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Medulloblastoma is the most common malignant paediatric brain tumour, with ~30% mediated by Sonic hedgehog signalling. Vismodegib-mediated inhibition of the Sonic hedgehog effector Smoothened inhibits tumour growth but causes growth plate fusion at effective doses. Here, we report a nanotherapeutic approach targeting endothelial tumour vasculature to enhance blood–brain barrier crossing. We use fucoidan-based nanocarriers targeting endothelial P-selectin to induce caveolin-1-dependent transcytosis and thus nanocarrier transport into the brain tumour microenvironment in a selective and active manner, the efficiency of which is increased by radiation treatment. In a Sonic hedgehog medulloblastoma animal model, fucoidan-based nanoparticles encapsulating vismodegib exhibit a striking efficacy and marked reduced bone toxicity and drug exposure to healthy brain tissue. Overall, these findings demonstrate a potent strategy for targeted intracranial pharmacodelivery that overcomes the restrictive blood–brain barrier to achieve enhanced tumour-selective penetration and has therapeutic implications for diseases within the central nervous system.

Polydopamine (PDA), a biomaterial inspired by marine mussels, has attracted interest in cancer nanomedicine due to its photothermal properties, nanoparticle coating, and pi-pi stacking-based drug encapsulation abilities. Despite numerous one-pot and post-polymerization modifications, PDA copolymers have not been sufficiently studied in the context of stabilizing hydrophobic drugs in the process of nanoprecipitation. In this study, we tested combinatorial panels of comonomers with PDA to optimize drug loading efficiency, particle size and stability of nano formulations made via drug nanoprecipitation. As a selection criterion for optimal comonomers, we used drug aggregation-induced emission (AIE). We identified 1,1,2-Trimethyl-3-(4-sulfobutyl)benz[e]indolium (In820) as a novel and highly useful comonomer for catecholamines and optimized the conditions for its incorporation into PDA copolymers used for drug nanoprecipitation. Surprisingly, it was superior to polyethylene glycol modifications in every aspect. The leading copolymer, poly(dopamine)-poly(L-dopa)-co-In820 (PDA-PDO-In820 1:1:1), was shown to be a good stabilizer for several hydrophobic drugs. The resulting nanoparticles showed stability for up to 15 days, high encapsulation efficiency of at least 80%, low toxicity, and high antitumor efficacy in vitro. Nanoprecipitation of hydrophobic drugs can be greatly enhanced by the use of PDA copolymers containing In820, which are easy-to-prepare and highly effective stabilizers.

Progress in the field of precision medicine has changed the landscape of cancer therapy. Precision medicine is propelled by technologies that enable molecular profiling, genomic analysis and optimized drug design to tailor treatments for individual patients. Although precision medicines have resulted in some clinical successes, the use of many potential therapeutics has been hindered by pharmacological issues, including toxicities and drug resistance. Drug delivery materials and approaches have now advanced to a point where they can enable the modulation of a drug’s pharmacological parameters, without compromising the desired effect on molecular targets. Specifically, they can modulate a drug’s pharmacokinetics, stability, absorption and exposure to tumours and healthy tissues, and facilitate the administration of synergistic drug combinations. This Review highlights recent progress in precision therapeutics and drug delivery, and identifies opportunities for strategies to improve the therapeutic index of cancer drugs and, consequently, clinical outcomes. Precision medicine identifies the optimal treatment strategy for an individual patient. Such personalized therapies can be greatly improved by targeted drug delivery approaches. This Review investigates the integration of targeted drugs, in particular, kinase inhibitors, with targeted drug delivery systems.

Nanoformulations of small molecule drugs are essential to effectively deliver them and treat a wide range of diseases. They are normally complex to develop, lack predictability, and exhibit low drug loading. Recently, nanoparticles made via co-assembly of hydrophobic drugs and organic dyes, exhibited drug-loading of up to 90% with high predictability from the drug structure. However, these particles have relatively short stability and can formulate only a small fraction of the drug space. Here, we developed an automated workflow to synthesize and select novel dye stabilizers, based on their ability to inhibit drug aggregation-induced emission (AIE). We first screened and identified 10 drugs with previously unknown strong AIE activity and exploited this trait to automatically synthesize and select a new ultra-stabilizer named R595. Interestingly, it shares several synthetic similarities and advantages with polydopamine. We found that R595 is superior to myriad types of excipients and solubilizers such as cyclodextrins, poloxamers, albumin, and previously published organic dyes, in both long-term stability and drug compatibility. We investigated the biodistribution, pharmacokinetics, safety and efficacy of the AIEgenic MEK inhibitor trametinib-R595 nanoparticles in vitro and in vivo and demonstrated that they are non-toxic and effective in KRAS driven colon and lung cancer models.

