Macromolecular NanotechnologyNanostructures based on ammonium-terminated amphiphilic Janus dendrimers as camptothecin carriers with antiviral activity
Graphical abstract
Introduction
The unspecific cytotoxicity of efficient drugs is a delicate issue in the treatment of infectious diseases, since drugs must target viral proteins without affecting host cell functions. Less developed than anticancer drug delivery systems, nanocarriers are also being explored to implement antiviral treatments. These can be used to lower drug doses [1] and/or to solve problems related with the limited solubility and stability in water of efficient antiviral drugs [2], [3].
In this respect, it was recently discovered that Camptothecin (CPT), a well-known drug used in cancer therapy [4], also presents high activity against the hepatitis C virus (HCV). Indeed, CPT is an allosteric inhibitor of the NS3 protease, which belongs to the HCV, and limits the HCV replication[5]. However, three important drawbacks have to be overcome to employ this drug as an anti-HCV agent: (i) its toxicity has to be controlled; (ii) its water-solubility has to be increased and (iii) its stability at physiological pH requires improvement since camptothecin contains a lactone ring that is hydrolyzed at physiological pH, and this lowers its activity. To tackle these issues, the encapsulation of camptothecin within polymeric nanocarriers appears as a good strategy, which has been widely explored for the implementation of antitumoral drug delivery systems.[6], [7], [8], [9], [10], [11] Nevertheless, the possibility of using CPT to treat the infection by HCV leads to the need to find suitable carriers [12], [13].
In a recent work, we designed a hybrid dendritic-lineal-dendritic block copolymer based on 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) dendritic blocks, which showed significant encapsulation efficiency for CPT within nanosized micelles, reducing its cytotoxicity without altering its antiviral activity [12]. In general, dendrimers offer key properties that explain their widespread use in drug delivery [14], [15], [16], [17]. In particular, dendritic structures derived from bis-MPA are good candidates to construct useful drug nanocarriers, since they can be water soluble, biocompatible and biodegradable [14], [18], [19], [20], [21]. In recent years, dendrons [22], globular dendrimers [23], ionic dendrimers [24], hyperbranched polymers [25], hybrid dendritic-linear polymers [26] and Janus dendrimers [27], [28], [29] derived from bis-MPA have been employed to implement drug or gene nanocarriers.
In this work, we focus our interest on Janus dendrimers, which are formed by two dendritic blocks that offer different and specific terminal groups. This makes possible to combine distinct properties in a single molecule in order to provoke a synergistic or an antagonist effect [30], [31], [32]. Accordingly, amphiphilic Janus dendrimers that combine a lipophilic and a hydrophilic dendron have been synthesized. Depending on the generation of each dendron, a wide variety of amphiphilic Janus dendrimers can be prepared with different lipophilic content (Lc), different molecular weight (MW) and different moieties at the periphery [33]. The different polarities of the two blocks of the Janus dendrimer can trigger its spontaneous self-assembly in water. Indeed, the poor affinity of the lipophilic block for the external aqueous medium favors intermolecular lipophilic interactions, thus leading to the formation of lipophilic domains surrounded and stabilized by hydrophilic ones. A large variety of different structures can be obtained such as micelles, vesicles, and lamellar and sponge phases and these can be used as drug nanocarriers [34], [35].
Furthermore, the inclusion of terminal amino groups at the periphery of the bis-MPA dendritic structures can trigger new properties in the amphiphilic Janus dendrimers. Amino-terminated amphiphilic Janus dendrimers can be used (i) to increase the cellular internalization [36], (ii) to complex polyanions of interest, like DNA [37], (iii) to deliver drugs [38], and (iv) in resistive antibacterial therapy [39] amongst other applications. Recently, we described two ammonium-terminated bis-MPA amphiphilic Janus dendrimers, namely (gly)4[2G]-[2G](C17)4 and (gly)8[3G]-[2G](C17)4 (Fig. 1a), which formed nanoaggregates in water that could effectively encapsulate chloroquine, a hydrophilic antimalarial drug [28].
With the aim of developing new nanocarriers for camptothecin (CPT) to fight against the hepatitis C virus (HCV), we have explored the potential of a series of four ammonium-terminated bis-MPA amphiphilic Janus dendrimers, (gly)4[2G]-[1G](C17)2, (gly)4[2G]-[2G](C17)4, (gly)8[3G]-[1G](C17)2 and (gly)8[3G]-[2G](C17)4, (Fig. 1). The dendrimers have different lipophilic contents associated with the different sizes of the lipophilic and hydrophilic dendrons, and this allows an evaluation of their compatibility with the poorly-water soluble drug, CPT. The self-assembly in water and the morphology of the resulting dendrimer aggregates are related to the different chemical structures of the dendrimers. Likewise, the encapsulation of CPT and the activity of the resulting systems against the virus replication and biocompatibility are discussed in terms of the dendrimer structure. The resulting delivery systems lead to the improvement of the therapeutic index of free CPT.
Section snippets
Materials
Solvents were purchased from Scharlab, S.L. All reagents and camptothecin, were purchased from Sigma-Aldrich® or Acros® and were used without further purification. TBTA catalyst and the saturated solution of HCl in ethyl acetate were prepared in our laboratory. DMEM (Dulbecco’s modified Eagle’s medium, 4.5 g L–1 glucose), DPBS (Dulbecco’s phosphate-buffered saline), l-glutamine, 1x non-essential amino acids, Geneticin (G418), and Alamar Blue reagent were purchased from Gibco. Penicillin/41
Synthesis
The lipophilic and hydrophilic dendrons were synthesized by a succession of Steglich esterification reactions in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridinium p-toluenesulfonate (DPTS) to increase the generation. Each esterification step was followed by deprotection of the hydroxyl groups by hydrolysis of the ketal protecting groups under mild conditions [46]. This process is compatible with the presence of alkyne and azide groups, which are sensitive to
Conclusions
The present work shows the possibility of tuning the morphology of nanoaggregates in water, which can be used as effective drug nanocarriers for anti-HCV therapy. Ammonium-terminated and stearic acid-terminated bis-MPA dendrons were linked in CuAAC reactions to form a series of four amphiphilic Janus dendrimers with different molecular weights (Mw) and lipophilic contents (Lc). All of the dendrimers form self-assembled nanostructures in water and the morphology is mainly influenced by the size
Acknowledgements
This work was financially supported by the MINECO-FEDER funds (under the projects CTQ2015-70174-P, MAT2015-66208-C3-1-P and BFU2013-47064-P), Miguel Servet Program from the Instituto de la Salud Carlos III (CP07/00289, CPII13/00017), Fondo de Investigaciones Sanitarias (PI15/00663) and the Gobierno de Aragón-FSE (E04, B89 and B01 research groups). A.L. and R.C-G thank the MECD for their FPU grants (FPU12/05210 and FPU13/3870). R.C-G thanks the DGA for the EPIF grant B136/13. The authors would
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