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This reinforces the LL18-loaded VD3-Dex micelles potential in improving infection eradication from osteoblasts, and to a lesser extent from BMMΦ, by shifting the internalization mechanism of the peptide to an endocytosis mechanism through lysosomal compartments.

Although LL18 encapsulation had no apparent influence on the intracellular distribution of the micelles, uptake by BMMΦ cells was considerably increased both at 0.5 mg/mL (*p < 0.05) and 1 mg/mL (***p

< 0.001) during 1 h incubation, in a dose-dependent manner (Figure 4.12). The loading of LL18 resulted in a lower surface charge and higher average size for the FICT-VD3-Dex micelles (Table 4.2), which may have contributed to the higher intake. This faster uptake when LL18 is present may lead to cytotoxicity, the micelles being unable to effectively provide a peptide shielding effect. The null hemolytic activity of both unloaded and loaded micelles – where uptake by endocytosis may be diminished – reinforces this uptake-dependent toxicity effect.

Figure 4.12 – Concentration dependence of the intracellular uptake of FITC-VD3-Dex and LL18-loaded FITC-VD3-Dex by BMMΦ cells, after 1 h incubation: (A) electronic microscope observation (20×

magnification), and (B) uptake quantification. Results presented as mean ± SD, n = 3, (*p < 0.05, **p <

0.01, ***p < 0.001). Formulation of 100 µg:1 mg peptide:micelle ratio.

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In our work, LL18 in the concentration range 50 – 500 µg/mL induced an overwhelming dose-dependent enhancing effect on albumin denaturation, which was drastically reduced by incorporation into VD3-Dex micelles (Figure 4.13A). As heat-induced denaturation is also time-dependent, significant albumin denaturation was also demonstrated for lower LL18 concentrations upon extended incubation periods.

LL18 has recently been shown to induce concentration-dependent tissue damage in a rat model of OM

27. Our results suggest that LL18-induced protein denaturation may also play an important role in increased inflammation in vivo.

LL18-loaded micelles revealed also a dose-dependent enhancing effect on albumin denaturation, though much less pronounced. Empty micelles revealed a dose dependent protective effect, reducing albumin denaturation compared to untreated condition. The protective effect could be ascribed to VD3 (probably the micellar structure being required as well for this effect) as dextrin per se seems to have a neutral effect on albumin denaturation. Albumin shares a considerable amino acid homology and structural similarity with Vitamin D3-binding protein (DBP). DBP, and to a lesser extent albumin, bind with high affinity to circulating, otherwise insoluble, VD3 metabolites through a specific protein binding site composed of hydrophobic residues ⁷⁴. The stability of DBP at 60 ºC was markedly enhanced in the presence of 25-hydroxyvitamin D3 ⁷⁵. Negatively-charged serum albumin displays a binding affinity for short cationic AMPs, a process exclusively driven by interactions with peptides´ hydrophobic residues rather than the (antimicrobial relevant) cationic residues ⁷⁶.

Overall, we propose that VD3-Dex micelles prevent LL18-induced albumin denaturation at high temperatures by means of a shield effect, leaving no free peptide molecules for albumin binding, and possibly also by a stabilizing effect provided by hydrophobic interaction with conjugated VD3, by a process requiring further investigation.

Cell migration is essential for bone formation. In a bone fracture of zebrafish model, osteoblasts were shown to move to the site of injury to replace damaged tissue, particularly by active migration rather than passive displacement caused by cell proliferation ⁷⁷. Up-regulation of MC3T3-E1 migration in vitro was associated to accelerated fracture healing in a rat femur ⁷⁸. In this work, cell migration capacity was studied (Figure 4.13B) in the presence of mitomycin C, to exclude cell proliferation (Figure 4.14).

The peptide significantly stimulated MC3T3-E1 migration after 24 h, resulting in lower open wound areas at the lowest concentrations tested of 6.25 (43.7 %) and 12.5 µg/mL (41.5 %) comparing to 25 (53.7 %) and 50 µg/mL (55.7 %). LL18 has previously demonstrated angiogenic and chemoattractant potential, improving burn wound healing in a rat model ²⁵. VD3-Dex micelles also reduced open wound area compared to the control from as low as 0.06 (59.5 %) and 0.125 mg/mL (57.4 %), but not from 0.25

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mg/mL upwards. It is interesting to note that 0.0625 mg/mL is below VD3-Dex calculated CMC (0.08 mg/mL), evidencing that the process of disassembly somehow does not abrogate the biological function.

Considering the apparent null effect of dextrin, this outcome may be attributed to conjugated VD3.

