Periodic Reporting for period 4 - NanoMOFdeli (Design of NanoMOFs Capsules for Drug Delivery and Bioimaging.)
Periodo di rendicontazione: 2022-03-01 al 2023-08-31
Cancer is a major health problem worldwide, being the most common cause of death after cardiovascular diseases. Every year, more than 12 million people are diagnosed with cancer and more than 1 in 3 people in Europe will develop some form of cancer during their lifetime. Fostering new capabilities for research in cancer therapy will be central to transform a critical sector of the pharmaceutical and medical European economy. The ultimate goal will be to create industrial opportunities in the commercialisation of new materials and therapies. The PI has developed two spin-put companies, Immaterial, for energy transition, and Vector Biosciences Cambridge, for macromolecule delivery.
One of the most promising opportunities is the use of novel treatments based on molecules capable of interfering the cell signalling system such as small interfering RNA (siRNA). Successful genetic manipulation via gene therapy and gene editing has the potential to revolutionise personalised medicine; it is estimated that the annual market value for effective gene delivery could exceed $30 billion. Despite its great potential, still, there is no feasible way of getting them delivered specifically to the tumour. The lifetime of such molecules is generally too short and therefore need to be protected and encapsulated in a drug delivery system until they are delivered into tumour target cells.
We have focussed on the use of biocompatible metal-organic framework (MOFs) for drug delivery. MOFs are a unique class of porous hybrid solids synthesised in a self-assembly process from metal corner units bridged by organic linkers. They combine vast structural and chemical diversity that make them extremely attractive for the encapsulation of siRNA. One of the most striking advantages of MOFs over more traditional porous materials used as carriers for drug delivery is the possibility to tune the host/guest interaction by functionalising the building blocks with chemical groups, providing the possibility to control the kinetic release of a therapeutic agent. Because of their intrinsic properties, MOFs can be in principle functionalised with further molecules with high affinity to target cells (e.g. antibodies).
There are four general objectives included in the proposal to achieve the main aim described above: i) the synthesis and characterisation of bio-compatible MOFs for drug delivery applications; ii) the post-synthesis modification of MOFs to enhance stability, controlled drug release, and targeting; iii) the identification of optimal textural properties and surface chemistry of MOFs; iv) the assessment of their performance.
One of the most promising opportunities is the use of novel treatments based on molecules capable of interfering the cell signalling system such as small interfering RNA (siRNA). Successful genetic manipulation via gene therapy and gene editing has the potential to revolutionise personalised medicine; it is estimated that the annual market value for effective gene delivery could exceed $30 billion. Despite its great potential, still, there is no feasible way of getting them delivered specifically to the tumour. The lifetime of such molecules is generally too short and therefore need to be protected and encapsulated in a drug delivery system until they are delivered into tumour target cells.
We have focussed on the use of biocompatible metal-organic framework (MOFs) for drug delivery. MOFs are a unique class of porous hybrid solids synthesised in a self-assembly process from metal corner units bridged by organic linkers. They combine vast structural and chemical diversity that make them extremely attractive for the encapsulation of siRNA. One of the most striking advantages of MOFs over more traditional porous materials used as carriers for drug delivery is the possibility to tune the host/guest interaction by functionalising the building blocks with chemical groups, providing the possibility to control the kinetic release of a therapeutic agent. Because of their intrinsic properties, MOFs can be in principle functionalised with further molecules with high affinity to target cells (e.g. antibodies).
There are four general objectives included in the proposal to achieve the main aim described above: i) the synthesis and characterisation of bio-compatible MOFs for drug delivery applications; ii) the post-synthesis modification of MOFs to enhance stability, controlled drug release, and targeting; iii) the identification of optimal textural properties and surface chemistry of MOFs; iv) the assessment of their performance.
To identify promising nanoporous MOF structures for siRNA delivery, a systematic study of the structure–adsorption performance relationship is essential. Yet to date, the discovery of promising novel porous materials for specific adsorption applications is happening by trial and error rather than by rational design. Tens of thousands of porous MOF structures have been reported in the literature and could potentially act as DDSs. However, most of them have only been tested for gas phase adsorption and studies of porous MOFs for drug delivery are extremely scarce, and generally do not include systematic drug adsorption, biocompatibility, targeting, and in vitro/in vivo studies.
