Rapid Expansion of Supercritical Solution Mediated Development of Resveratrol Solid Lipid Nanoparticles for Enhanced Radioprotective Effects

Abstract

Purpose Harmful effects of ionizing radiations were reported soon after the discovery of X-rays but the real magnitude was known later by physicians and scientists handling radioactivity and more specifically observed during the study of Japanese atomic bomb survivors of 1945. This effect is also seen during radiotherapy in various malignant conditions in which radiation causing damage to normal cells resulted in some severe adverse effects. With the potential application of ionizing radiation in medical practices (e.g. radiotherapy and nuclear medicine) and potential accidental exposure to radiation (e.g. industrial nuclear accident), the development of effective radiomodifiers is of great medical importance. Radioprotective agents are synthetic or natural compounds that are administrated before radio-exposure to reduce morbidity or mortality. Amifostine is the only radioprotector that has been clinically approved by the Food and Drug Administration (FDA) for mitigating side effects (xerostomia) in patients undergoing radiotherapy. This drug offers good protection, but is accompanied with side effects like nausea, vomiting and hypotension. Hence, the development of radioprotective agents with lower toxicity and an extended window of protection have attracted much attention. Natural compounds have been evaluated as radioprotectants and they seem to exert their effect through antioxidant and immunostimulant activities. Resveratrol (RVL) is one such molecule that acts via free radical scavenging/antioxidant mechanism to mitigate the radiation induced cell damage. But its poor bioavailability (> 1%) due to low solubility and extensive metabolism impedes its efficacy in vivo. So, the aim of present investigation is to develop resveratrol encapsulated long circulating solid lipid nanoparticles (SLN) for enhanced bioavailability and retention in vivo. Methods SLNs were produced by rapid expansion of supercritical fluid (RESS) technology in which drug-lipid-surfactant mixture (Lipid = Gelucire®50/02, Surfactant= Gelucire®50/13) was solubilized in supercritical CO 2 and rapidly expanded in aqueous phase (containing tween 80) to precipitate the particles. Temperature, Drug: lipid mix and Lipid: Surfactant ratios were optimized to get high yield (%Y), low particle size and high entrapment efficiency (%EE).In vivo pharmacokinetics studies were performed to show the increased circulation. Total antioxidant assay, superoxide radical scavenging activity, deoxyribose degradation assay, DPPH free radical scavenging assay, radiation induced lipid peroxidation and plasmid DNA relaxation assays were performed on the drug release samples taken out at different time interval for both optimized SLN and and pure resveratrol. Results The optimized formulation (F9) was found to have %Y, particle size, PDI, %EE and %drug release (after 72h) of 68.48±5.73%, 276.7±5.33nm, 0.18± 0.032, 62.66±4.52% and 70.05±3.003% respectively. Transmission and scanning electron microscopy showed non-spherical particles of size less than 300nm. DSC and XRD patterns inferred a molecular dispersion of drug in the lipid matrix. Antioxidant and radioprotective studies exhibited significant enhancement in activity of F9 as compared to RVL. In vivo pharmacokinetic study showed non-detectable RVL plasma concentration after 8h and 24h of single dose (20mg/KgBW) i.v. administration of RVL and Non-PEG SLN respectively. However, 172.99±8.600ng/mL of RVL was found even after 72h of F9 administration revealing its long circulation effect. Conclusion The results showed enhanced antioxidant profile and radioprotective ability of F9 against γ-radiation as compared to pure drug which is quite encouraging and thus subsequent phase of this work encompass comprehensive in vivo radioprotective studies and stability profiling of the developed system.

Full text

Rapid Expansion of Supercritical Solution Mediated Development of Resveratrol Solid Lipid
Nanoparticles for Enhanced Radioprotective Effects
I. Ahmad 1, S. Akhter 1, P. Thakur 2, R. Chawla 2, R. K. Sharma 2, A. Ali 1, F. J. Ahmad 1
1 Hamdard University, 2 Institute of Nuclear Medicine & Allied Sciences (INMAS)
Purpose
Harmful effects of ionizing radiations were reported soon after the discovery of X-rays but the real magnitude was known later by
physicians and scientists handling radioactivity and more specifically observed during the study of Japanese atomic bomb survivors of
1945. This effect is also seen during radiotherapy in various malignant conditions in which radiation causing damage to normal cells
resulted in some severe adverse effects. With the potential application of ionizing radiation in medical practices (e.g. radiotherapy and
nuclear medicine) and potential accidental exposure to radiation (e.g. industrial nuclear accident), the development of effective
radiomodifiers is of great medical importance. Radioprotective agents are synthetic or natural compounds that are administrated
before radio-exposure to reduce morbidity or mortality. Amifostine is the only radioprotector that has been clinically approved by the
Food and Drug Administration (FDA) for mitigating side effects (xerostomia) in patients undergoing radiotherapy. This drug offers
good protection, but is accompanied with side effects like nausea, vomiting and hypotension. Hence, the development of
radioprotective agents with lower toxicity and an extended window of protection have attracted much attention. Natural compounds
have been evaluated as radioprotectants and they seem to exert their effect through antioxidant and immunostimulant activities.
Resveratrol (RVL) is one such molecule that acts via free radical scavenging/antioxidant mechanism to mitigate the radiation induced
cell damage. But its poor bioavailability (> 1%) due to low solubility and extensive metabolism impedes its efficacy in vivo. So, the
aim of present investigation is to develop resveratrol encapsulated long circulating solid lipid nanoparticles (SLN) for enhanced
bioavailability and retention in vivo.
Methods
SLNs were produced by rapid expansion of supercritical fluid (RESS) technology in which drug-lipid-surfactant mixture (Lipid =
Gelucire®50/02, Surfactant= Gelucire®50/13) was solubilized in supercritical CO2 and rapidly expanded in aqueous phase
(containing tween 80) to precipitate the particles. Temperature, Drug: lipid mix and Lipid: Surfactant ratios were optimized to get high
yield (%Y), low particle size and high entrapment efficiency (%EE).In vivo pharmacokinetics studies were performed to show the
increased circulation. Total antioxidant assay, superoxide radical scavenging activity, deoxyribose degradation assay, DPPH free
radical scavenging assay, radiation induced lipid peroxidation and plasmid DNA relaxation assays were performed on the drug release
samples taken out at different time interval for both optimized SLN and and pure resveratrol.
Results
The optimized formulation (F9) was found to have %Y, particle size, PDI, %EE and %drug release (after 72h) of 68.48±5.73%,
276.7±5.33nm, 0.18± 0.032, 62.66±4.52% and 70.05±3.003% respectively. Transmission and scanning electron microscopy showed
non-spherical particles of size less than 300nm. DSC and XRD patterns inferred a molecular dispersion of drug in the lipid matrix.
Antioxidant and radioprotective studies exhibited significant enhancement in activity of F9 as compared to RVL. In vivo
pharmacokinetic study showed non-detectable RVL plasma concentration after 8h and 24h of single dose (20mg/KgBW) i.v.
administration of RVL and Non-PEG SLN respectively. However, 172.99±8.600ng/mL of RVL was found even after 72h of F9
administration revealing its long circulation effect.
Conclusion
The results showed enhanced antioxidant profile and radioprotective ability of F9 against
γ
-radiation as compared to pure drug which
is quite encouraging and thus subsequent phase of this work encompass comprehensive in vivo radioprotective studies and stability
profiling of the developed system.