITC (10 mg) with or without peptides (100 mg) was applied to the exposed abdominal skin of rats in a 100 ml volume made up by saline, except for SLA/PP treatment (administered in 100 ml of 1:1 PBS/ethanol solution containing 10 mg insulinFITC, 0.35% SLA and 0.15% PP). After 2 h the skin was carefully cleaned with 70% isopropyl alcohol, harvested and fixed with ice-cold 4% paraformaldehyde overnight. After washing, skin samples were immersed in 4.5% sucrose for 24 h and then dehydrated in 30% sucrose till deposition. Floating horizontal and vertical sections with a thickness of 20 mm were obtained on a freezing microtome (LEICA). Cryosections were mounted onto poly-L-lysine-coated glasses, dried at 25 1C and enveloped with 10 ml VECTASHIELD Mounting Medium (Vector Laboratories). Fluorescence photomicrographs of the sections were obtained with OLYMPUS IX-70 microscope using a filter set having excitation and emission length at 490–495 nm and 520–530 nm, respectively. The same procedure was used to visualize skin penetration of TD-1-FITC and SC-1-FITC. Direct interaction assessment between TD-1 and 125I-insulin. ELISA plate wells were coated with indicated amounts of TD-1 dissolved in 50 mM NaHCO3, PH 9.6 (37 1C, 2 h) and blocked with 1% BSA. 125I-insulin (B30,000 c.p.m.) was added to each well and incubated for 2 h at 37 1C. After extensive washing with PBST (PBS + 0.1% Tween-20), radioactivity was measured with Gamma Radioimmunoassay Counter. For the control, a guinea pig anti-porcine insulin antibody (Atom High-Tech) at various dilutions was coated on plates and assessed for binding to 125I-insulin in the same way as described above. Determination of octanol-water partition coefficient. n-Octanol and water were presaturated with each other by vigorous mixing and letting to stand at 25 1C for 24 h. 10 mg of TD-1 was transferred to a 15-ml screw-capped centrifuge tube, and pre-saturated octanol and water (2.5 ml each) were added. The mixture was vigorously shaken for 10 min, let to stand at 25 1C for 12 h, and then centrifuged (400g, 10 min) to achieve good separation of two phases. TD-1 concentrations in the two phases were determined by HPLC and used to compute log Pow. For HPLC, we used a 250 4.6 mm reverse C18 analytical column (COSMOSIL) with the mobile phase consisting of 30% acetonitrile/ 0.1% TFA and 70% water/0.1% TFA and a flow rate of 1 ml/min with an injection loop of 20 ml. Detection was at 230 nm. Note: Supplementary information is available on the Nature Biotechnology website.
ACKNOWLEDGMENTS We thank Yong Chen for help on sequence analysis. This work was supported by the One Hundred Talent Project and Nanomedicine Research Project (kjcx2sw-h12-01) of the Chinese Academy of Sciences, Anhui Talent Fund (2004Z023) and National Natural Science Foundation of China (30470871).
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COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/naturebiotechnology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Prausnitz, M.R., Mitragotri, S. & Langer, R. Current status and future potential of transdermal drug delivery. Nat. Rev. Drug Discov. 3, 115–124 (2004). 2. Thomas, B.J. & Finnin, B.C. The transdermal revolution. Drug Discov. Today 9, 697–703 (2004). 3. Scheuplein, R.J. & Blank, I.H. Permeability of the skin. Physiol. Rev. 51, 702–747 (1971). 4. Williams, A.C. & Barry, B.W. Penetration enhancers. Adv. Drug Deliv. Rev. 56, 603–618 (2004). 5. Purdon, C.H., Azzi, C.G., Zhang, J., Smith, E.W. & Maibach, H.I. Penetration enhancement of transdermal delivery–current permutations and limitations. Crit. Rev. Ther. Drug Carrier Syst. 21, 97–132 (2004). 6. Kalia, Y.N., Naik, A., Garrison, J. & Guy, R.H. Iontophoretic drug delivery. Adv. Drug Deliv. Rev. 56, 619–658 (2004). 7. Lavon, I. & Kost, J. Ultrasound and transdermal drug delivery. Drug Discov. Today 9, 670–676 (2004). 8. Prausnitz, M.R. Microneedles for transdermal drug delivery. Adv. Drug Deliv. Rev. 56, 581–587 (2004). 9. Bos, J.D. & Meinardi, M.M. The 500 Dalton rule for the skin penetration of chemical compounds and drugs. Exp. Dermatol. 9, 165–169 (2000). 10. Karande, P., Jain, A. & Mitragotri, S. Discovery of transdermal penetration enhancers by high throughput screening. Nat. Biotechnol. 22, 192–197 (2004). 11. Karande, P., Jain, A., Ergun, K., Kispersky, V. & Mitragotri, S. Design principles of chemical penetration enhancers for transdermal drug delivery. Proc. Natl. Acad. Sci. USA 102, 4688–4693 (2005). 12. Cwirla, S.E., Peters, E.A., Barrett, R.W. & Dower, W.J. Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87, 6378–6382 (1990). 13. Pasqualini, R. & Ruoslahti, E. Organ targeting in vivo using phage display peptide libraries. Nature 380, 364–366 (1996). 14. Arap, W., Pasqualini, R. & Ruoslahti, E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279, 377–380 (1998). 15. Raffii, S., Avecilla, S.D. & Jin, D.K. Tumor vasculature address book: identification of stage-specific tumor vessel zip codes by phage display. Cancer Cell 4, 331–333 (2003). 16. Frenkel, D. & Solomon, B. Filamentous phage as vector-mediated antibody delivery to the brain. Proc. Natl. Acad. Sci. USA 99, 5675–5679 (2002). 17. Duerr, D.M., White, S.J. & Schluesener, H.J. Identification of peptide sequences that induce the transport of phage across the gastrointestinal mucosal barrier. J. Virol. Methods 116, 177–180 (2004). 18. Guy, R.H. & Hadgraft, J. Transdermal Drug Delivery. (Marcel Dekker, New York, 2003). 19. Uversky, V.N. et al. Prediction of the Association State of Insulin Using Spectral Parameters. J. Pharm. Sci. 92, 847–858 (2003). 20. Montagna, W. The Structure and Function of Skin. (Academic Press, New York, 1956). 21. Grams, Y.Y. & Bouwstra, J.A. Penetration and distribution of three lipophilic probes in vitro in human skin focusing on the hair follicle. J. Control. Release 83, 253–262 (2002). 22. Rolland, A., Wagner, N., Chatelus, A., Shroot, B. & Schaefer, H. Site- specific drug delivery to pilosebaceous structures using polymeric microspheres. Pharm. Res. 10, 1738–1744 (1993). 23. Li, L. & Hoffman, R.M. The feasibility of targeted selective gene therapy of the hair follicle. Nat. Med. 1, 705–706 (1995). 24. Lauer, A.C., Lieb, L.M., Ramachandran, C., Flynn, G.L. & Weiner, N.D. Transfollicular drug delivery. Pharm. Res. 12, 179–186 (1995). 