Final Report Summary - NANOTAR (SELF-ASSEMBLY POLYMERIC NANO-BIOMATERIALS FOR DRUG DELIVERY AND TARGETING) NANOTAR was aimed to build up and consolidate a research program in Pharmaceutical Nanomaterials Science based on the design, synthesis, full characterization and preclinical evaluation of novel self-assembly polymeric nano-biomaterials as drug nanocarriers. The investigation of the relationship between the nano/microstructure and the properties and the exploration of new processing methods for the improvement of the biopharmaceutical performance of drugs for the therapy of diseases with high socioeconomic impact is the ultimate goal of NANOTAR. Our primary goal in this project was to investigate a robust and versatile nanotechnology platform that could be adjustable to a broad spectrum of active molecules and for the treatment of different diseases. The proposal was initially oriented to make a substantial contribution to improve the treatment of the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and tuberculosis (TB), two infetious diseases that mainly hit developing nations. Owing to the discoveries made during the project and the establishment of new strategic collaboration alliances, some of which are beginning now or will begin in the near future after the protection of new intellectual property generated during the project, we have also made breakthrough progresses in the treatment of solid pediatric cancers. This new research avenue emerged from a new collaboration with Dr. Angel Carcaboso of the Hospital Sant Joan de Déu-Barcelona (Spain), who is an expert in the development of pediatric patient-derived tumor models in mice. Collaborations with preclinical and clinical researchers expanded the applicability of these novel platforms, the socio-economic impact of our research, and enabled us to obtain additional funding to continue our investigations in new promising directions. In advance, the specific aims of the proposal and the progresses made in each one of them during the second half of the project are summarized:Specific Aim 1-Development of alkoxysilane-grafted polymeric amphiphiles. The goal was to investigate the use of the inorganic sol-gel chemistry to physically stabilize the preformed polymeric micelles and to produce a silicone release-controlling domains with tuned porosity and diffusion properties. For this, poly(ethylene oxide)-b-poly(propylene oxide) (PEO-PPO) amphiphilic block copolymers that form polymeric micelles in water were chemically modified with sol-gel precursor functional groups that after hydrolysis and condensation will form the silicone domains that will maintain the polymeric micelles assembled even after extreme dilution. We investigated two methods for the condensation/crosslinking stage: freeze-drying and spray-drying. Freeze-drying failed and thus, we focused on the spray-drying technology using the advanced Nano Spray-Dryer B-90. The produced nanoparticles were thoroughly characterized and the anti-HIV drug tipranavir encapsulated and its release in vitro assessed. The experimental work was conducted by the MSc student Julia Talal with the assistance of an undergraduate student. Julia finished her degree in late 2016 and the results of her research were published in the prestigious journal Chemistry of Materials in early 2017 (see details in publications list). In October 2016, the MSc student Vladi Kushnirov-Melnitzer joined the group to continue these investigations and, inspired by Julia’s results, he developed a completly novel method to synthesize polymer-ceramic hybrid nanocomposites formed by polymeric micelles and titanium oxide. He already characterized the nanostructure of these novel nanomaterials using different techniques, including an advanced transmission electron microscope recently installed in our faculty, and he successfully encapsulated a model drug. He is currently conducting release experiments and is expected to finish his degree later this year. The results of his research will be first protected and then, published in a high-impact journal. Specific Aim 2-Development of polymeric amphiphiles conjugated with mono- and oligosaccharide residues. Our hypothesis in this goal was that the conjugation of monosaccharides and oligosaccharides to the surface of polymeric micelles will increase their physical stability, the uptake of the polymeric micelle by sugar-avid cells and the inhibition of efflux pumps that remove drugs against a concentration gradient with respect to pristine micelles. In addition, we aimed to investigate the effect of the glycosylation extent on the physical stability, the drug encapsulation capacity and the active targeting of the polymeric micelles to sugar-avid cells. In this part of the project, we also used PEO-PPO block copolymers and glycocosylated. During the first period of the project, we confirmed our hypothesis and published these results in the journal Macromolecular Bioscience in 2014. To increase the glycosylation extent, we initially aimed to polymerize gluconolactone employing different polymer/gluconolactone ratios and using microwave radiation though, this approach failed. Thus, we changed the synthetic strategy and modified the copolymer with gluconic acid (the oxidized form of D-glucose, monosaccharide), lactobionic