(T1230-01-06) A Novel Approach Using an Organic Phase Nanosuspension to Enhance Drug Loading of Hydrophilic Compounds in Nanoparticles

Purpose: While hydrophobic compounds can be readily loaded into nanoparticles using the conventional oil-in-water (o/w) emulsion method, the encapsulation of hydrophilic substances within hydrophobic nanosized matrices presents a formidable challenge. Hydrophilic molecules are typically encapsulated using the double emulsion method, but one recurring drawback of this approach is its low drug loading capacity, often attributable to leakage[1]. In contrast to larger particles, the physical properties of nanoscale materials can undergo size-dependent variations. A hypothesis is that hydrophilic particles can show a hydrophobic properties at nanoscale particle sizes[2]. Building upon this hypothesis, we developed a novel strategy to achieve higher loading capacities for encapsulating hydrophilic salts within hydrophobic polymers. In this method, nanoscale salt crystals were prepared through emulsion and freeze-drying, then suspended in the oil phase to form a nanosuspension, rather than being dissolved in the water phase. Subsequently, the o/w emulsion method was employed to load these nanocrystals into nanoparticles. Trivalent arsenic salt is considered as an ideal hydrophilic model compound for testing this new method. Arsenic trioxide (ATO), which has been used for medicinal applications for more than a thousand years in traditional Chinese and Indian medicine. In modern medicine, it also has been shown to inhibit the development of cancer, for example, leukemia[3].For many of the anti-cancer drugs, preparing it into nanoparticles can reduce its severe toxicity and side effects. As a poor water and organic solvent soluble compound, ATO can be dissolved in alkali media to form soluble trivalent arsenic salt. However, this hydrophilic property makes it difficult to be loaded into nanoparticle compared to other hydrophobic drugs. In our study, trivalent arsenic salt was encapsulated into nanoparticles through both conventional double emulsion method and the new strategy. To address the time-consuming nature of nanosuspension preparation using the freeze-drying method, we also attempted to prepare nanosuspensions using the solvent-anti-solvent method in our study. The nanocrystals in the nanosuspension were promptly enveloped by the polymer in a subsequent phase separation step (nonaqueous phase separation method) and precipitated the nanoparticles.

Methods: Double emulsion method, o/w (nanocrystal) method, and nonaqueous phase separation method are used to prepare ATO nanoparticles. The preparation procedures are listed in Figure1. Dynamic light scattering(DLS) and Scanning electron microscope(SEM) were used to study the size distribution and morphology of the nanoparticles. The loading capacity and the release curve of the nanoparticles was measured by inductively coupled plasma mass spectrometry (ICP-MS).

Results: For the double emulsion method, loading capacities of two distinct protocols that tried were 0.009% and 0.030%, respectively (Figure2). The low loading capacity primarily results from the drug leaking from the inner to the outer water phase during nanoparticle production. [4]. Several batches of NPs were prepared by o/w (nanocrystal) method. The highest loading capacity can attend 12.63%(Figure2). To prepare nanosuspensions of trivalent arsenic salt in first step, a water-in-oil emulsion method is employed, using ultrasonic treatment to mix a trivalent arsenic salt solution with a polymer in chloroform, creating a stable emulsion. Post ultrasonic treatment, solvents are removed via freeze-drying, resulting in evenly dispersed hydrophilic arsenic salt nanocrystals within a hydrophobic polymer matrix (Figure2A). These nanocrystals remain suspended when the polymer matrix is dissolved by an added organic solvent, forming the nanosuspension. In o/w processes, the organic solvent forms droplets containing both the polymer and the drug, which solidify as the solvent evaporates, helped by the payload's hydrophobic properties. SEM analysis shows these nanocrystals encapsulated by the polymer, maintaining their structure within the nanoparticles (Figure2B). To streamline nanocrystal production, the "solvent-anti-solvent" method that exploiting ATO's solubility characteristics that highly soluble in basic solutions (pH>9) but precipitating rapidly as pH lowers was developed. 0.1 M sodium hydroxide solution and 0.1 M hydrochloric acid in dichloromethane are the solvent and anti-solvent respectively.Ultrasonic was used to maintain the uniform crystals size. Instead of traditional emulsion techniques, a phase separation method was applied, using methanol to precipitate polymer around the nanocrystals, forming coated nanoparticles. SEM analysis revealed these nanoparticles had wider particle size distribution compared to those prepared by emulsion method although their loading capacities were similar (Figure2). The study evaluated the cumulative release of nanoparticles prepared by different methods and batches. It was observed that the release rate of nanoparticles prepared via the nonaqueous phase separation method was slower compared to those prepared using the o/w method. This phenomenon can be attributed to the larger particle size of the nanoparticles prepared through the nonaqueous phase separation method. Additionally, it was noted that the release rate of nanoparticles prepared by the o/w method exhibited a direct proportionality to their loading capacity(Figure3).

Conclusion: This study to the best of the authors’ knowledge describes for the first time a new strategy that has a high loading capacity to encapsulate hydrophilic molecules rather than using the conventional W/O/W methods. Such a method has great potential for hydrophilic compound loading and delivery.

References: 1. Cohen-Sela, E., et al., A new double emulsion solvent diffusion technique for encapsulating hydrophilic molecules in PLGA nanoparticles. J Control Release, 2009. 133(2): p. 90-5.
2. Chiu, C.C., et al., Size-dependent hydrophobic to hydrophilic transition for nanoparticles: a molecular dynamics study. J Chem Phys, 2009. 131(24): p. 244706.
3. Glienke, W., et al., Down-regulation of wt1 expression in leukemia cell lines as part of apoptotic effect in arsenic treatment using two compounds. Leuk Lymphoma, 2006. 47(8): p. 1629-38.
4. Becker Peres, L., et al., Solid lipid nanoparticles for encapsulation of hydrophilic drugs by an organic solvent free double emulsion technique. Colloids Surf B Biointerfaces, 2016. 140: p. 317-323.

Acknowledgements: NoStopharm Inc. and Maryland Industrial Partnerships (MIPS) for technical assistance and financial support.


Figure. 1. Flowchart of three nanoparticles preparation methods (double emulsion method, o/w nanocrystal method, and nonaqueous phase separation method)


Figure.2. SEM picture and characterization data of ATO nanocrystal and nanoparticles prepared by different methods. A: ATO nanocrystal in porous structure formed by the PLGA-PEG; B: ATO nanoparticles prepared by o/w (nanocrystal) method; C: ATO nanoparticles prepared by nonaqueous phase separation method.


Figure. 3. Accumulative release and parameters of ATO nanoparticles in vitro