(M1330-06-34) Approaches toward Nanostructured-Lipid Carriers (NLCs) for Disulfiram Delivery

Purpose: Disulfiram (DSF) has a long history as an FDA-approved alcohol aversiveness drug for more than six decades now. Establish the repurposing of DSF as a potential anticancer drug. Such an approach is more feasible and reasonable compared to the time and fund-consuming approach of discovering and developing new anticancer moieties. DSF anticancer mechanisms haven’t been fully explored yet. However, DSF is always linked to copper (Cu) levels [1]. DSF acts as Cu-ionophore, yielding the anticancer substrate after being subjected to Glutathione reductase. The association of DSF and Cu, suggestively, will offer DSF the advantage of targeting cancer cells while sparing healthy ones. Since Cu levels were reportedly higher in cancer tissues compared to normal [2,3]. This work aims to use Nanostructured Lipid Carriers (NLCs) with their merits to overcome the DSF’s solubility issues, protect against degrading conditions, and achieve effective drug delivery. NLCs have a higher drug-loading capacity compared to Solid Lipid Nanoparticles (SLN), stable upon storage with no drug expulsion[4]. Moreover, it is reportedly effective in tumor-specific targeting by avoidance of Reticulum Endothelial System (RES) clearance[5]. One of the set goals was to sustain the NLC particle size below 300 nm for infiltration into cancer cells

Methods: NLCs components are primarily determined by screening for DSF best solubility in solid, liquid lipids and surfactants as well. The drug solubility is a critical factor in excipient selection. Hence, Saturation solubility studies of DSF in liquid lipids and surfactants were conducted by adding the excess drug to excipients in sealed vials, shaking horizontally, and periodically analyzing using HPLC until saturation was reached. Oils investigated included sesame oil, castor oil, oleic acid, labrasol®, and Labrafil® M 1944 CS. Drug solubility in solid lipids was evaluated by adding known amounts of DSF to the molten of these lipids and allowing the molten to stand for 24h, then inspected for any signs of precipitation or recrystallization. Compritol® 888 ATO, Dynasan® 118, and Percirol® ATO were the solid lipids evaluated. Based on previous literature and solubility screening we set factors for the Design of Experiment (DoE) and generated Full Factorial Design (4x2x3) using JMP software (Table 1); where the three factors namely Solid: liquid lipid ratio, the percentage of Surfactants mix (S.mix) and the amount of drug in the formulations were investigated for their effect on the responses. Responses were particle size (PS), Drug content (DC), and entrapment efficiency (EE). This resulted in 27 runs. The PS and Polydispersity index (PDI) were measured to ensure homogeneous size distribution and controlled size. During the preparation of DSF NLCs formulation, different manufacturing parameters such as the temperature applied during melting and mixing of the components and the homogenization time were assessed. Probe sonication was set to 10 minutes, 10 on/ 10 off with 40 % amplitude.

Results: The final parameters adopted were to raise the temperature of processing 20 degrees above the solid lipid melting point throughout the whole process. Upon our observations, the elevated temperature during manufacturing helped in the reduction and consistency of PS. Homogenization power was 12,000 rpm and for 10 minutes. Probe sonication was applied as mentioned in the methodology. These were the conditions with NLCs displaying optimum characteristics. The formulation composition consisted of Compritol® 888 ATO, Labrafil® M 1944 CS, Tween 20 and Poloxamer 188. The S. mix was (1:1) Tween 20: Poloxamer 188. Tween 20 was the surfactant with the highest solubility of DSF; however, for NLCs’ stability, Poloxamer 188 was required. The PS ranged from 195.9 and 362.1 nm, which meant that some formulations will be infiltrated into cancer cells as presumed. PDI (0.181-0.483) suggested the uniformity of NLCs. The 27 runs also showed DC (82.858- 105.042 %) and EE (80.3- 97.431 %). Meanwhile, the design suggested that solid: liquid lipid and the S. mix both have a significant effect on the PS with P-values (0.00001 and 0.00003), respectively (Figure 1a). However, these factors had no effect over the DC and EE responses. The drug amount had a negligible effect on all investigated responses as per design (Figure 1 b and c).

Conclusion: In conclusion, this study helped define manufacturing parameters that enhanced the NLCs' properties. Moreover, the use of DoE helped in setting the factors with a significant effect on the responses

References: 1. Chen D, Cui QC, Yang H, Dou QP. Disulfiram, a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and xenografts via inhibition of the proteasome activity. Cancer research. 2006 Nov 1;66(21):10425-33.
2. Santini C, Pellei M, Gandin V, Porchia M, Tisato F, Marzano C. Advances in copper complexes as anticancer agents. Chemical reviews. 2014 Jan 8;114(1):815-62.
3. Lu Y, Pan Q, Gao W, Pu Y, Luo K, He B, Gu Z. Leveraging disulfiram to treat cancer: Mechanisms of action, delivery strategies, and treatment regimens. Biomaterials. 2022 Feb 1;281:121335.
4. Taha IE, ElSohly MA, Radwan MM, Elkanayati RM, Wanas A, Joshi PH, Ashour EA. Enhancement of cannabidiol oral bioavailability through the development of nanostructured lipid carriers: In vitro and in vivo evaluation studies. Drug Delivery and Translational Research. 2024 Dec 30:1-1.
5. Han Y, Zhang Y, Li D, Chen Y, Sun J, Kong F. Transferrin-modified nanostructured lipid carriers as multifunctional nanomedicine for codelivery of DNA and doxorubicin. International journal of nanomedicine. 2014 Aug 25:4107-16.


Table 1. Summary of Investigated Factors and Responses in the Full Factorial Design.


Figure 2(a) Correlation between factor A and factor B and Particle Size. (b) Correlation between factor A and Factor C and Drug Content. (c) Correlation between factor A and factor C and Entrapment Efficiency.