Star-Graft Quarterpolymer-Based Polymersomes as Nanocarriers for Co-Delivery of Hydrophilic/Hydrophobic Chemotherapeutic Agents.

We report the fabrication of polymersomes, using as building blocks star-graft quarterpolymers, composed of hydrophobic polystyrene and pH-sensitive poly(2-vinylpyridine)-b-poly(acrylic acid) (P2VP-b-PAA) arms, emanated from a common nodule, enriched by thermosensitive poly(N-isopropylacrylamide) grafts covalently bonded on the PAA block-arms. These multicompartmental polymersomes were evaluated as nanocarriers for the encapsulation and controlled co-delivery of doxorubicin (hydrophilic) and paclitaxel (hydrophobic) chemotherapeutic agents. The polymersomes can load these drugs in different compartments and can efficiently be internalized in the human lung adenocarcinoma epithelial cells, delivering their cargo and inducing high cell apoptosis. The release kinetics of both anticancer agents was controlled differently by the environmental conditions (pH and temperature). Enhanced release was observed at the acidic pH 6.0 and under physiological temperature (37 °C). At the same total drug level, co-delivery of these drugs with the polymersomes caused enhanced cytotoxicity and induced significantly higher cell apoptosis in the cancer cell line compared to the polymersomes loaded with either of the two drugs.

Introduction

Star-shaped amphiphilic copolymers, exhibiting well-defined 3D architectures with various linear polymer arms emanating from a central core, constitute a unique class of macromolecular topology with exceptional functionality.[1,2] One of the most interesting properties of these amphiphilic macromolecules is that they can adopt the morphology of polymeric micelles in aqueous media, namely unimolecular micelles, by internal segregation of their hydrophobic block/arms. The advantage of the unimolecular micelles versus polymolecular ones is that they never dissociate making them suitable nanocarriers of therapeutic agents under sink conditions. Moreover, stars with high number of arms and hence bearing high number of functional end-groups, enabling thus conjugations with targeting molecules, diagnostics, and drugs, exhibit additional potentials for biomedical applications.[3−9]

Water-soluble star-shaped terpolymers bearing three kinds of block/arms, some of which exhibiting responsive properties, have been designed previously, aiming to offer novel, more advanced potentials for uses as “smart” nanocarriers.[10−13] Two are the main novel properties integrated to these star terpolymers, that is, compartmentalization of the star unimolecular nanostructure and responsiveness to pH. The first one enables encapsulation of different molecules in the different compartments of the star terpolymer and the second one allows controlled delivery upon varying pH.

Another strategy to take advantage of the compartmentalized responsive stars is to incorporate them, through layer-by-layer (LbL) techniques, into higher order assemblies, namely multicompartmental microcapsules, facilitating the ability for concurrent storage of hydrophobic and hydrophilic species in the hydrophobic compartments of the shells and in the aqueous lumen of the microcapsule respectively.[14−18] Additionally, the hydrophilic weak polyelectrolyte segments of the star building blocks of the microcapsule allow the pH-controlled loading and release of the different cargos by controlling the permeability of the microcapsule shell.

Among hollow nanocarrier systems used in co-delivery of multiple drugs, polymersomes composed of amphiphilic block copolymers and providing a bilayered membrane are favorable self-assembled polymeric systems, similar to lipids.[19,20] Because of their structure, polymersomes are characterized as advantageous systems affording the simultaneous encapsulation of hydrophilic compounds in their aqueous cavities and hydrophobic compounds in their membranes, such as drugs, genes, proteins, and diagnostic probes.[21−24] Furthermore, polymersomes facilitate selective tumor targeting by incorporating pH- and/or thermosensitive segments in the building copolymer. Stimuli-responsive polymersomes with pH[25−32] or thermosensitive[33,34] polymer blocks have been extensively studied as delivery systems of hydrophobic or hydrophilic therapeutic and diagnostic agents. Specifically, the variations of pH or temperature of the surrounding environment lead to changes on the conformation and/or integrity of the polymersome permitting the site-selective release of the polymersome-entrapped bioactive agent.[25−34] Li et al., developed recently glucose oxidase (GOD)-loaded vesicular nanoreactors based on a diblock copolymer, which could be activated by tumor acidity to produce H2O2 increasing tumor oxidative stress. High levels of H2O2 also induced self-destruction of the vesicles releasing quinone methide to deplete glutathione and suppress the antioxidant ability of cancer cells. This synergistic effect of the nanoreactors resulted in tumor ablation.[35] Also, GOD was efficiently loaded in polymersomes formed from diblock copolymers of poly(ethylene glycol) (PEG) and copolymer of camptothecin (CPT) and methacrylate modified with piperidine groups. In the environment of tumor tissues, the acidity of the tumor triggered the generation of H2O2, and as a consequence the release of CPT. The cancer cells were killed and the growth of tumor due to oxidation/chemotherapy was suppressed through the synergistic action of high oxidative stress of the tumor as well as the released drugs of CPT.[36]

In a previous work, our group reported the synthesis and characterization of novel star-graft quarterpolymers (SGQPs). These multiarmed and multiresponsive PS[P2VP-b-(PAA-g-PNIPAM)] macromolecules consisted of two types of arms, short hydrophobic polystyrene (PS) arm, and pH-responsive hydrophilic poly(2-vinylpyridine)-b-poly(acrylic acid) (P2VP-b-PAA) block copolymer arm with grafted poly(N-isopropylacrylamide) (PNIPAM) chains on the PAA outer block.[37] The SGQP polymers exhibited stimuli-responsive behavior in aqueous media owing to the pH-responsive weak P2VP and PAA polyelectrolyte segments as well the thermoresponsive PNIPAM grafting chains. Recently, PS[P2VP-b-(PAA-g-PNIPAM)] was used for the development of polymeric microcapsules of a few micrometer size for the co-delivery of hydrophobic and hydrophilic molecules.[38] In these microcapsules, the star-graft unimolecular micelles formed the multilayered shell, through the LbL technique, using tannic acid for H-bonding interconnection of the polymer layers. The compartmentalized layers served as the host area for incorporating the hydrophobic compound, while the hydrophilic macromolecules were encapsulated in the aqueous lumen of the microcapsules. The results obtained for the dual-loaded microcapsules justified further investigation on the potential of the PS[P2VP-b-(PAA-g-PNIPAM)] polymer to be used in designing stimuli-sensitive delivery systems for the co-delivery of chemotherapeutics agents.