Loading LL18 into micelles resulted in an intermediate effect between empty micelles and free LL18, reaching 52.9 % and 51.1 % at 0.065 and 0.125 mg/mL, respectively. Although peptide shielding seems to slightly diminish LL18 performance, this is counterbalanced by the positive stimulation of VD3 in the nanocarrier. As shown in our subcellular localization results, micelles changed the biodistribution of the peptide from the perinuclear region to lysosomes. The lower propensity for accelerated wound healing provided by loaded LL18 suggests that the presence of free peptide in the perinuclear region of osteoblasts is somehow required for the migration process. Thus, in this particular cell line, providing cathelicidin through lysosomes (where S. aureus has been traced) and perinuclear region simultaneously seems important to enhance both antimicrobial and tissue repair capabilities. LL18-loaded VD3-Dex may offer the targeting of exogenous cathelicidin to lysosomes allied to endogenous production of cathelicidin via VDR activation ^32,79.

Antimicrobial activity of LL18 against S. aureus reduced viability, at best, to 23.9 ± 5.0 % at 200 µg/mL, without further improvement up to 500 µg/mL (Figure 4.13C). LL18-loaded micelles, with LL18 concentrations of 15 and 17.5 µg/mL, reduced bacterial viability by 93.1 % and 100%, respectively.

Whereas dextrin displayed no activity, VD3-Dex micelles eradicated bacteria at 0.5 mg/mL concentration, an effect that may be assigned to VD3. The anti-microbial action of VD3 is drawing great attention, mostly on the basis of induced expression of cathelicidin via VDR-VDRE gene activation ^29,30. Compelling evidence of antimicrobial action of VD3 by direct contact is scarce in literature, with most studies reporting bactericidal improvements only by concomitant administration with antibiotics ^80,81. The underlying VD3 antimicrobial mechanism is likely to be related to its high lipophilicity ^80–82, solubility in the bacterial membrane and binding to efflux transporters, thus avoiding resistance mechanisms. It can alter the structure of the bacterial lipoprotein membrane, resulting in damage to components that are essential for the membrane integrity, thereby enhancing antibiotic uptake. Indeed, a study reported enhanced antibiotic activity by VD3 against resistant strains of S. aureus by affecting the bacterial of efflux systems

80.

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Figure 4.13 – (A) Albumin (1 % w/v) denaturation after 3h at 70 ⁰C (n=3), (B) quantification (%) of open wound area in MC3T3-E1 cells after 24 h (n=6), and (C) antimicrobial activity against S. aureus after 24 h (n=4). Results presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). Formulation of 100 µg:1 mg peptide:micelle ratio.

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24 72

0.0 0.1 0.2 0.3 0.4 0.5

Time (hours)

Absorbance (a.u.)

0 0.005 0.01 0.05 0.1 0.25 0.5 1

Figure 4.14 – Dose (µg/mL)-response results for MC3T3-E1 cells exposed up to 72 h to different concentrations of mitomycin C, assessed by the MTT reduction assay. Results are expressed in absorbance values. Data are presented as mean ± SD (n = 3).

Drug-shielding is usually accompanied by diminished drug performance. The advantage relies in the balance between cytotoxicity and function. Table 4.3 shows a summary of the balance between cell toxicity and antimicrobial activity attained by LL18-loaded micelles. The association between LL18 and VD3-Dex resulted in a synergistic partnership with highly improved MIC, absence of hemolysis, approximating antibacterial concentrations (≥ 0.15 mg/ml) towards formulations with accelerated cell migration (≤ 0.125 mg/mL). Since a functional concentration range is far below CC50, an increase in peptide loading could be considered to achieve an optimized formulation in the future.

Table 4.3 – Summary balance of cytotoxicity and antimicrobial activity.

CC⁵⁰, 50 % cytotoxic concentration; CI, confidence interval (n=3); MIC, minimal inhibitory concentration;

SI, Staphylococcus aureus, S. aureus; Selectivity Index = CC⁵⁰/MIC.

Sample LL18 Loaded LL18 LL18-Loaded

Micelles

Unloaded

Micelles Dextrin BMMΦ CC⁵⁰ 91.4 µg/mL

(CI 83.8 - 100.0) 123.4 µg/mL

(CI 116.5 - 130.7) 1.23 mg/mL

(CI 1.17 - 1.31) Cell viability > 50% up to 3 mg/mL MC3T3-E1

CC⁵⁰ 103.5 µg/mL

(CI 91.7 - 118.3) 153.4 µg/mL

(CI 132.9 - 191.0) 1.53 mg/mL (CI 1.33 - 1.91) MIC

S. aureus > 500 µg/mL 17.5 µg/mL 0.175 mg/mL 0.5 mg/mL - BMMΦ

SI - 7.05 7.05 - -

MC3T3-E1

SI - 8.77 8.77 - -

Hemolysis Hemolytic Non-hemolytic

Non-hemolytic

Non-hemolytic

Non-hemolytic

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