3.7.1. Synthesis of Solid Magnetoliposomes
Herein, a new preparation method for solid magnetoliposomes was developed. This new approach begins with the preparation of DPPC reverse micelles in which, by ultrasonication, the previously synthesized magnetic nanoparticles are forced to enter into. A second DPPC layer is injected after magnetic decantation, resulting in a lipid bilayer vesicle with a magnetic core in a solid magnetoliposome architecture. Since the new methodology involves the independent addition of DPPC layers, the lipid bilayer formation was confirmed by Förster Resonance Energy Transfer (FRET), following the previously reported procedure [
19]. The first lipid layer was labeled with Rhodamine B-DOPE (Rhodamine B as the energy acceptor), and the second lipid layer was labeled with NBD-C
12-HPC (NBD as the energy donor). Once the second layer was added, the two fluorophores become close to each other, and the conditions for FRET occurrence are satisfied.
Figure 8 shows the emission spectra of SMLs labeled with the energy donor NBD and the energy acceptor Rhodamine B, separately and together in a single system, which are all measured upon excitation of the donor (λ
exc = 470 nm). The pronounced decrease in the NBD emission, accompanied by an increase in Rhodamine B emission, evidences the energy transfer from excited NBD to Rhodamine.
The calculated FRET efficiency (Equation (3)) of 89.6% proves the probes’ proximity, as a strong energy transfer is observed from donor to acceptor. The lipid bilayer formation was corroborated by a donor–acceptor distance of 3.5 nm (from Equations (4) and (5)), since a lipid bilayer has a typical thickness between 7 and 9 nm [
47]. FRET efficiency value is very similar to the previously obtained for magnetoliposomes containing spherical Ca
0.25Mg
0.75Fe
2O
4 nanoparticles [
19], which reported an FRET efficiency of 87%. One of the main differences between the new synthesis method herein described and the one previously reported [
19,
25] is that the magnetoliposomes prepared by this new method do not present any amount of water in the inner compartment. As a result, the solid core makes the nanosystem less flexible (more rigid), which is translated into a decrease in lateral and rotational motion of lipids, improving the energy transfer between the probes.
3.7.2. Dynamic Light Scattering and Transmission Electron Microscopy
Considering the crucial role that the nanosystems’ inherent parameters (as size, polydispersity index, and shape) play on biological administration suitability and behavior, dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements were performed. DLS experiments allowed obtaining the size distribution of DPPC-based SMLs (
Table 3) exhibiting an average hydrodynamic size of 117.5 ± 0.5 nm and a generally small polydispersity index (PDI). Correlation curves are presented in
Figure S3 (Supplementary Material). The obtained average hydrodynamic size is slightly smaller than the previously obtained 127.3 ± 17 nm for SMLs containing spherical Ca
0.25Mg
0.75Fe
2O
4 nanoparticles [
19], PDI values also being slightly lower. The stability of DPPC-based SMLs in storage at 4 °C was also monitored for 30 days.
Figure 9 exhibits the size and PDI evolution of an aqueous solution of DPPC-based SMLs. This formulation is stable until day 15, with a small increase in size between days 15 and 20, to around 240 nm. A similar behavior was observed for PDI values, which increased to 0.25 on day 30 after storage. These results show that the nanosystems are completely stable for 15 days.
The encapsulation of low molecular weight molecules (as doxorubicin) in lipid vesicles increases the size of the administrated anticancer agent above the renal clearance threshold (≈40,000 Da), resulting in a reduction in kidney excretion and, consequently, blood half-life increase. It is reported that the encapsulation of doxorubicin in liposomes can improve its plasma half-life from 5–10 min (as a free drug) to 2–3 days (in liposomes) [
48]. This increase in circulation times takes better advantage of the EPR effect, resulting in a more efficient and selective accumulation in tumor sites [
49]. As mentioned before, the extravasation of liposomes to tumors is more effective at sizes below 200 nm [
4]. DLS measurements confirm that SMLs hydrodynamic size is in conformity to the preferred size range. Considering that modifications in liposomal and micellar formulations have shown promising results in preventing the aggregation and opsonization of plasma proteins, PEGylated magnetoliposomes were also studied, exhibiting an average hydrodynamic diameter of 153.8 ± 0.8 nm and a low polydispersity index. The average size and PDI of PEGylated formulations were slightly higher than those of non-PEGylated ones (prepared by the same method). In fact, these results were not as expected, since we reported a general size reduction trend in PEGylated liposomal formulations. This size reduction is usually explained by an increased lateral repulsion caused by PEG molecules, which induces curvature in the lipid bilayer and consequent reduction in vesicle size [
50]. The difference in size can be partially explained by a structural variation between liposomes and these magnetoliposomes, since the latter instead of an aqueous inner volume have a solid compartment constituted by a cluster of magnetic nanoparticles. The hydrodynamic diameter of SMLs with cholesterol (Ch) is similar to the values for PEGylated SMLs (151.4 ± 0.60 nm), however, with a lower PDI value (0.20 ± 0.01).
