Charge converting nanostructured lipid carriers containing a cell-penetrating peptide for enhanced cellular uptake


The aim of this study was the development of nanostructured lipid carriers (NLCs) decorated with a polycationic cell-penetrating peptide (CPP). A coating with polyphosphates (PP) enables charge conversion at target cells being triggered by the membrane bound enzyme intestinal alkaline phosphatase (IAP).


The CPP, stearyl-nona-L-arginine (R9SA) was obtained by solid phase synthesis. Formed nanocarriers were characterized regarding size, polydispersity index, zeta potential and charge conversion in the presence of IAP and on Caco-2 cells. The BCS class IV drug saquinavir (SQV) was loaded into NLCs in different concentrations. Mucus diffusion ability of the NLCs was evaluated by the rotating tube method. Furthermore, cellular uptake was evaluated on Caco-2 cells and endosomal escape properties were investigated using erythrocytes.


All NLCs were obtained in a size range between 146 nm and 152 nm and a polydispersity index of 0.2. Incubation of PP coated PP-R9SA-NLCs with IAP led to a charge conversion from −41.8 mV to 6.4 mV (Δ48.2 mV). After four hours of incubation with IAP, phosphate release reached a plateau, indicating a faster polyphosphate cleavage than on Caco-2. Drug load and encapsulation efficiency of SQV was obtained up to 80.6% and 46.5 µg/mg. Mucus diffusion was increasing in the following rank order: R9SA-NLCs < blank NLCs < PP-R9SA-NLCs. R9SA-NLCs and PP-R9SA-NLCs increased the cellular uptake 15.6- and 13.2-fold, respectively, compared to the control NLCs. Erythrocytes interaction study revealed enhanced endosomal escape properties for R9SA-NLCs and PP-R9SA-NLCs when incubated with IAP.

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Nona-L-arginine (≥98%) was purchased from Shanghai Hanhong Chemical Co. (Shanghai, China). Acetone was obtained from DonauChem (Vienna, Austria). Lumogen red (LGR) was a kind gift from BASF (Ludwigshafen, Germany). Miglyol® 840 (middle-chain triglycerides) was received by IOI Oleo GmbH (Witten, Germany). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, ≥ 99.5%) and N,N-dimethylformamide (DMF, ≥ 99.5%) was obtained from Roth GmbH (Karlsruhe, Germany). Precirol ® ATO 5 (glyceryl distearate) was a free sample from Gattefossé (St. Priest, France). D-Glucose anhydrous (≥99.5%), sodium chloride (≥99.5%), and acetonitrile (≥99.5%) was received from VWR Chemicals (Solon, USA). Phosphate buffered saline (PBS), penicillin, streptomycin, and fetal bovine serum (FBS) were obtained from Merck (Tutzing, Germany). Lipoid S100 (soy lecithin) was a gift from Lipoid GmbH (Ludwigshafen, Germany). Alkaline phosphatase from bovine intestinal mucosa (≥10 DEA U/mg), ammonium molybdate tetrahydrate (81.0–83.0%), magnesium chloride anhydrous (≥98%), sodium polyphosphate (Graham’s salt, ≥ 99.7%), zinc chloride anhydrous (≥97%), potassium phosphate monobasic (KH2PO4, ≥ 99%), phosphatase inhibitor cocktail 2, Triton X-100, malachite green oxalate salt (MLG), minimum essential medium eagle (MEM), resazurin sodium salt, stearic acid (≥98.5%), trypan blue (0.4%), trifluoroacetic acid (99%), 1-hydroxybenzotriazol (HOBT, ≥ 97%), dichloromethane (DCM, ≥ 99.8%), N,N-diisopropylethylamine (DIPEA, ≥ 99.5%), pepsin from gastric mucosa and pancreatin from porcine pancreas were purchased from Sigma-Aldrich (Vienna, Austria). Saquinavir was obtained from Biosynth (Staad, Switzerland). FaSSIF powder was received from Biorelevant (London, U.K.).

Patrick Knoll, Nikolas Hörmann, Nguyet-Minh Nguyen Le, Richard Wibel, Ronald Gust, Andreas Bernkop-Schnürch, Charge converting nanostructured lipid carriers containing a cell-penetrating peptide for enhanced cellular uptake, Journal of Colloid and Interface Science, Volume 628, Part A, 2022, Pages 463-475, ISSN 0021-9797,

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