Loading Efficiency of Polymersomes with Contrast Agents and their Intracellular Delivery: Quantum Dots Versus Organic Dyes

Results and Discussion

Loading efficiency and passive release of fluorophores from polymersomes. All investigated fluorophores were retained within polymer globules via electrostatic and hydrophilic–hydrophobic interactions, without chemical (covalent) conjugation with the polymer. The polymersomes were hydrophilic/amphiphilic and their surface was positively charged (ζ-potential was in the range 17-22 mV, depending on the type of incorporated fluorophore). Their average size was ~110 nm, which was determined by DLS. The nanoparticles were stable in high-salt physiological fluids such as 10 mM PBS and serum. Aggregation was not detected even after 24 h incubation with serum at 37°C (14).

The first step of the study aimed to investigate the loading capacity of polymersomes with small molecules (FITC) and to increase their retention in the polymer matrix by conjugating with dextran (FITC-dextran^10,000 and FITC-dextran^70,000) or by forming micelles with detergents. The data shown in Figure 2 demonstrate the dynamics of FITC fluorescence in the supernatant during 5-step ultrafiltration, which is representative of retention of an organic dye in the polymer matrix. FITC left the polymersomes very quickly – over 80% were out of polymer matrix after the first filtration (Figure 2). Better retention was detected for FITC-dextran^10,000 and FITC-dextran^70,000, at about 45% and 62%, respectively, after the first filtration (Figure 2). However, the rate of passive leakage of FITC-dextran from the polymersomes was also relatively high. The level of FITC fluorescence in the polymersomes was less than 20% of the initial level after the fifth filtration.

The use of detergent in the buffer (in which FITC-dextran^10,000 was dissolved) significantly increased the retention of fluorophore in the polymer matrix and reduced its leakage (Figure 2). Two detergents were applied, Triton-X100 and Brij. In the presence of Triton-X100, ~82% of FITC-dextran^10,000 was detected in the polymersomes after the first filtration, but only ~7% after the fifth filtration. In the presence of Brij, ~80% of FITC-dextran^10,000 was detected in the polymersomes after the first filtration, and ~60% after the fifth filtration. Both detergents formed micelles with the fluorophores and suppressed leakage from the polymer globules. The retention effect of Brij was much better than that of Triton-X100. Neither detergent significantly affected the size of polymersomes as analyzed by DLS.

The data suggest that FITC-dextran is not an appropriate fluorescent marker for tracking polymersomes in vitro and in vivo due to the release of the fluorophore from the polymer matrix. However, FITC-dextran is an appropriate and cheap model for investigating the rate of release of different substances from the polymer matrix, depending on their size.

The second step of the study aimed to investigate the loading capacity of polymersomes with QDs. The fluorescence signal in the supernatant was very high even after five filtrations of QD-labeled polymersomes (Figure 2). Fluorescence was not detected in the filtrate.

The data indicate that QDs are appropriate fluorescence markers for performing quantitative monitoring of pharmacodynamics of polymersomes in vitro and in vivo. In this case, we can be sure that the origin of the fluorescence signal will be entirely due to the QDs within the nanoparticles rather than free QDs. Figure 3 demonstrates the penetration of QD⁶⁰⁵_labeled polymersomes in cancer cells after 24-h incubation. The fluorescence signal was detected predominantly in the cytoplasm. QD⁶⁰⁵, which were not coated by polymersomes, did not penetrate into the cells – no fluorescence was detected in this case. Moreover QD⁶⁰⁵_labeled polymersomes were successfully delivered passively in solid tumors in vivo after their intravenous injection, which was demonstrated on colon cancer-grafted mice (16, 17).

Electro-induced release of fluorophore from polymersomes. The next step of the study aimed to verify whether the application of electrical pulses affects the ability of the polymersomes to retain or release the fluorescence marker, as well as the effect of electroporation on the nanoparticle size. The experiments were performed on QD⁶⁵⁵_labeled polymersomes and FITC-dextran^10,000_labeled Brij-treated polymersomes (Figure 4). The intensity of the electrical pulses varied in the range 200-1000 V/cm. The electroporation was applied after the second ultrafiltration, when the retention level was constant for FITC-dextran^10,000_labeled Brij-treated polymersomes. The fluorescence intensity was registered (recorded) simultaneously in the supernatant and filtrate after one additional ultrafiltration step.

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Figure 1.

Schematic representation of the experimental design. PES: Polyethylene-sulphonyl membrane.