Drug combination therapy is a main pillar of cancer therapy but the formation of an effective combinatorial standard of care (SOC) can take many years and its length of development is increasing with complexity of treatment. In this paper, we develop a path to extend the boundaries of complexity in combinatorial cancer treatments using text data mining (TDM). We first use TDM to characterize the current boundaries of cancer treatment complexity and find that the current complexity limit for clinical trials is 6 drugs per plan and for pre-clinical research is 10. We then present a TDM based assistive technology, cancer plan builder (CPB), which we make publicly available and allows experts to create literature-anchored high complexity combination treatment (HCCT) plans of significantly larger size. We develop metrics to evaluate HCCT plans and show that experts using CPB are able to create HCCT plans at much greater speed and quality, compared to experts without CPB. We hope that by releasing CPB we enable more researchers to engage with HCCT planning and demonstrate its clinical efficacy.

As knowledgebases become increasingly important for structuring vast amounts of scientific knowledge and making it accessible to researchers, their construction entails expensive multi-year projects involving teams of bio-curators, computer scientists, or both. This restricts the coverage of existing knowledgebases to a limited set of popular topics, leaving a long tail of more specialized interests uncovered.We present a methodology and a supporting tool to allow individual researchers or small teams, without background in bio-curation or computer science, to mine the scientific literature and construct ad-hoc, personalized, and literature-anchored knowledgebases, that are tailored around their specific research interests and support their scientific goals. The time investment involved in creating a knowledgebase ranges from a few hours to a few weeks, depending on the desired coverage and accuracy.We demonstrate the methodology by constructing knowledgebases for different purposes: a high-level overview of challenges and controversies in a field (the cancer frontiers knowledgebase); a mapping of main concepts and interactions in a field, to support lab-internal hypothesis generation (tissue engineering and regeneration, cancer surgery and radiotherapy knowledgebases); and a comprehensive and accurate knowledgebase designated as an online up-to-date resource for the wider research community (the cell specific drug delivery knowledgebase). In each case we show how the structured knowledgebase, coupled with effective visualizations, facilitates effective data exploration, hypothesis generation and meta-analysis.We implement the method as part of an open source web-based platform for knowledgebase construction, available publicly and freely at https://spike-kbc.apps.allenai.org.

Nanomedicine design is often a trial-and-error process, and the optimization of formulations and in vivo properties requires tremendous benchwork. To expedite the nanomedicine research progress, data science is steadily gaining importance in the field of nanomedicine. Recently, efforts have explored the potential to predict nanomaterials synthesis and biological behaviors via advanced data analytics. Machine learning algorithms process large datasets to understand and predict various material properties in nanomedicine synthesis, pharmacologic parameters, and efficacy. “Big data” approaches may enable even larger advances, especially if researchers capitalize on data curation methods. However, the concomitant use of data curation processes needed to facilitate the acquisition and standardization of large, heterogeneous data sets, to support advanced data analytics methods such as machine learning has yet to be leveraged. Currently, data curation and data analytics areas of nanotechnology-focused data science, or ‘nanoinformatics’, have been proceeding largely independently. This review highlights the current efforts in both areas and the potential opportunities for coordination to advance the capabilities of data analytics in nanomedicine.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor with ~30% mediated by Sonic hedgehog (SHH) signaling. While SHH effector Smoothened inhibition is promising, therapeutic index is reduced by high systemic doses required for sufficient intracranial drug concentrations. Treatment-related toxicities associated with radiation therapy result in lifelong morbidity, and SHH pathway inhibition in children notably results in permanent bone defects. Here, we found that nanoparticle-mediated P-selectin targeting on tumor vasculature induces caveolin-1-dependent transcellular passage across the blood-brain barrier. A vismodegib nanocarrier that targets P-selectin exhibited striking efficacy and markedly reduced both bone-related toxicities and drug exposure to healthy brain. These findings demonstrate a potent strategy and novel mechanism for targeted intracranial pharmacodelivery that overcomes the restrictive endothelial barrier to achieve enhanced tumor-selective penetration and has therapeutic implications for other diseases within the central nervous system.

