Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - NanoMOFdeli (Design of NanoMOFs Capsules for Drug Delivery and Bioimaging.)

Teaser

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...

Summary

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. Although cancer survival rates have doubled in the last 40 years, cancer – especially those with poor prognoses such as pancreatic cancer and mesothelioma – remains a key health concern. This presents a clear unmet need for the development of effective therapies. Fostering new capabilities for research in cancer therapy, which represents an imperative social challenge, 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 – something where the PI has experience through two spin-outs, Immaterial Labs, for the manufacture of monolithic MOFs and Tarsis Technologies, for the delivery of agrochemicals.

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. Nowadays, the efficient delivery of a siRNA to its designated site of action and the protection of novel treatment molecules is a challenging, multidisciplinary bioengineering problem, the benefits of which will extend not only to multiple cancer treatments but other diseases such as Alzheimer’s.

In order to solve this problematic, 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).

Aim and Objectives. Within NanoMOFdeli ERC proposal, we aim to design a novel MOF toolbox with immune-safe, biocompatible and cell-penetrable nanosystems that can protect the genetic material cargo from nuclease breakdown in vivo for high transfection efficiency. If successful, this proposal will have a direct impact in the society as a whole, overcoming the bottleneck problem of intracellular nucleic acid delivery, by designing novel MOF-drug delivery systems (DDS) capable of dramatically increasing transfection efficacy.

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 (i.e. pore size distribution, surface area, pore volume) and surface chemistry of MOFs for siRNA delivery using experiments and molecular simulation techniques; iv) the assessment of their performance in vi

Work performed

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. In this regard, the connection of advanced experiments, including in vitro and in vivo studies and super-resolution microscopy with molecular simulations provide a unique opportunity to show the potential of MOF materials and, like so, to select the optimal structures for a given application.

Careful reviews of the original work plan has allowed us to work according to the original proposal, achieving the work described in the individual tasks of each work package (WP): WP1 (Tasks A, B, C and D); WP2 (Tasks I and J); WP3 (Tasks M and N). The only exception is Task H (WP 2) – see below.

WP1. Synthesis and Engineering of Biocompatible MOFs
WP1.1. Synthesis and Characterisation of MOFs
We have achieved the synthesis of a series of biocompatible MOFs. In particular, the microporous Zr-based MOF UiO-66, using terephthalic acid as an organic linker plus 5 different sister structures with the same topology. We have also developed the synthesis of nano-sized Zr-MOFs with a wider pore size (> 2 nm, in the mesoporous range), 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. In particular, the synthesis of nanosized NU-1000 and NU-901 has been developed through collaboration with Prof. Omar Farha, Northwestern University, whereas the synthesis of the other MOFs has been developed in Cambridge. All the materials have been prepared with particle sizes smaller than 200 nm as required to ensure free circulation within the smallest capillaries.

The size of the synthesised nanoparticles has been determined using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and dynamic light scattering (DLS). 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 37ËšC and for different incubation times, using HPLC analysis to detect the concentration in solution of the different building blocks. We confirmed that PBS was able to degrade most of the relatively robust MOFs synthesised above but, interestingly, we found that alternative buffers such as Tris and His are compatible with the MOFs and therefore can be used to load sensible macromolecules (RNA, peptides) as well as to graft antibodies on the external surface.

In these 18 months, we have loaded different cancer drugs such as DCA and alfa-CHC as well as the fluorescent model molecule calcein. 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 – see WP3, below.

WP1.2. MOF Amorphization
We have extended the use amorphization, developed in our lab, to the new nano-sized MOFs synthesised now using both mechanical and temperature amorphization.

Final results

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.

Website & more info

More info: http://aam.ceb.cam.ac.uk/.