Regular Article
Synthetic ionizable aminolipids induce a pH dependent inverse hexagonal to bicontinuous cubic lyotropic liquid crystalline phase transition in monoolein nanoparticles

https://doi.org/10.1016/j.jcis.2020.12.060Get rights and content

Highlights

  • Nine ionizable aminolipids were synthesized and formulated into nanoparticles.

  • pH dependent mesophases of the nanoparticles were analyzed using synchrotron SAXS.

  • Nanoparticles are hexosomes at neutral pH and cubosomes at pH 5.5–6.5.

  • Phase transition pH can be fine-tuned by using an aminolipid mixture.

  • The nanoparticles can be used as anticancer and anti-infection drug carriers.

Abstract

A prospective class of materials for drug delivery is lyotropic liquid crystalline (LLC) nanoparticles, such as cubosomes and hexosomes. Efforts are being made to generate a pH dependent system, which exhibits slow release hexosomes (H2) at physiological pH and relatively fast release cubosomes (Q2) at acidic disease sites such as in various cancers and bacterial infection (pH ~ 5.5–6.5). Herein, we report the synthesis of nine ionizable aminolipids, which were doped into monoolein (MO) lipid nanoparticles. Using high throughput formulation and synchrotron small angle X-ray scattering (SAXS), the effects of aminolipid structure and concentration on the mesophase of MO nanoparticles at various pHs were determined. As the pH changed from neutral to acidic, mesophases, could be formed in an order L2 (inverse micelles) → H2 → Q2. Specifically, systems with heterocyclic oleates exhibited the H2 to Q2 transition at pH 5.5–6.5. Furthermore, the phase transition pH could be fine-tuned by incorporating two aminolipids into the nanoparticles. Nanoparticles with a pH dependent phase transition as described in this study may be useful as drug delivery carriers for the treatment of cancers and certain bacterial infection.

Introduction

The quest for smart nanomaterials has driven recent research toward the engineering of nanosystems with controllable functionality [1]. Nanoscale systems that selectively release drugs at pathological relevant pH may enhance the efficacy and reduce the side effects of various cancer and certain bacterial infection treatments [2]. These conditions are often characterized by a lower pH value such as in the extracellular tumor microenvironment and the infected areas compared to that in healthy tissues [3]. The extracellular pH values (pHe) of most solid tumors are reported to be varied from pH 5.8 to 7.6 with a median of 0.5 pH unit lower than that in normal tissues and in the blood [4]. Furthermore, drug loaded nanocarriers upon cell internalization can encounter an even lower pH environment in sub-cellular compartments, such as pH 5.5–6.0 in endosomes and 4.5–5.0 in lysosomes [5]. For certain infections, acidosis due to bacterial aerobic respiration and fermentation can lead to a lower pH at the infected sites. For example, Konttinen et al. measured a pH of 5.8 on an infected hip implant surface, which is significantly lower than the muscle pH of 7.4 [6]. Studies have demonstrated that pH-sensitive drug delivery systems can improve treatment efficacy and lower the side effects of drugs by slowing or accelerating the release of drugs at healthy tissues or disease sites respectively [7], [8].

Lyotropic liquid crystalline (LLC) materials are composed of amphiphiles which self-assemble in water into various mesophase structures. Bulk phase LLC materials are viscous, gel-like materials. The bulk self-assembled mesophases such as the lamellar (Lα), inverse bicontinuous cubic (Q2), inverse hexagonal (H2), or inverse micellar cubic (I2) phases are formed depending on the complex interplay between the molecular shape of the amphiphiles, the total free energy of the lipid-water system, and the environmental conditions [9]. The bulk phase materials can be dispersed in an aqueous solution to produce nanoparticles such as liposomes, cubosomes, hexosomes, and micellar cubosomes, respectively. For the non-lamellar dispersions, steric stabilizers are usually required. Several studies have demonstrated that self-assembled mesophase structures directly influence the release rate of small molecule drugs and biomolecules, particularly hydrophilic molecules, from these lipidic materials [10], [11], [12], [13], [14], [15], [16], [17]. Additionally, the mesophases of lipid nanoparticles were shown to affect the cell uptake, hemolysis and cytotoxicity [19], [20], [21], [22], [23]. Generally, it was reported that cubosomes exhibit faster drug release rate and stronger cell membrane interaction compared to hexosomes, micellar cubosomes, and inverse micelles (Fig. 1) [1], [18], [19], [20], [21]. A recent review by Tan et al. has summarized the influence of lipid mesophase nanostructures on the cytotoxicity of the nanoparticles [22]. The goal of our study is to create lipid nanoparticles that are hexosomes at neutral pH and switch to cubosomes at an acidic pH.

