Elsevier

Materialia

Volume 8, December 2019, 100516
Materialia

Full Length Article
Synergistic assembly of peptide amphiphiles with varying polarities for encapsulation of camptothecin

https://doi.org/10.1016/j.mtla.2019.100516Get rights and content

Abstract

Peptide amphiphiles (PA) are a group of peptide-based biomaterial design inspired by lipid molecules in the cell membrane. They possess distinct hydrophobic and hydrophilic regions which can self-assemble into highly ordered nanostructures based on the peptide sequence. In this work, the design strategy employed, explores the innate ability of PA to serve as a depository for hydrophobic drug camptothecin (CPT). CPT was encapsulated into two oppositely charged PA by hydrophobic attraction which develops into hybrid nanofibers by the interplay of electrostatic interactions and hydrogen bonding. This method of encapsulation averts the use of physical and chemicals factors like pH, temperature and ions to enable the self-assembly process. The CPT encapsulated PA nanofiber (PA-CPT) structure exhibits a high encapsulation efficiency of 84.83 ± 4.12%. After encapsulation, it was observed that the secondary structure of PA and active lactone conformation of CPT were perfectly retained. In vitro release studies reveal that 40% of the drug was released in the first 12 h. PA-CPT was effectively taken up by HepG2 cells with a lower IC50 value of 0.56 µM/L compared to free CPT having an IC50 of 3.75 µM/L. The developed PA-drug system enhances the bioavailability of CPT in an aqueous environment.

Introduction

Nature has served as an inspiration for the design of devices, systems and materials over a long period of time [1]. Biomimetics is a multidisciplinary field which utilizes the concepts of biology, materials chemistry and engineering for emulating biological systems [2]. Recently, advances in molecular biology and nanotechnology have led to a hybrid methodology which integrates naturally available molecular tools and synthetically developed nanostructures to form novel engineering systems having unique functional characteristics [3]. The genesis of such biologically inspired technologies has opened new avenues in the field of regenerative medicine, tissue engineering and drug delivery [4], [5], [6].

Inspired by nature, peptide amphiphiles (PA) are a versatile group of materials containing hydrophobic and hydrophilic segments mimicking the lipid molecules in the cell membrane [7]. The ability of the PA to self-assemble is capitalised to form supramolecular nanostructures by the subtle interplay of non-covalent forces like hydrophobic interaction, electrostatic interaction and hydrogen bonding [8]. Molecular design dictates the equilibrium between these forces to form ordered structures with a high degree of atomic precision, size and hierarchy [9]. These non-covalent forces permit the assembly and disassembly of nanostructures with ease in comparison to tedious covalent synthesis procedures [10].

Around 40% of the new chemical entities (NCE) developed in pharmaceutical industries are lipophilic in nature [11]. The key determinant of drug bioavailability is solubility and is one of the most challenging aspects of drug development. Drugs that are poorly soluble in aqueous solutions are dispensed at levels higher than the desired dose resulting in toxicity. Alternative strategies are being employed to improve the drug solubility and release rate [12]. Recently, PA with hydrophobic and hydrophilic structural motifs has attracted considerable attention to serve as a vehicle to transport hydrophobic drugs due to their propensity to form nanostructures of tunable size, shape and specificity [7], [13], [14]. By means of molecular design strategies, PA can be tuned to form high aspect ratio nanofibers from micelles [15]. Nanostructures with high aspect-ratio have been reported to exhibit enhanced targeting efficiency, longer circulation time and broader distribution [16], [17], [18]. Previously, work has been done on the encapsulation of hydrophobic drugs to PA molecules at high encapsulation efficiency and facilitate stimuli-responsive drug release [19], [20].