The aim of the present work was to evaluate the capability of these SGQPs to be integrated in stimuli-responsive vesicular assemblies (polymersomes) of nanometer size, in order to take advantage of the enhanced permeability and retention effect (EPR), which permits the accumulation of drug nanocarriers to inflammation and tumor sites following intravenous administration.[39,40] This multisegmental SGQP bears various segments conferring specific functionalities to the formed polymersomes such as amphiphilicity (hydrophobic and hydrophilic compartments) along with pH/thermo-sensitivity. The polymersomes were explored for their capability of loading simultaneously two anticancer drugs, namely doxorubicin (DOX) and paclitaxel (PCT). Co-delivery of these two anticancer agents using advanced nanocarrier systems has been shown to achieve enhanced anticancer efficacy.[41,42] The synergistic effect may result from the combination of individual antitumor mechanism of DOX and PCT.[41] DOX binds to DNA by intercalation, inhibiting further DNA and RNA biosynthesis, whereas PCT stabilizes microtubules preventing cell division. Both DOX and PCT lead finally to cancer cell apoptosis.[21,41] The hydrophilic DOX and the hydrophobic PCT chemotherapeutic agents were efficiently incorporated in the aqueous lumen and the membrane hydrophobic pockets of the polymersomes, respectively. Drug release from these polymersomes was controlled by pH and temperature. Interestingly, the drug-loaded polymersomes were readily internalized by human lung adenocarcinoma epithelial cells, inducing a high degree of cell apoptosis.

Results

The integration of the star-graft, polymeric species in polymersome assemblies was not obvious because of the complex topology of the building blocks. Hence, a number of methods used for polymersome and/or liposome preparation were attempted, namely, step dialysis, film hydration, phase transfer from immiscible solvent (CHCl3) to H2O, evaporation of immiscible solvent (CHCl3) from H2O using vacuum pump (see Figure S2), and miscible solvent evaporation [tetrahydrofuran (THF)/H2O] (see Figure S3). The latter method yielded nearly well-defined polymersomes as observed by transmission electron microscopy (TEM) and was adopted accordingly.

Physicochemical Characterization of Polymersomes

The size of the polymersomes was evaluated by dynamic light scattering and TEM microscopy and the morphology by TEM (Figure ). In Figure a, a characteristic autocorrelation function along with the intensity weighted distribution of the hydrodynamic diameter of the as-prepared polymersomes is demonstrated. A mean diameter was estimated at 122 nm (PDI 0.515). TEM verified the spherical or “ring-shaped” hollow morphology of the polymersomes, with no aggregates or clustered polymersomes being formed (Figures b and S3). In the schematic representation of the SGQP PS[P2VP-b-(PAA-g-PNIPAM)] (left of Figure c), it can be seen that the SGQP has two kinds of arms: one is the PS arm, whereas the other is the P2VP-b-PAA block copolymer, where the PAA blocks are grafted with PNIPAM chains. In basic conditions, PAA is deprotonated and behaves as a negatively charged polyelectrolyte, while the P2VP segments become hydrophobic and tend to collapse close to the PS/poly(divinylbenzene) (PDVB) core of the star. Segregation of the various blocks of the SGQP occurs during polymersome formation with the hydrophobic PS/P2VP (basic conditions) and the hydrophilic PAA/PNIPAM (room temperature) blocks forming the membrane and the inner/outer hydrophilic layers (shell), respectively (right of Figure c).

Figure 1

(a) Correlation function and intensity weighted hydrodynamic diameter of the SGQP polymersomes, (b) TEM micrographs of polymersomes formed by the SGQP (black scale bar 100 nm, blue scale bar 50 nm), and (c) schematic illustration of the involved star-graft polymers (left) and their segmental segregation in the wall of polymersomes, at physiological pH with the uncharged P2VP being hydrophobic (right). The different colors denote different arm segments: dark red PS, red P2VP, brown PAA, and blue PNIPAM grafts.

Colloidal Stability of Polymersomes

The size of the polymersomes (Figure a) did not change appreciably upon storage at ambient temperature for a period of 30 days. However, the ζ-potential exhibited some alteration with time suggesting possible charge surface rearrangements (Figure a). The size characteristics of the polymersomes during incubation in electrolyte (NaCl) solutions did not change significantly, even in high electrolyte concentrations (Figure a). The observed slight decrease should be attributed to the shrinkage of the shell of the polymersomes because of the electrostatic screening of the repulsive interactions among the PAA segments. However, the influence of pH is more pronounced. As can be observed in Figure b, the diameter of polymersomes decreased about 21% from pH 7.4 (94.81 ± 4.88 nm), to pH 6 (73.59 ± 0.76 nm). This might be ascribed to the conformational shrinkage of the corona PAA chain segments due to lowering of their degree of ionization. This might also be corroborated by attractions between the PAA negatively charged segments and the positively P2VP segments that have been partially protonated at pH 6. The ζ-potential of polymersomes initially increased (became less negative) and stabilized at low negative values (from −7.10 ± 0.66 mV up to −8.90 ± 0.36) at higher NaCl concentrations (Figure a), which justifies the size reduction (Figure a). Evidence for a steric stabilization mechanism is provided because the polymersomes did not aggregate even at high electrolyte levels wherein their ζ-potentials exhibit slightly negative values, probably due to repulsive interactions from the nonscreened, ionized AA moieties, preserved on the surface of the vesicle. The polymersomes after incubation at different pH for 24 h exhibited less negative values at pH 6 (−17.30 ± 0.82 mV) than pH 7.4 (−39.50 ± 3.29 mV) (Figure b). This effect should be attributed to the partially protonation of the deprotonated carboxylic groups (AA units), located on the surface of the polymersomes, upon lowering pH. We mention here that the same effect was observed for the same reason for the star-graft unimers (Figure b). The higher ζ-potential of the polymersome self-assemblies with respect to their unimers is reasonable because of the different surface area.

Figure 2

Size stability test of polymersomes at various conditions: (a) diameter vs time (magenta squares) and as a function of NaCl concentration (blue squares) and (b) in two different PB solutions (pH 6 and pH 7.4).

Figure 3

ζ-Potential stability test of polymersomes at various conditions: (a) ζ-potential vs time (magenta squares) and as a function of NaCl concentration (blue squares) and (b) ζ-potential in two different PB solutions (pH 6 and pH 7.4). Darker blue bars correspond to the ζ-potential of non-associated SGQP unimers.