Transmission electron microscopy (TEM) allows characterizing the size and shape of SMLs.
Figure 10A exhibits a single solid magnetoliposome, which is characterized by the presence of a nanoparticles cluster, highly contrasting with the bright thin DPPC lipid bilayer involving it. Using ImageJ software for image processing, several different segmented lines along the SML image were taken into account to measure the nanosystem mean diameter, resulting in mean diameter of 116.5 ± 5.5 nm. The same procedure was carried out to estimate the length of the lipid bilayer, resulting in a mean length of 8.3 ± 1 nm, complying with those reported in the literature and the obtained from FRET measurements. A schematic representation of solid magnetoliposomes was drawn from the clear TEM image and is presented in
Figure 10B. An additional TEM image, at lower magnification (and different contrast), is presented in the
Supplementary Material (Figure S2). The image reveals some dispersity in size, as inferred from DLS measurements (PDI near or above 0.2). It is also clear that the SMLs are not aggregated. The results prove the suitability of the new method here shown to originate magnetoliposomes with a typical structure, consisting of two DPPC monolayers surrounding an inner cluster core (with residual water content) of shape anisotropic nanoparticles.
3.7.3. Doxorubicin Encapsulation in Solid Magnetoliposomes
The photophysical properties of doxorubicin were here exploited to study its encapsulation in SMLs and its location in the nanosystems.
Figure 11 shows the emission spectra of doxorubicin, at the same concentration, in Ca
0.25Mg
0.75Fe
2O
4 based SMLs and in liposomes of the same lipid composition (without magnetic nanoparticles).
The pronounced decrease in fluorescence emission of DOX in SMLs, comparatively to liposomes, is due to a quenching effect by the nanoparticles and confirms the incorporation of doxorubicin in the nanocarriers. This effect of fluorescence quenching promoted by the magnetic nanoparticles has been reported for several fluorescent antitumor drugs encapsulated in magnetoliposomes containing different types of magnetic nanoparticles [
19,
51,
52,
53,
54]. The incorporation of doxorubicin in SMLs was further confirmed by fluorescence anisotropy measurements (Equation (7)) related to the rotational mobility of the fluorescent molecule. The results are shown in
Table 4.
The fundamental fluorophore anisotropy (
r0) corresponds to the maximum fluorescence anisotropy, and a value of 0.33 was reported for doxorubicin [
42]. As expected, upon the encapsulation of DOX in SMLs (DPPC), room temperature anisotropy measurements registered values below
r0 (
r = 0.137), but typical of a fluorophore in a lipid bilayer, which confirms the DOX encapsulation in SMLs. Since the melting transition temperature of DPPC occurs at 41 °C, an increase in membrane fluidity is expected when the liquid-crystalline phase is attained. A lower fluorescence anisotropy value was observed at 55 °C, since the temperature increase implies an increased mobility of DOX aglycone in the bilayer, providing evidence of DPPC phase transition from the gel to the liquid-crystalline phase.
Considering that the incorporation of cholesterol (present in most liposomal formulations used as drug carriers) and PEGylated lipids can significantly change the structural properties of a lipid bilayer, the anisotropy values of doxorubicin were measured for solid magnetoliposomes containing these modifiers. A considerable increase in anisotropy value, at 25 °C, in DPPC/DPSE-PEG SMLs was observed, indicating that DOX can be reliably retained in the membrane of PEGylated magnetoliposomes. At each condition, PEGylated SMLs showed higher anisotropy values than the other lipid formulations (
Table 4), indicating a DOX location in a more exterior environment. In fact, the lipid bilayer fluidity varies with depth due to changes in membrane-free volume, showing a tendency to decrease from the liposome surface to the interior [
55,
56]. So, the higher
r values may indicate that DOX is located more superficially, near PEG. Additionally, the significant decrease in anisotropy from 25 to 55 °C is an indicator that hyperthermia can enhance PEGylated SMLs’ ability to interact and release drugs in the target (due to the increase in membrane fluidity).
