Anna Morisco

Peptide-modified liposomes for selective targeting of bombesin receptors overexpressed by cancer cells: a potential theranostic agent

Objectives Drug delivery systems consisting of liposomes displaying a cell surface receptor-targeting peptide are being developed to specifically deliver chemotherapeutic drugs to tumors overexpressing a target receptor. This study addresses novel liposome composition approaches to specifically target tissues overexpressing bombesin (BN) receptors. Methods A new amphiphilic peptide derivative (MonY-BN) containing the BN(7–14) peptide, the DTPA (diethylenetriaminepentaacetate) chelating agent, a hydrophobic moiety with two C18 alkyl chains, and polyethylene glycol spacers, has been synthesized by solid-phase methods. Liposomes have been generated by co-aggregation of MonY-BN with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The structural and biological properties of these new target-selective drug-delivery systems have been characterized. Results Liposomes with a DSPC/MonY-BN (97/3 molar ratio) composition showed a diameter of 145.5 ± 31.5 nm and a polydispersity index of 0.20 ± 0.05. High doxorubicin (Dox) loading was obtained with the remote pH gradient method using citrate as the inner buffer. Specific binding to PC-3 cells of DSPC/MonY-BN liposomes was obtained (2.7% ± 0.3%, at 37°C), compared with peptide-free DSPC liposomes (1.4% ± 0.2% at 37°C). Incubation of cells with DSPC/ MonY-BN/Dox showed significantly lower cell survival compared with DSPC/Dox-treated cells, in the presence of 100 ng/mL and 300 ng/mL drug amounts, in cytotoxicity experiments. Intravenous treatment of PC-3 xenograft-bearing mice with DSPC/MonY-BN/Dox at 10 mg/kg Dox dose produced higher tumour growth inhibition (60%) compared with nonspecific DSPC/ Dox liposomes (36%) relative to control animals. Conclusion The structural and loading properties of DSPC/MonY-BN liposomes along with the observed in-vitro and in-vivo activity are encouraging for further development of this approach for target-specific cancer chemotherapy.

Micelles derivatized with octreotide as potential target-selective contrast agents in MRI†‡

New amphiphilic monomers (OCA‐DTPAGlu and OCA‐DOTA) containing, in the same molecule, three different functions: (i) the chelating agent (DTPAGlu or DOTA) able to coordinate gadolinium ion, (ii) the octreotide bioactive peptide able to target somatostatin receptors, and (iii) a hydrophobic moiety with two 18‐carbon atoms alkyl chains have been designed and synthesized by solid‐phase methods. The novel amphiphilic monomers aggregate, in water solution, giving stable micelles at very low concentration (cmc values of 2.3 × 10−6 mol kg−1 and 2.5 × 10−6 mol kg−1 for OCA‐DTPAGlu and OCA‐DOTA, respectively) as confirmed by fluorescence spectroscopy. Fluorescence studies and circular dichroism experiments indicate, for the two compounds as well as for their gadolinium complexes (OCA‐DOTA(Gd) and OCA‐DTPAGlu(Gd)), the complete exposure of octreotide on the micelle surface, and the predominant presence of an antiparallel β‐sheet peptide conformation characterized by a β‐like turn. The high relaxivity value (r1p = 13.9 mM−1 s−1 at 20 MHz and 25 °C), measured for micelles obtained by the gadolinium complex OCA‐DTPAGlu(Gd), indicates these aggregates as promising target‐selective magnetic resonance imaging (MRI) contrast agents. Copyright

Peptide modified nanocarriers for selective targeting of bombesin receptors

The present work describes new supramolecular aggregates obtained by co-assembling two different amphiphilic molecules, one containing the bioactive bombesin peptide (BN), or a scramble sequence, and the other, the DOTA chelating agent, (C18)(2)DOTA, capable of forming stable complexes with the radioactive (111)In(III) isotope. The peptide in the amphiphilic monomer is spaced by the lipophilic moiety through ethoxylic spacers of different length: a shorter spacer with five units of dioxoethylene moieties in (C18)(2)L5-peptide, or a longer spacer consisting of a Peg3000 residue in (C18)(2)Peg3000-peptide. Structural characterization by SANS and DLS techniques indicates that, independently from the presence of the peptide containing monomer in the final composition, the predominant aggregates are liposomes of similar shape and size with a hydrodynamic radius R(h) around 200 nm and bilayer thickness, d, of 4 nm. In vitro data show specific binding of the (111)In-(C18)(2)DOTA/(C18)(2)L5-[7-14]BN 90:10 liposomes in receptor expressing cells. However, the presence of the Peg3000 unit on the external liposomal surface, could hide the peptide and prevent the receptor binding. In vivo experiments using (111)In-(C18)(2)DOTA/(C18)(2)L5-[7-14]BN show the expected biological behavior of aggregates of such size and molecular composition, moreover there is an increase in concentration of the GRPR targeting aggregate in the tumors compared to control at the 48 h time point evaluated (2.4% ID/g versus 1.6% ID/g).

