ORDINARIAS SEGUIMIENTO LEY ESTADO ACTUAL TOTAL

All experiments were performed using biodegradable and synthetic microcapsules assembled upon CaCO3 microparticles, following the procedures described in

Chapter 3.2.5. Microcapsules constitution is shortly described in Table 5.1. For some samples (LucI, L1, LP1 and LS1), luciferase was encapsulated by co-precipitation into the cores, while for others it was sedimented as one of the layers into the shell provided by the fact that luciferase molecules have significant negative charge. For LucII.1 and LucII.2 microcapsules luciferase was situated both in the shell and in the cavity. For LucI.1 and LucI.2 samples the shells were labelled by adsorption of FITC- PLL instead of one of PLA layers.

Luciferase containment in microcapsules was quantified by measuring the amount of luciferase left in supernatants collected after layers adsorption and CaCO3 cores

dissolvation and subtracting this figure from amount of luciferase initially introduced. Quantification was performed for L1-LS3 samples both for co- precipitated and adsorbed luciferase and incorporation ratios were believed to stay the same for other samples due to similarity of preparation techniques.

Sample

name Core Shell structure containmentLuciferase

LucI.1 luciferase {PLA/DS}2/FITC-PLL/DS/PLA/DS 30 μg

LucI.2 luciferase {PLA/DS}2/FITC-PLL/{DS/PLA}2 30 μg

LucII.1 luciferase {PLA/DS}3/luciferase/DS 95 μg

LucII.2 luciferase {PLA/DS}3/luciferase/DS/PLA 95 μg

L1 luciferase {PLA/DS}3 250 μg

L2 - PLA/DS/PLA/luciferase/PLA/DS 250 μg

L3 - {PLA/DS}2/PLA/luciferase 250 μg

LP1 luciferase {PLA/DS}3/PEI 250 μg

LP2 - PLA/DS/PLA/luciferase/PLA/DS/PEI 250 μg

LP3 - {PLA/DS}2/PLA/luciferase/PEI 250 μg

LS1 luciferase {PAH/PSS}2/PAH/luciferase/PEI 250 μg

LS2 - PAH/PSS/PAH/luciferase/PAH/PSS/PEI 250 μg

LS3 - {PAH/PSS}2/PAH/luciferase/PEI 250 μg

Table 5.1: Description of microcapsules prepared for experiments with

luciferase. Estimated containment of luciferase in microcapsules (as prepared)

Quantification of luciferase containment in supernatant meant that a separate calibration curve was needed for each type of supernatant. Then it was discovered, that PAH, PSS and PEI even at low concentrations have a significant effect on luciferase activity (see Fig. 5.2). Despite extensively using polyelectrolyte-enzyme complexes229 _{(initially for purification reasons) and reports on high activity retention}

in these complexes229,230 _{and even in microcapsules,}111 _{it is obvious that activity}

retention depends both on the enzyme and polyelectrolyte used, and suppressing effect of polyelectrolyte on enzyme was also reported.231 _{This fact was taken into}

account when dealt with calculations of luciferase concentration in each of supernatants analysed.

For each of L1-LS3 samples (see Table 5.1), 2 mL of Na2CO3 and 2 mL of CaCl2 at

0.33M concentration were mixed to prepare templates, and to that amount of calcium carbonate microparticles 250 μg of luciferase was co-precipitated or deposited. The measurements have shown, that about 99% of initially introduced luciferase stayed in the capsules. Such high incorporation ratio proposes lack of luciferase to cover all the surface of microparticles and to fill all their pores. Thus, if the amount of luciferase introduced would be more, encapsulation ratio would decrease. If a mean diameter of microparticles is 4 μm, it gives S0=πD2/3=16 μm2

surface area of individual microparticle. In dispersion synthesized there was about 600 millions of them, which yields in ~0.01 m2 _{of total surface area. With 250 μg of}

luciferase adsorbed, it would give 25 mg/m2 _{mass/surface ratio. Despite the usual}

adsorption density is believed to be 0.1-1.5 mg/m,2,49 _{this figure varies for different}

substances used, and densities of up to 26 mg/m2 _{were reported previously.}232 _Also,

adsorption density for same material is known to decrease with ionic strength,233 _so

for conventional polyelectrolytes adsorbed from NaCl solution, it should be potentially lower. In case of co-precipitation, however, total surface area available for luciferase to adsorb, situated in the pores of CaCO3 microparticles, is obviously

Figure 5.2: PAH, PSS and PEI effect on luciferase activity, related to activity of

luciferase at the same concentration without doping.

much larger. At the same time, incorporation ratio is known to increase with molecular weight, and it was shown that for 66 kDa BSA incorporation of 92.6% can be achieved.115 _{Firefly luciferase has comparable 62 kDa molecular weight, and it is}

obvious that incorporation rate would depend on the amounts of substances used for co-precipitation and adsorption.

It is believed, that PEI is not fully protonated under physiological conditions, so can be still protonated in cells, when it enters increasingly acidic endosomal compartments, and may act as a “proton sponge”, adsorbing some of the entering protons.234,235 _{This proton sponge effect is considered to facilitate release of}

polyplexes from endosomes thus resulting in high transfection efficiencies. This behaviour of PEI was studied both experimentally and theoretically.236

As we were interested in measuring activity of luciferase in microcapsules internalised by cells, and ATP-dependant assay substrate capable of penetrating through cell membrane is used for such experiments, it was decided to employ PEI for creation of outermost layer in microcapsules shells to facilitate opening of endosomes and release of microcapsules to cytosol of cells upon internalisation, so that ATP from cytosol and luciferin from substrate added would be accessible for luciferase and light could be emitted. Single layer of PEI was adsorbed as last layer on microcapsules for most of samples investigated.

It was assumed, that the number of CaCO3 microparticles synthesized when co- precipitating luciferase in similar to that of empty ones. Thus, it allowed us to compare results obtained from using the same number of microcapsules for various samples. For that, microcapsules concentrations for each sample were measured by haemocytometry and adjusted to similar one. Then, equal volumes of samples taken for experiments provided possibility to compare the results quantitatively.

Luciferase assay is described in detail in Chapter 3.2.8.3. Briefly, 10 μL of microcapsules dispersion was added to wells of 96 well measurement plate. To the same wells, 50 μL of luciferase assay substrate (Promega) was added, and light emission was recorded for 10 seconds starting after 2 seconds of incubation. In first experiments activity of luciferase in microcapsules is compared to activity of free luciferase in solution of passive lysis buffer (PBL). The reason for using PBL is that when luciferase activity is measured from cells transfected luciferase encoding plasmid DNA (in experiments described in Chapter 5.2), the cells are lysed with PBL prior to measurements, therefore

luciferase is diluted in it. So, to make those two situations comparable, PBL was chosen as a solvent for free luciferase when its activity was compared to that in encapsulated state.