Monoolein (MO) has drawn much attention due to its biocompatibility and the ability to form a range of LLC mesophases in both bulk and dispersion [23]. However, pure MO does not respond to pH. MO nanoparticles stabilized by Pluronic F127 often give rise to a cubic phase (Q2) with a primitive symmetry (Im3m space group). A wide range of additives such as fatty acids, oils, cholesterol, charged lipids, vitamins and phospholipids have been added to both bulk and dispersed forms of MO to change the effective critical packing parameter (CPP) of the system and direct the transition into different mesophases [24], [25], [26], [27], [28], [29], [30]. CPP is defined as V/al, in which V is the effective volume of the hydrophobic tail of the amphiphile, a is the effective cross-sectional area of the headgroup, and l is the effective length of the hydrocarbon chain. This approach recently led to the development of a stimuli responsive cubosome-based drug delivery system with the capacity to release their contents in response to external triggers like magnetic fields, electrostatic interaction, temperature, ionic strength and pH [31], [32], [33], [34], [35]. A summary of stimuli responsive self-assembled lipid systems, including pH responsive lipid nanoparticles, can be found in a review by Fong et al. [36].

Self-assembled lipid nanoparticles with pH sensitive phase transitions can be achieved through the incorporation of ionizable amphiphiles into the lipidic dispersion. The protonation or deprotonation (ionization state) of the added amphiphiles drives a change in lipid packing, causing a phase transition upon a change in pH. To date, the incorporation of fatty acids has been the most common strategy to produce pH responsive self-assembled lipid nanoparticles. As pH increases, deprotonation of the carboxylic acid moiety of fatty acids leads to reduced headgroup repulsion and thereby reducing lipid membrane curvatures. These systems, exemplified by the study of oleic acid – MO dispersions by Salentinig et al., show phase transitions from L2 → H2 → Q2 → Lα as pH increases [37]. pH sensitive drug loaded nanoparticles can also be prepared by selecting drug molecules with a carboxylic acid group, such as in the case of MO nanoparticles containing anticancer drug 2-hydroxyoleic acid [38]. These transitions correspond to a decrease in CPP with an increase in pH.

The phase transition in the opposite direction (i.e. decrease in CPP as pH decreases), however, is less common as it usually requires synthetic amino group containing amphiphiles. Negrini et al. synthesized and added pyridinylmethyl linoleate to induce a phase transition in monolinolein bulk phase from H2 to Q2 as pH decreased to lower than 5.5. They also demonstrated a 10-fold faster release of doxorubicin at pH 5.5 compared to at pH 7.4. However, the study was conducted in bulk phase, not in nanoparticles, which are generally more suitable for injectable drug delivery [39]. The Yamazaki group has published several studies on low-pH-induced phase transition in dioleoylphosphatidylserine (DOPS)/MO vesicles [40]. They observed a transition from a Lα to Q2 phase via an intermediate H2 phase as the pH dropped. The transition pH values, however, were around 2.5 and maybe more suitable for delivering drugs orally. Additionally, since no stabilizer was used in these studies, instead of cubosomes, bulk cubic phase was found after the phase transition. Recent developments of pH sensitive cubosomes for drug delivery applications have been summarized in a review by Mertins et al. [41].

To our knowledge, there are no reported synthetic material libraries, which can fine-tune the internal nanostructures of MO based lipid nanoparticles to be a Q2 phase at acidic pH (fast drug release) and H2 at physiological pH (slow drug release). For this purpose, we designed and synthesized nine novel ionizable aminolipids with either one or two oleyl tails covalently bonded to headgroups containing tertiary amines via an ester linker (Fig. 2). Out of the nine synthesized aminolipids, four of them are pyridine derivatives, two are heterocyclic, one is an aniline derivative, and two are di-oleates. We used a high throughput formulation approach and synchrotron SAXS technique to analyze the mesophase structures of MO nanoparticles doped with the nine aminolipids in a pH range of 2.5–10. In this study, several aminolipid-doped MO nanoparticle systems exhibited a desirable hexagonal to cubic phase transition within the pathologically relevant pH range of 5.5–6.5. The novel pH-responsive nanosystems developed in this study hold great promise to pinpoint the drug release at disease sites with a different pH environment than the healthy tissues. This has implications for the next generation of nanomedicine.

Section snippets

Material

Aminoalcohols: 4-pyridinemethanol (purity > 99%), 2-Pyridineethanol (purity > 99%), 6-Methyl-2-pyridinemethanol (purity > 98%), 2, 6-Pyridinedimethanol (purity > 98%), 4-(2-Hydroxyethyl) morpholine (purity > 99%) 1-(2-Hydroxyethyl) piperidine (purity > 99%), 2-(Methylphenylamino) ethanol (purity > 98%), 2-Benzimidazolemethanol (purity > 97%), were obtained from Sigma Aldrich. N-3-Dimethylaminopropyl-N′-ethylcarbodiimidehydrochloride (EDCl) (purity > 90%), Hydroxybenzotriazole (HOBt)

Synthesis, purification and analysis of the ionizable aminolipids

The physical state of 7 synthesized aminolipids (Lipid-1 to Lipid-7) were freely flowing liquid at room temperature. Di-oleates (Lipid-8 and Lipid-9) were semi-solid at room temperature. In general, the yield of the reactions was in a range of 60–75% and the purity of the final compounds was around 85–95%. The structures and purities of all synthesized lipids were confirmed by NMR (see Supplementary Information Fig. S1) and GCMS (data not provided).