25. Agarwal, R., Katare, O.P. & Vyas, S.P. The pilosebaceous unit: a pivotal route for topical drug delivery. Methods Find. Exp. Clin. Pharmacol. 22, 129–133 (2000). 26. Meidan, V.M., Docker, M., Walmsley, A.D. & Irvin, W.J. Low intensity ultrasound as a probe to elucidate the relative follicular contribution to total transdermal absorption. Pharm. Res. 15, 85–92 (1998). 27. Joliot, A. & Prochiantz, A. Transduction peptides: from technology to physiology. Nat. Cell Biol. 6, 189–196 (2004). 28. Rothbard, J.B. et al. Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation. Nat. Med. 6, 1253–1257 (2000). 29. Schutze-Redelmeier, M.-P.M., Kong, S., Bally, M.B. & Dutz, J.P. Antennapedia transduction sequence promotes anti tumour immunity to epicutaneously administered CTL epitopes. Vaccine 22, 1985–1991 (2004). 30. Lopes, L.B. et al. Comparative study of the skin penetration of protein transduction domains and a conjugated peptide. Pharm. Res. 22, 750–757 (2005).
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Transdermal protein delivery by a coadministered peptide identified via phage display Yongping Chen1,2, Yuanyuan Shen1,2, Xin Guo1,2, Caoshou Zhang1, Wenjuan Yang1, Minglu Ma1, Shu Liu1, Maobin Zhang1 & Long-Ping Wen1 Efficient transdermal drug delivery of large hydrophilic drugs is challenging. Here we report that the short synthetic peptide, ACSSSPSKHCG, identified by in vivo phage display, facilitated efficient transdermal protein drug delivery through intact skin. Coadministration of the peptide and insulin to the abdominal skin of diabetic rats resulted in elevated systemic levels of insulin and suppressed serum glucose levels for at least 11 h. Significant systemic bioavailability of human growth hormone was also achieved when topically coadministered with the peptide. The transdermal-enhancing activity of the peptide was sequence specific and dose dependent, did not involve direct interaction with insulin and enabled penetration of insulin into hair follicles beyond a depth of 600 lm. Timelapse studies suggested that the peptide creates a transient opening in the skin barrier to enable macromolecular drugs to reach systemic circulation. As the largest organ of the human body, skin provides a painless interface for systemic drug delivery1,2. However, the permeability of foreign molecules, especially large hydrophilic molecules, across the stratum corneum in the outer skin surface is extremely low3. In the last 50 years a large number of chemical penetration enhancers (CPEs), belonging to several different classes such as surfactants, fatty acids, fatty esters and Azone-like compounds, have enabled limited skin permeation enhancement4,5. CPEs have two major limitations: skin irritation and inability to deliver large molecules. Without physical enhancement, mediated by iontophoresis6, ultrasound7 or microneedles8, CPEs are generally unable to deliver therapeutic levels of relatively large drugs (molecular mass 4500 Da) through intact skin to the systemic circulation9. Recent progress in high-throughput screening to identify enhancer combinations10 and application of CPE design principles11 may help overcome these two limitations, but transdermal delivery of large hydrophilic proteins remains a formidable task. Phage display peptide libraries are commonly used to obtain defined peptide sequences that interact with a particular molecule. As many as 109 different peptides are expressed on the phage surface by fusion to one of the phage surface proteins, and the desired peptides are selected on the basis of binding to the target molecule12.
1Hefei 2These
Phage libraries have been used to select for peptides that bind to immobilized proteins, carbohydrates, cultured cells and even inorganic material. An extension of this technique is in vivo phage display, which entails the injection of a peptide display phage library into the bloodstream of an animal and subsequent isolation and identification of phage that have the ability to home to a particular organ or tissue13–15. Filamentous phage, known to reach the brain after intranasal administration, have been used as a carrier to deliver antibodies against amyloid plaques16. Furthermore, a recent study used in vivo phage display to identify peptide sequences that induced the transport of phage across the gastrointestinal mucosal barrier17. These studies prompted us to study whether in vivo phage display could identify peptides with transdermal capability. We applied Ph.D.-C7C, a disulfide-constrained, phage-display, peptide library, onto the abdominal skin of BALB/cA nude mice and recovered phage particles from the blood circulation. Recovered phage were amplified and used for the next round of in vivo selection. Amplified phage from the first round exhibited a transdermal efficiency two orders of magnitude higher than that of the library phage. After the second round, twelve clones were randomly picked and sequenced. Eight of them contained an identical nucleotide sequence insert (termed PH-1), which coded for the displayed peptide CSSSPSKHC. When tested in Wistar rats, a representative phage carrying this sequence consistently crossed the skin barrier and reached the bloodstream, achieving an average titer of 4,556 infective phage particles per milliliter of blood 1 h after topical administration. In contrast, a control phage randomly selected from the library exhibited an average titer of 6.8 infective phage particles per milliliter of blood, with zero infective phage particles detected in the majority of the experiments (n ¼16). As PH-1 differs from the random phage only in the displayed peptide sequence, the phage-displayed peptide most likely interacts with the skin to enable skin barrier penetration of PH-1. If the displayed peptide interacts with the skin in a specific manner that can be saturated, an excess of the displayed peptide would inhibit PH-1’s transdermal activity . To test this model, we synthesized a cyclic peptide named TD-1 (ACSSSPSKHCG; the flanking A and G were derived from the M13 coat protein and included to make the peptide more stable). As expected, coadministered TD-1 inhibited the
National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, Hefei, Anhui 230027, China. authors contributed equally to this work. Correspondence should be addressed to L.-P. Wen ([email protected]).