Loading Drugs to the Polymersomes

The drug loading characteristics of the polymersomes are presented in Table . As can be seen, the polymersomes displayed DOX loading capacity 2.57% (w/w) for a theoretical (feed) loading 9.92% (w/w) and PCT loading capacity 5.98% (w/w) for a theoretical loading 10% (w/w). The loading capacity of the dual DOX/PCT polymersomes was nearly 4% (w/w) for both drugs for a theoretical loading of 9% (w/w). From TEM observation of the DOX- or PCT-loaded SGQP polymersomes, it was confirmed that the drug loading procedure (rotary vacuum evaporation, centrifugation, and redispersion in water) did not affect the formation of polymeric vesicles (Figure S4).

Table 1

Loading Characteristics of the DOX-Loaded, PCT-Loaded, and DOX/PCT-Loaded Polymersomes

sample	theoretical loading (%)	loading capacity (%)	entrapment efficiency (%)
SGQP-DOX	9.92	2.57 ± 0.98	23.95 ± 2.69
SGQP-PCT	10	5.98 ± 1.26	57.28 ± 3.48

Indeed, both the DOX-loaded (Figure S4a,b) and PCT-loaded (Figure S4c,d) SGQPs self-assembled in a vesicular nanostructure. The average size of the DOX-loaded and the PCT-loaded polymersomes was 133.9 ± 13.7 and 175.7 ± 14.7 nm, respectively.

Drug Release Profiles from Polymersomes

In Figure , the release profiles of DOX and PCT are demonstrated for 0–6 h (Figure S5 also presents the data up to 48 h). Sustained drug (DOX or PCT) release from the single- or dual-loaded polymersomes was observed, which was influenced by the pH and temperature experimental conditions. The results of DOX release in the first 3 h, in both single- and dual-loaded polymersomes, show some burst effect. Because of ionic interactions between the positively charged DOX (amino group; pK, 8.6) and the negatively charged PAA,[43] a portion of DOX seems to be trapped in the outer shell of the polymersomes, which is delivered rapidly. The change of ζ-potential between the blank polymersomes (−39.50 ± 3.29 mV) and the DOX-loaded ones (−22.1 ± 1.69 mV) is consistent with this assumption. At longer time (see also Figure S5), DOX was released faster at pH 6 than at pH 7.4, especially at 37 °C (Figure a,c). At pH 7.4, DOX release was faster at 25 °C than at 37 °C, especially with the single-loaded system, whereas at pH 6 the temperature did not affect DOX release (Figure a,c). PCT release from the single-loaded polymersomes was faster at pH 6 than at pH 7.4 at both temperatures and faster at 37 °C in both pHs (Figure b). PCT release was slower in the case of the dual-loaded polymersomes compared to the single-loaded ones (Figure b,c). The pH affected significantly PCT release form the dual-loaded polymersomes only in the case of 37 °C, with the pH 7.4 leading to much slower release compared to pH 6 (Figure b,c).

Figure 4

(a) Release profiles of DOX from the single-loaded polymersomes at PB of pH 7.4 and 6 at 37 and 25 °C, (b) release profiles of PCT from single-loaded polymersomes at PB of pH 7.4 and 6 at 37 and 25 °C, and (c) co-release profiles of DOX (solid symbols) and PCT (open symbols) from dual-loaded polymersomes at PB of pH 7.4 and 6 at 37 and 25 °C.

Cytotoxicity and Cellular Uptake

The viability of A549 cancer cells was assessed after 24 and 48 h of incubation with blank (without drug) and drug-loaded polymersomes. With the blank polymersomes, high cell viability values were obtained at all concentrations tested (almost 100% at 24 h and above 80% at 48 h) (Figure S6). The drug-loaded polymersomes exhibited dose- and time-dependent cytotoxicity against the A549 cancer cells (Figure ). Τhe cytotoxicity of free DOX and DOX-loaded polymersomes was increased with DOX concentration and incubation time (Figure ). The DOX-loaded polymersomes exhibited higher cytotoxicity than free DOX in equivalent drug concentrations, the difference in cytotoxicity being more pronounced at 24 h (Figure , Table ). In the case of PCT, the cytotoxicity of both the free and polymersome-entrapped drug was significantly increased with incubation time (Figure ), but the effect of drug concentration was less pronounced than in the case of DOX for the range of concentrations tested here. At both incubation times, the free and polymersome-entrapped PCT exhibited comparable cytotoxicities at equivalent PCT levels (Figure , Table ). At equivalent drug concentrations, the dual drug-loaded polymersomes exhibited higher cytotoxicity than the mixture of the two drugs (free DOX plus free PCT) at both 24 and 48 h (Figure , Table ). At relatively high concentrations, the dual drug-loaded polymersomes, that is, those carrying both DOX and PCT, exhibited enhanced cytotoxicity compared to the single drug-loaded polymersomes (Figure ). The cytotoxicity differences between the dual drug-loaded polymersomes and the respective single drug-loaded polymersomes were statistically significant (p < 0.05) at drug concentrations higher than 5 μg/mL for the 24 h incubation and higher than 1 μg/mL for the 48 h incubation.

Figure 5

Viability of A549 cancer cells following incubation with different concentrations of free (a,b) and polymersome (c,d) entrapped drugs for 24 (a,c) and 48 h (b,d).

Table 3

Estimated IC50 Values of Free Drugs and Drug-Loaded Polymersomes and CI Values of the Combined Free Drugs (Free DOX/PCT) and DOX/PCT-Polymersomes at 24 and 48 h at 50% of Viability

	24 h				48 h
sample	DOX IC50 (μg/mL)	PCT IC50 (μg/mL)	DOX/PCT IC50 (μg/mL)	CI50	DOX IC50 (μg/mL)	PCT IC50 (μg/mL)	DOX/PCT IC50 (μg/mL)	CI50
free DOX	10.000				2.120
free PCT		3.180				0.140
free DOX/PCT			14.500	0.940			0.750	2.385
DOX-polymersomes	1.340				1.190
PCT-polymersomes		14.350				0.120
DOX/PCT-polymersomes			4.960	1.028			0.610	1.790

The induction of A549 cell apoptosis by the drug-loaded polymersomes was assessed at a total drug concentration of 5 μg/mL (Figure ). The dual drug-loaded polymersomes (DOX/PCT 1:1 by weight) induced apoptosis in more than double population (%) of cells compared to the single drug-loaded polymersomes at the same total drug concentration. The differences in the degree of apoptosis between the dual-loaded polymersomes and the single-loaded polymersomes were highly significant (p < 0.01). As might have been expected from the lack of cytotoxicity (Figure S6), the blank polymersomes did not induce cell apoptosis (Figure ). The concentration of the blank polymersomes in the apoptosis test was 80 μg/mL, that is, equal to the carrier concentration used to generate the respective drug concentration of 5 μg/mL in the case of the drug-loaded polymersomes.