In the case of systems containing Ch, a general decrease in anisotropy was registered compared to non-modified vesicles. It is reported [
57] that the addition of cholesterol in bilayers increases the apparent microviscosity of phosphatidylcholine (PC) membranes (up to 1.4 times) as a result of membrane-induced ordering of the PC acyl chains in the liquid phase. However, this effect is only verified to concentrations up to 20 mol% Ch. At higher content, its admixture decreases the microviscosity in the polar region of liposomes. Above 20 mol%, the region where cholesterol OH groups are located has a larger free volume available than in cholesterol-free membranes, resulting in a decreased fluorophore anisotropy [
57]. This explains the smaller anisotropy values obtained for cholesterol-modified membranes.
Doxorubicin encapsulation efficiencies (EE%) were determined by the percentage of incorporated drug into SMLs relative to the initial amount of drug added (Equation (8)). Different lipid formulations with two different initial DOX concentrations were studied. As shown in
Table 5, EE% decreased with increasing drug/lipid ratio. The EE% of non-modified DPPC-based SMLs varied between 72% ± 3% (for the initial concentration of 1 × 10
−4 M) and 65% ± 8% (for the initial concentration of 2 × 10
−4 M). At the same conditions, a slight decrease was evidenced from non-modified SMLs to PEGylated SMLs. These results suggest that PEGylation has a negligible effect on drug encapsulation efficiency. The lowest encapsulation efficiency was found in DPPC:Ch-based SMLs, which varied between 50% ± 2% and 48% ± 8%. The general high encapsulation efficiencies observed in all systems evidence the suitability of the new method herein presented for DOX encapsulation and the use of these nanocarriers for dual cancer therapy (by combining magnetic hyperthermia and chemotherapy).
Non-specific interaction between drug-loaded magnetoliposomes and GUVs (giant unilamellar vesicles, used as membrane models) was investigated to assess the ability of magnetoliposomes to release the content by fusion. Since doxorubicin fluorescence emission suffers a quenching effect from the magnetic nanoparticles that compose the SMLs core, this assay was performed by analyzing doxorubicin fluorescence variations before and after interaction with GUVs. Upon interaction, if fusion between the systems occurs, the DOX emission spectrum will reveal an unquenching effect due to the formation of a larger membrane and a corresponding increase in distance between the magnetic nanoparticles and DOX. The interest in DPPC-based SMLs is due to DPPC melting transition temperature, which is closely similar to the temperatures used in mild hyperthermia therapy. Therefore, the assays were also conducted at 55 °C to conclude the potential of magnetic hyperthermia to enhance fusion with cell membranes and the release capability of the solid magnetoliposomes. In all spectra of DPPC-based SMLs, DPPC:DSPE-PEG-based SMLs, and DPPC:Ch-based SMLs, an unquenching effect of DOX fluorescence is noticed upon SMLs’ interaction with GUVs (
Figure 12). When the interaction takes place at 55 °C, the spectra revealed a more pronounced unquenching effect. These results prove that membrane fusion occurs and that the fusogenic capability is enhanced with an increase in temperature.
Doxorubicin anisotropy was also measured upon SMLs interaction with GUVs to corroborate the results (
Table 4). It is important to notice that before interaction, DOX presented a lower anisotropy value in DPPC and DPPC:Ch lipid formulations than in DPPC:DSPE-PEG. Upon interaction with GUVs, DOX shows a slightly higher anisotropy in DPPC and DPPC:Ch-based SMLs than before interaction. In the case of DPPC:DSPE-PEG, a decrease in anisotropy was noticed. Since doxorubicin is located more superficially in PEGylated SMLs, anisotropy has an intermediate value between soy lecithin GUVs and DPPC:DSPE-PEG, proving interaction and fusion. These results can also explain the more prominent unquenching effect noticed in DPPC:DSPE-PEG spectra upon interaction with GUVs at 25 °C. In addition, above the DPPC phase transition temperature, an enhanced fusion between all systems occurs. The anisotropy values registered at 55 °C are rather low (around 0.07), which is the anisotropy value of doxorubicin in a saline PBS buffer [
58]. These values point to a possible DOX release to the aqueous medium.