Received 22 August 2005; accepted 3 January 2006; published online 26 March 2006; doi:10.1038/nbt1193
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b 4.5
TD-1 (µg) 0 4 8 16
Serum insulin (ng/ml)
4.0
*
3.5
***
** **
3.0 2.5 2.0 1.5 1.0 0.5 0.0
5
0
10
15
20
25
0
2
4
Time (h)
6
8
10
12
Blood glucose (% initial)
Pretreatment 5h
3 2 1
20
100 TD-1 (µg)
*** ***
***
500
1,000
***
***
*** 2
4
6
8
10
12
Time (h)
f
100 80 60 40 20
AP-1 100 µg TD-1 500 µg TD-1 100 µg Saline
1.8 1.6
*
1.4 1.2 1.0 0.8
*
0.6 0.4 0.2
0
0 0
TD-1 + insulin AP-1 + insulin SLA/PP + insulin Insulin TD-1 Insulin subcutaneous
**
0
e 120
5 4
120 110 100 90 80 70 60 50 40 30 20 10 0
Time (h)
Serum growth hormone (ng/ml)
d
c TD-1 + insulin AP-1 + insulin SLA/PP + insulin Insulin TD-1 Insulin subcutaneous
Blood glucose (% initial)
2,400 2,200 2,000 1,800 1,600 1,400 1,200 1,000 800 600 400 200 0
Serum insulin (ng/ml)
© 2006 Nature Publishing Group http://www.nature.com/naturebiotechnology
Blood radioactivity (cpm/ml)
a
0.0 0
20
100 TD-1 (µg)
500
1,000
0
2
4 Time (h)
6
8
Figure 1 Systemic protein drug delivery mediated by TD-1. (a) 125I-Insulin delivery. 125I -insulin (5,000,000 c.p.m.) was topically coadministered to normal rats with various amounts of TD-1. Radioactivity in the whole blood samples was measured at various time points after administration. (b,c) Delivery of therapeutic levels of insulin. Streptozotocin-induced diabetic rats were topically administered as shown, with the following dosing: TD-1, 500 mg; AP-1, 500 mg ; SLA/PP, 0.5% (wt/vol); porcine insulin, 70 mg, with the exception of subcutaneous treatment (14 mg). At various time points after administration, serum insulin concentration (b) and blood glucose level (c) were measured. Glucose levels were normalized against the initial (0 h) value. Mean ± s.e.m. (n ¼ 6 for glucose and n Z 3 for insulin). *P o 0.05, **P o 0.01, ***P o 0.001. (d,e) Dose-response of TD-1. Porcine insulin (70 mg) and various doses of TD-1 were topically coadministered to streptozotocin-induced diabetic rats. Serum insulin (d) and blood glucose (e) levels were measured before and 5 h after administration. Mean ± s.e.m. (n ¼ 6 for glucose and n Z 3 for insulin). (f) Transdermal delivery of human growth hormone. 500 mg of recombinant human growth hormone (495% pure) was topically coadministered to dexamethasone-treated rats with AP-1, two different doses of TD-1, or saline as indicated. At various time points after administration, serum growth hormone levels were measured. Mean ± s.e.m. (n Z 3) *P o 0.05.
transdermal activity of PH-1 in a dose-dependent manner and at amounts over 5 mg, completely abolished PH-1’s transdermal activity (Supplementary Fig. 1 online). A control peptide named AP-1 (ACNATLPHQCG; a cyclic 11-mer with the same flanking amino acids and an unrelated internal sequence) had no effect on PH-1’s ability to traverse the skin. This result strongly suggested that PH-1’s transdermal activity involved a specific interaction between the displayed PH-1 peptide and an unknown skin component. In contrast to the effect on PH-1 phage, coadministered TD-1 enhanced the transdermal penetration of the control phage, but the activity was relatively low and somewhat inconsistent, as we observed this enhancing activity on only 20% of the tested rats (data not shown). This finding led us to investigate whether TD-1 could deliver protein drugs transdermally. Insulin was selected as the first test candidate owing to the high market demand to deliver this drug noninvasively. We coadministered 125I-insulin and three different doses of TD-1 to the abdominal skin (an area B4 cm2) of rats and measured radioactivity in the systemic circulation. Significant levels of 125I were detectable in the whole blood 2 h after administration and continued to increase for 24 h (Fig. 1a). All three doses of TD-1 were effective. In the absence of TD-1, a low 125I level was detectable above background in the bloodstream, especially 24 h after administration. In principle, 125I detected in the blood could be a result of any of the following: (i) 125NaI contamination (125NaI, a small molecule present as a contaminant (o5%) in the 125I-insulin preparation, may penetrate the skin barrier without the aid of TD-1); (ii) skin degradation of 125I-insulin and subsequent delivery of the breakdown products to the blood; (iii) systemically delivered 125I-insulin without degradation in the skin and with degradation in the blood; (iv) systemically delivered 125I-insulin without any degradation