Bimetallic nanostructures can be attractive contrast-enhancing materials for dual modal imaging, allowing improved diagnosis ability using two different physical phenomena, following administration...

Preclinical measurements of drug exposure to specific organs and tissues is normally performed by destructive methods. Tissue-specific measurements are important, especially for drugs with intractable dose-limiting toxicities, such as doxorubicin-mediated cardiotoxicity. We developed a method to rapidly quantify doxorubicin exposure to tissues within living organisms using an implantable optical nanosensor that can be interrogated noninvasively following surgical implantation. The near-infrared fluorescence of single-walled carbon nanotubes functionalized with DNA was found to respond to doxorubicin via a large and uniform red-shift. We found this to be common to DNA-intercalating agents, including anthracycline compounds such as doxorubicin. Doxorubicin was measured in buffer and serum, intracellularly, and from single nanotubes on a surface. Doxorubicin adsorption to the DNA-suspended nanotubes did not displace DNA but bound irreversibly. We incorporated the nanosensors into an implantable membrane which allowed cumulative detection of doxorubicin exposure in vivo. On implanting the devices into different compartments, such as subcutaneously and within the peritoneal cavity, we achieved real-time, minimally invasive detection of doxorubicin injected into the peritoneal cavity, as well as compartment-specific measurements. We measured doxorubicin translocation across the peritoneal membrane in vivo. Robust, minimally invasive pharmacokinetic measurements in vivo suggest the suitability of this technology for preclinical drug discovery applications.

Objective. In this perspective paper, we aim to highlight the potential of sleep as an auspicious time for diagnosis, management and therapy of non-sleep-specific pathologies. Approach. Sleep has a profound influence on the physiology of body systems and biological processes. Molecular studies have shown circadian-regulated shifts in protein expression patterns across human tissues, further emphasizing the unique functional, behavioral and pharmacokinetic landscape of sleep. Thus, many pathological processes are also expected to exhibit sleep-specific manifestations. Modern advances in biosensor technologies have enabled remote, non-invasive recording of a growing number of physiologic parameters and biomarkers promoting the detection and study of such processes. Main results. Here, we introduce key clinical studies in selected medical fields, which leveraged novel technologies and the advantageous period of sleep to diagnose, monitor and treat pathologies. Studies demonstrate that sleep is an ideal time frame for the collection of long and clean physiological time series data which can then be analyzed using data-driven algorithms such as deep learning. Significance.This new paradigm proposes opportunities to further harness modern technologies to explore human health and disease during sleep and to advance the development of novel clinical applications – from sleep medicine to medicine during sleep.

Multifunctional nanocarriers have attracted considerable interest in improving cancer treatment outcomes. Poly(lactide-co-glycolide) (PLGA) nanospheres encapsulating copper oxide nanoparticles (CuO-NPs) are characterized by antitumor activity and exhibit dual-modal contrast-enhancing capabilities. An in vitro evaluation demonstrates that this delivery system allows controlled and sustained release of CuO-NPs. To achieve localized release on demand, an external stimulation by laser irradiation is suggested. Furthermore, to enable simultaneous complementary photothermal therapy, polydopamine (PDA) coating for augmented laser absorption is proposed. To this aim, two formulations of CuO-NPs loaded nanospheres are prepared from PLGA polymers RG-504 H (H-PLGA) and RG-502 H (L-PLGA) as scaffolds for surface modification through in situ polymerization of dopamine and then PEGylation. The obtained CuO-NPs-based multifunctional nanocarriers are characterized, and photothermal effects are examined as a function of wavelength and time. The results show that 808 nm laser irradiation of the coated nanospheres yields maximal temperature elevation (T = 41°C) and stimulates copper release at a much faster rate compared to non-irradiated formulations. Laser-triggered CuO-NP release is mainly depended on the PLGA core, resulting in faster release with L-PLGA, which also yielded potent anti-tumor efficacy in head and neck cancer cell line (Cal-33). In conclusion, the suggested multifunctional nanoplatform offers the integrated benefits of diagnostic imaging and laser-induced drug release combined with thermal therapy., GRAPHICAL ABSTRACT

