Chondroitin Sulfate Nanovectorized by LC-PUFAs Nanocarriers Extracted from Salmon (Salmo salar) by Green Process with Decreased Inflammatory Marker Expression in Interleukin-1β-Stimulated Primary Human Chondrocytes In Vitro Culture

Abstract

Chondroitin sulfate (CS), a glycosaminoglycan, supports health through various physiological functions, including tissue protection, bone growth, and skin aging prevention. It also contributes to anticoagulant or anti-inflammatory processes, with its primary clinical use being osteoarthritis treatment. This study presents the results of the valorization of lipids and CS, both extracted from salmon co-products through enzymatic processes. The polar lipids, naturally rich in long-chain fatty acids (docosahexaenoic acid DHA C22:6 n-3 and eicosapentaenoic acid EPA C20:5 n-3), and the CS, primarily located in the nasal cartilage, were separated and concentrated before being characterized using various techniques to determine functional and lipid composition. These compounds were then used to formulate liposomes of 63 to 95 nm in size composed of 19.38% of DHA and 7.44% of EPA and encapsulating CS extract with a Δdi-4S/Δdi-6S ratio of 0.53 at 2 weight masses (10-30 kDa and >30 kDa) or CS standard all at two different concentrations. Liposomes were tested on human chondrocytes in inflamed conditions. Thus, compatibility tests, the expression of various inflammation markers at transcriptional and molecular levels, nitrites, and the amount of collagenase produced were analyzed. The results showed that CS, in synergy with the liposomes, played a positive role in combating chondrocyte inflammation even at a low concentration.

Keywords: Salmo salar; anti-inflammatory; chondroitin sulfate; encapsulation; enzymatic hydrolysis; liposome; phospholipid.

Figure A1

Calibration range of 4S and 6S isomers.

Figure 1

FT-IR spectra of (a) CS and (b) lipids all extracted from Salmo salar heads by enzymatic hydrolysis.

Figure 2

Monitoring Δdi-4S and Δdi-6S disaccharides in (a) reference mixture of hydrolyzed commercial marine CS by UV₂₄₀ in panel 1 and by MS² in panels 2 (specific screening of delta Δdi-4S with daughter ion m/z = 300) and 3 (specific screening of delta Δdi-6S with daughter ion m/z = 282); and in (b) sample of interest by UV₂₄₀ for semi-quantitative evaluation.

Figure 3

Biocompatibility of liposomes with CS with human chondrocytes. Human chondrocytes exposed to nanoliposomes (250 μg/mL) or nanoliposomes and CS extracts (250 μg/mL + 125 or 25 μg/mL) for 1 days. (A) Lactate Deshydrogenase (LDH) release determined as described under Section 3. Metabolic activity assessed using MTT assay. (B) Cell metabolic activity results on different membranes presented in % vs. control results (as 100%). (C) DNA concentrations measured to estimate proliferation of cells. Results shown are mean ± SD of at least four individual experiments. *: p < 0.01, compared to control.

Figure 4

Effect of nanoliposomes/CS exposure on IL-1β-stimulated Cox/mPGES pathways. Human chondrocytes stimulated or not with 1 ng/mL IL-1β and exposed to nanoliposomes (250 µg/mL) or nanoliposomes and CS extracts (250 µg/mL + 25 µg/mL). For (A,B,D,F), culture conditions performed for 6 h for early inflammation markers (COX-2 and iNOS mRNA) and 24 h (Aggrecan and mPGES-1 mRNA). Total RNA extracted, then reverse transcribed into cDNA and analyzed by Real-Time Polymerase Chain Reaction (RT-PCR). Relative abundance of Cox-2, mPGES, iNOS, and Aggrecan mRNAs normalized to Retinitis Pigmentosa 29 (RP29) mRNA. Comparison made by using ΔCt method with fold value of reference = 1. Results shown are mean ± SD of at least four individual experiments (*: p < 0.01 vs. control; #: p < 0.01 versus IL-1β). For (C,E), culture conditions performed for 48 h for PGE2 and of nitrites (*: p < 0.01 vs. control; #: p < 0.01 vs. IL-1β).

Figure 5

The effect of nanoliposomes/CS exposure on IL-1β-stimulated MMPs. Human chondrocytes were stimulated or not with 1 ng/mL IL-1β and exposed to nanoliposomes (250 µg/mL) or nanoliposomes and CS extracts (250 µg/mL+ 25 µg/mL). For (A,B,E), total RNA was extracted after 24 h stimulation then reverse transcribed into cDNA and analyzed by RT-PCR. The relative abundance of MMP1, MMP3, and MMP13 mRNAs was normalized to RP29 mRNA. Comparison was performed using the ΔCt method with the fold value of reference = 1. The results shown are the mean ± SD of at least four individual experiments (*: p < 0.01 vs. control; #: p < 0.01 vs. IL-1β). For (C,D,F) levels of MMP1, 3, and 13 after 48 h of stimulation (*: p < 0.01 vs. control; #: p < 0.01 vs. IL-1β).

Figure 6

Recapitulative graphical abstract of study design and objectives.