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Theranostic Injectable Hydrogel for Glioblastoma

Periodic Reporting for period 2 - HyGlio (Theranostic Injectable Hydrogel for Glioblastoma)

Reporting period: 2018-09-30 to 2019-09-29

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Its infiltrative nature, ability to mutate into newer and more aggressive tumor variants, and the location behind a partially-intact blood brain barrier, make this tumor extremely difficult to treat via systemic chemotherapy and invariably fatal.
This project aimed at designing a biomaterials-based approach for direct intra-cranial (IC) drug delivery to brain tumors.
To achieve this goal, a multi-component system composed of nanoparticles and a hydrogel for enhanced intra-cranial treatment retention, was designed and characterized.
Drug and particle distribution and retention in the brain after IC injection were also characterized in vivo on tumor-free and tumor-bearing mice, using fluorescent drug surrogates and fluorescent-tagged nanoparticles.

The overall objectives of HyGlio were:

1.To optimize nanoparticles design to maximize drug loading and tumor targeting
2. To design IC drug delivery systems
3. To analyze brain distribution and retention of IC-injected nanoparticles or nanoparticles embedded in injectable hydrogels
4. To validate the system in an intra-cranial model of GBM in mice

In conclusion, the results achieved in this project showed that hydrogels enhance the retention of nanoparticles in the brain of tumor-bearing mice, and increased the tumor coverage. Moreover, tumor treatment with drug loaded nanoparticles resulted in enhanced survival of tumor bearing mice and in reduced toxicity of the encapsulated drug. We also determined that after drug treatment, modifications in number and structure of tumor blood vessels as well as over-expression of TWIST (indicative of epithelial to mesenchymal transition) occurred.
"1. Polymer nanoparticles with a core-shell structure have been designed and characterized.
I used 3 different polyurethanes to compose the particle core and a mixture of phospholipids to obtain a biocompatible shell.
I demonstrated that these particles can be used to co-encaspulate multiple drugs and imaging agents with high entrapment efficiency and are able to co-deliver them to tumors to a significantly higher extent as compared to free drugs.
This work has been published in Acta Biomaterialia (2018).
the same particle design was exploited in the return phase to encapsulate and release in a controlled fashion microRNA molecules for cell reprogramming

2. Once demonstrated the validity of this nanoparticle approach, I designed and characterized an intracranial drug delivery system for the delivery of a potent proteasome inhibitor. This drug has shown high cytotoxicity in vitro on different GBM cell lines, including GBM stem cells (GSCs).

3. Intracranial retention and distribution of fluorescent nanoparticles loaded with a fluorescent drug surrogate was studied in vivo on tumor-free and tumor-bearing mice. We demonstrated that intracranial injection of nanoparticles or nanoparticles embedded in a commercial polymer hydrogel is feasible and safe for animals. We showed that the hydrogel enhances intra-tumor retention of nanoparticles and drug, by using in vivo imaging system. We also analyzed drug distribution in the brain after 24h, 5days, 10 days and 20 days post-injection, by fluorescence analysis and quantification on at least 20 coronal sections of the brains (4 mice/group). We showed that nanoparticles remain in the tumor, are internalized by cancer cells in vivo, and migrate through the corpus callous, main route of GBM cells invasion.

4. I demonstrated that intra-cranial treatment with nanoparticles significantly extends the survival of GBM bearing mice (injected with a GSCs-derived tumor or U87 tumors). Some preliminary investigations on the reasons for tumor recurrence after intra-cranial treatment have also been performed, to identify possible combination therapies that may further extend survival. Periostin secretion, enhanced vascularization, and enhanced TWIST expression was detected after treatment with drug-loaded nanoparticles. Strategies addressed at the concomitant inhibition of these factors may further improve treatment of these tumors.
The use of the HG only partially extended the survival of mice with respect to free nanoparticles, but not in a significant fashion.

Results have been disseminated at international and national events:

a. IV workshop ""RESEARCH AND NANOMEDICINE"" organized in Pavia (Italy) for Ph.D. students (18/06/2019) - Oral Presentation for master and Ph.D. students (50 participants)
b. Keystone Symposium ""Cancer Metastasis: The Role of Metabolism, Immunity and the Microenvironment"" Florence, March 15 - March 19, 2019 - Poster Presentation (approx. 500 participants)
c.30th EUROPEAN SOCIETY FOR BIOMATERIALS CONFERENCE, 09-13 September 2019 Dresden (Germany) - Poster Presentation and Flash Oral Presentation (approx. 900 participants)
d.Invited lecture for students of the Master Course in Bioengineering and Mechanical Engineering (course of Materials Process and Manufacturing )(University of Newcastle, UK) Feb. 28 2019 Oral Presentation for master students (100 participants) and short lecture for post doctoral fellows (10 participants)
e. Three peer-reviewed publications have been published in high-impact journals. One publication is currently under review in Acta Biomaterialia."
"An intra-cranial treatment for GBM has been designed and characterized. In depth biodistribution after intra-cranial injection of drug-loaded nanoparticles is only poorly investigated, so this work sheds light on the validity/potential/and drawbacks of such approach.
I achieved extended survival of animals bearing an invasive intracranial GBM model, which is a great advancement with respect to the state of the art where less relevant models are used.

Most anticancer drugs are screened out for GBM treatment in spite of their promising in vitro results, because they do not accumulate in the brain after systemic administration. With this system, t is possible to inject drugs directly in the brain, by-passing the biological barriers that hamper efficient IC drug delivery. I showed IC injection of nanoparticles is feasible, safe and strongly reduces toxicity vs. free drugs.

Based on the results, we have identified enhanced vascularization and Periostin secretion as two mechanisms associated with resistance to treatment. Addressing such modifications with a combined treatment strategy, may further enhance survival.

Carrier impact of the fellowship.

Three Scientific publications in high impact journals (2 of which as senior author) and one currently under review.
I have been assigned the role of contract professor for the master course ""hands on training in biomedical nanotechnologies and advanced therapies"" and for the Ph.D. course ""Advanced Therapies (Nanomedicine, Gene and Cell Therapy) in Surgery""
Beginning Dec 1st 2019: I will be hired as tenure track assistant professor in the Department of Mechanical and Aerospace Engineering (Politecnico di Torino, Italy)."
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