TY - JOUR
T1 - Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide
AU - Ramanathan, Srinivasan
AU - Qiu, Bo
AU - Pooyan, Shahriar
AU - Zhang, Guobao
AU - Stein, Stanley
AU - Leibowitz, Michael J.
AU - Sinko, Patrick J.
N1 - Funding Information:
This research was supported by NIH grant AI42007 (P.J.S.) and a grant from the Campbell Foundation (M.J.L., S.P.). Partial support was provided to S.R. by the New Jersey Center for Biomaterials.
PY - 2001/12/13
Y1 - 2001/12/13
N2 - We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large (∼1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 μM) and the bioconjugate (0.1-30 μM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 μM and 0.003-30 μM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (Km=6.61 μM) and CHO/hSMVT cells (Km=1.26 μM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (Km=6.13 μM) and CHO/hSMVT cells (Km=8.19 μM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide therapeutic agents.
AB - We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large (∼1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 μM) and the bioconjugate (0.1-30 μM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 μM and 0.003-30 μM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (Km=6.61 μM) and CHO/hSMVT cells (Km=1.26 μM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (Km=6.13 μM) and CHO/hSMVT cells (Km=8.19 μM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide therapeutic agents.
KW - Caco-2
KW - Drug targeting
KW - PEG conjugates
KW - Peptide delivery
KW - SMVT
KW - Tat
UR - http://www.scopus.com/inward/record.url?scp=0035856813&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035856813&partnerID=8YFLogxK
U2 - 10.1016/S0168-3659(01)00474-6
DO - 10.1016/S0168-3659(01)00474-6
M3 - Article
C2 - 11733088
AN - SCOPUS:0035856813
SN - 0168-3659
VL - 77
SP - 199
EP - 212
JO - Journal of Controlled Release
JF - Journal of Controlled Release
IS - 3
ER -