Generating a targeted, brain-permeable and stable polymeric nanoparticle for systemic gene delivery to glioblastoma

Periodic Reporting for period 1 - TargetGBM (Generating a targeted, brain-permeable and stable polymeric nanoparticle for systemic gene delivery to glioblastoma)

Berichtszeitraum: 2019-07-01 bis 2021-06-30

Glioblastoma multiforme (GBM) is the most prevalent deadly brain tumour and is currently incurable. Patients die from recurrent tumours that grow from cells surviving the first round of treatment. This is mainly due to the narrow therapeutic window of current drugs and to the low permeability of these drugs across the blood-brain barrier (BBB). Targeted non-viral delivery systems have shown great promise to decrease side effects of therapeutics and to enable the transport across biological barriers. The main objective in this proposal is to generate a targeted nanoparticle loaded with genetic material to treat brain cancer. The nanoparticle has been modified with two ligands: an antibody for targeting tumour cells and a peptide that enables transport across the BBB. In addition, a coating has been developed to enhance the stability of the polyplex in blood and be released upon internalization. A variety of formulations have been screened in vitro to evaluate their stability and transfection efficiency, and BBB permeability has been assessed in a cell-based BBB model. In the next stage of the project, the efficacy of the most promising candidates will be tested in a GBM mouse model.

The research project proposed in this action aimed to develop targeted and BBB-permeable nanoparticles aiming to provide a new treatment for glioblastoma multiforme. To guide my way toward this aim I set four specific objectives: 1) generation of site-specifically modified antibodies and peptides, 2) preparation and characterization of the targeted polyplexes, 3) in vitro assessment of safety, transfection efficiency, selectivity and BBB transport, and 4) in vivo study of targeting capacity and therapeutic efficacy. In the 24 months given for this action, I have completed goals 1-3. Given the COVID-19 pandemic many of the experiments were delayed and, as a result, objective 4, which involves in vivo experiments has not been accomplished yet. However, the remaining experiments will be conducted within the research projects awarded.

PROGRESS BY OBJECTIVES

Objective 1: Generation of site-specifically modified antibodies and peptides

Antibodies capable of targeting EGFR that can be anchored on the nanoparticles were produced. The extensive research conducted toward this end, led to the production of a review article published in the high impact journal (IF: 14.5) ACS Central Science. As for peptides intended to cross the BBB (BBB-shuttle peptides), not only we synthesized sequences reported in the literature but also we developed novel cyclic protease-resistant anti-transferrin receptor peptides. A research article reporting the novel BBB shuttles developed and a review are in preparation and will be submitted in the last trimester of 2021.

Objective 2: Development of the targeted polyplexes

Acrylate-terminated pBAEs were produced. As mentioned in the project, several strategies were explored in order to coat the pBAEs. Overall, polyplexes with adequate physicochemical properties have been developed.

Objective 3: In vitro assessment of safety, transfection efficiency and BBB transport

pBAEs polyplexes were proved to have negligible toxicity on a variety of cells. After a long process of optimization, the new coating enabled reducing the transfection efficiency of pBAEs by over 24-fold. Upon ligand modification, selective transfection to cancer cells overexpressing the transferrin receptor was enhanced by over 42-fold. Therefore, nanoparticles with selective transfection capacity have been prepared. These results are the basis for a manuscript in preparation. Moreover, we have assayed the capacity of coated nanoparticles in a BBB cell-based model and identified the most suitable candidates. The preliminary results obtained for the transport capacity of the final conjugates is currently being verified.

Objective 4: In vivo study of targeting capacity and therapeutic efficacy

Due to the COVID-19 pandemic all experiments were delayed and we intend to start the in vivo stage in late 2021. We have obtained funding for three additional projects well aligned with this proposal that will enable pursuing these experiments.

DISSEMINATION AND EXPLOITATION

Regarding scientific audiences, so far, we have published a review article at ACS Central Science (IF: 14.5) and we are currently preparing three more manuscripts. Moreover, results from this Project have been/will be shared at four international conferences and a seminars.

As planned activities for the general public, I have given a conference to IQS, delivered a talk followed by a colloquium with high school teachers in the Biosciences talks organized by IQS, and given another talk to pre-university students invited by the Catalan Chemical Society. Additionally, a webpage devoted to the research program that has emerged from this project has been created and an article for the IQS community and affiliated institutions has been prepared and will be released in September.

Regarding exploitation of the results, we have been contacting several companies who could be interested either in the technologies we have developed in the project or in the methods we have set up in the process. We have applied for a competitive project with one of the companies and we are negotiating a contract for the development of peptides against a particular target with another company.

Last but not less important, building on the preliminary results of this project and the experience acquired with this action, I have obtained the three competitive projects. In these projects, we will develop more sophisticated and efficient systems to target the nanomedicine developed in the MSCA-IF fellowship, which should enable higher degree of selectivity.

In this project we have developed a systemic non-viral gene delivery with the potential to increase the efficacy and safety of the current treatments for glioblastoma, which have i) low transfection efficiency, ii) little stability, iii) inadequate targeting capacity, and/or iv) insufficient BBB transport. We have built the system on the state-of-the-art oligopeptide-end-modified poly(beta-aminoester) (OM-pBAE) technology developed at the Materials Engineering Group (GEMAT) at IQS, which has exceptional transfection capacity and low toxicity. We have engineered coatings that enable controlling the promiscuity of OM-pBAEs. Moreover, we have modified them with appropriate targeting ligands to overcome the BBB and reach tumour cells. The modularity of the system makes it easily amenable to the targeted delivery of any oligonucleotide therapeutics to specific cancer cell populations and to provide a timely dosed and personalized therapy. This highly interdisciplinary project, encompassing chemistry, bioengineering, material sciences and drug delivery, has maximized the synergies between my background and the expertise of the host laboratory to engineer a novel multimodal platform against GBM. This project has been the basis of a new research program on protein and peptide targeted nanotherapeutics at IQS, for which we have already obtained considerable additional funding, and for several collaborations in academia and industry.

Toward brain-permeable nanomedicines to treat brain tumors

Periodic Reporting for period 1 - TargetGBM (Generating a targeted, brain-permeable and stable polymeric nanoparticle for systemic gene delivery to glioblastoma)

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