Development of multi-responsive phase change contrast agents with hybrid shells of potential theranostic relevance

Recently, in medicine, there has been much focus on the early screening of diseases such as tumours. Screening refers to the use of simple tests across an apparently healthy population in order to identify individuals who have risk factors or early stages of disease, but do not yet have symptoms. This technique has some limitations [1]: - It can involve cost and use of medical resources on a majority of people who do not need treatment; - Adverse effects of screening procedure (e.g. stress and anxiety, discomfort, radiation exposure, chemical exposure); - Test result may show false positive or false negative. Ultrasound diagnostic technique can overcome some of these limitations, being a non-invasive and low-cost real-time imaging tool. In order to improve the quality of the ultrasound image, contrastographic techniques have been introduced, based on the use of gas microbubbles (MBs) capable of reflecting the ultrasound signal and therefore, once injected intravenously, increasing the echogenicity of the blood. The “phase-change” contrast agents, also called microdroplets (MDs), have aroused great interest because of their small size and improved stability, in fact they are able to extravasate into tumor tissues, constituting a potential diagnostic and therapeutic agents. MDs with a liquid perfluorocarbon (PFC) core exposed to a proper ultrasound field transform into microbubbles (MBs) by a process known as Acoustic Droplet Vaporization (ADV). The properties of the phase-change ultrasound contrast agents rely on this process [2]. The main asset of such systems is the possibility to conjugate their drugs and nanoparticles cargo capability with the dramatically increased ultrasound echogenicity after ADV. A number of evidences have highlighted the role of nanotechnologies in the field of biomedicine. The introduction of phase-change MDs in the vast drug delivery and theranostic scenario is evaluated through the key opportunity they offer to conjugate multi-drugs intracellular cargo capability with echogenicity by medical ultrasound promoted core vaporization [3]. Significant improvement in this respect mainly passes through the optimization of the MDs shell with respect to stability, chemical versatility and synthesis efficiency. Recently, our group defined a novel class of MDs (named DexMA MDs) based on a biocompatible double shell made up of a surfactant monolayer coated by biodegradable cross-linked dextran-methacrylate (DexMA), and filled with a high boiling decafluoropentane (DFP) core [4, 5]. The elastic properties of these systems are comparable to those of the lipid-shelled ones, with a gain in stability and reversibility with respect to the ADV event [5]. However, more efforts are needed to increase the size control and the ADV efficiency of DexMA MDs, and to demonstrate their actual potential as theranostic candidate. Specifically, the polydispersity of the size distribution in the 2–5 µm range results in the formation upon ADV of large microbubbles with sizes of ∼ 10µm. Such bubbles are too large to actually be employed in vivo since they would cause occlusion of capillaries, whose typical size is of 5–10 µm. Moving from these results, in the first part of this PhD work, we focused our efforts on the optimization of the MDs preparation protocol, by varying the emulsification parameters such as time and speed, with the aim to obtain more monodispersed size distributions, centred at lower values. The obtained size ∼ 1 µm with a polydispersion index of 0.1, however, has a limited stability over time which worsens at physiological temperature of about 37 . In particular, they tend to grow over time mainly due to aggregation and coalescence phenomena. The shell undoubtedly plays a crucial role in this respect. Consequently we have worked on the MDs shell design allowing to improve the stability of the system as well as to integrate the MDs shells with gold nanoparticles (AuNPs). In fact, the well-known ability of AuNPs to release near-field photothermal energy could be exploited to facilitate the phase transition of MDs core (yielding a high vaporization efficiency), to induce the photoacoustic effect (enhancing the imaging contrast) and to support photothermal therapies [6]. The effect of vaporization using photothermal energy is called Optical Droplet Vaporization (ODV). Proceeding from literature we introduced the dioctadecyldimethylammonium bromide (DDAB), a cationic lipid characterized by a very small polar head and an apolar tail, as a novel component of the internal shell, replacing the surfactant used in the previous studies [4]: Epikuron200 (a heterogeneous mixture of surfactants with a short/medium hydrophobic chain and low negative charge). The positive charge of the surfactant positively affects both the stability of the colloidal system, avoiding the collision between the drops, and the electrostatic adsorption of the AuNPs. Consecutively we performed a systematic characterization, focused on the stability of MDs as well as on their acoustic response. A good stability over time and in temperature was observed. Furthermore, the additional elastomeric coating (dextran methacrylate) confers greater elasticity and strength to the system. The DDAB MDs show a clear acoustic response both at room temperature and at physiological temperature. A simple strategy for functionalising the MDs shell with AuNPs, based on electrostatic adsorption, was developed and then was carried out a stability study over time and temperature. MDs implemented with AuNPs cannot withstand the stress caused by ultrasound, in fact, AuNPs amplify the effect of the ADV [7], causing the rupture of the drops. This limit is overcome with the addition of the outer shell of polymerized dextran methacrylate (DexMa), which makes the system more resistant to compressions and expansions generated during irradiation. Such hybrid MDs with a double (lipidic and polymeric) shell show, in addition to an acoustic response, an important photothermal response: irradiating with a laser at 34 mW/cm2 for 2 minutes we can observe the MD-MB transition at physiological temperature. MDs, and in general capsules, are also an interesting toll for drug delivery application, in fact they can act as cargo and allow the release of drugs in concentrations locally superior to those obtainable from the single non-encapsulated drug, thus increasing the efficiency and decreasing the side effects [8, 9]. Therefore, in the last part of PhD work we have studied the MDs drug cargo capability and the in vitro biological applications of DDAB MDs on different cell lines, exploiting the biological attractiveness of DDAB shell, that lies in the high affinity with cell plasma membrane, and the ability to bind proteins and DNA, for remarkable gene therapy and antitumor action [10, 11]. In order to obtain a significant interaction between the MDs and the cells it has been necessary to further implement the MDs preparation protocol using a pulsed high-power insonation method. In this way 3 · 1010 MDs/mL can be readily provided in a few seconds, resulting in low polydispersed 1 µm sized MDs. These DDAB MDs act as efficient reservoir for doxorubicin (Dox) in vitro with a entrapment efficency of ∼ 30% of the total Dox. Furthermore, we have carried out a cytotoxicity study on different cell lines as: NIH/3T3 (murine fibroblasts), RAW 264.7 (murine macrophoges), MDAMB231 (human breast adenocarcinoma). The last one is ER, PR and E-cadherin negative and expresses mutated p53, and represents a good model of triplenegative breast cancer. So, we have studied cytotoxicity for different MDs concentration and consequently for different Dox concentration and for different time of incubation: 24, 48 and 72h. In general the interaction between the MDs shell and the cell membrane favours the drug internalization within cells to kill them up to 45% in only 24h. Equivalent concentrations of either free doxorubicin or unloaded MDs do not produce any effect on the cell viability. More important, the transition from doxorubicin loaded MDs to microbubbles can be used for in real-time imaging. These results may be relevant in greatly amplifying the benefit-to-risk ratio of chemotherapeutics as well as in facilitating real-time monitoring of the treatments. Summing up we obtained a stable and versatile multimodal “phase-change” contrast agent which can also act as a drug and/or nanoparticles carrier suitable for its synergistic effect with drugs (Dox).