Liposomal nanotechnology provides a versatile platform for exploring several approaches that can potentially enhance the delivery and targeting of therapies to tumors. As a biodegradable and essentially nontoxic platform, liposomes can be used to encapsulate both hydrophilic and hydrophobic materials and be utilized as drug carriers in drug delivery systems (DDSs). In addition, liposomes can be used to carry radioactive moieties, such as radiotracers, which can be bound at multiple locations within liposomes, making them attractive carriers for molecular imaging applications. In this study, gelatinase-binding peptides were attached to liposomes for synthesizing a targeted drug delivery vehicle.
For active targeting or drug delivery applications or both, intraliposomal encapsylation of multiple targeting agents or therapies can be (i) to the lipid bilayer, which can bind hydrophobic conjugates; (ii) to hydrated compartments for water-soluble components; (iii) by covalent binding directly or by utilizing spacer to the outer lipid leaflet [1
]. Delivery of these nanoformulations to the reticuloendothelial system (RES) is readily achieved since, given their larger size, the RES traps most conventional liposomes that are not shielded by polyethylene glycol chains (PEGs) or other similar steric water carrying substance. RES uptake can be increased by altering particle surface chemistry and charge, for instance, by adding positively charged lipids or biologically activating proteins or sugars on the surface of the liposomes. For purposes of agent delivery to target organs other than the RES, long-circulating liposomes have been developed by modifying the liposomal surface [2
]. Determination of the in vivo
biodistribution and targeting kinetics of liposome-encapsulated drugs is required for the assessment of drug bioavailability.
The most commonly used nanoformulated drug is Caelyx/Doxil, a liposomal doxorubicin product. It has nearly supplanted doxorubicin in the therapy of ovarian cancer, breast cancer, and Kaposi's sarcoma. It differs from the former generation liposomal delivery systems, as the outer surface of Caelyx/Doxil is coated with PEG chains that protect the liposomes from being opsonized by components of the immune system in the circulation. These stealth-type liposomes have longer circulation half-times than those for uncoated liposomes. In addition, they are safer than the native drugs themselves (e.g., Caelyx/Doxil is not cardiotoxic, a major concern for native doxorubicin delivery).
For cancer-based applications, peptides that can selectively detect and target metastatic disease and tumor invasive potential may offer critical prognostic information. Metastatic invasion is promoted by the attachment of tumor cells to the extracellular matrix, the degradation of matrix components by tumor-associated proteases, and the cellular movement into the area modified by protease activity. Matrix metalloproteases (MMPs) represent a family of enzymes capable of degrading the basement membrane and extracellular matrix (ECM), thus contributing to tissue remodeling and cell migration [3
]. This family of enzymes can cleave ECM proteins, as well as alter the integrity of basement membranes that serve as barriers to cell movement. This is normally a tightly regulated process, with the presence of activated metalloproteinases occurring only under specific conditions.
MMPs may be divided into subgroups, one comprised of type IV collagenases (gelatinases) such as MMP-2 and MMP-9, which play major roles in tumor growth, angiogenesis, and metastatic disease. These gelatinases degrade type IV collagen (and its breakdown product, gelatin) and comprise the primary structural component of the ECM, enabling tumor cells to gain access to the rest of the body. Overexpression and/or prognostic significance of gelatinases have been examined in a range of cancer types, including ovarian cancer [6
], endometrial and cervical cancer [10
], and breast cancer [12
]. High expression levels of gelatinases in breast and ovarian cancers, for instance, are known to be associated with unfavorable prognoses.
In this study, binding peptides (BPs) extracted from MMP-9 were attached to liposomes for synthesizing a targeted drug delivery vehicle. Downregulation of MMP-9 is known to exert inhibitory effects on endothelial cell migration and tube formation [14
]. Intriguingly MMP-2 has been shown to dock on tumor cell-surface integrins, which makes gelatinases even more interesting as a target [15
]. Adenoviral-mediated MMP-9 downregulation was shown to retard endothelial cell migration in cell wounding and spheroid migration assays, resulting in reduced capillary-like tube formation [16
]. MMP-2- or MMP-9-deficient mice were found to exhibit abnormal angiogenic properties [17
]. Further, in human gliomas, immunohistochemical findings suggested that neoplastic and endothelial cells expressing MMP-9 protein may be associated with tumoral angiogenesis [18
One of the first known specific gelatinase inhibitors, a cyclic MMP-9-binding peptide identified by random phage display libraries (i.e., CTTHWGFTLC peptide later CTT1), has previously been shown to have high affinity not only to MMP-9, but also to MMP-2 [19
]. The peptide actively inhibits endothelial and tumor cell migration in vitro
and tumor progression in in vivo
murine models [19
]. Specifically, CTT-displaying phages block the formation of new blood vessels, resulting in tumor size reductions and prolonged overall survival. These findings highlight the potential of CTT peptide for targeting chemotherapeutics or other imaging probes to the tumor neovasculature. CTT-peptide has been used for liposomal drug delivery in vitro
and in vivo
] and has been shown to be effective in improving selective localization of chemotherapies such as doxorubicin in human tumor cells. In this work we modified a CTT peptide with a peptide linker that bears a tyrosine moiety for iodination procedures and attached several additional amino acids (GRENYHG) to enhance freedom of peptide binding in order to create GRENYHGCTTHWGFTLC-peptide (i.e., CTT2-peptide) (). The synthesis of CTT2-peptide enabled us to retain bioactivity that would otherwise not be present if CTT peptide itself was directly linked to lipids or PEG spacers. This is the first study, to our knowledge, that has utilized a peptide derived from a synthetic phage display library for constructing a more selective liposomal delivery system for targeting extracellular target molecules.
Figure 1 Chemical structure of 125I-CTT2-peptide. CTT2-peptide is a 17-amino acid peptide with a disulphide bridge between the two cysteines. The amino terminal end of the peptide is amidated to increase its stability. Upon iodination, peptide labeling occurs (more ...)
We initially present the synthesis of PEG-PE-CTT2 peptide-bound micelles and liposomes. The feasibility of utilizing micellar and liposomal nanoformulations as therapeutic delivery vehicles to achieve efficacy in ovarian carcinoma models was explored by attaching the radioiodinated CTT peptide tracer, 125I-CTT2 peptide, to these platforms and loading them with doxorubicin, an inherently fluorescent chemotherapeutic agent. Biodistribution studies of both targeted nanoformulations were performed in normal and immunosuppressed subcutaneous human xenograft models using the CTT2-peptide.