Nanoscale. 2015 Aug 14;7(30):12943-54.
Novel stable dendrimersome formulation for safe bioimaging applications.
Filippi M1, Patrucco D1, Martinelli J2, Botta M2, Castro-Hartmann P3, Tei L2, Terreno E1.
1Dipartimento di Biotecnologie Molecolari e Scienze della Salute, Centro di Imaging Molecolare e Preclinico, Università degli Studi di Torino, Via Nizza 52, Torino, 10126, Italia.*Corresponding author: E-mail: email@example.com, Telephone: +39 011 6706452, Fax: +39 011 6706487.
2Dipartimento di Scienze ed Innovazione Tecnologica, Università del Piemonte Orientale “Amedeo Avogadro”, Viale T. Michel 11, Alessandria, 15121, Italia.*Corresponding author: E-mail: firstname.lastname@example.org, Telephone: +39 0131 360 208, Fax: +39 0131 360250.
3Servei de Microscòpia. Universitat Autònoma de Barcelona, 08193, Cerdanyola del Vallès, Spain.
Dendrimersomes are nanosized vesicles constituted by amphiphilic Janus dendrimers (JDs), which have been recently proposed as innovative nanocarriers for biomedical applications. Recently, we have demonstrated that dendrimersomes self-assembled from JDG1G2(3,5) dendrimers (Figure 1) can be successfully loaded with hydrophilic and amphiphilic imaging contrast agents (1). Here, we present two newly synthesized low generation isomeric JDs: JDG0G1(3,5) and JDG0G1(3,4). Though less branched than the above-cited dendrimers, they retain the ability to form self-assembled, almost monodisperse vesicular nanoparticles. This contribution reports on the characterization of such nanovesicles loaded with the clinically approved MRI probe Gadoteridol and the comparison with the related nanoparticles assembled from more branched dendrimers. Special emphasis was given to the in vitro stability test of the systems in biologically relevant media, complemented by preliminary in vivo data about blood circulation lifetime collected from healthy mice. The results point to very promising safety and stability profiles of the nanovesicles, in particular for those made of JDG0G1(3,5), whose spontaneous self-organization in water gives rise to a homogeneous suspension. Importantly, the blood lifetimes of these systems are comparable to those of standard liposomes. By virtue of the reported results, the herein presented nanovesicles augur well for future use in a variety of biomedical applications.
PMID: 26167654 DOI: 10.1039/c5nr02695d
A growing interest has been recently dedicated to the investigation of natural membranes in order to elucidate their properties, reproduce their architectures, and possibly exploit them for several biomedical applications. Indeed, the capability of several natural or novel synthetic amphiphiles to self-assemble into organized structures in aqueous environments became a domain of great impact. Liposomes, the best-known example of artificial nanosized vesicles based on phospholipid bilayer, are already successfully used for a number of biomedical purposes: from model systems for the study of biological membranes to efficient vectoring carriers for the delivery of bioactive substances or other molecules to pathological sites (2). Besides liposomes, other nanoparticles have been investigated aiming at optimizing certain specific membrane physico-chemical characteristics (e.g. mechanical resistance, wall thickness, chemical groups exposed on the outer surface). Two examples are represented by polymersomes (3) and dendrimersomes (4), which are obtained through the self-assembly of amphiphilic block co-polymers or Janus dendrimers (JDs), respectively. Intriguingly, the dendrimersomes share a characteristic vesicular shape with the liposomes, with an internal aqueous core surrounded by a double-layered membrane. Whereas the potential of liposomes and polymersomes in biomedical studies has been already demonstrated, dendrimersomes have been proposed only very recently, and therefore their behaviour in the biological environments is largely unexplored. A relevant exception are glycodendrimersomes, which were primarily proposed as nanotools to investigate specific protein functionalities (e.g. lectins), as well as the binding to biomolecules from plants and bacteria (5).
Our interest in using liposomes or other nanosized systems is mainly for molecular imaging applications as carriers for Magnetic Resonance Imaging (MRI) contrast agents, drugs or other imaging probes. Indeed, the stealth liposomes have been extensively used as drug delivery systems with convenient properties such as biodegradability, biocompatibility and non-toxicity for molecular imaging applications, as they can be combined with specific targeting ligands and probes, and reach the disease tissue with high specificity. Based on this consideration, we argued that dendrimersomes could represent a relevant alternative to liposomes, by virtue of the lower cost of the starting materials. Furthermore, reporting in a previous work that dendrimersomes assembled from JDG1G2(3,5) Janus dendrimer (Figure 1) could be loaded with contrast agents (CAs) for both optical imaging and MRI (1), we also discovered that, due to the high water permeability of the dendrimeric membrane, the longitudinal relaxivity (r1, i.e. a measure of the ability to generate the MRI contrast) of the paramagnetic agents encapsulated in the inner aqueous core is not severely limited, suggesting a diagnostic/theranostic potential even higher than liposomes endowed with a standard membrane formulation.