acid (the oxidized form of lactose, disaccharide) and the sequential conjugation of gluconic acid and lactobionic acid (trisaccharide). After the complete characterization that confirmed the chemical modification of the different derivatives, the effect of the glycosylation extent on the critical micellar concentration (CMC) was characterized by dynamic light scattering. Conjugation of a D-gluconic acid in each terminal block of the copolymer resulted in a dramatic decrease of the CMC by 8- to 11-fold. In contrast, further glycosylation with lactobionic acid and gluconic acid + lactobionic acid had a detrimental effect due to the increase of the amphiphile hydrophilicity. Then, glycosylated PMs successfully encapsulated the tyrosine kinase inhibitor imatinib (an anticancer drug of the novel group of “molecularly targeted” anticancer agents), increasing its aqueous solubility by up to 100 times. Glycosylated polymeric micelles showed very good cell compatibility in the rhabdomyosarcoma cell line Rh30 that overexpresses glucose transporter-1 that is targeted by the sugar residues. Rhabdomyosarcoma is a type of solid pediatric cancer that attacks different tissues such as muscle. Finally, we compared the efficacy of free and the nano-encapsulated drug to inhibit the proliferation a in which tyrosine kinase signaling promotes growth and glucose transporter-1 is overexpressed. Nano-encapsulation in the glycosylated micelles increased the anti-cancer efficacy in vitro up to 84-fold. The experimental work was conducted by the MSc student Alexandra Bukchin that finished her degree in late 2016 and the results of her research were published in the journal Applied Materials Today in early 2018 (see details in publications list). Based on these results, we focused our investigations on the polymeric micelles modified with gluconic acid and the encapsulation of the second-generation tyrosine kinase inhibitor dasatinib which is currently being clinically trialed in different pediatric sarcomas. Our interest in this application stemmed from the fact that most pediatric sarcomas overexpress glucose transporter-1 and overexpress tyrosine kinase pathways that can be inhibited by these drugs. The glucosylated PEO-PPO nanocarrier encapsulated significantly higher amount of drug and showed a 9-fold increase of the drug efficacy in the Rh30 in vitro with respect to the free drug. In addition, using immunodeficient mice bearing the glucose-avid Rh30 xenograft, we revealed that the glucosylated polymeric nanomicelles increased the delivery of dasatinib to the tumor parenchyma and that the exposure of off-target tissues and organs to the drug was substantially minimized. Upon experimental confirmation that most patient-derived xenograft (PDX) models of pediatric sarcomas overexpress glucose transporter 1 (GLUT-1), we demonstrated the selective accumulation of dasatnib in a patient-derived rhabdomyosarcoma model in vivo. Conversely, the reference dose administered by the oral route was not tumor-selective. Finally, the improved nanocarrier pharmacokinetics led to prolonged median survival of mice bearing a clinically relevant PDX model of alveolar rhabdomyosarcoma from 19 days for the untreated controls to 27 days for the targeted therapy. These results were recently published in the Journal of Controlled Release (see details in publications list). Following the European regulations, all the animal studies were conducted in the laboratory of our collaborator Dr. Angel Carcaboso after the approval of the corresponding protocols. Alexandra Bukchin continues her PhD studies in my laboratory and she is now employing the polymeric micelles designed in Specific Aim 3 to target a deadly pediatric brain tumor called diffuse intrinsic pontine glioma; this research is being funded by a Euronanomed2 project (Cure2DIPG). The chemical modification of PEO-PPO polymeric micelles with glucose is still tricky and the yields are not optimal. Thus, another MSc student (Anna Zaritski) who joined the group in March 2016 and is expected to finish her degree later this year, is employing the same concept of targeting glucose transporters with polymeric micelles but she is using the synthetic pathways developed in Specific Aim 3. In this framework, she synthesizes polymeric micelles in which the hydrophilic domain is completely formed by a polymer that targets these transporters. Her results are very promising and we are now pondering the submission of a patent application. Specific Aim 3. Development of mucoadhesive polymeric micelles. The concept in this aim was that the grafting of hydrophobic polymer blocks of different chemical nature to mucoadhesive polysaccharides confers them self-assembly properties, improve the encapsulation capacity of model drugs and prolong the residence time of the nanocarrier in different mucosal tissues. We initially worked with chitosan as mucoadhesive template and modified its side-chain with different hydrophobic blocks. After characterizing the produced copolymers and demonstrating the formation of nanometric polymeric micelles, we investigated encapsulation of different drugs. We confirmed good cell compatibility in different cell types and the mucoadhesion in vitro with mucin in solution. A new idea that emerged was during our work was