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Binding of the fibronectin-mimetic peptide, PR_b, to alpha5beta1 on pig islet cells increases fibronectin production and facilitates internalization of PR_b functionalized liposomes.

ABSTRACT: Islet transplantation is a promising treatment for type 1 diabetes. Recent studies have demonstrated that human islet allografts can restore insulin independence to patients with this disease. As islet isolation and immunotherapeutic techniques improve, the demand for this cell-based therapy will dictate the need for other sources of islets. Pig islets could provide an unlimited supply for xenotransplantation and have shown promise as an alternative to human islet allografts. However, stresses imposed during islet isolation and transplantation decrease islet viability, leading to loss of graft function. In this study, we investigated the ability of a fibronectin-mimetic peptide, PR_b, which specifically binds to the alpha(5)beta(1) integrin, to re-establish lost extracellular matrix (ECM) around isolated pig islets and increase internalization of liposomes. Confocal microscopy and Western blotting were used to show the presence of the integrin alpha(5)beta(1) on the pig islets on day 0 (day of isolation) as well as on different days of islet culture. Islets cultured in medium supplemented with free PR_b for 48 h were found to have increased levels of ECM fibronectin secretion compared to islets in normal culture conditions. Using confocal microscopy and flow cytometry, we found that PR_b peptide-amphiphile functionalized liposomes delivered to the pig islets internalized into the cells in a PR_b concentration dependent manner and nonfunctionalized liposomes showed minimal internalization. These studies proved that the fibronectin-mimetic peptide, PR_b, is an appropriate peptide bullet for applications involving alpha(5)beta(1) expressing pig islet cells. Fibronectin production stimulated through alpha(5)beta(1) PR_b binding may decrease apoptosis and therefore increase islet viability in culture. In addition, PR_b peptide-amphiphile functionalized liposomes may be used for targeted delivery of different agents to pig islet cells.

SUBMITTER: Atchison NA

PROVIDER: S-EPMC2932789 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Binding of the fibronectin-mimetic peptide, PR_b, to alpha5beta1 on pig islet cells increases fibronectin production and facilitates internalization of PR_b functionalized liposomes.

Atchison Nicole A NA Fan Wei W Papas Klearchos K KK Hering Bernhard J BJ Tsapatsis Michael M Kokkoli Efrosini E

Langmuir : the ACS journal of surfaces and colloids 20100901 17

Islet transplantation is a promising treatment for type 1 diabetes. Recent studies have demonstrated that human islet allografts can restore insulin independence to patients with this disease. As islet isolation and immunotherapeutic techniques improve, the demand for this cell-based therapy will dictate the need for other sources of islets. Pig islets could provide an unlimited supply for xenotransplantation and have shown promise as an alternative to human islet allografts. However, stresses i ...[more]

PMID: 20704278

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Project description:Integrin alpha(5)beta(1) is expressed on several types of cancer cells, including colon cancer, and plays an important role in tumor growth and metastasis. The ability to target the integrin alpha(5)beta(1) using an appropriate drug delivery nano-vector can significantly help in inhibiting tumor growth, reducing tumor metastasis, and decreasing deleterious side effects associated with different cancer therapies. Liposomes are nano-sized phospholipid bilayer vesicles that have been extensively studied as drug delivery carriers. The goal of this study is to design stealth liposomes (liposomes covered with polyethylene glycol (PEG)) that will target colon cancer cells that express the integrin alpha(5)beta(1). The PEG provides a steric barrier allowing the liposomes to circulate in the blood and the functionalizing moiety, PR_b peptide, will specifically recognize and bind to alpha(5)beta(1) expressing cells. PR_b is a novel peptide sequence that mimics the cell adhesion domain of fibronectin, and includes four building blocks, RGDSP (the primary recognition site for alpha(5)beta(1)), PHSRN (the synergy site for alpha(5)beta(1)), a (SG)(5) linker, and a KSS spacer. In this study we have demonstrated that by varying the amount of PEG (PEG750 or PEG2000) and PR_b on the liposomal interface we can engineer nano-vectors that bind to CT26.WT, HCT116, and RKO colon cancer cells in a specific manner and are internalized through most likely alpha(5)beta(1)-mediated endocytosis. GRGDSP-targeted stealth liposomes bind to colon cancer cells and internalize, but they have much lesser efficiency than PR_b-targeted stealth liposomes, and more importantly they are not as specific since many integrins bind to RGD peptides. PR_b-targeted stealth liposomes are as cytotoxic as free 5-Fluorouracil (5-FU) and exert the highest cytotoxicity on CT26.WT cells compared to GRGDSP-targeted stealth liposomes and non-targeted stealth liposomes. Thus, the proposed targeted delivery system has the great potential to deliver a therapeutic load directly to colon cancer cells, in an efficient and specific manner.

Project description:Advancements in liposomal drug delivery have produced long circulating and very stable drug formulations. These formulations minimize systemic exposure; however, unfortunately, therapeutic efficacy has remained limited due to the slow diffusion of liposomal particles within the tumor and limited release or uptake of the encapsulated drug. Here, the carboxyl-terminated CRPPR peptide, with affinity for the receptor neuropilin-1 (NRP), which is expressed on both endothelial and cancer cells, was conjugated to liposomes to enhance the tumor accumulation. Using a pH sensitive probe, liposomes were optimized for specific NRP binding and subsequent cellular internalization using in vitro cellular assays. Liposomes conjugated with the carboxyl-terminated CRPPR peptide (termed C-LPP liposomes) bound to the NRP-positive primary prostatic carcinoma cell line (PPC-1) but did not bind to the NRP-negative PC-3 cell line, and binding was observed with liposomal peptide concentrations as low as 0.16mol%. Binding of the C-LPP liposomes was receptor-limited, with saturation observed at high liposome concentrations. The identical peptide sequence bearing an amide terminus did not bind specifically, accumulating only with a high (2.5mol%) peptide concentration and adhering equally to NRP positive and negative cell lines. The binding of C-LPP liposomes conjugated with 0.63mol% of the peptide was 83-fold greater than liposomes conjugated with the amide version of the peptide. Cellular internalization was also enhanced with C-LPP liposomes, with 80% internalized following 3h incubation. Additionally, fluorescence in the blood pool (~40% of the injected dose) was similar for liposomes conjugated with 0.63mol% of carboxyl-terminated peptide and non-targeted liposomes at 24h after injection, indicating stable circulation. Prior to doxorubicin treatment, in vivo tumor accumulation and vascular targeting were increased for peptide-conjugated liposomes compared to non-targeted liposomes based on confocal imaging of a fluorescent cargo, and the availability of the vascular receptor was confirmed with ultrasound molecular imaging. Finally, over a 4-week course of therapy, tumor knockdown resulting from doxorubicin-loaded, C-LPP liposomes was similar to non-targeted liposomes in syngeneic tumor-bearing FVB mice and C-LPP liposomes reduced doxorubicin accumulation in the skin and heart and eliminated skin toxicity. Taken together, our results demonstrate that a carboxyl-terminated RXXR peptide sequence, conjugated to liposomes at a concentration of 0.63mol%, retains long circulation but enhances binding and internalization, and reduces toxicity.

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Dataset Information

Binding of the fibronectin-mimetic peptide, PR_b, to alpha5beta1 on pig islet cells increases fibronectin production and facilitates internalization of PR_b functionalized liposomes.

Publications

Binding of the fibronectin-mimetic peptide, PR_b, to alpha5beta1 on pig islet cells increases fibronectin production and facilitates internalization of PR_b functionalized liposomes.

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