Fig 1: Assessment of the cellular toxicity and haemo and immune nano-bio interface of human phenylalanine hydroxylase (hPAH) chitosan/tripolyphosphate (CS/TPP) nanoparticles (NP) modified with hyaluronic acid (HA) and cyclodextrin (CDs) derivatives. (A) Cellular viability of HEK293T (light and dark blue) and HepG2 (light and dark green) upon incubation with empty (light blue and light green) and hPAH loaded (dark blue and dark green) NP; Control refers to the cells assays performed only with culture medium. (B) Haemolysis degree monitored by the cyanomethaemoglobin method after incubation of empty (light orange) and hPAH-loaded (dark orange) NP in whole blood; TritonX100 represents total haemolysis and PBS buffer in the negative control assay; (C) Platelet activation monitored by the release of CD62P incubation of empty (light orange) and hPAH-loaded (dark orange) NP in platelet-rich plasma. (D) Complement activation monitored by the release of C3a desArg fragment after incubation of empty (light orange) and hPAH-loaded (dark orange) NP in plasma; (E) Coagulation assays of PT (bars) and APTT (symbols) after incubation of empty (light orange) and hPAH-loaded (dark orange) NP in plasma. In (C,D), plasma was used as the control. In all assays the naked hPAH (hPAH) was also used as a control. NPs were obtained using 5:1 CS-TPP and CS:HA-300 at 200:1 mass ratio; CS:HP-ß-CD 0.99 and CS:CM-ß-CD at 2:1 mass ratio; hPAH encapsulation at a final concentration of 250 µg/mL. Values represent mean ± S.D. (n = 3).
Fig 2: Haemocompatibility profile of the chitosan.tripolyphosphate (CS-TPP) nanoparticles modified with hyaluronic acid (HA) and cyclodextrins (CDs). The CS-TPP matrix (CS:TPP at 5:1 mass ratio) was modified with low (CS:HA 200:1) and high loading (CS:HA 66:1) of HA with different molecular masses (HA-50, HA-300 and HA-1000) and different CD (ß-CD, ?-CD, HP-ß-CD 0.65, HP-ß-CD 0.99 and CM-ß-CD) at high (CS:CD 2:1) and low loading (CS:CD 1:1). (A) Haemolysis percentage monitored by the cyanomethaemoglobin method after nanoparticle incubation in whole blood; (B) Coagulation assays of PT (bars) and APTT (symbols) after nanoparticle incubation in plasma; (C) Platelet activation monitored by the release of CD62P after nanoparticle incubation in platelet-rich plasma; (D) Complement activation monitored by the release of C3a desArg fragment after nanoparticle incubation in plasma. (CS) CS-TPP non-modified nanoparticles. Values represent mean ± S.D. (n = 3). See text for details. In (B–D) the lines represent the reference values of control plasma.
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