Final Report Summary - NANOSMART (Smart nanosystems for advanced cancer therapy)
The NanoSmart project was designed to advance the current knowledge in the preparation of drug-loaded nanosystems able to provide a triggered release of the therapeutic agents under stimulation. The advantages associated to these devices are mainly the on-demand dosing once the drug is located at the target site as a result of either passive accumulation or active delivery. This strategy was proposed in order to enhance the drug pharmacokinetics and minimize off-target effects of potentially cytotoxic substances. In fact, the administration of these molecules (e.g. antitumoral drugs) poses severe limitations to achieve effective dosing patterns able to maximise efficacy.
In the context of drug delivery systems, the binding of the drug to the carrier should be strong enough to avoid premature leakage, but labile to allow for an efficient release in the target site. Even in those cases in which a drug-loaded nanocarrier is able to reach a tumor region, an insufficient release of the bound drug may hinder therapeutic efficacy. Overall, given the various constraints affecting drug accumulation in tumors, it is not surprising that most currently approved drugs do not substantially increase performance after formulating them in nanocarriers.
In NanoSmart, these drawbacks were tackled with the development of nanometric matrices investigated to provide a platform able to protect the drug cargo for tunable periods of time. Most importantly, the ability to achieve site- and time- controlled release of therapeutics has been evaluated with the aim to entrap the drug and avoid premature release at undesirable places or time in the body. With a main focus on heat-activated nanocarriers, robust inorganic particles of mesoporous silica have been surface-functionalised with thermosensitive layers of biodegradable polymers. The behaviour of these polymers is such that a bulky hydrophilic conformation helps retaining the drug within the porous network while at temperatures above physiological they undergo a phase transition towards a collapsed structure that facilitates drug leakage. In this project we have employed biodegradable copolymers based on mono- and dilactate hydroxypropylmethacrylamide in which varying ratios of both monomers give rise to different temperature of response. A commonly used anticancer agent, doxorubicin, was released from these nanoparticles under thermal stimulation. In addition, the attached copolymers were chemically end-modified to allow longer circulation times and tumor targeting capabilities.
To realise the full potential of the thermosensitive copolymers synthesised in the project, the ability to form micellar nanocarriers upon heating was also assessed. The phase transition implies a shift to a hydrophobic behaviour in a portion of the polymer, enabling the formation of micelles. Interestingly, we successfully demonstrated two different pathways to stably encapsulate non-toxic doxorubicin prodrugs within the lipophilic core of the micelles. In this way, a pH-sensitive prodrug was loaded and further crosslinked to provide a responsive release once the nanocarriers are uptaken by tumor cells. On another tested approach, an enzyme-activated prodrug of doxorubicin was chemically attached to the polymer backbone before micelle formation. The in vitro evaluation with carcinoma cell lines showed a specific activation triggered by the presence of the enzyme, β-glucuronidase, in the tumor environment. Further in vivo studies in mice demonstrated the feasibility of this strategy to enhance the efficacy of the drug. In parallel experimentation with a different tumor model, neuroblastoma in mice, we managed to validate the enzyme-responsive release and prove its superiority in comparison with free drug treatment.
In an attempt to modify the release kinetics of the prodrug from the polymeric micelles, we studied the effect of the application of high intensity focused ultrasound (HIFU) in two different modes, continuous and pulsatile. With this technology, we managed to heat the nanocarriers in a specific manner and slightly accelerate the hydrolysis of the prodrug to yield a faster kinetics. The ability to tune the release of the therapeutic agents by an external application of ultrasounds opens exciting possibilities to remotely adjust the dosing to the patient needs.
The possibility to endow our matrices with a theranostic potential was examined through the encapsulation of iron oxide magnetic nanoparticles in the mesoporous silica network. The magnetic nanoparticles have the ability to enhance contrast in magnetic resonance imaging (MRI). In our experiments, we evaluated the structural changes caused by the incorporation of different amounts of contrast agents within the silica. It was shown that there is a threshold concentration under which the mesoporous particles keep the structural properties and an intact porosity. The efficiency of iron oxide encapsulation was optimized through the functionalisation of the magnetic particles with hydrophobic moieties (i.e. oleic acid), which were effectively transferred to a hydrophilic phase by the silica structure-directing agent in the synthesis.