Figure 6

Apoptosis of A549 cells after 24 h of incubation with blank polymersomes, dual drug-loaded polymersomes (DOX/PCT 1:1 by weight), DOX-loaded polymersomes, and PCT-loaded polymersomes at a total drug concentration of 5 μg/mL. The concentration of blank nanoparticles was 80 μg/mL and corresponded to the carrier concentration used to generate the drug concentration tested in the case of the drug-loaded polymersomes.

The uptake of DOX-loaded polymersomes which had been labeled with fluorescein isothiocyanate (FITC content 3.21 ± 0.36%, DOX content 2.57 ± 0.98%) by the A549 cancer cells was evaluated at 1, 4, and 24 h by FACS (Figure a). The uptake of polymersomes by the cancer cells was increased from 30% at 1 h to 40% at 24 h. As the cell uptake of total DOX was measured at the different incubation times, that is, both the leaked from the carrier fraction of DOX and the (still) entrapped in the carrier fraction of DOX, cell uptake of DOX was faster than the carrier (FITC-labeled polymersomes) (Figure a). This effect suggests that part of DOX has been entrapped in the outer PAA layer of the polymersomes (ionic interactions), which is released before the nanocarriers enter into the cell. This is in consistent with the DOX release profiles in the first 3 h. For the incubation times tested, an inverse linear correlation between DOX uptake and cell viability was observed (Figure b).

Figure 7

Cellular uptake of DOX (DOX) and DOX-loaded polymersomes labeled with FITC (FITC) by A549 cells after 1, 4, and 24 h of incubation (a) and relation of A549 cells viability with the uptake of the DOX-loaded polymersomes by these cells (b).

The uptake of the FITC-labeled, DOX-loaded polymersomes was visualized by confocal laser microscopy (Figure ). At early times (1 h), red and green colors were mostly co-localized in the merged picture, indicating that the main fraction of DOX (red) still remained within the polymersomes (green), which were localized in the area around the nuclei of the cells. At longer incubation times (4 and 24 h), the polymersomes were surrounding the nuclei of the cells. At 4 h, a portion of DOX (liberated from the polymersomes) was surrounding cell nuclei or was within the nuclei. At 24 h a higher amount of DOX can be seen intracellularly compared to 4 h, with the DOX located mainly around cell nuclei and only a small part of it being within cell nuclei (Figure ). For clarity, the confocal images of FITC label and DOX are also given separately in Supporting Information (Figures S7 and S8).

Figure 8

Confocal fluorescence microscopy images of the uptake of FITC-labeled DOX-polymersomes by the A549 cancer cells at 1, 4, and 24 h. From left to right, the panels in each row show fluorescence from DAPI (nuclei stained blue), FITC-polymersomes (polymersomes stained green), DOX (red fluorescence of the drug), and merged images. In merged pictures, co-localization of FITC and DAPI gives yellow-green color, co-localization of DOX and DAPI gives red-purple color, and co-localization of FITC, DOX, and DAPI gives orange color. The scale bars are 20 μm.

Discussion

Lately, the combination therapy has been recognized as an efficient strategy in cancer treatment, as more than one drug is applied with different action sites. Among the advantages of combination therapy, the most important is the diminution of side effects of drugs because decreased doses are required, and in most cases, a synergistic therapeutic effect is achieved, which has resulted in suspending cancer cell mutations and cancer adjustment process.[41,44−47] However, an optimum synergistic anticancer activity is difficult to be achieved as the therapeutic result of a combination of two or more free drugs is highly affected by the varied biochemical activities and pharmacokinetic behavior of drugs.[42,48] Thus, the simple combination of free drugs commonly resulted in severe toxic adverse effects in cancer patients making their clinical application problematic.[49] To address this problem, advanced nanomedicine can be utilized for the co-administration of different drugs into tumor cells with a delivery system, such as nanoparticles, micelles, and polymersomes.[41,45−47]

Polymersomes exhibit larger mechanical stability and lower permeability than their structural lipid analogues, the liposomes. Their more robust, less leaky, and tunable membrane is very useful for improved stability in circulation.[24] In this work, pH- and temperature-sensitive polymersomes of the SGQP PS[P2VP-b-(PAA-g-PNIPAM)] for the co-delivery of DOX and PCT were prepared and characterized. The aim is to evaluate these (multi)stimuli-responsive polymersomes as nanocarriers to co-deliver two anticancer agents of different pharmacological activity mechanisms with a predefined releasing profile. These polymersomes can simultaneously encapsulate hydrophilic and hydrophobic compounds at different compartments and release their drug selectively under the effect of varied pH and temperature conditions. Co-delivery of PCT and DOX has been also investigated using PLA–PEG nanoparticles[41] and a PEG-polypeptide nanovehicle,[42] and the co-delivery nanosystems have been found to exhibit higher antitumor efficiency in reducing tumor size compared to free drug combination or single drug-loaded nanoparticles.

The SGQP polymersomes were prepared by the organic solvent evaporation method.[50] This method provided polymeric vesicles of average sizes of 120 nm (depending on the environment) with a bilayered membrane and the ability to dual-encapsulate hydrophilic DOX in their aqueous lumens and hydrophobic PCT in their membranes (Scheme ). The relative small size of the polymersomes (Figure ) and their hydrophilic coating may represent suitable requirements for their accumulation in the tumor area following intravenous administration. Tumor accumulation of long-circulating nanocarriers is based on the EPR effect (passive targeting).[39,40]

Scheme 1

Schematic Representation of the DOX/PCT Dual-Loaded Polymersome and the Different Migration Pathways of the Hydrophobic/Hydrophilic Drug Transmembrane Diffusion

Colloidal stability, that is, the avoidance of aggregation on storage or in the physiological environment, is a prerequisite for nanocarriers intended for drug delivery applications. The SGQP polymersomes synthesized here showed an exceptional colloidal stability during storage and, in the presence of high concentration of electrolyte (Figures and 3), much higher than the physiological one of 0.15 M.