Figure 1. Chemical structures of the 3,5 bisfunctionalized Janus dendrimers investigated in this work: the already reported JDG1G2(3,5) (top) and the novel JDG0G1(3,5) (bottom) dendrimer self-assemble into double-layered membranes, thus originating nano-vesicles that can encapsulate the clinically approved MRI contrast agent Gadoteridol.
Because in a concrete scenario a novel biocompatible, stable, and effective nanosystem is likely to be systemically administered in living organisms, the investigation of the dendrimersomes interaction with the main components present in blood and cells is of fundamental relevance before their involvement in pre-clinical research. We believed that this study can be efficiently carried out in vitro by incubation assays where nanoparticles are put into contact with proteins and cells at appropriate temperature, pH, and environmental conditions. The results can give helpful indications to optimize the further in vivo investigation. In particular, our preliminary tests allowed the selection of certain types of dendrimersomes as the most stable and biocompatible, before they became the subject of more complex experiments in animal models.
Encouraged by our preliminary data (1), we extended our research to other forms of dendrimeric constituents and we synthesized two novel low-generation isomeric JDs (coded JDG0G1(3,4) and JDG0G1(3,5)) bearing only one hydrophobic and one hydrophilic dendron linked together by an ethylene glycol residue (Figure 1).
We assessed the ability of such dendrimers to form nanovesicles and to entrap the Gadoteridol (Gd-HPDO3A, ProHance®, Figure 1), and then compared with the results obtained with the analogue higher generation dendrimers. The dendrimersomes herein reported were prepared by thin film hydration method with the addition of 5% of a negatively charged pegylated phospholipid (DSPE-PEG2000-COOH) to increase stability by steric/electrostatic repulsion and to prolong the blood residence lifetime by pegylation. Briefly, a thin homogeneous film of the amphiphilic components was formed by slow evaporation of a chloroform solution and then the film was hydrated with a buffered solution (with physiological pH and osmolarity) containing highly concentrated Gadoteridol (0.25 M). High temperature (50°C) and vigorous shaking detached the film from the glass surface, letting the amphiphilic material organize into a raw suspension of multilamellar vesicular structures with different dimension. To obtain dimensional homogeneity and unilamellarity, the samples were forced to pass through several extrusion polycarbonate filters with pore diameters decreasing from 1 μm to 200 nm, producing vesicular suspensions with low Polydispersity Index (PDI) values, and a mean hydrodynamic diameter ranging from 105 to 195 nm according to the molecular shape of the dendrimeric components, as measured by Dynamic Light Scattering and Cryo-Transmission Electron Microscopy (Figure 2A). Finally, exhaustive dialysis purified samples from the non-encapsulated Gadoteridol.
Figure 2. Cryo-TEM images of JDG0G1(3,5)-based and JDG1G2(3,5)-based dendrimersomes at 40000X magnification, showing unilamellar vesicular architectures with different diameters for the two dendrimeric constituents (A). In vitro stability and payload release from different dendrimersomes in presence of human serum: residual Gd3+ concentration in the dendrimersome suspension before and after repeated dialysis (B). In vitro stability of (3,5)JD-based dendrimersomes: residual Gd3+ concentration (C) in the sample after exposition to the reconstituted human serum at 37°C for variable time ranges, and changes in the suspension dimensional homogeneity, assessed by percentage variation of the Polidispersity Index (PDI) as a function of incubation times with Human Serum Albumin (D).
In the presence of Human Serum (HS) or Human Serum Albumin (HSA) at 37°C (i.e. physico-chemical conditions mimicking the biological environments), the vesicles based on the novel dendrimer JDG0G1(3,5) displayed the greatest ability to retain the transported material, and to maintain the structural integrity and the low dimensional polydispersity of the suspension (Figure 2B, C and D). We argued that there is a direct correlation between the molecular structure of the dendrimer and the morphology and stability of the supramolecular assemblies. Indeed, the low generation dendrimers with the hydrophobic chains substituting the aromatic ring in position 3,5 produce larger dendrimersomes with high interdigitation patterns and thin walls, that are generally described as tougher and more stable (4). Intriguingly, the vesicles based on the novel 3,5 dendrimer retain the content and the structural integrity more efficiently also than those obtained from the higher generation analogue (Figure 2B, C and D). Nevertheless, even if at a moderate extent, a release of the payload occurred as stimulated by the interaction of the particles with plasma proteins, and correlated also to a modest increase of the nanosystem suspension polydispersity (Figure 2D). This phenomenon, typically presenting a double-phased kinetic, is well-known to characterize also other conventional vesicular nanoplatforms, such as liposomes (6). However, after a first “burst” release of the content (about 25% after 1 h), our dendrimersomes displayed an excellent stability, and the amount of encapsulated Gadoteridol did not change within 2 days of incubation in HS at 37°C. This observation was further proved by the more sophisticated experimentation carried out in living animals to estimate the blood circulation lifetime of dendrimersomes, being this one of the most important properties to determine the in vivo potential of a nanoparticle. The plasma residence was estimated by injecting vesicles intravenously in healthy mice and then quantifying the residual fraction of Gd3+metal ion over the time by ICP-MS (Figure 3). Interestingly, pegylated dendrimersomes remain in blood vessels and circulate for time periods comparable to those of conventional liposomes with similar size (half-life ≈ 90-120 min). The amount of Gd3+ found in the blood five minutes post-injection was ca. 25-30% lower than the total amount injected, suggesting that the mechanisms responsible for the “burst” release of Gadoteridol required a much shorter timescale in vivo.