3.7.4. Interaction with Human Serum Albumin
The interaction between nanocarriers and Human Serum Albumin (HSA) has significance in investigating and designing new nanocarriers in vitro. HSA intrinsic fluorescence comes from the emission of tryptophan, tyrosine, and phenylalanine residues. However, HSA emission arises mainly from the Trp214 residue alone, which is located in the hydrophobic cavity. The interaction between HSA and lipid vesicles induces changes in protein conformation that result in a quenching effect of Trp fluorescence. Therefore, fluorescence quenching assays can give an insight into the interactions of doxorubicin and doxorubicin-loaded liposomes with plasma proteins that affect stability of the different lipid formulations under physiological conditions.
The titration of an HSA aqueous solution (0.2 mM) with 1 μL of the different freshly prepared solutions (free doxorubicin, DOX-loaded DPPC liposomes, and DOX-loaded DPPC:DSPE-PEG liposomes) revealed a gradual HSA fluorescence drop, implying an interaction between the protein and doxorubicin or drug-loaded liposomes. The variation of HSA fluorescence quenching (%) with ligand concentration is shown in
Figure 13, evidencing an apparent reduction of interaction between DOX and HSA when the drug is encapsulated in PEGylated nanocarriers.
Fitting the results according to the nonlinear regression given by Equation (8), it was possible to calculate the dissociation constant (K
d) and the number of specific binding sites and then estimate the binding constant (K
b) (
Table 6). The results revealed a large binding constant between DOX and HSA and 1.20 binding sites, indicating a significant binding of free doxorubicin to the blood plasma protein. A significant decrease in binding constants is observed upon the encapsulation of doxorubicin in DPPC-based and DPPC:DSPE-PEG-based liposomes. The obtained results suggest that DPPC and DPPC:DSPE-PEG nanosystems effectively protect DOX from interaction with plasma proteins. Even though the binding constant presents a higher value in PEGylated liposomes when compared to non-PEGylated ones, the number of available binding locations decreases. Thakur et al. [
59] demonstrated that HSA penetrates DPPC liposomes, inducing an alteration in their packing order. This penetration is caused by hydrophobic interactions, which lead to the release of encapsulated payload. Thus, PEGylated vesicles can better protect the payloads, enhancing their bioavailability for longer time intervals, which is especially relevant for the effectiveness of EPR effect in tumors.
3.7.5. Drug Release Kinetics and Mathematical Modeling of Release Profile
Considering the complexity of cancer cells’ biology and the difficulties found along the treatments, it is essential to integrate combined and synergistic approaches to ensure a more effective, localized, and controlled therapy. The DOX release profiles from DPPC-based and DPPC:DSPE-PEG-based SMLs at different pH values (5.5 and 7.4) and temperatures (37 °C and 42 °C) are presented in
Figure 14. For DPPC SMLs (
Figure 14A), the results show that the drug release rate is highly dependent on pH and temperature. The release profile at 42 °C and pH = 5.5 stands out compared to the other combinations, exhibiting a burst release of 21.0 ± 0.4% in the initial six hours. The burst release is followed by a linear release phase, presenting a slow and controlled release profile that achieved the maximum release of 25 ± 2% at 28 h. Considering that 42 °C is above the DPPC transition temperature, there was an expected higher release rate at this temperature, considering the increase in permeability of magnetoliposomes membrane, which was verified at pH = 5.5. However, at pH = 7.4, the maximum release percentage is only 6.5 ± 0.2%, which is 3.5 times lower than the values obtained at pH = 5.5. These results provide evidence that acidic pH increases the hydrophilicity of DOX, contributing to an accelerated drug release, that is also verified at 37 °C. At physiological temperature, both pH conditions present slow-release kinetics, achieving a maximum release of 9 ± 1% at pH = 5.5 and 4 ± 1% at pH = 7.4. At physiological pH, DOX is protected by magnetoliposomes, and its release is delayed. In therapeutic conditions, the cardiotoxicity of the anthracyclines is dose-limiting, often leading to heart failure due to dilated cardiomyopathy years after exposure [
60]. Willis et al. [
61] demonstrated dose-dependent cardiac atrophy in mice at relatively low dose exposure. By delaying DOX release at physiological conditions, magnetoliposomes offer a protective effect over DOX-associated cardiomyopathy and other systemic adverse side effects. Liposomal doxorubicin was associated with a cardiac and gastrointestinal toxicity reduction, while maintaining a similar antitumor efficacy [
62]. Recently, Swietch et al. [
63] demonstrated that the medium pH has an impact on the affinity of DOX toward the carrier, the protonation of the anthracycline at acidic pH facilitating drug release. In addition, Chai et al. [
64] observed an increased release rate of DOX at a decreased medium pH in DOX-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles. The authors attributed the results to a strongly pH-dependent solubility, facilitating DOX release at low pH values.