The release of DOX or PCT from the single-loaded polymersomes exhibited different kinetics owing to their different hydrophilicity and in turn to their encapsulation in different compartments of the polymersomes. More importantly, their release profiles depend on environmental conditions such as pH (6 or 7.4) and temperature (25 or 37 °C) (Figure a,b). Concerning pH, both drugs display enhanced release at lower pH 6.0, which could be attributed mainly to the pH-sensitive P2VP segments, incorporated in the hydrophobic membrane. The ionic interactions between the positively charged DOX and the negatively charges of PAA segments do not change between 7.4 and 6,[43] and thus, they are not taken into consideration as far as the pH effect on the DOX release behavior is concerned.[51] Thus, slight protonation (ionization) of the 2VP moieties at the lower pH 6 likely induces partial swelling of the membrane, increasing thus its permeability.[30,31] The effect of temperature on release kinetics appears more complicated for the different drugs and depends on pH. DOX release at pH 7.4 slowed down upon heating to 37 °C (Figure a). At this temperature, PNIPAM segments turn into hydrophobic. However, because of the charged PAA arms, on where they are grafted, they do not exhibit intermolecular association.[37,38] At 37 °C, therefore, DOX seems to meet another obstacle to overcome during its diffusion out of the wall. This is probably due to the creation of hydrophobic domains in the inner and outer hydrophilic layers of the polymersome walls. On the other hand, no appreciable temperature effect was observed at pH 6, which might be attributed to the fact that the determining factor for delivery of DOX (which is more hydrophilic in this pH) is the permeability of the hydrophobic P2VP membrane, which is mainly affected by pH. The hydrophobic PCT follows different migration pathways (Figure b). It is already located within the hydrophobic membrane and upon heating its delivery seems to be facilitated by the creation of the PNIPAM hydrophobic domains. Thus, the PCT release was enhanced upon heating and this appears independent of the pH of the release medium (Figure b). This result is in very good agreement with the heating-induced fast release of a hydrophobic probe (Nile red) from microcapsules fabricated by the same SGQP building blocks.[38]

In the dual DOX/PCT-loaded polymersomes, the release of the hydrophilic DOX follows profiles similar to those of single-loaded polymersomes (Figure c). On the other hand, PCT release was restrained when released from the DOX/PCT-polymersomes, suggesting possible antagonistic effects between the two different drugs (Figure c).[21,42,52] As we have mentioned above, it may also be possible that DOX, interacting via ionic interactions with the hydrophilic PAA blocks of the polymersome shell, will pose an additional obstacle in the diffusion of hydrophobic PCT through the membrane. The release rate of both drugs from the dual-loaded polymersomes was much higher at pH 6 than in pH 7.4 under physiological temperature (37 °C) (Figure c), and this is encouraging because it indicates that the dual system would not lose a significant fraction of its drug load in the blood, that is, before it reaches the tumor site where the pH is lower than the physiologic one, and even more so before being internalized within the acidic compartments (lysosomes) of the cancer cells in the tumor. That is, the system would exhibit a degree of selectivity of drug release in the site of drug action.[53,54]

The blank polymersomes did not affect the viability (Figure S6) and did not induce apoptosis of A549 cancer cells (Figure ), which is suggestive of the cytocompatibility of the nanocarrier, under the experimental conditions applied and the polymer concentrations used. At the same total drug level, the dual-loaded polymersomes (i.e., those carrying DOX and PCT) exhibited higher cytotoxicity against the A549 cancer cells compared to the single-loaded polymersomes (i.e., those carrying only DOX or only PCT) (Figure , Table ) and induced a significantly higher degree of cell apoptosis (Figure ), suggesting an enhanced anticancer effect due to the co-delivery of the two drugs.[21,42,52,55,56] DOX and PCT have been found to induce apoptosis in A549 cell line,[57,58] and our results show that the polymersome-entrapped DOX and/or PCT can also induce A549 cells apoptosis (Figure ). The combination index values for 50% cells survival CI50, estimated based on the cells viability data presented in Figure , suggest that co-delivery to cells of DOX and PCT, either as free drugs or polymersome-entrapped, does not result to synergistic effect between the two drugs (Table ). Hahn et al. also observed that exposure of A549 cells to DOX and PCT produced a less-than-additive effect and related it with the effects of these drugs on cells cycle.[59] It becomes apparent that the time and dose schedule of DOX/PCT co-delivery has to be optimized in order to obtain synergistic anticancer activity.

Cell uptake experiments showed that the SGQP polymersomes were capable of entering the A549 cancer cells (Figures and 8). The cytotoxicity of the drug-loaded polymersomes, either single- or dual-loaded, increased with incubation time (Figure ), probably because of the increasing internalization within the cells of the drug with time, as the cell uptake experiment with the DOX-loaded polymersomes shows (Figure ). The confocal studies (Figures , S7 and S8) indicated that the polymersomes accumulated with time around cell nuclei and that their drug load (DOX) was liberated from the carrier with time, accumulating in the nuclei of the cells where it exerts its pharmacological activity.

Lack of the biodegradability of the nanocarrier is a drawback. For instance, we could replace PAA arms with the corresponding poly(aspartic acid) which is a biodegradable polypeptide. Thus, retro-design of the polymer building blocks is suggested before investigating the system further toward potential dual-anticancer drug delivery applications.

Conclusions

In this work, pH/thermosensitive amphiphilic PS9(P2VP-b-PAA-g-PNIPAM)9 SGQPs were used as building blocks to form hollow vesicles (polymersomes) attempting various preparation methods. To the best of our knowledge, we show for the first time that such topologically complex multisegmented SGQPs can be integrated to polymersomes, bearing complex pH-sensitive (P2VP block arms) hydrophobic membrane and thermosensitive (PNIPAM grafts) hydrophilic inner and outer corona.

The as-prepared polymersomes were evaluated as nanocarriers to carry anticancer model drugs (DOX and PCT) to human lung adenocarcinoma epithelial cells (A549). The polymersomes exhibited satisfactory single and dual drug loading efficiency and excellent colloidal stability. Drug release showed pH and temperature dependencies manifested differently for the hydrophobic and hydrophilic drugs, carried at different compartments. For the single-loaded and dual-loaded polymersomes, enhanced release was observed at low pH 6 and at physiological temperature (37 °C).

In the dual DOX/PCT-loaded polymersomes, although the release of the hydrophilic DOX follows profiles similar to those of single-loaded polymersomes, the PCT release was restrained. However, at the same total drug level, the dual-loaded polymersomes exhibited higher cytotoxicity and induced significant higher degree of cell apoptosis (about 125% enhancement in 24 h) in the A549 cancer cell line compared to the single-loaded polymersomes.

The obtained results indicate the potential of the SGQP polymersomes to be simultaneously loaded with two anticancer drugs (hydrophobic/hydrophilic carried at different compartments) and release them slowly and in a pH-dependent fashion in cancer cells, provoking a high degree of cell apoptosis.

Experimental Section

Materials

N-Ethyl-N′-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC·HCl, >98%, Mw: 191.7 g/mol) and FITC (>90% HPLC) were obtained from Sigma-Aldrich. PCT was purchased from LC Laboratories, USA. DOX hydrochloride (DOX·HCl) was purchased from Pfizer. All other chemicals and solvents were of analytical grade. Ultrapure 3D-water was obtained by means of an ELGA MEDICA apparatus.