to physically stabilize the polymeric micelles using mild (non-covalent) chemistries. Following this concept, these polymeric micelles were stabilized in a controlled manner using ionotropic crosslinking to form amphiphilic nanogels that were stable in dispersion for several weeks. The first work in this aim was undertaken by the MSc student Maya Menaker Raskin who finished her degree in May 2016 and the results were published in the journal Nanomedicine (Lond.) in late 2016 (see details in publications list). This work also resulted in one patent application that has been first filed in PCT and in 2017 submitted to the USPTO. At the same time, we investigated the use of protoporphyrin, a highly aromatic molecule that tends to aggregate by pi-pi interactions, as stabilizer of the polymeric micelles. We successfully modified the polymeric amphiphiles with the red fluorescent molecule and demonstrated that amounts as low as 2% w/w stabilized the polymeric micelles. Moreover, we capitalized on the red fluorescence of protoporphyrin to study the permeability of the nanoparticles in an in vitro model of the intestinal epithelium, Caco2 monolaters. Our results indicated that despite the relatively large size of the micelles (200-300 nm), they cross the epithelial monolayer mainly by a paracellular pathway due to the opening of tight junctions. Complementary uptake studies by flow cytometry indicated that no endocytosis, though due to passive or facilitated diffusion, some internalization takes place. This work was conducted by the student Inbar Schlachet who joined the group in April 2016 and was transferred directly to the PhD program in late 2017. The results were published in the journal Biomaterials Science in 2017 (see details in publications list). Inbar is currently employing these polymeric micelles for the intranasal delivery of anti-cancer drugs in pediatric brain tumors in a project funded by the Israel Science Foundation. At the same time, the permeability of these chitosan-made polymeric micelles before and after crosslinking and without and with thiolation (modification with thiol groups) was investigated in a more reliable in vitro model of intestinal epithelium that comprised Caco2 cell line and HT29-MTX cell line that produces mucin. This research was conducted by the MSc student Imrit Noi who joined the group in April 2016 and is expected to graduate during the next month. The results of this thesis will be submitted for publication in the open-access journal Polymers during the coming weeks. In parallel, we also investigated poly(vinyl alcohol) (PVA) was hydrophilic component of the polymeric micelles; PVA can be crosslinked with boric acid, a very mild and biocompatible chemistry. For this, PVA-g-poly(N-isopropylacrylamide) amphiphiles were synthesized and fully characterized. Then, preformed polymeric micelles were crosslinked with boric acid. The nanomaterials showed sizes in the 100-250 nm range and a spherical morphology. We also demonstrated that the size of the polymeric micelles could be fine-tuned by changing the concentration (and the aggregation pattern) of the polymeric amphiphile in water. Upon crosslinking, the polymeric micelles turned into physically stable amphiphilic nanogels that displayed size and size distribution similar to the micellar precursor for up to two weeks, even under disfavored conditions of concentration and temperature that, in the case of non-crosslinked counterparts, resulted in fast disassembly. In addition, we show for the first time the feasibility of the spray-drying technology to consolidate the 3D network formed between PVA and boric acid and to produce stable powders that can be reconstituted upon use at any desired concentration. Moreover, the formation of a borated surface conferred the nanogels with good mucoadhesiveness in vitro. Finally, these novel nanomaterials showed optimal cell compatibility in a model of the intestinal epithelium, the Caco2 cell line. This work was conducted by the student Hen Moshe who joined the group in April 2016 and was transferred directly to the PhD program in early 2018. The results were also published in the journal Biomaterials Science in 2017 (see details in publications list). Hen is currently investigating cell compatibility and targeting capabilities with these nanoparticles. Intriguingly, the research on PVA polymeric micelles opened a new avenue that pertains to the use of these nanocarriers in the production of more complex (hierarchical) nanoparticle-in-microparticle systems. We have received additional funding from the HaLevy Fund to pursue this new research line and Hen will incorporate this part of the work to her thesis. This funding will enable us to conduct the first preclinical studies in rat for which a protocol of animal welfare has been submitted to the Ministry of Health of Israel. After the completion of this study, we will consider the protection of the results by a patent application to then, submit them to publication. As explained above, the capabilities developed in this Aim enabled us to synthesize other copolymers employing for example polysaccharides that target carbohydrate pathways such as glucose receptors. The connection between Specific Aims 2 and 3 is in hands of the MSc student Anna Zaritski. After