In the case of QD_labeled polymersomes, the fluorescence signal in the supernatant remained constant after electroporation (Figure 4A, left). The fluorescence intensity in the supernatant was same as before electroporation and no fluorescence was detected in the filtrate (Figure 4B, left). The data suggest that electroporation with pulse intensity up to 1000 V/cm did not induce a release of QDs from the polymer matrix. We also established that increase of electroporation intensity (2000 V/cm) resulted in the appearance of QD fluorescence in the filtrate, which indicates a disruption of the integrity of polymersomes and leakage of QDs from the matrix.

The effect of electroporation on the size of QD⁶⁵⁵_labeled polymersomes is shown in Table I. The size was not altered significantly up to 1000 V/cm, which indicates that the applied pulses did not affect the structure of the nanoparticles. However, two peaks were recorded in the DLS histograms at 1500 V/cm, at 88 nm and 313 nm. The fraction with small size could be explained by fragmentation and the fraction with large size by aggregation of the nanoparticles.

The fluorescence signal of FITC-dextran^10,000_labeled Brij-treated polymersomes in the supernatant decreased after electroporation (Figure 4B, right) and FITC fluorescence appeared in the filtrate (Figure 4A, right). The electroporation accelerated the leakage of fluorophore from the polymer matrix.

The size of FITC-dextran^10,000_labeled Brij-treated polymersomes did not change significantly up to 750 V/cm (Table II). However, the nanoparticle size decreased significantly after electroporation with 1000 V/cm and higher, which indicates fragmentation of the polymersomes.

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Figure 2.

Fluorescence intensity, obtained from the upper phase (supernatant) of Vivaspin filter, after 5-step ultrafiltration of fluorophore-loaded polymersomes. The data are means±SD from three independent experiments for fluorescein isothiocyanate (FITC); FITC-dextran^10,000; FITC-dextran^70,000; FITC-dextran^10,000 plus Triton-X100 (1:32, mol:mol); FITC-dextran^10,000 plus Brij (1:128, mol:mol); QD⁶⁵⁵. In the case of FITC: λ_ex=492 nm, λ_em=525 nm. In the case of QD⁶⁵⁵: λ_ex=420 nm, λ_em=655 nm.

In conclusion, the data demonstrate that the loading efficiency of FITC in the polymersomes was below 20%, accompanied by slow or faster release of fluorophores, depending on the size of conjugated dextran. Electroporation increased the release of FITC-dextran from the nanoparticles. In contrast, the loading efficiency of non-tagged QDs in the polymersomes was 100%. QDs were retained in the polymer globule even after 5-step ultrafiltration. Electroporation (at the selected conditions) did not induce a release of QD from the polymersomes. The results suggest that QD_labeled polymersomes are appropriate fluorescent probes and nano drug-delivery system with high tracing opportunities for in vitro and in vivo applications. Moreover, loading polymersomes with organic dyes of different molecular weights (such as FITC-dextrans) is a simple model for visualizing and predicting the rate of release of small organic molecules (e.g. conventional drugs, other contrasts, stabilizers and supplements) from the polymer matrix.\

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Figure 3.

Intracellular delivery of QD⁶⁰⁵_labeled polymersomes to HeLa cells after 24-h incubation in a humidified atmosphere, as detected by fluorescence confocal microcopy (excitation – 420 nm; emission filter – 575-630 nm). QD⁶⁰⁵ concentration, 2 nmol/10⁵ cells.

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Figure 4.

A: Fluorescence intensity (upper panel), obtained from the upper phase (supernatant) of a Vivaspin filter, from fluorophore-loaded polymersomes: QD⁶⁰⁵_labeled (left) and fluorescein isothiocyanate (FITC)-dextran^10,000_labeled Brij-treated polymersomes (right) after electroporation and subsequent ultrafiltration. The data are means±SD from three independent experiments. B: Representative fluorescence spectra of the supernatant and filtrate, obtained after electroporation of fluorophore-loaded polymersomes and subsequent ultrafiltration: left: QD⁶⁰⁵_labeled polymersomes, treated with 1000 V/cm (λ_ex=420 nm); right: FITC-dextran^10,000_labeled Brij-treated polymersomes, treated with 500 V/cm (λ_ex=492 nm). In the case of FITC: λ_ex=492 nm, λ_em=525 nm. In the case of QD⁶⁵⁵: λ_ex=420 nm, λ_em=655 nm.