Radiolabelling and SPECT/CT imaging of lipid-based nanosystems for development of nanoformulated antibiotic or growth factors embedded wound dressings

Objectives: Development of novel complementary systems for local topic delivery of antibiotics provide significant advantages over systemic treatments for healing of infected complex wounds. Nanoformulated antibiotics embedded in an adequate sanitary dressing would additionally permit prolonged residence time and slow, controlled release of the antibiotic (and additionally growth factors) for increased efficacy and safety. To ensure safety of the sanitary product and permit its approval by the competent authorities it must be demonstrated that the nanosystem itself is not absorbed. The objective of the current work was to radiolabel a lipid-based nanosystem under development and the of use molecular imaging technology to provide evidence of its safety

Methods: Lipid nanoparticles were prepared by hot melt homogenisation followed by high-pressure homogenisation step. Precirol ATO 5 and Miglyol 182 were selected as core lipid and Dextran 40 as cryopreservative, as reported elsewhere¹. Lipid-based hybrid nanosystems were radiolabelled with 99mTc by direct reaction with SnCl₂-reduced ^99mTc-pertechnetate with high yield (>95% as per radioTLC) and used without need for further purification. An excision open wound rat model was used for in vivo experiments. A 10-mm wide punch was used to create (under topic anaesthesia) wounds extending through panniculus carnosus in the shaved back of female Wistar rats (n=17). Wounds were covered with TegadermTM dressing and a sanitary bandage for 24 h and then 5 mg of ^99mTc-radiolabelled NP (3 MBq, n=9) or free 99mTc-pertechnetate (negative control, 6 MBq, n=8) in 20 µL were carefully placed inside the wound. New dressing and bandage were placed and animals imaged at 1, 2, 4, 8, 12 and 24 h in a SPECT/CT system. Rats were then euthanized, organs excised and radioactivity measured in a gamma-counter. SPECT/CT datasets were exported to PMOD and VOIs drawn over the CT and transferred to SPECT images for numerical calculations.

Results: Nanoparticles displayed 144.7±6.32 nm size, negative Zeta potential (~-20 mV) and 0.22±0.01 polydispersity index showing that the particle population was very homogeneous. Images (fig. 1) showed that virtually all radioactivity (>98% at all times, from 1 to 24 h; see fig. 2, bar graph) remained in the wound for ^99mTc-NP while it was quickly absorbed for control animals and only around 7% remained at the wound at 24 h. Data from the gamma counter obtained from different organs at 24 h showed a similar pattern. Furthermore, the area around the wound was dissected into dressing, skin, subcutaneous tissue and subjacent muscle and counted separately. In ^99mTc-NP treated animals, the dressing retained 41 % of the radioactivity, while 57 % was in the surrounding skin, 1 % in the subcutaneous tissue and 0.02 % in the muscle. For control animals results were 6 %, 13 %, 0.2 % and 0.01 %, respectively, for the same areas, while most of the activity was found in the stomach (20.5 %), small intestines (11.2 %), large intestine (21.8 %), caecum (20.1 %) and kidneys (4.4 %).

Conclusion: Radiolabelling of the used nanosystems showed that they are not absorbed at all and remain in the place of administration for more than 24 h. These results provide sufficient evidence about the safety of the topic administration of the nanosystems, and to use them in the development of nanoformulated antibiotic or growth factors embedded wound dressings for human use. 1. G Gainza, M Pastor, JJ Aguirre et al. J Control Release 2014;185:51-61 Research Support: This research was financed by Praxis Pharmaceutical $$graphic_E50A5EFD-A673-4D53-8C86-095AFA100D12$$ $$graphic_22294D9E-3C84-448D-9C57-CE39FC638B76$$