Molecular simulation have been used widely for studying adsorption processes in porous materials, allowing the explanation and prediction of new experimental results. From a pool of ca. 1M entries of crystal structures at the CCDC, we looked for “look-for-MOF” criteria. From about 90,000 existing MOFs, only 9,000 were porous. In addition, we were able to filter only biocompatible metals such as Fe, Zr, Zn and Mg, reducing the number of MOFs to about 400 structures. The idea is to get a landscape of structure-property relationships to filter the best structures for RNA delivery.
Following MOF identification, we have achieved the synthesis of a series of biocompatible MOFs. We have also developed the synthesis of nano-sized Zr-MOFs with a wider pore size (> 2 nm), such as NU-1000, NU-901, PCN-222, PCN-224, PCN-333 and PCN-777, capable of encapsulating macromolecules such as siRNA. The nanoparticulate form of these MOFs has been optimised using solvothermal methods and the use of synthesis modulators, using monodentate capping ligands during the self-assembly of the MOFs. All the materials have been prepared with particle sizes smaller than 200 nm as required to ensure free circulation within the smallest capillaries.
The degradation of the MOFs has been studied under physiological conditions in phosphate buffered saline (PBS) as well as other different buffers (His, Tris, etc.) at 37˚C and for different incubation times. We have measured release times and have de-risked the experimental protocols to work with MOFs in drug delivery applications. We have also achieved the successful loading and protection from enzymatic degradation of a siRNA fragment – with a sequence of 22 nucleotides in length for HEK-293 cell lines expressing mCherry – on nano-sized NU-1000, allowing the delivery of siRNA effectively in the cytoplasm to knockdown gene expression.
We have worked on magnetic induction technology using Fe-based, magnetic nanoparticles (NPs). We have achieved the synthesis of core@shell Fe@Fe3O4 NPs with core diameters of ca. 8 nm. On the other hand, we have demonstrated the tuneable and versatile room-temperature incorporation of gold nanorods (AUNRs) into the NU-901 MOF by using the AuNRs as seeds for the MOF growth. We were able to achieve an excellent core-shell yield (>99%), with the first preliminary evidence for an NU-1000 phase grown at room temperature.
Performance of selected materials for drug delivery in cancer therapy has been evaluated by using in vitro studies. We evaluated the toxicity of the materials developed to better understand how MOFs cross the cell membranes. We have used an array of in vitro techniques, such as MTS and Annexin V cytotoxicity tests to study the biocompatibility of the Zr-MOFs named above and half-maximal inhibitory concentration (IC50) of siRNA/drug/calcein-loaded MOFs and free siRNA/drug/calcein on HeLa cells, J774.A1 and HEK-293. The basic idea is to use the MOF DDS to entrap and internalise the siRNA into the cellular endosomes, where the MOF will be subsequently degraded, causing the release of the siRNA into the cytosol. Once in the cytosol, the siRNA will bind the RNA-induced silencing complex (RISC).18 The activated siRNA-RISC complex will recognise the complementary mRNA, and the catalytic components of the complex, the endonucleases called Argonaute 2 (AGO2) proteins, will cleave the target mRNA sense strand. This will leave the antisense strand bound to the newly activated RISC-siRNA unit, which then will travel around the cells to find, bind to, and cleave any other complementary mRNA strands.
Molecular simulation have been used widely for studying adsorption processes in porous materials, allowing the explanation and prediction of new experimental results. From a pool of ca. 1M entries of crystal structures at the CCDC, we looked for “look-for-MOF” criteria. From about 90,000 existing MOFs, only 9,000 were porous. In addition, we were able to filter only biocompatible metals such as Fe, Zr, Zn and Mg, reducing the number of MOFs to about 400 structures. The idea is to get a landscape of structure-property relationships to filter the best structures for RNA delivery.