In this work, CPT was chosen as the model hydrophobic drug for encapsulation. Currently, its analogues are being used in cancer chemotherapy for the treatment of lung, prostate and colon cancer. These analogues impose certain clinical constraints as they undergo inactivation of their lactone form in the blood and need prolonged infusions to thwart reversal of DNA cleavage [21]. Nanoparticles and diblock copolymers have been previously used as vehicles to encapsulate CPT but they are associated with longer preparation times due to dialysis [22], [23]. Lately, liposomes are also being used for CPT encapsulation, but they suffer from lower encapsulation efficiency [24]. These shortcomings can be overcome by incorporation of CPT in the hydrophobic core of PA. Of late, studies have been done on the encapsulation of CPT in PA at high encapsulation efficiency [25]. Codelivery of CPT and paclitaxel to the target site was enabled utilizing PA to bring about a synergistic effect [26]. The release of CPT from PA has been modulated by linker groups for an effective drug release efficiency [27]. Targeted release of CPT at the overexpressed CD44 cancer cells was possible by HA incorporated PA-CPT nanostructures [28]. Thus, several strategies using a single tail PA has been explored to enhance the drug delivery capabilities of PA. In addition to the encapsulation efficiency of the drug and its desired effects, it is necessary to investigate if the functionality of the encapsulating material is conserved before and after encapsulation.

Herein, we propose a method for CPT encapsulation by the collective efforts of coassembly and solvent evaporation method utilising two single tail PA. In literature, several cases of coassembly have been reported to form hybrid nanofibers [29], [30], [31]. This technique was hypothesised to harbour hydrophobic drugs and provide enhanced functionality due to the duality of the hybrid nanofibers. The present work mainly focusses on validating the encapsulation of CPT using PA with varying polarities. To our knowledge, there was no prior work which employs PA of opposing charges for hydrophobic drug encapsulation. Two oppositely charged PA sequences enable CPT encapsulation by hydrophobic attraction. The charges on the PA are mutually screened by electrostatic interaction. Further, hydrogen bonding along the fiber axis serves as the third driving force to form CPT encapsulated hybrid nanofibers. The use of external factors like pH, temperature and ions for self-assembly was negated by this method. This system offers the advantage of preserving the nature of the drug and peptide during the encapsulation process. Besides, the influence of varying amounts of PA on secondary structure formation in hybrid nanofiber was also explored. Further, the antitumor activity of the CPT encapsulated peptide nanofibers was validated by performing in vitro studies using HepG2 cells.

Section snippets

Materials

PA sequences were procured from Biopeptek Pharmaceuticals, LLC (Malvern, PA, USA) and stored at −20 °C. They were protected by a palmitoyl and amino group at the N and C terminal respectively. The sequences are C16-AAAAAEEEEGGK (E-PA) and C16-VVVAAAKKKGGK (K-PA) with molecular weights of 1369.10 and 1394.05 respectively and a purity of 95%. Fmoc solid phase peptide synthesis method was employed for the synthesis of the peptides [32]. The purity and molecular weight of the synthesized peptides

Design and synthesis of peptide amphiphiles

PA sequences with varying amino acid residues in the β sheet region and charged region were synthesized by solid state peptide synthesis followed by alkylation with C16 palmitoyl group from Biopeptek Pharmaceuticals, LLC (Malvern, PA, USA) [35]. The molecular weight of the peptides was characterized by electrospray ionization mass spectrophotometer (ESI MS) and was found to be consistent with the desired calculated value. Further, HPLC was used to analyze the purity of the synthesized PA and

Discussion

In this work, E-PA and K-PA sequences were designed to facilitate the hydrophobic drug encapsulation and nanofiber formation majorly by hydrophobic interaction and electrostatic interaction respectively [31], [50]. According to the Kyte Doolittle hydropathy index, valine is more hydrophobic than alanine and thus K-PA will be more hydrophobic than the E-PA and allows greater encapsulation of CPT [51]. The oppositely charged amino acids in the charged region of E-PA and K-PA brings about the

Conclusion

A strategy for encapsulation of hydrophobic drug CPT at high encapsulation efficiency by the synergistic assembly of two oppositely charged PA sequences was developed in this work. The incorporation of CPT in the hydrophobic core of PA was by the collaborative efforts of hydrophobic attraction, electrostatic interaction and hydrogen bonding. This method improved the solubility of CPT in aqueous solutions by 60-fold. CD spectroscopy and FTIR studies reveal that PA-CPT nanofibers retain their

Author contributions

S.M. performed the research and analysed the data. Y.W.T. supervised the research. S.M. wrote the original manuscript and Y.W.T. helped to revise it.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Ms. Steffie Mano would like to thank the Department of Chemical and Biomolecular Engineering at National University of Singapore for her scholarship. Additionally, she is thankful to Chua Khong Jui, Hui Xian Gan, Yan Shan Ang, Pon Janani Sugumaran and You Kang Lim for the help provided during this work.

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