Nanoparticles containing octreotide peptides and gadolinium complexes for MRI applications

New mixed nanoparticles were obtained by self‐aggregation of two amphiplic monomers. The first monomer (C18)2L5‐Oct contains two C18 hydrophobic moieties bound to the N‐terminus of the cyclic peptide octreotide, and spaced from the bioactive peptide by five units of dioxoethylene linkers. The second monomer, (C18)2DTPAGlu, (C18)2DTPA or (C18)2DOTA, and the corresponding Gd(III) complexes, contains two C18 hydrophobic moieties bound through a lysine residue to different polyamino‐polycarboxy ligands: DTPAGlu, DTPA or DOTA. Mixed aggregates have been obtained and structurally characterized by small angle neutron scattering (SANS) techniques and for their relaxometric behavior. According to a decrease of negative charges in the surfactant head‐group, a total or a partial micelle‐to‐vesicle transition is observed by passing from (C18)2DTPAGlu to (C18)2DOTA. The thicknesses of the bilayers are substantially constant, around 50 Å, in the analyzed systems. Moreover, the mixed aggregates, in which a small amount of amphiphilic octreotide monomer (C18)2L5‐Oct (10% mol/mol) was inserted, do not differ significantly from the respective self‐assembled systems. Fluorescence emission of tryptophan residue at 340 nm indicates low mobility of water molecules at the peptide surface. The proton relaxivity of mixed aggregates based on (C18)2DTPAGlu(Gd), (C18)2DTPA(Gd) and (C18)2DOTA(Gd) resulted to be 17.6, 15.2 and 10.0 mM−1 s−1 (at 20 MHz and 298K), respectively. The decrease in the relaxivity values can be ascribed to the increase in τM (81, 205 and 750 ns). The presence of amphiphilic octreotide monomer exposed on mixed aggregate surface gives the entire nanoparticles a potential binding selectivity toward somatostatin sstr2 receptor subtype, and these systems could act as MRI target‐specific contrast agent. Copyright

Bombesin peptide antagonist for target-selective delivery of liposomal doxorubicin on cancer cells

Purpose: This study addresses novel peptide modified liposomal doxorubicin to specifically target tissues overexpressing bombesin (BN) receptors. Methods: DOTA-(AEEA)2-peptides containing the [7–14]bombesin and the new BN-AA1 sequence have been synthesized to compare their binding properties and in serum stabilities. The amphiphilic peptide derivative (MonY-BN-AA1) containing BN-AA1, a hydrophobic moiety, polyethylenglycole (PEG), and diethylenetriaminepentaacetate (DTPA), has been synthesized. Liposomes have been obtained by mixing of MonY-BN-AA1 with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Results: Both 111In labeled peptide derivatives present nanomolar Kd to PC-3 cells. 177Lu labeled peptide DOTA-(AEEA)2-BN-AA1 is very stable (half-life 414.1 h), while DOTA-(AEEA)2-BN, shows a half-life of 15.5 h. In vivo studies on the therapeutic efficacy of DSPC/MonY-BN-AA1/Dox in comparison to DSPC/MonY-BN/Dox, were performed in PC-3 xenograft bearing mice. Both formulations showed similar tumor growth inhibition (TGI) compared to control animals treated with non-targeted DSPC/Dox liposomes or saline solution. For DSPC/MonY-BN-AA1/Dox the maximum effect was observed 19 days after treatment. Conclusions: DSPC/MonY-BN-AA1/Dox nanovectors confirm the ability to selectively target and provide therapeutic efficacy in mice. The lack of receptor activation and possible acute biological side effects provided by using the AA1 antagonist bombesin sequence should provide safe working conditions for further development of this class of drug delivery vehicles.

Peptide-labeled supramolecular aggregates as selective doxorubicin carriers for delivery to tumor cells.

New liposomal aggregates, prepared by combining together, in a 90:10 molar ratio, two amphiphilic monomers, one containing two hydrocarbon chains in the hydrophobic region and the anionic DOTA chelating agent as hydrophilic moiety, and the other containing the same hydrophobic moiety and the CCK8 peptide, are described. The liposomal aggregates because of the presence of the specific moiety, constituted by the CCK8 peptide, which selectively recognizes CCK receptors on tumor cells are used as drug carriers with the aim to deliver into tumor cells the appropriate antitumor drug. The drug loading content and the releasing properties of the liposomal aggregates are studied by the use of the cytotoxic doxorubicin as drug model. The doxorubicin loading content determination reveals that above 95% of the total drug was uptaken with a corresponding drug/lipid w/w ratio of 0.134. The cellular uptake of the targeted liposomal doxorubicin with respect to the self‐assembled, nonspecific, liposomal doxorubicin is evaluated using flow cytometry assays. The doxorubicin cell content for two types of cell systems, namely, A431 and HuVEC cells, for peptide derivatized liposomes was 70‐ and 8‐fold higher, respectively, than for nontargeted liposomes, indicating that the bioactive CCK8 peptide is able to enhance the doxorubicin uptake into the carcinoma cells in vitro. The cytotoxicity effect of liposomal doxorubicin on A431 cells has been assessed by MTT assays: in presence of drug amounts ranged between 250 and 1000 ng/ml, incubation with peptide derivatized liposomes showed significantly lower cell survival compared with nontargeted liposomes.

Role of Positron Emission Tomography/Computed Tomography

Colorectal cancer (CRC) incidence and mortality rates are extremely variable around the world. CRC is the second most commonly diagnosed cancer in both genders [1]. Many patients are cured with initial surgery for primary disease and postoperative chemotherapy. Nevertheless, recurrent locoregional or distant metastases occur in approximately 40% of patients with stage II and stage III CRC [2]. A significant proportion of CRC recurrences occur in a single location, such as pelvis, liver, or lung [3, 4]. Surgery may be curative in some patients with localized recurrent disease and has an impact on 5-year overall survival (OS), which is 27% among patients who undergo surgery vs. 6% in patients who do not [3, 4]. Therefore, accurate and early identification of recurrent and/or metastatic disease is a critical and challenging issue in terms of improving OS of CRC patients. The measurement of circulating carcinoembryonic antigen (CEA) is the most widely accepted test in clinical practice for screening for recurrent CRC. Additionally, periodic colonoscopy, ultrasound (US), and multi-detector computed tomography (MDCT) for localization of recurrent CRC in the early stages are often performed during follow-up. None of these imaging modalities is extremely accurate [5].