The novel calix[4]resorcinarene-PEG conjugate: Synthesis, self-association and encapsulation properties

https://doi.org/10.1016/j.colsurfa.2019.03.021Get rights and content

Abstract

The synthesis and study of self-association and encapsulation ability of new low toxic conjugate of tetraphenyleneoxypentylcalix[4]resorcinarene and methoxy-poly(ethylene glycole) (C5OPh-mPEG) were described. The conjugate is characterized by 1H, 13C NMR, FT-IR, MS, SLS methods. In an aqueous solution the conjugate form multimicellar nanoparticles with low polydispersity value (0.1, DLS method). The self-association of the conjugate was studied by DLS, fluorescence, fluorescence anisotropy method, TEM, and SAXS methods. The SAXS data were detailed analyzed, and the conjugate associates structure as ellipsoidal core-shell particles was proposed. The encapsulation efficiency is a result of the affinity of the conjugate low rim substituents to substrates and reaches of 39.8, 26.7, and 28.9% toward naproxen, ibuprofen and doxorubicin drugs, respectively.

Introduction

The drug delivery systems should supply a number of requirements: low toxicity, optimal size and monodispersity, high solubility and biocompatibility, good biodistribution, еnhanced targeting via multivalent binding of drug. The polymer conjugates offer a powerful opportunity to develop new drug delivery systems due to their size scale, combination of the qualities mentioned above, and the possibilities for non-covalent or covalent decoration by drugs, targeting ligands, and imaging agents [1]. One of the methods to construct the branched or dendritic polymers, providing nanoscopic spherical structure and good biocompatibility, is the conjugation of synthetic macrocycle and polymer by convergent synthesis or by attachment of polymer fragment to the macrocyclic framework. Most examples of macrocycle-polymer conjugates include the cyclodextrines-polymer compounds due to their natural origin and low toxicity [2].

On the other hand, the conjugation of macrocyclic molecules with biocompatible polymers fragments is aimed to create a polymer conjugate with the topology of macrocycle, which has the additional binding sites. Polymeric groups of such conjugate provide its high solubility and biocompatibility. Thus, the low toxic poly(ε-caprolactone)-block-poly(ethylene glycol) – calix[4]resorcinarene conjugate [3], calix[4]arene - poly(ethylene glycol) conjugate [4], pillararenes conjugates with poly(ethylene glycol), poly(N-iso-propylacrylamide), poly(glutamamide), and poly(caprolactone) [5] were successfully synthesized for potential using as drug delivery and theranostic materials.

Among all macrocycles calixresorcinarenes attract much attention as the framework and building blocks for drug delivery system formation due to their capabilities of multidecoration by functional groups and as a consequence multiple noncovalent interactions of macrocycle molecules with each other and with encapsulated substrates.

Herewith the choose of calixresorcinarene in cone or boat rccc configuration provides the possibility to intro of various (hydrophilic and hydrophobic) groups on the different (lower and upper) rims of macrocycle cavity. Such structure imparts the amphiphilicity to macrocycle molecules due to the clear separation of their hydrophilic and hydrophobic zones and ensures their usage in the formation of supramolecular nanocontainers.

Opportunities and achievements in the use of amphiphilic calixresorcinarenes as supramolecular nanocontainers are reported in some reviews [6,7]. As a rule, the formation of mixed associates of amphiphilic calixresorcinarene with substrates leads to the high encapsulation efficiency values (EE) in the condition of their complementarity. For example, the effectiveness of loading of therapeutic carboxy-acids (naproxen, ibuprofen, ursodeoxycholic acid) in aminocalixresorcinarenes associates reached on 45%, that is explained by the deprotonation of carboxy-acids as a results of interaction with amino-groups of macrocycles and by the binding of substrates due to CH-π, π-π, hydrophobic effect and electrostatic interactions [8]. But high toxicity of aminocalixresorcinarenes is proved to be an obstacle to their further use for the potential drugs delivery application. Therefore we focused on the synthesis of the calixresorcinarene conjugates with fragments of biocompatible polymer. Previously, we have synthesized tetraundecylcalixresorcinarene-methoxypolyethylene (mPEG) conjugate, which has a low hemolytic activity [9]. With the help of DLS method, it was shown the bimodal particles size distribution in the aqueous solutions of the conjugate with an intensity-averaged hydrodynamic diameters of particles of 9–18 and 59–531 nm and PDI (ratio of the mean deviation squared to the mean diameter squared) values of 0.365–0.654. It was suggested that particles with a smaller diameter are the conjugate micelles, and with a larger diameter - their multimicellar aggregates which are formed by hydrophobic interaction between ethylene groups of conjugate molecules.

