Población y muestra del estudio

IC50 values can only be estimated for the poly(L-lysine) samples during the evaluation of the poly(amidoamine)s and the results obtained are comparable with the literature (Sgouras, 1990), consequentially, its usefulness as a control is not compromised. When CCRF-CEM cells are used to evaluate the IC50 of poly(L- lysine), (Mw 57 000 Da) Sgouras, (1990) reported a value of 23.90pg/ml. The IC50 value obtained herein using the same experimental conditions gave a value of 12.00±80.00pg/ml. If the experiment is repeated using Hep G2 cells, Sgouras (1990) reports an IC50 value of 59.90pg/ml and the value obtained herein is 50.00+10.OOpg/ml.

In general the human melanoma cell line seemed to be more sensitive to the effects of poly(amidoamine)s than the mouse melanoma B16 FIO cells, though, the apparent differences were not statistically significant. When the mechanisms of polycation mediated membrane rupture documented in Nevo et al, (1955), Kachalsky et al, (1959) and Hartmann and Galla, (1978) are considered (Chapter 1), this result is consistent with the literature (Sgouras, 1990). The IC50 values for poly(L-lysine) are approximately 100 times less than those of ISA 22 and ISA 23. Poly(ethylenimine) (Mw 70 000) has been shown to have an IC50 value of 6.5pg/ml when incubated with Hep G2 cells (Sgouras, 1990) and it is nearly 1000 times more toxic than the poly(amidoamine)s assayed against Hep G2 cells herein. When PAMAM dendrimers are compared with the linear poly(amidoamine)s evaluated herein, generation 3 and 4 dendrimers have been reported to be cytotoxic at concentrations above lOOpg/ml against B 16 FIO

Chapter 3 Biocompatihility of Chitosan and Poly(amidoamine)s

Figure 3.23 Scanning electron micrographs sowing typical RBC morphology following either 1 or 5h exposure to chitosans at a concentration of Img/ml

Panel (a) RBCs following Ih exposure to Nl at Img/ml (x2 140)

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Panel(b) RBCs following 5h exposure to Nl at Img/ml (x2 137)

Panel (c) RBCs following Ih exposure to N2 at Img/ml (x2 749)

Panel (d) RBCs following 5h exposure to N2 at Img/ml (xl 068)

Panel (e) RBCs following Ih exposure to N3 at Img/ml (x2 473)

Panel (f) RBCs following 5h exposure to N3 at Img/ml (xl 068)

Figure 3.24 Scanning electron micrographs showing typical control RBC morphology

Panel (a) RBCs following Ih exposure to poly(L-lysine) at Img/ml (x4 216)

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a o o k v - t o i w i t i x L * 103 i ' o î r f l l y s m e K - ^ l m ^ m l b l i o m

Panel (b) RBCs following 5h exposure to poly(L-lysine) at Img/ml (xl 066)

Panel (c) RBCs following Ih exposure to dextran at Img/ml (x2 108)

Panel (d) RBCs following 5h exposure to dextran at Img/ml (x2 141)

Panel (e) RBCs following Ih exposure to PBS pH 7.4 (xl 054)

Panel (f) RBCs following 5h exposure to PBS pH 7.4 (xl 065)

Chapter 3 Biocompatibility o f Chitosan and Poly(amidoamine)s

cells (Malik et al., 1997). Other families of poly(amidoamine)s such as BPI (Mn 6

200) have been found to be quite cytotoxic (IC50 of SOqg/ml using CCRF-CEM cells) at relatively low concentrations, however poly(amidoamine)s such as EPF (Mw 6 300) have been shown to have IC50 values in excess of SOOpg/ml when tested on both CCRF-CEM cells and Hep G2 cells (Sgouras, 1990). This is encouraging as it has shown that it is possible to alter the degree of cytotoxicity associated with poly(amidoamine)s by altering the structure and relative charge of the poly(amidoamine) backbone.