As an attractive option to provide nanoparticles targeting to the disease, we also designed injectable carrier gels composed of chitosan and β-glycerophosphate mixed in acid solution. The thermoresponsiveness of the gel formulation allows an efficient entrapment of drug delivery systems upon co-injection in the target area. With the aim to carry out a proof-of-concept of this multicomponent system (defined as lipogel), we prepared doxorubicin-loaded thermosensitive liposomes with a transition over physiological temperature. The retention of these nanomedicines in the hydrogel was assessed after thermal treatment at body temperature, with the intention to trigger gelation without inducing doxorubicin release. The mechanical properties of the gel were not significantly affected by the addition of thermosensitive liposomes, and also the gelation temperature was kept within levels suitable for clinical application. Purposefully, the liposomal formulation was selected to provide a quick release of the drug upon heating. In this way, a pulsed release was observed when the system was submitted to temperature above the phase transition of the liposomes. The leakage of liposomes from the carrier was shown to be below 20% of the total doxorubicin amount during one week. Additionally, the combined effect of the release of free doxorubicin from the hydrogel followed by thermally triggered release from the loaded liposomes was shown to be effective to stop proliferation in carcinoma cell cultures in vitro. In a following step, an animal study has been designed in which the intratumoral injection of the lipogels will be assayed under HIFU treatment.
Finally, and in order to expand the potential applications of thermosensitive nanosystems for local delivery, we designed hard collagen scaffolds doped with a calcium phosphate phase. This kind of scaffolds is widely used in bone regeneration applications due to their ability to induce cell proliferation while replacing the mechanical and structural characteristics of the damaged tissue. Improved release kinetics of pro-regenerative cues mimicking natural cascades of growth factors is highly desirable for the success of these bone substitutes. We suggested a chemical route to covalently attach thermosensitive liposomes loaded with an active peptide, and subsequently tested a heat-responsive release from the scaffolds. This device was evaluated in pre-osteoblastic cell lines and demonstrated an enhanced capability for bone formation under external stimulation.
The results delivered by NanoSmart constitute a solid proof of the strong capabilities of triggered release systems to meet the needs of current clinical applications. The spatiotemporal control of the administration of highly cytotoxic drugs by using smart nanocarriers will offer translatable alternatives able to expand their therapeutic window and provide cost-effective treatments.
In the context of drug delivery systems, the binding of the drug to the carrier should be strong enough to avoid premature leakage, but labile to allow for an efficient release in the target site. Even in those cases in which a drug-loaded nanocarrier is able to reach a tumor region, an insufficient release of the bound drug may hinder therapeutic efficacy. Overall, given the various constraints affecting drug accumulation in tumors, it is not surprising that most currently approved drugs do not substantially increase performance after formulating them in nanocarriers.
In NanoSmart, these drawbacks were tackled with the development of nanometric matrices investigated to provide a platform able to protect the drug cargo for tunable periods of time. Most importantly, the ability to achieve site- and time- controlled release of therapeutics has been evaluated with the aim to entrap the drug and avoid premature release at undesirable places or time in the body. With a main focus on heat-activated nanocarriers, robust inorganic particles of mesoporous silica have been surface-functionalised with thermosensitive layers of biodegradable polymers. The behaviour of these polymers is such that a bulky hydrophilic conformation helps retaining the drug within the porous network while at temperatures above physiological they undergo a phase transition towards a collapsed structure that facilitates drug leakage. In this project we have employed biodegradable copolymers based on mono- and dilactate hydroxypropylmethacrylamide in which varying ratios of both monomers give rise to different temperature of response. A commonly used anticancer agent, doxorubicin, was released from these nanoparticles under thermal stimulation. In addition, the attached copolymers were chemically end-modified to allow longer circulation times and tumor targeting capabilities.