OBJECTIVE The Sonic Hedgehog (SHH) medulloblastoma subgroup accounts for ~25% of all cases and has an intermediate prognosis. Current conventional therapies result in devastating morbidities including intellectual disability and secondary malignancies. Although molecularly targeted agents that inhibit the SHH pathway have demonstrated clinical efficacy, recent studies have shown on-target secondary toxicities on bone development suggesting new therapeutic approaches are needed. METHODS We investigated the efficacy of the SHH pathway inhibitor, Vismodegib packaged in a fucoidan-based nanoparticle (Fi-Vis) that targets P-selectin, a protein overexpressed on vascular endothelial cells and induced by low-dose ionizing radiation (XRT) in a time- and dose-dependent manner. This P-selectin targeting nanoparticle drug delivery system shows selectivity toward tumor vasculature and not normal brain vasculature in a genetic SHH medulloblastoma mouse model as assessed by ex vivo infrared imaging and two-photon intravital imaging. RESULTS Quantitative RT-PCR analysis of SHH medulloblastoma tissue following single dose XRT and Fi-Vis treatment (10mg/kg) showed a synergistic inhibition of Gli1 expression (>90% target inhibition). Furthermore, we demonstrate that very low dose XRT (0.25Gy) can induce P-selectin expression specifically within MB tumor vasculature and synergize with low dose Fi-Vis (10mg/kg) to significantly enhance mouse survival (p CONCLUSIONS These data suggest applicability of combined XRT and tumor vasculature-targeted nanotherapeutic dose de-escalation strategies for SHH medulloblastoma with implications for other pediatric and adult brain tumors.

Preclinical measurements of drug exposure to specific organs and tissues is normally performed by destructive methods. Tissue-specific measurements are important, especially for drugs with intractable dose-limiting toxicities, such as doxorubicin-mediated cardiotoxicity. We developed a method to rapidly quantify doxorubicin exposure to tissues within living organisms using an implantable optical nanosensor that can be interrogated noninvasively following surgical implantation. The near-infrared fluorescence of single-walled carbon nanotubes functionalized with DNA was found to respond to doxorubicin via a large and uniform red-shift. We found this to be common to DNA-intercalating agents, including anthracycline compounds such as doxorubicin. Doxorubicin was measured in buffer and serum, intracellularly, and from single nanotubes on a surface. Doxorubicin adsorption to the DNA-suspended nanotubes did not displace DNA but bound irreversibly. We incorporated the nanosensors into an implantable membrane which allowed cumulative detection of doxorubicin exposure

OBJECTIVE The Sonic Hedgehog (SHH) medulloblastoma subgroup accounts for ~25% of all cases and has an intermediate prognosis. Current therapies result in devastating morbidities including intellectual disability and secondary malignancies. Although molecularly targeted agents against the SHH pathway have demonstrated efficacy, on-target bone toxicities suggest new therapeutic approaches are needed. METHODS We investigated the SHH pathway inhibitor, vismodegib, packaged in a fucoidan-based nanoparticle (Fi-Vis) that targets P-selectin expressed on endothelial cells and induced by low-dose ionizing radiation (XRT) in a time- and dose-dependent manner. This P-selectin targeting nanoparticle shows selectivity toward tumor and not normal brain vasculature in a GEM SHH medulloblastoma model as assessed by ex vivo infrared imaging and molecular studies. RESULTS Quantitative RT-PCR analysis of SHH medulloblastoma following single dose XRT and Fi-Vis treatment (10mg/kg) showed synergistic reduction of Gli1 expression (>90% target inhibition). We demonstrate that low-dose XRT (0.25Gy) can induce P-selectin expression specifically on medulloblastoma tumor endothelium and synergize with low-dose Fi-Vis (10mg/kg) to significantly enhance mouse survival (p CONCLUSIONS These data suggest applicability of combined XRT and tumor vasculature-targeted nanotherapeutic dose de-escalation strategies for SHH medulloblastoma with implications for other pediatric brain tumors.

