Nanostructured lipid carriers loaded with resveratrol modulate human dendritic cells

Abstract

Dendritic cells (DCs) are promising targets for drug delivery, as they can induce immunity or tolerance. The current study aims to examine the potential of using nanostructured lipid carriers (NLC) as delivery systems for human DC by evaluating nanoparticle internalization, cell labeling, and drug activity. NLC were formulated incorporating the fluorochrome fluorescein isothiocyanate (FITC-NLC) or the natural anti-inflammatory molecule resveratrol (rsv-NLC). Primary human DCs were differentiated from peripheral blood monocytes, and the innovative imaging flow cytometry technique was used to examine FITC-NLC internalization. The capacity of rsv-NLC to inhibit DC activation in response to proinflammatory cytokine tumor necrosis factor-α (TNF- α) was investigated by conventional flow cytometry. A combination of imaging and conventional flow cytometry was used to assess NLC cytotoxicity. The results obtained indicate that both NLC formulations were stable over time, with mean diameter <200 nm and highly negative zeta potential (about −30 mV). When DCs were placed in contact with NLC, imaging flow cytometry clearly showed that DCs efficiently internalized FITC-NLC, with nearly 100% of cells internalizing nanoparticles upon 1 hour of incubation. Both immature and mature DCs internalized NLC to high and comparable levels, and without cytotoxicity. Stimulating DC with TNF-α in the presence of rsv-NLC revealed that, using these nanoparticles, very small concentrations of rsv were sufficient to significantly decrease surface expression of activation marker CD83 (5 µM) and major histocompatibility complex-class II molecule human leukocyte antigen – antigen D related (10 µM), both upregulated in response to TNF-α stimulation. Rsv-NLC were compared with free rsv; at 5 µM, rsv-NLC were able to inhibit nuclear factor κ beta phosphorylation and significantly decrease the level of interleukin-12/23, both upregulated in response to TNF-α, while 10 µM free rsv were needed to promote a similar effect. Taken together, the results presented show that NLC are suitable carriers of fluorescent labels or bioactive molecules for human DCs, leading to inflammation modulation.

Keywords:

• immunomodulation
• dendritic cell
• solid lipid nanoparticle
• imaging flow cytometry
• resveratrol
• TNF-α

Supplementary materials

Figure S1 Workflow for determination of percentage of FITC-NLC internalization.

Notes: Gates define population in focus (top left), excluding cell duplets or other complexes (top right), defining CD1a positive cells (bottom left) and FITC intensity (bottom right). Gate R1 shows positive events for NLC internalization.

Abbreviations: FITC, fluorescein isothiocyanate; NLC, nanostructured lipid carriers.

Figure S2 Effect of time of storage on resveratrol entrapment efficiency of NLC.

Notes: The analysis was performed after production (day 0) and along 3 months of storage. All data represent the mean ± SD (n=3). No statistically significant differences were found (P>0.05).

Abbreviations: SD, standard deviation; NLC, nanostructured lipid carriers.

Table S1 Preparation of FITC and rsv-loaded NLC

Acknowledgments

The authors would like to thank the Serviço de Imunohemoterapia of Centro Hospitalar de São João, for kindly donating buffy coats and b.IMAGE – the Bioimaging Center for Biomaterials and Regenerative Therapies. This work was financed by UP and Santander (PP_IJUP2011_220); and FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274).

Disclosure

ARN and AMS were funded by PhD fellowships from FCT-POPH (SFRH/BD/73379/2010 and SFRH/BD/85968/2012). The authors declare no other conflicts of interest in this work.