Polymer Synthesis

The synthesis of the SGQP polymer [PS9(P2VP-b-PAA-g-PNIPAM)9] with nine heteroarms has been described in a previous work.[37] Briefly, the star terpolymer precursor, PS9(P2VP-b-PtBA)9, was synthesized through a one-pot/four-step sequential “living” anionic polymerization method.[12,13,60] Initially, the PS arms were synthesized in THF using sec-BuLi as the initiator and the “living” PS chains were used to polymerize a small quantity of DVB. Polymerization of 2-vinyl pyridine (2VP) was initiated from the active sites in the PDVB core of the “living” star precursors (PS9) leading to a PS9P2VP9 star polymer. Finally, tert-butyl acrylate (tBA) was polymerized at the end sites of the P2VP arms. The PS9(P2VP-b-PAA)9 terpolymer was obtained after acidic hydrolysis of the PtBA blocks in 1,4-dioxane with a 6-fold excess of HCl at 80 °C. The synthesis of the PS9(P2VP-b-PAA-g-PNIPAM)9 SGQP was performed according to the following steps. First, the PS9(P2VP-b-PAA)9 star terpolymer was dissolved in dimethylformamide (DMF), and aqueous HCl solution (0.01 M) was added until a 3/7 volume ratio of DMF/H2O. The solution was dialyzed against HCl solution (0.01 M) to remove DMF [dialysis membrane; molecular weight cutoff (MWCO): 12 000 Da]. Further dialysis of the resulting solution with a pH 2 was followed using aqueous solutions of pH 8. At the next stage, the grafting of amine-terminated PNIPAM chains onto the neutralized PAA of the star terpolymer was achieved through carbodiimide chemistry, using EDC as a condensing agent. Nongrafted PNIPAM chains and excess of EDC were removed by dialysis against H2O (dialysis membrane; MWCO: 25 000 Da). Finally, the SGQP was recovered by freeze drying. Detailed characterization of the polymer is reported elsewhere,[37] and the basic molecular characteristics of the SGQP are presented in Table .

Table 2

Molecular Characteristics of the Heteroarm SGQP

polymera	Mw of star precursorb (g mol–1)	Mw of SGQPc (g mol–1)	average PNIPAM chains per PAA armd	PNIPAM weight fractiond
SGQP:S339(V126-b-A69-g-N484.5)9	199 000	426 194	4.48	0.53

The numbers next to the letters denote the degree of polymerization of each block. The subscripts symbolize the number of arms and/or the grafted chains per arm.

Calculated by light scattering of the heteroarm PS9(P2VP-b-PtBA)9 precursor and assuming quantitative deprotection of tBA monomer units.

Calculated from the Mw of the PS9(P2VP-b-PAA)9 and the PNIPAM weight fraction.

Calculated by 1H NMR.

Synthesis and Characterization of Polymersomes

Polymersomes were prepared by the organic solvent evaporation method.[50] In brief, predetermined amounts of the heteroarm SGQP polymer (9 mg) were added in 0.5 mL of THF and stirred for 24 h. Then, the mixture was added dropwise in 5.5 mL of ultrapure water under gentle agitation. Next, the organic solvent was evaporated in a rotary vacuum evaporator (35 °C) for 2 h, wherein the polymersomes were hardened. Then, mild centrifugation at 3000 rpm for 5 min was followed in order for the aggregates to be precipitated and the supernatant was collected. The purified polymersomes were stored at room temperature until further use. The yield was determined by freeze-drying of purified polymersomes and weighing the solid residues obtained.

The morphology of polymersomes was characterized by TEM using a JEOL JEM-2100 microscope at an acceleration potential of 200 kV, equipped with a GATAN camera Erlanashen ES500W, model 782. For TEM specimens, an aqueous polymersomes sample of 0.16 mg/mL was spread onto a carbon-coated cooper MS200 grid and air-dried before observation. A ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd, Malvern, UK) equipped with a He–Ne Laser beam at a wavelength of 633 nm and a fixed backscattering angle of 173° was used for the study of hydrodynamic diameter and ζ-potential of the polymersomes in water.

The colloidal stability of the polymersomes was assessed by recording the average size and ζ-potential with time (for 30 days) and the resistance of the polymersomes to aggregation induced by increasing concentrations of electrolyte (NaCl) and by phosphate buffer (PB) solutions of pH 6 and 7.4.

Synthesis and Characterization of FITC-Labeled Polymersomes

In order to prepare FITC-polymersomes, the SGQP polymer was labeled with the fluorescent dye FITC prior polymersomes preparation. The conjugation of FITC on the polymer was implemented between the carboxyl groups of the PAA arms and the amine moieties of FITC in the presence of EDC as the coupling agent. Particularly, 5 mg of the polymer (1.17 × 10–8 mol SGQP) was dissolved in 4 mL of ultrapure water, and 1.5 mg of EDC predissolved in 0.2 mL of cold ultrapure water (4 °C) was added dropwise. The mixture was sonicated in an ice bath for 1 h, and the pH was adjusted to 8.0 with Et3N (100 μL of 0.35% solution). Then, FITC 0.1 mg (2.6 × 10–7 mol FITC) from a stock solution (1 mg/mL in ultrapure water) was added dropwise in the dark, and the reaction solution was left under stirring at ambient temperature for 24 h in shade. The final ratio of FITC/SGQP was 22 (in moles). The product was purified by extended dialysis against 3D-H2O. The purified FITC-SGQP polymer was freeze-dried, in order to obtain the final product, and stored in a desiccator until further use. The amount of FITC label on SGQP was evaluated spectrophotometrically (excitation 480 nm, emission 520 nm) using a Shimadzu UV-1800 UV spectrophotometer. For the measurement, a standard curve within a range of FITC concentrations 5–50 μg/mL (R2 = 0.983) was constructed and a modification rate of 3.01 ± 0.89% w/w was achieved.

For the FITC-polymersomes, the FITC-SGQP polymer was used and the process was followed as described above (in section Synthesis and Characterization of Polymersomes).

Loading of Polymersomes with DOX and PCT

For DOX-loaded polymersomes, the above method was used. The hydrophilic DOX (1 mg) was added in the aqueous phase of 5.5 mL ultrapure water and gently agitated in the dark for 30 min to completely homogenize. Then, the mixture of the polymer (9 mg) in THF (0.5 mL) was added dropwise in the dark and left under gentle agitation for 30 min. The ratio of DOX/SGQP was 1/9 (in mg). The dispersion was rotary vacuum-evaporated (35 °C) for 2 h. In order to remove free (not encapsulated) DOX, the dispersion was centrifuged at 15 000 rpm for 1 h at 8 °C, the precipitate with the DOX-loaded polymersomes was resuspended in the initial water volume, and the supernatant with the free DOX was discarded. The purified DOX-loaded polymersomes were stored in a fridge (4 °C).

