br Morphological analysis of nano liposomes br TEM
3.2. Morphological analysis of nano-liposomes
TEM images detailed the formation of regular, consistent NLS as illustrated in Fig. 1(I). Fluorescence microscopy was employed to con-firm the restoration of NLS structure and subsequent conversion to NLBs. The fluorescence micrographs of the CHO-NLBs and DSPE-NLBs are displayed in Fig. 1(II), respectively, highlight the resilience of the bi-layer lipid membrane structure and the restoration of the spherical NLB structure.
3.3. Establishing the efficiency of camptothecin incorporation
The efficiency of CPT incorporation for candidate CHO- and DSPE-NLS demonstrated an 82.41% and 59.19% CPT entrapment for the CHO-NLS and DSPE-NLS systems respectively. Conversion of NLS to NLB did not result in significant change in drug entrapment efficiency (DEE). However, lyophilization followed by reconstitution resulted in ˜2% decrease in DEE for the CHO-NLB and DSPE-NLB systems.
Whilst the application of polymeric coating significantly slowed the release of CPT from the CHO-NLBs at both physiologic and tumoral pH, the disparity in release characteristics was considerably more acute at pH 7.4 (Fig. 2). The bi-phasic release pattern observed with the NLBs was distinctly absent, with the release profile taking on a more constant linear shape (Fig. 2A). The cumulative release of CPT achieved at 24 h was < 50%. This is a considerable extension of the less than 1 h V5 Epitope Tag Peptide of CPT in aqueous medium as reported in the literature . In addi-tion, less than 7% of CPT was released from the NLB-DDS in the first hour of analysis, highlighting the absence of burst release. The achievement of the aforementioned release characteristics of CPT from the CHO- NLBs has the potential to address the toxicity profile of CPT which is one of the major drawbacks limiting the clinical application of this broad-spectrum antineoplastic agent.
revealed a favorable decrease in the release of CPT over the period of investigation. Moreover, the release profile of CPT from the CHO-NLB formulation had a notably more linear appearance suggesting a more controlled manner of release. However, beyond the first hour the re-lease of CPT from the polymer coated NLB-DDS was substantially more rapid than observed at physiological pH. A cumulative release of ˜63% CPT was achieved over the 24-hour period, which was more than 14% higher than CPT release attained from the CHO-NLBs over the same period. This was attributed to the pH-responsive property of CHT which was incorporated in the polymeric layers coating the NLBs. CHT is a linear polysaccharide that demonstrates aqueous solubility up to pH 6.2, due to the protonation of glucosamine units at this lower pH .
Evaluation of the release characteristics of CPT from the DSPE-NLBs bore a strong resemblance to that obtained from the CHO-NLBs in both a physiologic and tumoral pH release medium. This observation was attributed to lower stability of the DSPE-NLBs, due to the less anionic surface charge, as well as the higher permeability of DSPE-NLBs through the lipid membrane. Release of CPT from DSPE-NLBs was faster over the first 8 h of evaluation. The cumulative release of CPT from the DSPE-NLBs at physiological and tumoral pH was approximated to be 50% and 58%, respectively.
SB is a naturally occurring polyphenol antioxidant extracted from milk thistle (Silybum marianum) and has an excellent safety profile, being well tolerated at considerably high doses following different routes of administration [18–21]. It is also known for its anti-cancer properties where the mechanism of its anti-tumor effect has been at-tributed to factors such as the promotion of cellular antioxidant defense mechanisms, angiogenesis antagonism by a reduction in VEGF secre-tion, induction of apoptosis and antiproliferative effects due to cell cycle arrest [18,20,22]. SB, however, has a limited aqueous solubility and undergoes metabolism by phase II conjugation. The incorporation of silibinin in the formulated NLB-DDS was therefore undertaken to enhance the cytotoxic activity of the formulations and provide a means of effective delivery of this poorly soluble phytochemical. Incorporation of silibinin into the formulated NLB-DDS revealed a size of 137.56 nm and 93.65 nm, a zeta potential of -27.58 mV and -8.24 mV and a drug incorporation efficiency of 65.59% and 52.75% for the CHO-NLB and DSPE-NLB systems respectively.
The release pattern of CPT from CHO-NLBs containing SB (CHO-NLB + SB) demonstrated no outstanding differences to that of SB for-mulations for the first 10 h, except for an evident burst release of CPT over the first hour (Fig. 2A). A similar burst release of SB was observed over this period, displayed in Fig. 2B, suggesting association of both compounds to a certain degree with the surface of the NLBs. Further-more, the presence of the additional SB compound may have altered the surface tension of the formulated CHO-NLBs, leading to the initial burst release of both CPT and SB. From 10 h the release of CPT from CHO-NLB + SB is approximately 7–9% higher than that observed for CHO-NLB without SB. An effect of the different pH of release medium (7.4 and 6.0) employed during analysis only became evident after 10 h, when the release of CPT at pH 6.0 appeared to be slightly higher than that at pH 7.4. However, the effect of tumoral pH on the release characteristics of CPT from CHO-NLB + SB was still considered negli-gible. A cumulative CPT release of 82–86% was achieved for CPT from CHO-NLB + SB over the 24-hour investigation.