Characterization and stability evaluation of nanoencapsulated epoxylignans

Introduction

The hedgehog (Hh) pathway is required for the growth and proliferation of various cancers¹. The signaling begins with the binding of Hh protein ligand to its membrane receptor Ptch, which represses the activity of Smoothened (Smo). Smo in turn promotes the expression of the GLI (Glioma-associated oncogene) family of transcription factors, leading to tumor development². The direct association of GLI with a specific binding site (5’-GACCACCCA-3’) in the promoter region of the target gene has been reported to contribute to pancreatic and prostate cancer cells³. We previously reported Hedgehog/GLI inhibitors from various plants^4–6. We also isolated five lignans ((8R*,8’R*)-9-hydroxy-3,4-dimethoxy-3’,4’-methylenedioxy-9,9’-epoxylignan, kusunokinin, haplomyrfolol, dihydroclusin) including a new epoxylignan dmeo from Piper nigrum using the immobilization of GLI-GST on carboxylic acid magnetic dynabeads. DMEO was confirmed to have Hh signaling inhibitory activity and to be selectively cytotoxic against PANC1. Meanwhile the synthetic epoxylignan of DMEO related compound was reported to inhibit the mRNA expression of protein patched homolog (Ptch) in human pancreatic cancer cells (PANC1) and thus is considered to be a prospective drug candidate to treat cancer related to the GLI signaling pathway. However, poor solubility remains the main limitation of DMEO⁷, hence making drug administration in vivo difficult. The nanoencapsulation of DMEO is one of the ways of overcoming the problem.

Nanoencapsulation techniques are particularly important to protect drugs from degradation in biological fluids and improve their penetration into cells. The techniques are also beneficial for hydrophobic molecules because the ultra-dispersed pharmaceutical dosage forms that nanoencapsulation provides allow rapid drug dissolution⁸. The nanoencapsulation of DMEO within a suitable polymer is considered to be a good way to ameliorate its poor solubility, because the polymer acts as a rate-controlling membrane to obtain the desired controlled release. The physical stability of pure compounds remains the greatest challenge for pharmaceutical scientists seeking to exploit higher solubility properties. A gold standard revealed by the International Council for Harmonization (ICH) stated that physical stability tests of compounds should be performed within accelerated (6 months) and/or long-term (12 months) storage conditions⁹. The physical stability was examined using powder X-ray diffraction (PXRD) analysis.

The objective of this study was to characterize the physical stability of DMEO-loaded nanocapsules, which were optimized by varying the polymer concentration to obtain stable spherical particles. The most stable particles would result from the best concentration ratio between polymers and stabilizers, ultimately improving the dissolution rate of poorly water soluble drugs.

Methods

Results

SEM analysis reveals that all DMEO-loaded nanocapsules are mostly spherical (Figure 1). As the polymer content increased, the encapsulation efficiency and mean particle size also increased, as seen in Table 1, suggesting that nanoencapsulation minimized deactivation of the drugs during the delivery process due to the protection from the polymer shell. This might ensure sufficient amounts of drug reaching the targeted areas. ERL is a copolymer of partial esters of acrylic acid containing low amounts of a quaternary ammonium group. ERL is a water-soluble polymer that plays a crucial role in controlling drug release because its water uptake characteristic contributes to the swelling of the polymers.

Figure 1.

SEM images of sprayed-dried powder in formulations (A) F1 (B) F2 and (C) F3.

In crystalline materials, atoms are periodically arranged, but in non-crystalline materials, atoms are randomly arranged. Figure 2 shows that the peaks of DMEO do not possess that periodicity, while the characteristic peak falls at 19.3° 2θ. After the physical mixtures were subjected to various concentrations of polymers and stabilizers, all the peaks were re-observed at 0 and 6 months of storage. No crystalline peaks were observed in the diffraction patterns, as shown in Figure 2a, 2b and 2c (upper, down), suggesting that the particles remain spherical throughout the duration of the stability study. The unchanged position of the largest peak of DMEO, which remains at 19.3° 2θ (Figure 2a, 2b and 2c (upper, down)), indicates that there is no change in the diffraction pattern of DMEO after 6 months of storage. It is important for the DMEO particles to remain stable during 6 months of storage to ensure that the resulting DMEO-loaded nanocapsules meet the minimal standard requirements of drug formulation stability.

Figure 2.

(A) X-ray diffraction patterns of DMEO-loaded nanocapsules of F1 after 0 and 6 months of storage (B) X-ray diffraction patterns of DMEO-loaded nanocapsules of F2 after 0 and 6 months of storage (C) X-ray diffraction patterns of DMEO-loaded nanocapsules of F3 after 0 and 6 months of storage.

Discussion

The polymer-based encapsulation may be beneficial regarding improved short-term physical stability. The nanoencapsulation of DMEO yielded entrapment efficiencies of 89.53±0.307, 89.46±0.211, 90.31±0.352% with particle sizes of 227.55±0.231, 253.96±0.012, and 255.09±0.455 nm, respectively. It is generally considered that higher molecular weight polymers have less entropy loss related to their fast motion, which results in a higher affinity to drug surfaces¹⁰, and they therefore perhaps have better steric formation. The charge along the polymer chains can build a strong double layer surrounding the particles, granting electrostatic stabilization and therefore smaller particle size¹¹.

The results showed that the polymer concentration influenced the particle size and entrapment efficiency. The 1% polymer concentration formula (F1) fulfilled the requirement of stable nanocapsules, including spherical and uniform surface morphology. This formula exhibited the greatest percentage of nanocapsules’ weight (10.24%) but had the smallest particle size (227.55 nm). Meanwhile, F3 showed the greatest entrapment efficiency (90.31%). We chose F1, F2 and F3 for the further physical stability study using XRD to see whether the different concentrations of polymer interfered with the stability of DMEO in nanocapsules. Polymers were expected to adsorb to the surface of the nanocapsules, providing both electrostatic and steric repulsion among particles, therefore producing nanoparticles with decreased particle sizes and improved physical stability. The polymers may adsorb to the drug surfaces through several points along the polymer chains, enabling the loops and tails of the polymers to extend into the liquid medium, providing a steric effect.

The key finding in this work was that higher concentrations of polymer in the stirring solution during the production process yielded higher encapsulation efficiencies and smaller particle sizes. Tuning the polymer ratio was essential to obtaining spherical particles, without necessarily affecting the stability of the obtained nanocapsules.

Data availability

Dataset 1: Raw data for particle size, particle yields, nanoencapsulation efficiencies and X-ray diffraction pattern values. 10.5256/f1000research.13047.d195348¹²