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in the skin and blood. As insulin cannot traverse the skin barrier, the low level of 125I detected in the blood of the rat treated without TD-1 was most likely due to the delivery of either 125NaI or degraded 125I-insulin. To confirm insulin delivery and determine whether TD-1–delivered insulin can elicit therapeutic effects, we used streptozotocin-induced diabetic rats. In untreated diabetic rats the measured levels of insulin were relatively constant, ranging between 0.2 and 0.7 ng/ml, and were most likely due to low levels of endogenous insulin or a cross-reacting component in the radioimmunoassay. Upon topical coadministration of a saline solution of TD-1/porcine insulin (500:70 mg), we detected serum insulin at an elevated level at 2 h (0.66–1.18 ng/ml, P ¼ 0.100, compared to TD-1 alone), peaked at 5 h (2.85–4.76 ng/ml, P ¼ 0.002) and returned to basal level at 11 h (0.37–0.81 ng/ml, P ¼ 0.169) after administration (Fig. 1b). Correspondingly, blood glucose was decreased at 2 h (65.7–84.4%, P ¼ 0.004, compared to TD-1 alone) after administration, reached its lowest level at 8 h (16.2–38.4%, P ¼ 6.7710e-005) and was sustained at a significantly reduced level for at least 11 h (25.4–52.9%, P ¼ 0.000149) (Fig. 1c). The control peptide AP-1 was inactive. The CPE combination sodium laureth sulfate and phenyl piperazine (SLA/PP), shown to enhance transdermal delivery of a variety of large molecular weight drugs11, was also found to be ineffective in our system. This discrepancy could be due to the different model systems used to assess transdermal delivery: in vitro studies with isolated porcine skin and an in vivo study with hairless rats11 versus our in vivo studies on shaved rats. Alternatively, SLA/PP may show drug selectivity and may work for some protein drugs but not for insulin. TD-1 without insulin had no effect on either blood glucose or serum insulin level, indicating that the glucoselowering effect observed with TD-1 and insulin coadministration was
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Meanc (%)
Range (%)
S.e.m. (%)
P valued
Meanc (ng/ml)
Range (ng/ml)
S.e.m. (ng/ml)
P valued
TD-1 TD-2
ACSSSPSKHCG CSSSPSKHC
38.68 68.44
38.05–44.48 58.76–70.31
2.04 6.64
1 0.0017
3.9224 1.7480
2.874–4.451 1.654–1.881
0.45633 0.0685
1 0.0092
TD-11 TD-24
SSSPSKH ACSASPSKHCG
92.93 82.17
86.28–102.6 76.63–85.50
3.07 2.79
0.0001 0.0002
0.1938 0.7442
0.162–0.252 0.607–0.873
0.0291 0.077
0.0012 0.0024
TD-3 TD-6
ACSSSASKHCG ACSSSPAKHCG
89.80 66.65
80.60–100.0 58.55–76.87
5.62 5.58
0.0012 0.0158
0.4017 1.6198
0.311–0.429 1.416–1.899
0.0677 0.1446
0.0014 0.0086
TD-22 TD-23
ACSSSPSAHCG ACSSSPSKACG
85.90 81.36
82.05–90.38 74.17–85.53
2.42 3.61
0.0001 0.0006
0.7939 1.0422
0.761–0.841 0.813–1.265
0.0241 0.1305
0.0024 0.0037
TD-10 TD-4
ACSSSSSKHCG ACSSSPSDHCG
53.79 89.68
28.10–90.36 87.46–93.50
6.86 1.92
0.6654 5.58E-5
3.4505 0.7765
3.221–3.604 0.718–0.850
0.1165 0.0389
0.3730 0.0024
Peptide
h value, expressed as % of the 0 h value. b5 h value minus 0 h value. cn ¼ 3, with the exception for the blood glucose of TD-1,TD-11,TD-10 and TD-4 (n ¼ 6). dCompared to TD-1.
due to the delivered exogenous insulin and not a physiological response elicited by TD-1 (Fig. 1b,c). In comparison, subcutaneous injection of insulin (14 mg) resulted in a rapid and short-lasting insulin-elevating and glucose-lowering effect, with a peak effect between 2 h (insulin 2.44–4.40 ng/ml, P ¼ 0.031, glucose 6.6– 44.0%, P ¼ 1.85e-007, compared to 0 hour value) and 3 h (insulin 3.62–4.05 ng/ml, P ¼ 0.001, glucose 3.9–42.4%, P ¼ 7.06e-008) after administration, and a duration of less than 5 h (Fig. 1b,c). The ability of TD-1 to transdermally deliver insulin is dose dependent. 20 mg of TD-1 (topically coadministered with 70 mg of insulin) significantly elevated insulin levels (0.92–1.17 ng/ml, P ¼ 0.016 compared to 0 mg TD-1 group) without affecting glucose levels (89.726–125.379%, P ¼ 0.569 compared to 0 mg TD-1 group) (Fig. 1d,e). However, 100 mg of TD-1 had both insulin-elevating (2.33–2.65 ng/ml, P ¼ 0.002) and glucose-lowering (30.8–91.0%, P ¼ 0.006) effects. Maximum insulin-elevating (3.29–4.76 ng/ml, P ¼ 0.001) and glucose-lowering (27.5–44.5%, P ¼ 3.41e-005) effects were observed at 500 mg. A dose-dependent effect was also observed with insulin. With coadministered TD-1 fixed at 500 mg, 35 mg of insulin was sufficient to significantly enhance serum insulin (1.86–2.03 ng/ml, P ¼ 7.10e-005 compared to 0-mg insulin group) and lower blood glucose (49.5–61.6%, P ¼ 0.00026 compared to 0-mg insulin group), whereas 70 mg of insulin was needed to achieve the maximum
a
b
Pretreatment
4.0
response (insulin 3.29–4.76 ng/ml, P ¼ 0.013, glucose 27.5–44.5%, P ¼ 0.00039) (Supplementary Fig. 2 online). To assess whether TD-1 can deliver other protein drugs, we coadministered recombinant human growth hormone (hGH, 500 mg) and TD-1 at two different doses to Wistar rats pretreated with dexamethasone (12.5 mg/kg) for 2 d. Dexamethasone pretreatment effectively lowered the endogenous growth hormone of the rats to undetectable levels (data not shown), enabling easy detection of the delivered hGH. TD-1 (100 mg) enabled hGH to reach systemic circulation, with significant amounts at 2 h after administration (0.57–1.68 ng/ml, P ¼ 0.016 compared to hGH alone) (Fig. 1f). The effect was similar with 500 mg of TD-1 (0.33–0.91 ng/ml, P ¼ 0.025) but was somewhat less effective than 100 mg. Although the difference between 100 mg and 500 mg was not statistically significant (P ¼ 0.189 for the 2-h values), it nevertheless indicates that the dose-response profile of TD-1 may reach a plateau beyond which TD-1’s effect could diminish. This suggests that the optimum amount of TD-1 for transdermal use may depend on the particular drug to be delivered. TD-1’s effect on hGH delivery was specific, as the control peptide AP-1 was inactive. To assess the sequence specificity of TD-1, we chemically synthesized a series of peptide variants of TD-1 (Table 1). Removing the terminal A and G residues (TD-2) led to a significant loss of activity.
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Serum insulinb
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Figure 2 Exploring TD-1’s mode of action. (a) TD-1 does not bind insulin directly. 125I-insulin was added to ELISA microwell plates precoated with increasing amounts of TD-1 (left panel) or an insulin antibody (right panel), and bound radioactivity was determined after washing. (b) Delivery of different molecular forms of insulin by TD-1. 500 mg of TD-1 and 200 mg of insulin were administered to the abdominal skin of streptozotocin-induced diabetic rats in 100 ml saline (after adjusting the pH to 2.0, 3.0 and 7.0, respectively), and serum insulin was measured before and 5 h after administration. (c,d) Timelapse effect of TD-1. TD-1 (500 mg) in 100 ml of saline was administered to the abdominal skin of streptozotocin-induced diabetic rats and left for 5 min. The skin area was then carefully washed with an excess of saline. After various waiting periods, porcine insulin (70 mg in 100 ml saline) was administered to the same skin site. Serum insulin (c) and blood glucose (d) levels were measured before TD-1 treatment and 5 h after insulin administration. Coadministration (CO) of TD-1 (500 mg) and insulin (70 mg) served as the control. Mean ± s.e.m., (n ¼ 6 for glucose and n Z 3 for insulin).
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TD-11, a peptide consisting of only the internal seven-amino acid sequence of TD-1, was totally inactive for delivering insulin, suggesting that the disulfide constrained nature of TD-1 was important for transdermal activity. A partial alanine scan revealed that nearly every amino acid of the internal sequence is important for TD-1’s transdermal activity, as all alanine scan mutants, including TD-24, TD-2, TD-6, TD-22 and TD-23, resulted in a statistically significant loss of activity when compared to TD-1 (see Table 1), but some amino acids are more important than others. Notably, K-D (TD-4) and P-A (TD-3) substitutions had the most effect among the point mutants and resulted in an almost complete loss of activity. On the other hand, the P-S substitution (TD-10) led to only a slight, insignificant decrease in activity. These results demonstrated that TD-1’s transdermal activity is highly sequence specific. How could a peptide as small as TD-1 achieve transdermal delivery of a protein drug such as insulin? To address this question, we studied the hydrophilic nature of TD-1. As the octanol/water partition coefficient (log Pow) for TD-1 was equal to –3.03, TD-1 is predicted to be highly hydrophilic with poor skin permeability based on various models on log Pow18. Thus, the mechanism of TD-1 might be quite different from that of a typical CPE. One possibility is that TD-1 binds to insulin to facilitate its delivery. To test this, we conducted two types of direct binding assays. In the first assay, 125I-insulin was added to enzyme-linked immunosorbent assay (ELISA) microwell plates precoated with increasing amounts of TD-1 (up to 1 mg per well), and bound radioactivity was determined after washing. No significant radioactivity above the background level was detected at any TD-1 amount (Fig. 2a). In the control, precoating with increasing amounts of an insulin antibody resulted in increasing amounts of 125I-insulin
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Figure 3 Hair follicle penetration of insulin-FITC. After topical administration to the abdominal skin of rats, vertical and horizontal (taken at B600 mm below the skin surface) skin sections were examined. Light and fluorescent microscope pictures are shown in tandem. (a–h) Follicular penetration of insulin-FITC facilitated by TD-1. 10 mg of insulin-FITC was coadministered with 100 mg TD-1 (a and e), 100 mg AP-1 (b and f), nothing (c and g) or 0.5% SLA/PP (d and h) in 100 ml of saline solution. Microscopy of vertical (a–d) and horizontal (e–h) skin sections 2 h after administration was shown. (i–l) Time-lapse effect of TD-1 on follicular penetration of insulin-FITC. TD-1 (100 mg) was topically administered for 5 min and then carefully washed away with an excess of saline. After a waiting period of 0 (i), 5 (j), 15 (k) or 60 (l) min, insulin-FITC (10 mg) was administered to the same skin site. Microscopy of horizontal skin sections was shown. Magnification 200. Bar ¼ 100 mm.
bound to the wells, indicating that the coating and detection procedure was working. In the second assay, microwell plates were coated with increasing amounts of TD-1 and then incubated with insulin. Any bound insulin was then detected with a guinea pig anti-insulin antibody coupled with anti-guinea pig IgG conjugated with peroxidase. No significant amount of insulin was found to bind to precoated TD-1 (Supplementary Fig. 3a online), whereas as little as 1 ng of insulin directly coated on the microwell plate could be detected by the same procedure. We also performed the reverse scheme of this experiment, in which we coated the plate with increasing amounts of insulin and then incubated with TD-1-AngII, a fusion peptide composed of TD-1 and angiotensin II sequences. Any bound TD-1AngII was then detected with a rabbit anti-angiotensin II antibody coupled with anti-rabbit IgG conjugated with peroxidase. No significant amount of TD-1-AngII was found to bind to precoated insulin (Supplementary Fig. 3b online), whereas as little as 10 ng of TD-1AngII was detected by the same procedure. We conclude from these results that TD-1 does not bind insulin directly. TD-1 could conceivably mediate skin penetration by altering the molecular form of insulin. Insulin is known to exist as a hexamer under physiological pH and as lower-molecular-weight forms under acidic pH conditions19. To test this possibility, we conducted dynamic light-scattering studies. In the absence of TD-1, insulin at pH 7.0 exhibited an apparent molecular weight of 32,328, consistent with a predominant hexamer form (Supplementary Table 1 online). At pH 2.0, the observed molecular weight of insulin was 14,585, suggesting mostly a dimer configuration, whereas at pH 3.0 (which is close to the pH value for an unadjusted TD-1/insulin mixture in saline) an intermediate molecular weight (20,343) was seen. Importantly, TD-1 did not significantly alter the apparent molecular weights of insulin under any of the three pH conditions. The radius data further supported this conclusion. The transdermal efficiency of insulin (mediated by TD-1) was similar at pH 2.0 (2.95–3.84 ng/ml) and 3.0 (2.85–3.42 ng/ml, P ¼ 0.39 compared to pH 2.0 group) and was about 20% lower at pH 7.0 compared with the pH 3.0 group (2.08– 2.45 ng/ml) (Fig. 2b). However, some insulin precipitated at pH 7.0, which may have affected delivery of the drug at this pH. Overall, the different molecular forms of insulin did not appear to have a major effect on transdermal delivery facilitated by TD-1. To further study the mechanism of TD-1, we performed time-lapse experiments in which we pretreated the skin of diabetic rats with TD-1 for 5 min, washed off the peptide and waited for a period of time before the administration of insulin to the same skin site. As judged by serum insulin levels (Fig. 2c), when there was no waiting period (the washing process took about 2 min), the transdermal efficiency of insulin was about the same as that observed under the TD-1 and insulin coadministration (3.26–4.36 ng/ml, P ¼ 0.805 compared to