Finally, the novel vesicles displayed a very high biocompatibility, such that no negative effects on cell viability and proliferation rate were observed on fibroblasts and on two populations of murine macrophages (Figure 3). Thus, this finding augurs well for the use of dendrimersomes in biomedicine, being also supported by the lack of acute episodes of toxicity and other side effects on general health conditions of animals during in vivo experimentation.
Figure 3. Left: blood circulation lifetime. Kinetics of gadolinium concentration in blood normalized to the injected dose ([Gd]t/[Gd]0) in healthy BALB/c mice after systemic injection of Gadoteridol (yellow circles, error bars in black), JDG0G1(3,5) dendrimersomes (Gd-DS, blue squares, error bars in blue) and control liposomes (Gd-Lipo, orange squares, error bars in orange) both loaded with Gadoteridol. All animals received equivalent Gadoteridol doses (0.16 mmol/kg bw). Right: cell viability estimated by Trypan Blue assay on RAW 264.7 macrophages after incubation along different incubation times with PBS or JDG0G1(3,5)-based vesicles at high concentration (10 mg/ml), encapsulating Gadoteridol or not (Gd-DS and DS respectively).
The relevance of the present study stems from the fact that even if detailed studies about the structural variability of dendrimersomes have already been reported (4), these did not consider specifically and thoroughly the role of biological variables. Thus, it was demonstrated that the novel JDs with 3,5bis(dodecyloxy)benzene hydrophobic groups produce vesicles with advantageous properties in terms of stability and content retention. Overall, these promising results suggest that JDG0G1(3,5)-based dendrimersomes are good candidates for the development of effective nanosystems for medical bioimaging. As a consequence, future work will be directed to test the diagnostic/therapeutic/theranostic potential of this new class of soft nanoparticles at the preclinical level. Indeed, our research team already reported their first in vivo use for MRI tumour diagnosis (7) after the incorporation into the dendrimeric membrane of an innovative, highly efficient, amphiphilic Gd-complex with several favourable properties and improved imaging potential, which legitimize these new nanoobjects as reliable alternative to liposomes in the advanced diagnostic protocols.
Notes and references
- M. Filippi, J. Martinelli, G. Mulas, M. Ferraretto, E. Teirlinck, M. Botta, L. Tei and E. Terreno, Chem. Commun., 2014, 50, 3453.
- E. Sezgin and P. Schwille, Mol. Membr. Biol., 2012, 29, 144.
- D.E. Discher and A. Eisenberg, Science, 2002, 297, 967.
- V. Percec, D. A. Wilson, P. Leowanawat, C. J. Wilson, A. D. Hughes, M. S. Kaucher, D. A. Hammer, D. H. Levine, A. J. Kim, F. S. Bates, K. P. Davis KP, T. P. Lodge, M. L. Klein, R. H. DeVane, E. Aqad, B. M. Rosen, A. O. Argintaru, M. J. Sienkowska, K. Rissanen, S. Nummelin and J. Ropponen, Science, 2010, 328, 1009.
- V. Percec, P. Leowanawat, H. J. Sun, O. Kulikov, C. D. Nusbaum, T. M. Tran, A. Bertin, D. A. Wilson, M. Peterca, S. Zhang, N. P. Kamat, K. Vargo, D. Moock, E. D. Johnston, D. A. Hammer, D. J. Pochan, Y. Chen, Y. M. Chabre, T. C. Shiao, M. Bergeron-Brlek, S. André, R. Roy, H. J. Gabius and P. A. Heiney, J. Am. Chem. Soc., 2013, 135, 9055.
- M. H. Gaber, K. Hong, S. K. Huang, D. Papahadjopoulos, Pharm. Res., 1995, 12, 1407.
- M. Filippi, D. Remotti, M. Botta, E. Terreno, and L. Tei., Chem Commun., 2015, 51, 17455.