In order to prepare PCT-loaded polymersomes, the SGQP (9 mg) was dissolved in THF (0.5 mL) and to this mixture PCT (1 mg) dissolved in 0.1 mL of ethanol was added to an analogy of PCT/SGQP 1/9 (in mg). The mixture was stirred for 24 h in the dark and then added dropwise in 5.5 mL of ultrapure water under gentle agitation. The dispersion was rotary vacuum-evaporated (35 °C) for 2 h to remove organic solvents (THF and ethanol). Then, in order to remove PCT aggregates and free PCT, the mixture was centrifuged (3000 rpm for 15 min at 8 °C). The supernatant dispersion, of PCT-loaded polymersomes in water, was kept, while free PCT and PCT aggregates were precipitated and the precipitate was discarded. The purified product was stored in the fridge (4 °C).

The dual DOX/PCT-loaded polymersomes were prepared by a combination of the above processes. In detail, 9 mg of SGQP was dissolved in THF (0.5 mL), and 1 mg of PCT dissolved in 0.1 mL of ethanol was added. The PCT/SGQP mixture was allowed to stir for 24 h in the dark. Then, DOX (1 mg) was added in 5.5 mL of ultrapure H2O under gentle agitation for 30 min in the dark and then the mixture of PCT/SGQP was added dropwise. The reaction mixture was left under gentle stirring for 30 min. The ratio of DOX/PCT/SGQP was 1/1/9 (in mg). The dispersion was rotary vacuum-evaporated (35 °C) for 2 h. In order to remove free (not encapsulated) PCT and possible PCT aggregates, the mixture was centrifuged at (3000 rpm, 15 min, 8 °C) and the supernatant was kept. In order to remove free (not encapsulated) DOX, the supernatant was centrifuged (15 000 rpm, 1 h, 8 °C). After this centrifugation, the precipitate with the DOX/PCT-loaded polymersomes was resuspended in the initial water volume and the supernatant with the free DOX was discarded. The purified DOX/PCT-loaded polymersomes were stored in the fridge (4 °C).

The quantity of drug loaded in polymersomes was determined by UV–vis spectroscopy, in a UV-1800 Shimadzu spectrophotometer. The DOX-loaded polymersomes were analyzed at 495 nm. The blank polymersomes (with no drug) at 495 nm had zero absorbance; thus, the DOX signal was clear and not overlapped. The amount of loaded DOX in the polymersomes was calculated based on a calibration curve (R2 = 0.9989). The limit of quantification was 0.1 μg/mL, and the linear part of the standard curve used for DOX assay was from 0.1 to 50 μg/mL.

For the PCT-loaded polymersomes, freeze-dried samples were dissolved in 1 mL of a 60:40 (v/v) mixture of acetonitrile/water and analyzed using a UV-1800 Shimadzu spectrophotometer at 229 nm. The absorbance of blank polymersomes (with no drug) at 229 nm was extracted before every measurement. The amount of loaded PCT in the polymersomes was calculated based on a calibration curve (R2 = 0.9988). The limit of quantification was 1 μg/mL, and the linear part of the standard curve used for PCT assay was from 1 to 50 μg/mL.

In the dual drug-loaded samples, the DOX loading was first determined by UV–vis analysis at 495 nm. Then, the polymersomes were freeze-dried and the PCT loading was determined at 229 nm as described above. The DOX and PCT contents were calculated based on separate calibration curves under the conditions described above.

The loading capacity (LC %), entrapment efficiency (EE %), and theoretical (feed) loading (Lth %) of DOX and PCT in polymersomes were calculated from the equations:where W, WC, and W0 were the amount (mg) of entrapped drug according to the UV–vis spectroscopy, the amount of polymersomes, and the amount of initially added drug, respectively.

In Vitro Drug Release

The drug release from the polymersomes (DOX, PCT, and DOX/PCT) was evaluated under sink conditions in PB solutions (10 mM, pH = 7.4 and pH = 6) at 37 and at 25 °C. The ionic strength of the PB was 154 mM, similar to the 149.5 mM of human blood plasma.[61] The polymersomes were enclosed in dialysis membranes (MWCO of 12 kDa) and dialyzed against 7 mL of PB buffer for both pHs (pH = 7.4 and pH = 6) at 37 and at 25 °C (ambient temperature) in a water bath under gentle agitation. At predetermined time intervals (30 min, 1, 2, 3, 4, 5, and 6 h), the release medium was completely removed and replaced with fresh buffer of 37 or 25 °C. The release medium containing DOX was assayed by UV–vis spectroscopy at 495 nm. The amount of released DOX for each release medium (pH = 7.4 and pH = 6) was calculated based on separate calibration curves (for pH = 7.4: R2 = 0.9990 and for pH = 6: R2 = 0.9981). The limit of quantification for pH = 7.4 was 0.1 μg/mL and for pH = 6 was 0.05 μg/mL. The linear part of the standard curve used for DOX assay for pH = 7.4 was from 0.1 to 50 μg/mL and for pH = 6 was from 0.05 to 50 μg/mL.

The release medium containing PCT for both buffers was treated with 2 mL of dichloromethane (DCM) in order to extract the drug. Then, the dichloromethane was collected and allowed to evaporate at room temperature, and the solid residue was dissolved in 1 mL of a 60:40 (v/v) mixture of 3D-H2O/MeOH. The samples of released PCT were assayed by UV–vis spectroscopy at 229 nm. The PCT content was calculated based on a calibration curve (R2 = 0.9999), wherein the limit of quantification was 1 μg/mL and the linear part of the standard curve was from 1 to 50 μg/mL.

The release medium containing both DOX and PCT was first assayed by UV–vis spectroscopy at 495 for DOX release, as described above. Then, PCT was extracted from the samples following the above extraction process.

Cellular Studies

The human lung adenocarcinoma epithelial cells A549 (ATCC) were cultured routinely in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and a mixture of antibiotic agents (1.1% v/v penicillin/streptomycin and 0.15% v/v amphotericin). Cultures were retained at 37 °C in a humidified atmosphere with 5% (v/v) CO2. The culture medium was changed every 48 h and cells were harvested with 0.25% (v/v) trypsin in phosphate-buffered saline (PBS).