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LETTERS coadministered group). A waiting time of 5 min still enabled a significant amount of insulin to reach blood circulation (2.35– 2.61 ng/ml, P ¼ 0.059). But when the waiting period increased to 15 min (1.48–1.86 ng/ml, P ¼ 0.022) or more, the transdermal efficiency of insulin dramatically decreased. Similar results were obtained with blood glucose measurement (Fig. 2d). These results indicated that TD-1 may create a transient opening on the skin barrier that enables insulin to pass through and reach the systemic circulation. To investigate the route of skin penetration for TD-1–mediated protein drug delivery, we used fluorescent microscopy to examine skin sections after topical coadministration of TD-1 and insulin-fluorescein isothiocyanate (insulin-FITC). Vertical sectioning (sectioning perpendicular to the skin surface) revealed green fluorescence, and thus penetration of insulin-FITC deep into hair follicles (Fig. 3a). Little fluorescence was seen outside hair follicles. The control peptide AP-1 or saline did not lead to follicular penetration of insulin-FITC (Fig. 3b,c). In comparison, SLA/PP enabled penetration of insulinFITC into both the hair follicles and the surrounding dermal tissue, but the amount and depth of follicular penetration were much less than that observed under TD-1 coadministration (Fig. 3d). These findings were backed up by the results of horizontal sectioning (sectioning parallel to the skin surface) at ~600 mm depth (Fig. 3e–h). We estimated the length of hair follicles on the abdominal skin of rats to be about 800 mm, thus at 600 mm-depth it would be close to the bottom portion, or the bulb region, a region known to be important for providing nutrient supply from the blood circulation to the growing hair follicles. It was also known that there are more blood vessels surrounding the lower half of hair follicles20. Deep follicular penetration of insulin appears to be well correlated with systemic delivery of insulin. Coadministration of AP-1, which did not facilitate transdermal insulin delivery, did not lead to follicular penetration of insulin-FITC (Fig. 3b,f). The time-lapse studies further supported this notion, as the length of the waiting period was inversely proportional to both the extent of follicular penetration of insulin-FITC (Fig. 3i–l) and the amount of insulin delivered to the systemic circulation (Fig. 2c). Time-course studies indicated that follicular penetration of insulin-FITC facilitated by TD-1 was detectable as early as 30 min after administration, reached a maximum at 2 h and was sustained for at least 24 h (Supplementary Fig. 4 online). To assess whether TD-1 itself also enters hair follicles, we conducted fluorescent microscopy on skin sections after topical administration of FITC-labeled TD-1 (TD1-FITC). Follicular penetration of TD-1-FITC, similar to that observed under insulin-FITC and TD-1 coadministration, was clearly seen, but a control peptide (SC-1-FITC; FITC-HPGARPVFPWPG) did not show follicular penetration (Supplementary Fig. 5 online). Various lipophilic molecules21, microspheres22 and liposome formulations23 are known to exhibit follicular penetration, and hair follicles are increasingly being recognized as an important route of entry for transdermal drug delivery24,25. However, definitive proof of transfollicular delivery has been difficult to obtain26. Additional work is needed to ascertain whether the transdermal protein drug delivery facilitated by TD-1 indeed involves the transfollicular route. Furthermore, human skin has lower density of hair follicles than rat skin, so the applicability of TD-1 for transdermal drug delivery in humans remains to be determined. A class of membrane-permeable peptides, called protein transduction domains (PTDs)27, has recently been reported to facilitate epicutaneous delivery of protein and peptide molecules28–30. PTDs, however, deliver cargo only locally and not systemically, and they require physical association (usually achieved through covalent linkage) with the cargo to fulfill the delivery function. In contrast, TD-1
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achieves efficient delivery of a cargo to systemic circulation without linkage or direct association with the cargo. Further research may lead to the development of a new class of peptide-based enhancers with the ability to transdermally deliver hydrophilic macromolecular drugs. METHODS Peptide synthesis. All peptides were synthesized by Shanghai GL Biochemicals, using standard solid-phase FMOC method with automatic peptide synthesizer (CS Bio) and purified to 495% by high-performance liquid chromatography (HPLC). FITC was linked to the N terminus of TD-1 and SC-1 through an aminocaproic (Acp) spacer. The identity of the peptides was independently verified by us, using a mass spectrometer (BIFLEX). Library screening. BALB/cA nude mice (Slaccas) were maintained in the SPF grade animal house under a 12-h light/dark cycles at 24–25 1C with a relative humidity of 50–55%. Institutional guidelines were followed in handling the animals. Mice were anesthetized using 5 ml/kg of 20% solution of urethane. 1011 plaque forming units (pfu) of Ph.D.-C7C phage library (New England Biolabs) in 100 ml saline was applied to the abdominal skin of nude mice and spread evenly over an area of B3 cm 3 cm using the side of a pipet tip. One hour after the phage administration, 1 ml of blood was withdrawn from the heart and mixed with 0.5 ml of rapidly growing Escherichia coli ER 2738. After 30 min incubation, the recovered phage were amplified in 20 ml of LuriaBertani (LB) medium for 6 h. Amplified phage were resuspended in PBS and used for the second round of screening, following the same procedure. Phage recovered from the blood sample of the second round were plated out on LB plates containing X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactoside) and IPTG (isopropyl-b-D-thiogalactoside). Twelve blue plaques were randomly picked and subjected to DNA sequencing. Phage delivery. Male Wistar rats (180 g–220 g) were housed at a constant temperature (22 1C) and relative humidity (60%) with a fixed 12-h light/dark cycle, and free access to food and water. The hair in a B3 cm 3 cm area on the abdomen was carefully trimmed with scissors (rats with any visible sign of skin damage were not used). 1012 pfu (phage forming unit) of PH-1 phage or control phage (randomly picked from the library and encoding a displayed peptide of CDQSHPQQC) were applied to the center of the area and spread evenly over the entire area. One hour later, blood was withdrawn from the tail vein and immediately assayed for phage titer. 125I-insulin