Following MOF identification, we have achieved the synthesis of a series of biocompatible MOFs. We have also developed the synthesis of nano-sized Zr-MOFs with a wider pore size (> 2 nm), such as NU-1000, NU-901, PCN-222, PCN-224, PCN-333 and PCN-777, capable of encapsulating macromolecules such as siRNA. The nanoparticulate form of these MOFs has been optimised using solvothermal methods and the use of synthesis modulators, using monodentate capping ligands during the self-assembly of the MOFs. All the materials have been prepared with particle sizes smaller than 200 nm as required to ensure free circulation within the smallest capillaries.
The degradation of the MOFs has been studied under physiological conditions in phosphate buffered saline (PBS) as well as other different buffers (His, Tris, etc.) at 37˚C and for different incubation times. We have measured release times and have de-risked the experimental protocols to work with MOFs in drug delivery applications. We have also achieved the successful loading and protection from enzymatic degradation of a siRNA fragment – with a sequence of 22 nucleotides in length for HEK-293 cell lines expressing mCherry – on nano-sized NU-1000, allowing the delivery of siRNA effectively in the cytoplasm to knockdown gene expression.
We have worked on magnetic induction technology using Fe-based, magnetic nanoparticles (NPs). We have achieved the synthesis of core@shell Fe@Fe3O4 NPs with core diameters of ca. 8 nm. On the other hand, we have demonstrated the tuneable and versatile room-temperature incorporation of gold nanorods (AUNRs) into the NU-901 MOF by using the AuNRs as seeds for the MOF growth. We were able to achieve an excellent core-shell yield (>99%), with the first preliminary evidence for an NU-1000 phase grown at room temperature.
Performance of selected materials for drug delivery in cancer therapy has been evaluated by using in vitro studies. We evaluated the toxicity of the materials developed to better understand how MOFs cross the cell membranes. We have used an array of in vitro techniques, such as MTS and Annexin V cytotoxicity tests to study the biocompatibility of the Zr-MOFs named above and half-maximal inhibitory concentration (IC50) of siRNA/drug/calcein-loaded MOFs and free siRNA/drug/calcein on HeLa cells, J774.A1 and HEK-293. The basic idea is to use the MOF DDS to entrap and internalise the siRNA into the cellular endosomes, where the MOF will be subsequently degraded, causing the release of the siRNA into the cytosol. Once in the cytosol, the siRNA will bind the RNA-induced silencing complex (RISC).18 The activated siRNA-RISC complex will recognise the complementary mRNA, and the catalytic components of the complex, the endonucleases called Argonaute 2 (AGO2) proteins, will cleave the target mRNA sense strand. This will leave the antisense strand bound to the newly activated RISC-siRNA unit, which then will travel around the cells to find, bind to, and cleave any other complementary mRNA strands.
The NanoMOFdeli project aims to develop a new fundamental capability for the design of novel MOF-based systems for drug delivery and bio-imaging for cancer therapies. Our team aims to develop a rational design of nanoMOFs and the optimisation of their structure and functionalisation to maximize siRNA carrying payloads, release kinetics, targeting and stability. Our recent advances have been able to show the potential to go beyond the state of the art in this topic, designing new DDs with extended-release times and able to cross the cell membrane during in vitro studies, using molecular simulations for high-throughput screening of MOFs. By using the materials described in the proposal as a platform technology, we plan to combine the specific tissue targeting of biologic medicines such as monoclonal antibodies with the potent intracellular mode of action of small molecule medicines (siRNA) – currently meeting with significant success in the field of antibody drug conjugates (ADCs). It will also advance the integration of in silico molecular simulations, in vitro and in vivo experiments, and super-resolution microscopy to understand the delivery process. This project will cross-fertilise capabilities: drug delivery on one hand through the design of targeting, uptake and release systems, and technology on the other hand: I will use live organism models to look at MOFs and their function, something that has not been attempted before. I am able to bring together leading-edge expertise and infrastructure in the underpinning fields to make this happen. This will feed into a rigorous programme of simulations, which in turn will permit us to understand and optimise MOFs to impart desirable characteristics such as release profiles and MOF stability, etc.