Poly(amidoamine) ISA 1, 4 and 9 red blood cell interactions at pH 7.4 were concentration- and time-dependant. Figure 3.8 and 3.9 show a high degree of haemoglobin release relative to poly(L-lysine). This result is likely to indicate that these polymers may not be suitable for systemic administration at high doses. ISA 22 and 23 were less lytic than ISA 1, 4 and 9 at concentrations below 3mg/ml after 24h incubation (Figure 3.10 and 3.11). This is likely to be due to the differences in the structure of the polymer backbone and the less cationic nature of ISA 22 and 23 relative to ISA 1, 4 and 9 as described in Chapter 1.

At concentrations of 1 mg/ml all of the poly(amidoamine)s tested demonstrated pH-responsive haemoglobin release. This is best exemplified with ISA 22 and 23, which caused approximately 65% and 90% haemoglobin release, respectively after 24h incubation at pH 5.5 (Figure 3.15). As this response is not seen with cationic polymers such as poly(L-lysine), it may be attributable to the protonation of the poly(amidoamine) backbone. This observation was supported morphalogically with SEM (Figure 3.16). The difference in morphology between the rat RBCs that have been exposed to ISA 23 at 1 mg/ml for 24h at pH 7.4 (Figure 3.16 panel (b)) and those at pH 5.5. (panel (c)) is apparent. Panel (c) shows a total loss of RBC morphology as well as a high degree of membrane fusion mediated by ISA 23 in response to a drop in pH (5.5) after 24h incubation. This is not evident in panel (b) (pH 7.4, ISA 23 1 mg/ml, 24h incubation) where only a small degree of crenation is observed.

Chitosan has been widely reported to be a biocompatible substance though again the intended application of the material needs to be carefully considered

(Hirano et a i, 1988; Aspden et al, 1996; Lee et al, 1995; Heller et al, 1996). Although chitosan is an approved pharmaceutical excipient in Japan, this does not necessarily mean that it will not demonstrate toxicity if administered i.v. (Bodmer

et al, 1989; Carreno Gomez and Dunean, 1997). Although specific chitosan molecules have been reported to be cytotoxic in a concentration-, molecular weight-, charge- and counter-ion- dependent manner (Carreno Gomez and Duncan, 1997), the molecular weight fractions examined here in did not demonstrate any significant toxicity within the experimental parameters examined (Figure 3.19, 3.20 and Table 3.1). This may be because of the molecules reduced degree of deacetylation and molecular weight in comparison to the experimental molecules previously examined (Carreno Gomez and Duncan, 1997) which in the instance of CL210 had a 100% degree of deacetylation and a molecular weight of approximately 100 000 Da. It is also of note that only one polymer, of the five compounds tested in Carreno Gomez and Duncan, (1997) demonstrated any cytotoxicity below 1 mg/ml (Cl 210). Figure 3.17 and 3.18 show that there is a pronounced alteration in media pH at chitosan concentrations above 1 mg/ml. This could possible be responsible for the drop in cellular viability seen (Figure 3.20) at high chitosan concentrations both here and in the study of Carreno Gomez and Duncan, (1997).

All the chitosan preparations showed very little haemolysis at concentrations up to 5mg/ml at all of the time points tested (Figures 3.21 and 3.22). These findings are in agreement with previous experiments (Heller et al,

1996) and these molecules (Nl-3) demonstrate a better biocompatibility profile than chitosan Cl 210 manufactured by Pronova. This is because chitosans Nl-3 show a reduction in molecular weight and degree of deacetylation. Both these polymer attributes have been reported to be responsible for the red blood cell lysis and cytotoxicity. The RBC lytic profile of the ehitosans N l-3 examined herein were assayed at time points Ih and 5h as opposed to Ih and 24h as reported by (Carreno Gomez and Duncan, 1997). This is because of the likely plasma residence time of these moleeules as detailed in Chapter 5.

Chapter 3 Biocompatibility o f Chitosan