delivery. 125I-insulin (specific activity: 170 mCi/mg) was prepared (Beijing Atom High-Tech) using Bolton-Hunter procedure, and purified by HPLC. Five million c.p.m. of 125I-insulin was coadministered with different amounts of TD-1 or 16 mg of AP-1 to the exposed abdominal skin (approximately 2 cm 2 cm) of anesthetized Wistar rats in 100 ml of saline. Blood was withdrawn from the tail vein at the various time points and assayed for 125I radioactivity with Gamma Radioimmunoassay Counter (Chuangxin). Insulin delivery. Male Wistar rats were given an intraperitoneal injection of streptozotocin (90 mg/kg; Sigma). Seven to 10 days after injection blood glucose levels were measured using One-Touch Ultra Glucometer (LifeScan). Rats with a blood glucose level of over 20 mmol/l (normal rats are between 4 and 6 mmol/l) were selected and randomly assigned to treatment groups, with six rats for each group. One treatment group received 70 mg of pharmaceutical-grade (498%, 30 IU/mg, containing 0.35% zinc) porcine insulin (Xuzhou Pharmaceuticals) and 500 mg of TD-1 per rat, coapplied to the exposed abdominal skin (B2 cm 2 cm) in 100 ml saline. The chemical enhancer group received topical administration of 100 ml of 1:1 PBS/ethanol solution containing 70 mg insulin, 0.35% (wt/vol) SLA and 0.15% (wt/vol) PP. Other transdermal treatment groups were all in 100 ml saline. For the subcutaneous treatment group, 14 mg of insulin per rat was injected. At various time points after treatment (0, 2, 5, 8 and 11 h for transdermal and 0, 1, 2, 3 and 5 h for subcutaneous), blood was drawn from the tail vein and assayed for blood glucose as described above and serum insulin by an insulin IRMA kit (Immunotech). The significance of the difference in the serum insulin and blood glucose levels between a test group and the control (TD-1 alone) group at each time point was determined by two-tailed independent samples t-test,
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with the exception of the subcutaneous treatment group (we used two-tailed paired samples t-test to compare each time point after administration with the 0 h data for this group, since the time points for this group were unique). Growth hormone delivery. Male Wistar rats were given a daily subcutaneous injection of dexamethasone (12.5 mg/kg; Sigma). After 2 d treatment they were randomly assigned to treatment groups, with 3 to 4 rats for each group. One treatment group received 500 mg of pharmaceutical-grade recombinant human growth hormone (21.5 kDa full-length protein expressed in E. coli and purified to 495%, with a specific activity of 2.5 IU/mg; Rising Bio-tech) and 500 mg of TD-1 per rat, coapplied to the exposed abdominal skin (B2 cm 2 cm) in 100 ml saline. Other treatment groups were as indicated in the figure legend. At various time points (0, 2, 5 and 8 h) after treatment, blood was drawn from the tail vein and the amount of growth hormone in the serum assayed by ELISA (Diagnostic Systems Laboratories). For statistical analysis, the various groups were compared to the saline+hGH group on each time point, using a two-tailed independent samples t-test. Microscopy of skin sections. Insulin-FITC (Xi-an Huacheng) was dissolved in a small amount of DMF and subsequently diluted in saline for topical administration (final DMF concentration was o0.2%). Insulin-FITC (10 mg) with or without peptides (100 mg) was applied to the exposed abdominal skin of rats in a 100 ml volume made up by saline, except for SLA/PP treatment (administered in 100 ml of 1:1 PBS/ethanol solution containing 10 mg insulinFITC, 0.35% SLA and 0.15% PP). After 2 h the skin was carefully cleaned with 70% isopropyl alcohol, harvested and fixed with ice-cold 4% paraformaldehyde overnight. After washing, skin samples were immersed in 4.5% sucrose for 24 h and then dehydrated in 30% sucrose till deposition. Floating horizontal and vertical sections with a thickness of 20 mm were obtained on a freezing microtome (LEICA). Cryosections were mounted onto poly-L-lysine-coated glasses, dried at 25 1C and enveloped with 10 ml VECTASHIELD Mounting Medium (Vector Laboratories). Fluorescence photomicrographs of the sections were obtained with OLYMPUS IX-70 microscope using a filter set having excitation and emission length at 490–495 nm and 520–530 nm, respectively. The same procedure was used to visualize skin penetration of TD-1-FITC and SC-1-FITC. Direct interaction assessment between TD-1 and 125I-insulin. ELISA plate wells were coated with indicated amounts of TD-1 dissolved in 50 mM NaHCO3, PH 9.6 (37 1C, 2 h) and blocked with 1% BSA. 125I-insulin (B30,000 c.p.m.) was added to each well and incubated for 2 h at 37 1C. After extensive washing with PBST (PBS + 0.1% Tween-20), radioactivity was measured with Gamma Radioimmunoassay Counter. For the control, a guinea pig anti-porcine insulin antibody (Atom High-Tech) at various dilutions was coated on plates and assessed for binding to 125I-insulin in the same way as described above. Determination of octanol-water partition coefficient. n-Octanol and water were presaturated with each other by vigorous mixing and letting to stand at 25 1C for 24 h. 10 mg of TD-1 was transferred to a 15-ml screw-capped centrifuge tube, and pre-saturated octanol and water (2.5 ml each) were added. The mixture was vigorously shaken for 10 min, let to stand at 25 1C for 12 h, and then centrifuged (400g, 10 min) to achieve good separation of two phases. TD-1 concentrations in the two phases were determined by HPLC and used to compute log Pow. For HPLC, we used a 250 4.6 mm reverse C18 analytical column (COSMOSIL) with the mobile phase consisting of 30% acetonitrile/ 0.1% TFA and 70% water/0.1% TFA and a flow rate of 1 ml/min with an injection loop of 20 ml. Detection was at 230 nm. Note: Supplementary information is available on the Nature Biotechnology website.