There is currently no effective means to prevent or control metastatic dissemination of cancer cells. E-selectin, an adhesion molecule expressed exclusively on inflamed and angiogenic blood vessels, plays an important role in several rate-limiting steps of cancer metastasis. In this study, we assessed the in vivo antitumor efficacy of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers conjugated to an E-selectin binding peptide (Esbp, DITWDQLWDLMK) and equipped with the chemotherapeutic drug doxorubicin (P-(Esbp)-DOX) or with the proapoptotic peptide D(KLAKLAK)2 (P-(Esbp)-KLAK). Following a single intravenous injection, P-(Esbp)-DOX reduced tumor growth rate and prolonged the survival of mice bearing primary Lewis lung carcinoma (3LL) tumors significantly more than treatment with a non-targeted copolymer (P-DOX) or with free DOX. In an experimental B16-F10 lung metastasis model, a single intravenous dose of P-(Esbp)-DOX or P-(Esbp)-KLAK prolonged mice survival time significantly more than the non-targeted copolymers or the free drugs, and the percentage of complete tumor regression increased with increasing doses and with dosing frequency. In addition, mice pretreated with an E-selectin-targeted "drug-free" copolymer (P-(Esbp)-FITC) exhibited significantly fewer B16-F10 tumor foci in the lungs as compared with non-treated mice, demonstrating the anti-metastatic properties of the copolymer and its ability to control cancer spread through E-selectin-mediated interactions. Biodistribution analysis further confirmed the preferential accumulation of the E-selectin-targeted near-infrared fluorescently-labeled copolymer P-(Esbp)-IR783 in B16-F10 lung metastases. Taken together, this study demonstrates, for the first time, that the E-selectin targeted copolymer-drug conjugates can inhibit primary tumor growth and prevent metastases in vivo.

Alterations in PIK3CA, the gene encoding the p110α subunit of phosphatidylinositol 3-kinase (PI3Kα), are frequent in head and neck squamous cell carcinomas. Inhibitors of PI3Kα show promising activity in various cancer types, but their use is curtailed by dose-limiting side effects such as hyperglycaemia. In the present study, we explore the efficacy, specificity and safety of the targeted delivery of BYL719, a PI3Kα inhibitor currently in clinical development in solid tumours. By encapsulating BYL719 into P-selectin-targeted nanoparticles, we achieve specific accumulation of BYL719 in the tumour milieu. This results in tumour growth inhibition and radiosensitization despite the use of a sevenfold lower dose of BYL719 compared with oral administration. Furthermore, the nanoparticles abrogate acute and chronic metabolic side effects normally observed after BYL719 treatment. These findings offer a novel strategy that could potentially enhance the efficacy of PI3Kα inhibitors while mitigating dose-limiting toxicity in patients with head and neck squamous cell carcinomas., Head and neck squamous cell carcinomas (HNSCC) often harbour PIK3CA mutations but PI3Kα inhibitors can cause some side effects. Here, the authors develop P-selectin targeted nanoparticles to enhance tumour-specific delivery of a PI3Kα inhibitor to HNSCC PDX and orthotopic xenograft models.

Disseminated tumors are poorly accessible to nanoscale drug delivery systems because of the vascular barrier, which attenuates extravasation at the tumor site. We investigated P-selectin, a molecule expressed on activated vasculature that facilitates metastasis by arresting tumor cells at the endothelium, for its potential to target metastases by arresting nanomedicines at the tumor endothelium. We found that P-selectin is expressed on cancer cells in many human tumors. To develop a targeted drug delivery platform, we used a fucosylated polysaccharide with nanomolar affinity to P-selectin. The nanoparticles targeted the tumor microenvironment to localize chemotherapeutics and a targeted MEK (mitogen-activated protein kinase kinase) inhibitor at tumor sites in both primary and metastatic models, resulting in superior antitumor efficacy. In tumors devoid of P-selectin, we found that ionizing radiation guided the nanoparticles to the disease site by inducing P-selectin expression. Radiation concomitantly produced an abscopal-like phenomenon wherein P-selectin appeared in unirradiated tumor vasculature, suggesting a potential strategy to target disparate drug classes to almost any tumor.

The near-infrared photoluminescence from non-covalently dispersed single-walled carbon nanotubes (SWCNT) is optimally suited to probe distinct classes of biological environments. With aspect ratios of >100:1, nanotubes present an opportunity to observe atypical interactions between linear filamentous particles and three-dimensional biological environments. A pressing concern in therapeutic biomedicine is the inability of drug-carrying nanoparticles that function well in two dimensional cell cultures, to show efficacy in vivo. The inability of spherical nanoparticles to penetrate dense three-dimensional structures in a well defined point of failure. By exploiting the ability to image single carbon nanotubes via widefield fluorescence microscopy, we quantified the relative permeability of multi cellular tumor spheroids to a linear nanoparticle1. The results indicate the ability of nanoparticles to penetrate tumor spheroids depends strongly on the nanoparticle shape. Additionally, this platform portends a new approach to characterize the permeability of living multicellular environments. References: Photoluminescent carbon nanotubes interrogate the permeability of multicellular tumor spheroids, Yosef Shamay*, Prakit V. