Difference between revisions of "Part:BBa K1074000"

Latest revision as of 00:03, 20 October 2016

TD1, Transdermal peptide

TD1 is a short synthetic peptide(ACSSSPSKHCG)identified by in vivo phage display, facilitated efficient transdermal protein delivery through intact skin. Studies suggested that the peptide creates a transient opening in the skin barrier to enable macromolecular material to reach systemic circulation.

As the largest organ of the human body, skin provides a painless interface for systemic drug delivery. However, the permeability of foreign molecules, especially large hydrophilic molecules, across the stratum corneum in the outer skin surface is extremely low.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 enabledlimited skin permeation enhancement. But CPEs have the limitations to deliver large molecules,especially large hydrophilic proteins. Now, TD1 has fulfilled this formidable task.

Small, efficient, easy implementation etc, TD1 offer a powerful experimental methodology for the transdermal protein delivery . And its nature of polypeptide make it an undoubtedly nice biobrick in synthetic biology . In our project,we fuse TD1 with a varity of antigens(HBsAg(Hepatitis B virus),Ag85b(Mycobacterium tuberculosis),PAD4(Bacillus Anthracis)) and adjuvant so they can be delievered through the skin to provoke the immune response to achieve the goal of "Transdermal vaccine".

Usage and Biology

TD1 can facilitated transdermal protein delivery by Coadministration of it with target protein or fused with the target protein(always N-terminal).Studies shows that 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(figure 1).

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 normalrats 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.

The transdermal-enhancing activity of the peptide was sequence specific(figure 2) and dose dependent, did not involve direct interaction with target proteins(figure 3).

figure 2 Transdermal activity of TD-1 peptide variants

figure 3 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).

When you fuse TD1 with your target proteins, N-terminal modified and a linker of GGGS are recommended.

References

Chen, Y.P., et al., Transdermal protein delivery by a coadministered peptide identified via phage display. Nature biotechnology, 2006. 24(4): p. 455-460.

Sequence and Features

Further Characterization - UofC_Calgary 2016

The UofC_Calgary 2016 team used TD1 fused to the N-terminus of the modified version of the Bowman-Birk Protease Inhibitor (BBI), a small radioprotective peptide 14 amino acids in length (https://parts.igem.org/Part:BBa_K2008005). We performed in vivo trials in mice to determine if our peptide fused to the TD1 tag could pass from our delivery system, an adhesive patch, through the skin of the mice and into the blood. Patches were adhered to mice for a period of five days before the mice were euthanized and blood samples were taken. Mass spectrometry was used to analyze blood and serum samples to detect the presence of TD1-BBI.

Methods
1) Peptides were extracted from mouse blood plasma or serum via purification on a C18 Zip-Tip column from Thermo; eluted with 50% acetonitrile, 0.1% formic acid
2) Samples were concentrated 10x via evaporation and resuspended in 0.1% formic acid
3) Extracted samples were injected on to a 10 cm C18 column and eluted with a 30 min, 5-40%B gradient at 300 nL/min
4) MS spectra were acquired on a Thermo LTQ Orbitrap Velos in ion trap mode; MS/MS spectra were acquired in Orbitrap mode with CID
5) Spectra were manually analyzed for the presence of BBI (monoisotopic mass = 2577.1 Da) at various charge states; pure sample of BBI, synthesized by BioBasic, was used as a reference for identification

We acquired mass spectra for a synthetic BBI standard [tagged with TD1 and KSCI]. The characterization of a BBI standard will be of value to future experiments when detection of BBI is necessary. MS spectrum of intact BBI in multiple charge states is shown in Figure 4; fragmentation of the 3+ and 4+ charge states, via CID, is shown in figures 5 and 6, respectively.

Fig 4. Standard mass spectra with states denoted.

Fig 5. Mass spectra of BBI in a +3 state

Fig 6. Mass spectra of BBI in a +4 state

From these results, it was shown that TD1-BBI was not present in our mouse blood sample. This may indicate that TD1 does not facilitate the ease of transdermal transport, or that our TD1-BBI peptide was degraded in mouse blood before it could be. Further studies for characterization are required.