Our initial liposome preparations were composed of DPPC: Lyso PC: DSPE-MaL-PEG2000 and DiR, a lipophilic probe with excitation/emission in near infrared region was incorporated into liposomes (, ), for future in vivo
imaging application. Similar lipid compositions have been reported previously for thermo-sensitive liposomes (19
). However, we were unable to reproducibly entrap calcein (a water-soluble fluorescent probe, λex/em 490/515 nm) into these liposomes. Therefore, we tested formulations without lyso-PC (a destabilizing lipid). We entrapped calcein at its self-quenched concentration (0.1–0.15 M) in liposomes containing DPPC/DSPE-MaL-PEG2000/DiR (, and Methods section). Kinetics of temperature-induced release of entrapped calcein from liposomes was determined by monitoring increase in fluorescence due to relief of self-quenching by spectrofluorometry. Calcein was released from Thermosensitive liposomes (Control-II and Control-III, ) in a temperature-dependent manner, with 90–100% leakage at 41 °C (). Non-thermosensitive liposomes prepared from egg PC (Control-I, ), however, under similar conditions released only 5–10% calcein at 41°C. These data confirmed suitability of our liposomes for triggered release application. Analysis of hydrodynamic size distribution of these liposome preparations was conducted by dynamic light scattering measurements (see below, for details). All our formulations exhibited an average size distribution of 80–100 nm. We also observed that incorporation of the DSPE-MaL-PEG2000 lipid in liposomes neither altered thermosensitivity nor calcein entrapment efficiency of our liposomes (data not shown). Next, these liposomes were tested for conjugation of ZHER2:342
-Cys Affibody molecules.
ZHER2:342-Cys Affibody Conjugation to Thermosensitive Liposomes
Steps involved in liposome-affibody conjugation are summarized in . The conjugation of affibody to the liposome surface was based on chemical reaction between maleimide of the lipid with the thiol group on the C-terminus of Affibody, as previously described for conjugation for anti-HER2 antibodies to the liposome surface (16
). We used a ratio of 1:5 or 1:10 of Affibody:DSPE-MaL-PEG2000 for conjugation (see Methods section) to achieve optimal affibody coupling. Initial analysis of affibody-liposome conjugation was done by separation of unconjugated affibody from the conjugated product on a size exclusion column. Elution profile of affibody-conjugated liposomes (Affisomes) on a BioGel A0.5m column (1×40 cm) is shown in . All fractions were analyzed for entrapped calcein and DiR fluorescence, representing liposomes that are eluted into the void-volume (fractions 8–12) of the column. On the other hand, free affibody (even in its dimer form) was fractionated in the included volume of the column (fraction 20 and beyond, data not shown).
Steps involved in Conjugation of Affibody Molecule to Liposome Surface.
Our initial attempts to directly analyze liposome-conjugated affibody quantity in various fractions by using protein assay kits were unsuccessful because of high background and extremely low protein ratios in the liposomes. Therefore, presence of affibody in various liposome fractions was assayed by analyzing the fractions by gel electrophoresis (see Methods section). For quantitation of affibody, various concentrations (0.25 –1 μg) of reduced affibody were used and a calibration curve was generated (not shown, see Methods section). It is clear from the elusion profile of liposomal lipid and affibody () that affibody co-eluted in the liposome fraction only when DSPE-MaL-PEG2000 was incorporated in liposomes (Affisomes, see ). Non-targeted-SUV prepared without DSPE-MaL-PEG2000-lipid (control-II, see ), only co-eluted a very small concentration of affibody, presumably due to non-specific association of affibody with liposomes. The affibody conjugation in the liposome fractions was further confirmed by gel electrophoresis (). A ~3 kDa increase in molecular weight of affibody (M.W. 8.3 kDa) in Affisomes due to the conjugation with DSPE-MaL-PEG2000 (M.W. 2.9 kDa) was present (, lane 6). Treatment of Affisomes with dithiothreitol (DTT) did not affect the mobility of conjugated affibody (, lane 7). In contrast, free affibody (lane 3, ) upon treatment with DTT was reduced to monomer (, Lane 4). Control-III liposomes (without affibody conjugation) did not show any bands corresponding to the affibody (, Lane 8). Additional controls (lipid compositions without maleimide functional group (Control-II) or incubation of Affisomes with non-reduced affibody) were included to determine specificity of affibody-conjugation via the maleimide-SH reaction. In either case we did not observe any association of affibody to the liposomes above background levels confirming the specificity of this reaction (data not shown). Analysis of population size distribution of the liposomes before and after conjugation with affibody showed that conjugation did not have any significant effect ().
Since we aimed at developing affibody-conjugated liposomes to improve targeting potential without compromising their thermosensitivity, we evaluated the effect of affibody-conjugation on temperature-dependent release of entrapped calcein (see ). Calcein loaded Affisomes and non-conjugated Thermosensitive liposomes (Control III) were examined for temperature-triggered calcein release from liposomes (due to relief of self-quenching) at λexcitation/emission of 490/515nm in a Fluorimeter (see Methods section). The results presented in show that Control-III liposomes show ~100% release of encapsulated calcein at temperatures at and above 40°C, with only a slight calcein release at 39°C. We made similar observations when Affisomes were tested. Both liposomes released calcein only at or above 40°C, confirming that affibody conjugation did not alter thermosensitivity of liposomes.
Effect of Affibody Conjugation on thermo-sensitivity of Liposomes
For future medical applications, it is critical that physical properties of Affisomes are not altered upon storage or upon interactions with plasma components prior to targeting to permissive cells and/or tissues. Therefore, we examined stability of Affisomes in the presence of serum by monitoring release of calcein. Affisomes were incubated in PBS supplemented with 10% heat-inactivated serum at 37°C for various time periods, and extent of calcein release was measured. Results are shown in . We did not observe any significant release of calcein from Affisomes or non-conjugated thermo-sensitive liposomes (Control-III) in the presence of serum up to 4 hours incubations. Therefore, affibody-conjugation to thermosensitive liposomes had no significant effect on the natural leakage of entrapped calcein. In addition, we also did not observe any change in thermosensitivity of liposomes following incubation with 10% serum (data not shown).
Stability of Affisomes in the presence of serum at 37°C
The susceptibility to aggregation of Affisomes and control liposomes was assessed after incubation for 0, 5 and 7 days at 25 °C. The inorganic Pi, average diameter and calcein release was determined following a low speed spin to remove any aggregated material (see Methods section). The results are summarized in . We observed only slight differences (±7.5%) in the amounts of lipids in the supernatants at days 0, 5 or 7 for Affisomes and Control-III liposomes. Similarly, there was no significant difference in the population size distribution (data not shown) and average diameter of Affisomes (86–94 nm) or control-III liposomes (82–86 nm). Next, we assayed the amount of entrapped calcein in these liposomes. It is evident from the data (shown in ) that affibody-conjugation did not have an adverse effect on the time-dependent integrity of liposomes (control-III liposomes, 2.8–3.73%, Affisomes, 3.0–4.0%, days 0–18 respectively). Stability of affibody-DSPE-MaL-PEG2000 conjugates, was further analyzed by gel electrophoresis (see Methods section). The results presented in show that there was no difference in the mobility of 11.3 kDa band for Affisomes at day 0 (Lane B, left panel) or day 7 (Lane B, right panel). The free affibody, before (Lane A) and after (treatment with reducing agent (Lane D) and Control-III liposomes (Lane C) are also shown in for comparison.
Physical Properties of Affisomes: Effect of incubation at 25°C
Stability of Protein Conjugation of Affisomes