To realise the full potential of the thermosensitive copolymers synthesised in the project, the ability to form micellar nanocarriers upon heating was also assessed. The phase transition implies a shift to a hydrophobic behaviour in a portion of the polymer, enabling the formation of micelles. Interestingly, we successfully demonstrated two different pathways to stably encapsulate non-toxic doxorubicin prodrugs within the lipophilic core of the micelles. In this way, a pH-sensitive prodrug was loaded and further crosslinked to provide a responsive release once the nanocarriers are uptaken by tumor cells. On another tested approach, an enzyme-activated prodrug of doxorubicin was chemically attached to the polymer backbone before micelle formation. The in vitro evaluation with carcinoma cell lines showed a specific activation triggered by the presence of the enzyme, β-glucuronidase, in the tumor environment. Further in vivo studies in mice demonstrated the feasibility of this strategy to enhance the efficacy of the drug. In parallel experimentation with a different tumor model, neuroblastoma in mice, we managed to validate the enzyme-responsive release and prove its superiority in comparison with free drug treatment.
In an attempt to modify the release kinetics of the prodrug from the polymeric micelles, we studied the effect of the application of high intensity focused ultrasound (HIFU) in two different modes, continuous and pulsatile. With this technology, we managed to heat the nanocarriers in a specific manner and slightly accelerate the hydrolysis of the prodrug to yield a faster kinetics. The ability to tune the release of the therapeutic agents by an external application of ultrasounds opens exciting possibilities to remotely adjust the dosing to the patient needs.
The possibility to endow our matrices with a theranostic potential was examined through the encapsulation of iron oxide magnetic nanoparticles in the mesoporous silica network. The magnetic nanoparticles have the ability to enhance contrast in magnetic resonance imaging (MRI). In our experiments, we evaluated the structural changes caused by the incorporation of different amounts of contrast agents within the silica. It was shown that there is a threshold concentration under which the mesoporous particles keep the structural properties and an intact porosity. The efficiency of iron oxide encapsulation was optimized through the functionalisation of the magnetic particles with hydrophobic moieties (i.e. oleic acid), which were effectively transferred to a hydrophilic phase by the silica structure-directing agent in the synthesis.
As an attractive option to provide nanoparticles targeting to the disease, we also designed injectable carrier gels composed of chitosan and β-glycerophosphate mixed in acid solution. The thermoresponsiveness of the gel formulation allows an efficient entrapment of drug delivery systems upon co-injection in the target area. With the aim to carry out a proof-of-concept of this multicomponent system (defined as lipogel), we prepared doxorubicin-loaded thermosensitive liposomes with a transition over physiological temperature. The retention of these nanomedicines in the hydrogel was assessed after thermal treatment at body temperature, with the intention to trigger gelation without inducing doxorubicin release. The mechanical properties of the gel were not significantly affected by the addition of thermosensitive liposomes, and also the gelation temperature was kept within levels suitable for clinical application. Purposefully, the liposomal formulation was selected to provide a quick release of the drug upon heating. In this way, a pulsed release was observed when the system was submitted to temperature above the phase transition of the liposomes. The leakage of liposomes from the carrier was shown to be below 20% of the total doxorubicin amount during one week. Additionally, the combined effect of the release of free doxorubicin from the hydrogel followed by thermally triggered release from the loaded liposomes was shown to be effective to stop proliferation in carcinoma cell cultures in vitro. In a following step, an animal study has been designed in which the intratumoral injection of the lipogels will be assayed under HIFU treatment.
Finally, and in order to expand the potential applications of thermosensitive nanosystems for local delivery, we designed hard collagen scaffolds doped with a calcium phosphate phase. This kind of scaffolds is widely used in bone regeneration applications due to their ability to induce cell proliferation while replacing the mechanical and structural characteristics of the damaged tissue. Improved release kinetics of pro-regenerative cues mimicking natural cascades of growth factors is highly desirable for the success of these bone substitutes. We suggested a chemical route to covalently attach thermosensitive liposomes loaded with an active peptide, and subsequently tested a heat-responsive release from the scaffolds. This device was evaluated in pre-osteoblastic cell lines and demonstrated an enhanced capability for bone formation under external stimulation.
The results delivered by NanoSmart constitute a solid proof of the strong capabilities of triggered release systems to meet the needs of current clinical applications. The spatiotemporal control of the administration of highly cytotoxic drugs by using smart nanocarriers will offer translatable alternatives able to expand their therapeutic window and provide cost-effective treatments.