Usually, for the achievement of the optimal clinical results nanocontainers should have a constant and narrow size distribution [10]. Particle size affects the rate of drug release, and a narrow particle size distribution ensures that the drug substrate delivered by the particles will affect the body in a single manner [11]. Therefore, the development of new conjugates as well as the investigation of their morphology is an intriguing task.

Here we report the synthesis and study of toxicity, self-association and encapsulation properties of the conjugate of mPEG and tetra-p-phenyleneoxypenthylcalix[4]resorcinarene. This calix[4]resorcinarene has the small alkyl chains on the lower rim, attached to the macrocycle platform via phenyleneoxy fragments. Earlier it was shown that the calix[4]resorcinarenes, bearing p-phenyleneoxyalkyl groups on the lower rim, can be incorporated into lipid bilayer and form ion channels for K+ ions by forming the dimeric structures. It was concluded that the additional rigidity of the macrocycle framework, which is provided by phenyleneoxy fragments of the molecules lower-rim substitutents, promotes the formation of such channels [12]. We proposed that the introduction of phenyleneoxy fragments on the lower rim of macrocyclic platform will provide the additional rigidity of the conjugate structures as well as the additional binding site of the conjugate, which will reflect on the morphology of the conjugate particles and their size distribution and also their interaction with substrates. So, the conjugate self-association data with the help of DLS, fluorescence pyrene probe and anisotropy probe methods, and TEM were collected, as well as detailed analysis of SAXS method to assess the conjugate association model was performed. By the study of interaction with some hydrophobic substrates it was suggested the binding sites of the conjugate self-associates.

Section snippets

Materials

Poly(ethylene glycol)methyl ether (average Mn 550, HO-(CH2CH2O)n-CH3, n = 8÷20, Aldrich), Naproxen (Sigma-Aldrich, purity ≥98%), Ibuprofen (Sigma-Aldrich, purity ≥ 98%), Orange OT (Sigma-Aldrich, dye content 75%), Pyrene (Sigma-Aldrich, purity ≥ 99%), 1,6-diphenyl-l,3,5-hexatriene (DPH, Sigma, purity 98%), and Quercetin (Sigma, purity ≥95%) were used as received. Doxorubicin hydrochloride (Sigma-Aldrich, purity 98.0–102.0 %) was hydrophobized by the procedure reported in ref. [13]. Tosylated

The synthesis, characterization and hemotoxicity of the tetra-p-phenyleneoxy-penthyl-calixresorcinarene-mPEG conjugate C5OPh-mPEG

For the synthesis of the amphiphilic conjugate C5OPh-mPEG the rccc conformer of tetra-p-phenyleneoxy-penthyl-calix[4]resorcinarene C5OPh-OH was taken (Fig. 1). Rccc isomer of any calix[4]resorcinarenes is pre-organized to obtain of amphiphilic derivatives due to the clear separation of hydrophobic and hydrophilic part in the molecule. If rctt isomer of the calix[4]resorcinarene is characterized by doubling of signals of aromatic platform protons in the 1H NMR spectrum, the rccc isomer has more

Conclusion

In total, the synthesis and characterization of new conjugate of tetraphenyleneoxypentylcalix[4]resorcinarene and methoxy-poly(ethylene glycole) are described. The conjugate is demonstrated low hemotoxicity. The bulky hydrophilic mPEG fragments and short hydrophobic fragments of the conjugate molecules lead to their association into core-shell ellipsoidal micelles formation in an aqueous solution. It is found, that the conjugate micelles spontaneous aggregate in small multimicellar associates

Acknowledgements

This work was funded by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities (4.1493.2017/4.6 and 4.5151.2017/6.7). The authors are grateful to the staff of CSF-SAC FRC KSC RAS for their research and assistance in discussing the results.

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