considering the protection of the intellectual property, we will begin a new collaboration with a Singaporean research of the National University of Singapore for the targeting of antibacterial peptides in TB and continue our fruitful collaboration with Dr. Carcaboso. As part of the aim, we also began a collaboration with a research group at the Czech Academy of Sciences in Prague where with the assistance of Slovak and Norwegian researchers, we are investigating the self-assembly pattern of chitosan and PVA polymeric micelles employing techniques that are not available here (e.g. SANS). Interestingly, regardless of the hydrophobic component, chitosan and PVA show different aggregation pattern. We are beginning the writing of two manuscripts that will report one these results and where Inbar Schlachet and Hen Moshe will be first authors. Specific Aim 4. Development of core-anchored polymeric micelles. This Specific Aim was not in the original proposal and it was incorporated in the first biannual report. The goal was to investigate a completely new approach to produce amphiphilic drug nanocarriers that resemble the core-shell structure of a polymeric micelle though that do not form by self-assembly; by doing so, we expected to overcome the physical instability of polymeric micelles upon extreme dilution. Thus, we designed and synthesized amphiphilic hybrid nanomaterials utilizing two inorganic multifunctional nanomaterials, namely carboxylated nanodiamonds and boron nitride nanotubes as particulate anchors to covalently link amphiphilic diblock copolymers consisting of poly(epsilon-caprolactone) and poly(ethylene glycol) monomethyl ether as hydrophobic and hydrophilic components, respectively. After confirming the core-corona nanostructure using various characterization techniques, we evaluated the cell compatibility in Caco2 cell monolayers, an in vitro model of the intestinal epithelium, and encapsulated different hydrophobic molecules such as the anti-helmintic drug nitazoxanide (in nanodiamond-based carriers) and curcumin (in boron nitride carriers). Cargoes as high as 31% w/w were achieved. This work was conducted by Doaa Abu Saleh who joined the group for MSc in October 2015 and after finishing the degree, stayed for PhD. It is important to remark that this work enabled the beginning of a collaboration with Dr. Olga Shimoni (U of Technology Sydney, Australia) and Prof. Francoise Winnik (U of Montreal, Canada). Doaa spent a 1-month internship in Prof. Winnik’s lab in late 2016. In her PhD, she is investigating other boron nitride particulate anchors and their doping to produce quantum dots that could carry a drug and also be tracked by imaging techniques. The results of the works on nanodiamonds and boron nitride nanotubes were published in two manuscripts in the journal Materials Today Chemistry during 2017 (see details in publications list).Specific aim 5. Characterization of the in vivo performanceAs described above, animal studies were completed with glucosylated polymeric micelles of Specific Aim 2, while studies with those of Specific Aim 3 are only beginning and they will be continued in the framework of projects that are continuation of the current one. The delay in the preclinical studies with the latter nanoparticles stemmed from the very extensive research done using in vitro models (cells and cell monolayers seeded on membranes) what enabled us to optimize the properties of the nanocarriers to prevent any toxicity. Owing to organizational delays, this milestone will be carried out between the second semester of 2018 and the first semester of 2019. The goal is to teach an intensive course (20 h in 5 days) on the field of “Biomaterials and Drug Delivery” in an recently founded academic institution of a low-income African country that lacks of teachers and researchers; I am still working on its organization. Partial outreach activities that comprised conferences to undergraduate students in the framework of Science Clubs and other informal activities have been already presented. In any event, my goal is to continue the dissemination of my activities to the general public and professionals at different academic levels and of different sectors. The overall goal of establishing a new world recognized laboratory dedicated to the research of polymeric biomaterials for drug delivery applications has been achieved, even beyond the initial expectations. Now, the Laboratory of Pharmaceutical Nanomaterials Science is one of the largest in the Department of Materials Science and Engineering and became a magnet for undergraduate students of Technion and other Israeli universities who want to continue their studies to higher degrees. In my view, this is one of the most relevant achievements. When I joined this faculty, now research at the interface of materials and biology was taking place. This situation has changed completely. Regarding our research, I anticipate that our lines are very sound and they will have impact on the quality of life of patients for whom we dedicate much efforts to improve disease treatments. The most outstanding results were achieved in the field of pediatric cancer where we established a solid strategic collaboration and a sound nanomedicine program to explore novel approaches to improve their therapy.