ACKNOWLEDGMENTS We thank Yong Chen for help on sequence analysis. This work was supported by the One Hundred Talent Project and Nanomedicine Research Project (kjcx2sw-h12-01) of the Chinese Academy of Sciences, Anhui Talent Fund (2004Z023) and National Natural Science Foundation of China (30470871).
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COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/naturebiotechnology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Prausnitz, M.R., Mitragotri, S. & Langer, R. Current status and future potential of transdermal drug delivery. Nat. Rev. Drug Discov. 3, 115–124 (2004). 2. Thomas, B.J. & Finnin, B.C. The transdermal revolution. Drug Discov. Today 9, 697–703 (2004). 3. Scheuplein, R.J. & Blank, I.H. Permeability of the skin. Physiol. Rev. 51, 702–747 (1971). 4. Williams, A.C. & Barry, B.W. Penetration enhancers. Adv. Drug Deliv. Rev. 56, 603–618 (2004). 5. Purdon, C.H., Azzi, C.G., Zhang, J., Smith, E.W. & Maibach, H.I. Penetration enhancement of transdermal delivery–current permutations and limitations. Crit. Rev. Ther. Drug Carrier Syst. 21, 97–132 (2004). 6. Kalia, Y.N., Naik, A., Garrison, J. & Guy, R.H. Iontophoretic drug delivery. Adv. Drug Deliv. Rev. 56, 619–658 (2004). 7. Lavon, I. & Kost, J. Ultrasound and transdermal drug delivery. Drug Discov. Today 9, 670–676 (2004). 8. Prausnitz, M.R. Microneedles for transdermal drug delivery. Adv. Drug Deliv. Rev. 56, 581–587 (2004). 9. Bos, J.D. & Meinardi, M.M. The 500 Dalton rule for the skin penetration of chemical compounds and drugs. Exp. Dermatol. 9, 165–169 (2000). 10. Karande, P., Jain, A. & Mitragotri, S. Discovery of transdermal penetration enhancers by high throughput screening. Nat. Biotechnol. 22, 192–197 (2004). 11. Karande, P., Jain, A., Ergun, K., Kispersky, V. & Mitragotri, S. Design principles of chemical penetration enhancers for transdermal drug delivery. Proc. Natl. Acad. Sci. USA 102, 4688–4693 (2005). 12. Cwirla, S.E., Peters, E.A., Barrett, R.W. & Dower, W.J. Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87, 6378–6382 (1990). 13. Pasqualini, R. & Ruoslahti, E. Organ targeting in vivo using phage display peptide libraries. Nature 380, 364–366 (1996). 14. Arap, W., Pasqualini, R. & Ruoslahti, E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279, 377–380 (1998). 15. Raffii, S., Avecilla, S.D. & Jin, D.K. Tumor vasculature address book: identification of stage-specific tumor vessel zip codes by phage display. Cancer Cell 4, 331–333 (2003). 16. Frenkel, D. & Solomon, B. Filamentous phage as vector-mediated antibody delivery to the brain. Proc. Natl. Acad. Sci. USA 99, 5675–5679 (2002). 17. Duerr, D.M., White, S.J. & Schluesener, H.J. Identification of peptide sequences that induce the transport of phage across the gastrointestinal mucosal barrier. J. Virol. Methods 116, 177–180 (2004). 18. Guy, R.H. & Hadgraft, J. Transdermal Drug Delivery. (Marcel Dekker, New York, 2003). 19. Uversky, V.N. et al. Prediction of the Association State of Insulin Using Spectral Parameters. J. Pharm. Sci. 92, 847–858 (2003). 20. Montagna, W. The Structure and Function of Skin. (Academic Press, New York, 1956). 21. Grams, Y.Y. & Bouwstra, J.A. Penetration and distribution of three lipophilic probes in vitro in human skin focusing on the hair follicle. J. Control. Release 83, 253–262 (2002). 22. Rolland, A., Wagner, N., Chatelus, A., Shroot, B. & Schaefer, H. Site- specific drug delivery to pilosebaceous structures using polymeric microspheres. Pharm. Res. 10, 1738–1744 (1993). 23. Li, L. & Hoffman, R.M. The feasibility of targeted selective gene therapy of the hair follicle. Nat. Med. 1, 705–706 (1995). 24. Lauer, A.C., Lieb, L.M., Ramachandran, C., Flynn, G.L. & Weiner, N.D. Transfollicular drug delivery. Pharm. Res. 12, 179–186 (1995). 25. Agarwal, R., Katare, O.P. & Vyas, S.P. The pilosebaceous unit: a pivotal route for topical drug delivery. Methods Find. Exp. Clin. Pharmacol. 22, 129–133 (2000). 26. Meidan, V.M., Docker, M., Walmsley, A.D. & Irvin, W.J. Low intensity ultrasound as a probe to elucidate the relative follicular contribution to total transdermal absorption. Pharm. Res. 15, 85–92 (1998). 27. Joliot, A. & Prochiantz, A. Transduction peptides: from technology to physiology. Nat. Cell Biol. 6, 189–196 (2004). 28. Rothbard, J.B. et al. Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation. Nat. Med. 6, 1253–1257 (2000). 29. Schutze-Redelmeier, M.-P.M., Kong, S., Bally, M.B. & Dutz, J.P. Antennapedia transduction sequence promotes anti tumour immunity to epicutaneously administered CTL epitopes. Vaccine 22, 1985–1991 (2004). 30. Lopes, L.B. et al. Comparative study of the skin penetration of protein transduction domains and a conjugated peptide. Pharm. Res. 22, 750–757 (2005).
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