Viability

The cytotoxicity of blank polymersomes, drug-loaded (DOX, PCT, and dual DOX/PCT) polymersomes, and free drug (DOX, PCT, and dual DOX/PCT) against the A549 human lung cancer cells was investigated by the MTT assay.[62] A549 cells were seeded into 24-well plates at a density of 5 × 104 cells/well and allowed to grow and proliferate as a monolayer under standard conditions for 24 h. Then, the supernatant in each well was completely removed and replaced with 500 μL of fresh medium comprising different concentrations of free or polymersome-entrapped drug. The concentrations were 1, 5, 10, and 15 μg/mL of DOX, PCT, or a physical mixture of DOX and PCT (DOX/PCT ratio approx 1:1 by weight). The cytotoxicity of blank (without drug) polymersomes (carrier) was also tested at concentrations corresponding to the concentrations of carrier used in the case of drug-loaded polymersomes in order to generate the drug concentrations mentioned above in the cells incubation medium. The concentrations of blank (without drug) polymersomes were (8, 40, 80, and 120 μg/mL) evaluated as control to the drug-loaded polymersomes. After the predetermined time periods (24 and 48 h) of incubation at 37 °C, the supernatant was removed and the cells were washed with PBS. Then, 200 μL of MTT solution (10% v/v in PBS, pH 7.4) was added in each well and plates were incubated at 37 °C for 2 h in order the formazan crystals to be formed. The medium was completely removed, and 200 μL of DMSO was added in each well and incubated at 37 °C for 15 min to fully dissolve the formazan crystals. The fluorescence of the crystals (corresponding to cell viability) was determined with a UV spectrophotometer at a wavelength of 490 nm. The experiments were performed in triplicate and repeated three times. To calculate the background fluorescence of the cells, unlabeled cells without addition of any polymersomes or drug were used as a negative control in every measurement. Cytotoxicity was expressed as reduction in cell viability (%). The IC50 values (Table ) were calculated from the % cell viability versus concentration data by fitting the dose–effect curves according to a four-parameter logistic model with OriginPro 8 software (Origin Lab Corp, Northampton, MA). To discriminate synergistic, additive, or antagonistic cytotoxic effects,[63] the next equation was usedwhere (D)1 and (D)2 represent the IC value of drug 1 (free DOX) and drug 2 (free PCT) alone, respectively. (D)1 and (D)2 represent the concentration of drug 1 (DOX) and drug 2 (PCT) in the combined delivery (free DOX/PCT or polymersome-entrapped DOX/PCT) at the IC value. CI > 1 indicates antagonism, CI = 1 indicates additive effects, and CI < 1 indicates synergism.

In Vitro Cellular Uptake

The uptake of FITC-labeled DOX-polymersomes by A549 cells was quantitatively evaluated by flow cytometry.[64] A549 cells, following the above seeding process, were grown as a monolayer, harvested by trypsinization (0.25% w/v trypsin), plated in 24-well plates (density of 5 × 104 cells per well), and incubated. After 24 h, the supernatant in each well was completely removed and substituted with fresh medium containing FITC-labeled DOX-polymersomes at a DOX concentration of 1 μg/mL (8 μg/mL polymersomes) and incubated for 1, 4, and 24 h. Then, the supernatant was removed; the cells were washed twice with PBS and harvested with 0.25% (w/v) trypsin. The fluorescence was measured by flow cytometry using appropriate filters for FITC (excitation λ = 495 nm, emission λ = 519 nm) and DOX (excitation λ = 550 nm, emission λ = 573 nm) in a FACS Calibur, Coulter Epics XL-MCL apparatus. To calculate the background fluorescence of unlabeled cells, cells without any addition of studied particles were used as a negative control in every measurement. Data analysis was performed with the WinMDI cytometry analysis software.

In order to visualize cellular uptake of the FITC-labeled DOX-polymersomes, confocal laser microscopy was used with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for the nuclear, DOX for the drug and FITC for the polymersomes staining.[65] A549 cells were grown on coverslips seeded in 6-well plates. Sterilized cover slips (Borosilicate Glass, VWR) were placed in each well followed by the addition of cell suspensions (5 × 104 cells/mL). After 24 h, the cells were incubated with FITC-labeled DOX-polymersomes (1 μg/mL DOX content) for 1, 4, and 24 h. The cells were washed thrice with PBS and fixed directly onto the glass coverslips using 4% paraformaldehyde (PFA) dissolved in PBS (300 μL) and incubated for 15 min. After washing the cells thrice with PBS, 300 nM DAPI stain solution (nucleic acid staining) was added to cover the cells. The cells were then washed with PBS to remove the stain solution. The slides were drained and mounted with Mowiol 4-88. The obtained specimens were imaged using a Leica SP5 confocal microscope (Germany) using appropriate filters for DAPI (excitation 359 nm, emission 457 nm), FITC (excitation λ = 495 nm, emission λ = 519 nm), and DOX (excitation λ = 550 nm, emission λ = 573 nm).

Apoptosis

In brief, phosphatidylserine externalization follows the loss of cellular membrane integrity, which is related with apoptotic or necrotic processes leading to the later stages of cell death.[66] Annexin V protein (FITC Annexin V, BD Pharmingen) was used to detect the externalization of phosphatidylserine as a marker of apoptosis. A549 cells were seeded into 24-well plates (density 5 × 104 cells/well) and allowed to grow and proliferate as a monolayer, for 24 h. The supernatant in each well was then replaced with medium containing 40 μg/mL blank, PCT-loaded, DOX-loaded, and DOX/PCT-loaded polymersomes (in the case of the drug-loaded polymersomes, the total drug concentration was 5 μg/mL). After 24 h incubation, the supernatant in each well was completely removed, the cells were washed with PBS, detached with trypsin (0.25% w/v trypsin), delivered to FACS tubes, and centrifuged (1600 rpm for 5 min); the obtained pellet was washed and resuspended in 1 mL 1× Annexin V binding buffer. Then, the cells were centrifuged (1600 rpm for 5 min), and the obtained pellet was resuspended in 100 μL of 1× Annexin V binding buffer. The cells were incubated with 5 μL of FITC-Annexin V in the dark for 15 min at room temperature. At the end of the incubation, the cells were washed with 0.5 mL Annexin V binding buffer. Cell fluorescence due to Annexin V (% cell apoptosis) was determined by flow cytometry (excitation λ = 495 nm, emission λ = 519 nm), in a FACS Calibur, Coulter Epics XL-MCL apparatus. The background fluorescence of unlabeled cells was determined and used as negative control. Data analysis was performed with the WinMDI cytometry analysis software.