Particle size of the nanoparticles

In this study, we mixed various amounts of

Conclusions

In this study, nine novel ionizable aminolipids were synthesized and formulated into stable MO nanoparticles. High throughput SAXS experiments demonstrated that both aminolipid composition and pH influenced the internal mesophase of the nanoparticles. Out of nine aminolipid–MO nanoparticle systems, eight of them displayed a pH dependent H2 → Q2 phase transition at pathologically relevant pH (pH 3.5-pH 6.5). The phase transition pH can be further fine-tuned by co-doping two aminolipids into MO

CRediT authorship contribution statement

Sarigama Rajesh: Investigation, Methodology, Data curation, Writing - original draft, Writing - review & editing, Visualization, Formal analysis. Jiali Zhai: Investigation, Methodology, Writing - review & editing, Data curation. Calum J. Drummond: Supervision, Resources, Data curation, Writing - review & editing. Nhiem Tran: Conceptualization, Investigation, Methodology, Data curation, Writing - review & editing, Visualization, Formal analysis, Supervision, Resources.

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

This research includes work undertaken on the SAXS/WAXS beamline at the Australian Synchrotron, Victoria, Australia. We thank Dr Stephen Mudie, Dr Tim Ryan, and Dr Nigel Kirby of the SAXS/WAXS beamline for their assistance with SAXS experiments. N. T. is supported by a RMIT Vice Chancellor’s Research Fellowship.

References (56)

  • B.J. Boyd et al.

    Lyotropic liquid crystalline phases formed from glycerate surfactants as sustained release drug delivery systems

    Int. J. Pharm.

    (2006)
  • E. Nazaruk et al.

    Charged additives modify drug release rates from lipidic cubic phase carriers by modulating electrostatic interactions

    J. Electroanal. Chem.

    (2018)
  • N. Tran et al.

    Manipulating the ordered nanostructure of self-assembled monoolein and phytantriol nanoparticles with unsaturated fatty acids

    Langmuir

    (2018)
  • K. Hida et al.

    Tumor blood vessels as targets for cancer therapy

  • I.F. Tannock et al.

    Acid pH in tumors and its potential for therapeutic exploitation

    Cancer Res.

    (1989)
  • A. El-Kenawi et al.

    Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer

    Br. J. Cancer

    (2019)
  • Y.T. Konttinen et al.

    Acidic cysteine endoproteinase cathepsin K in the degeneration of the superficial articular hyaline cartilage in osteoarthritis

    Arthritis Rheum.

    (2002)
  • F. Yang et al.

    Nanoparticle drug delivery can reduce the hepatotoxicity of therapeutic cargo

    Small

    (2020)
  • X. Xu et al.

    Ultra-pH-responsive and tumor-penetrating nanoplatform for targeted siRNA delivery with robust anti-cancer efficacy

    Angew. Chem. Int. Ed. Engl.

    (2016)
  • J. Zhai et al.

    Paclitaxel-loaded self-assembled lipid nanoparticles as targeted drug delivery systems for the treatment of aggressive ovarian cancer

    ACS Appl. Mater. Interfaces

    (2018)
  • T.G. Meikle et al.

    Predicting the release profile of small molecules from within the ordered nanostructured lipidic bicontinuous cubic phase using translational diffusion coefficients determined by PFG-NMR

    Nanoscale

    (2017)
  • C. Fong et al.

    Lyotropic liquid crystal engineering–ordered nanostructured small molecule amphiphile self-assembly materials by design

    Chem. Soc. Rev.

    (2012)
  • I. Koltover et al.

    An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery

    Science

    (1998)
  • Y. Tian et al.

    Folic acid-targeted etoposide cubosomes for theranostic application of cancer cell imaging and therapy

    Med. Sci. Monit.

    (2017)
  • J. Zhai et al.

    Amphiphilic brush polymers produced using the RAFT polymerisation method stabilise and reduce the cell cytotoxicity of lipid lyotropic liquid crystalline nanoparticles

    Faraday Discuss.

    (2016)
  • N. Tran et al.

    Nanostructure and cytotoxicity of self-assembled monoolein–capric acid lyotropic liquid crystalline nanoparticles

    Roy. Soc. Chem. Adv.

    (2015)
  • S. Assenza et al.

    Curvature and bottlenecks control molecular transport in inverse bicontinuous cubic phases

    J. Chem. Phys.

    (2018)
  • A. Tan et al.

    Self-assembled nanostructured lipid systems: is there a link between structure and cytotoxicity?

    Adv. Sci.

    (2019)
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