A similar release kinetics behavior was found for DPPC:DSPE-PEG-based SMLs (
Figure 14B), with a notable delayed release rate at 42 °C. PEGylated SMLs did not exhibit a burst release in the early hours, only achieving a maximum DOX release of 13 ± 1% at 28 h. These results indicate that the PEG matrix on SMLs’ surface acts as a barrier by reducing the diffusion of DOX into the surroundings. The increased sustained DOX release in PEGylated SMLs is indicative of a prolonged delivery period of the drug.
Overall, the results point to a preferable DOX release at hyperthermia temperatures and acidic conditions, meaning that drug release may occur more readily in tumors. These are preliminary results of the behavior of the novel nanocarriers with a temperature and/or pH trigger, not considering the mechanical effect that superparamagnetic nanoparticles (subjected to an alternating magnetic field) would have on the active drug release. Considering that the purpose of controlled release systems is to maintain drugs in target sites at the desired concentration and control the drug release rate and duration, the sustained release ability of SMLs suggests a novel controlled release nanosystem.
The doxorubicin release profile was fitted to three kinetic models, specifically Weibull, first-order, and Korsmeyer–Peppas (see
Supplementary Material, Tables S3 and S4). The coefficients of determination (
R2) indicate that Weibull is the best-fitting model to describe the overall release of DOX from both formulations. Since the Weibull model is empirical, it lacks kinetic basis information and its parameters do not present a physical meaning. Papadopoulou et al. [
65] studied a link between the values of
b and the diffusional mechanisms of the release, proposing that for
b > 1, the drug transport follows a complex release mechanism;
b ≤ 0.75 indicates Fickian diffusion (in either fractal or Euclidian spaces); 0.75 <
b < 1 indicates a combined mechanism (Fickian diffusion and Case II transport).
Table S3 (DPPC SMLs) shows that at 42 °C, both pH conditions present a
b value higher than 1, evidencing a complex mechanism for DOX release. At 37 °C, it is verified that 0.75 <
b < 1, evidencing a Fickian diffusion.
Table S4 (DPPC:DSPE-PEG SMLs) indicates that
b < 0.75 in all the conditions, pointing to a Fickian diffusion. The results are indicative of a change in the mechanism of DOX release when functionalizing DPPC SMLs with PEG.
Similarly to the Weibull model, the first-order model revealed a higher
R2 for the assays performed at 42 °C and pH = 5.5 in both formulations, suggesting that the amount of released DOX is proportional to the remaining drug in the nanocarrier. The Korsmeyer–Peppas model presents the worst coefficient of determination values. The
n values are under the threshold value of 0.43, unless in the PEGylated SMLs at 42 °C and pH = 5.5. However,
n < 0.5, indicating a diffusion-controlled release mechanism [
66]. Since DOX is encapsulated in nearly spherical-shaped nanosystems, values of
n ≤ 0.45 are representative of a Fickian diffusion mechanism [
67]. The large variations of
R2 found at 42 °C in DPPC SMLs (when compared to the ones obtained by the Weibull model) indicate a complex drug release mechanism instead of only a diffusion-controlled release.
The morphology evolution of DOX-loaded DPPC and DPPC/DSPE-PEG SMLs as a function of temperature at both pH values were studied by DLS measurements (
Figure 15). Overall, it was noticed that the SMLs size tends to gradually decrease at pH = 7.4 with increasing temperature. At pH = 5.5, both formulations revealed an opposite variation in size, with a tendency to increase with rising temperature. These results show that SMLs become more unstable at acidic conditions and above the DPPC transition temperature, corroborating the DOX release profiles presented above.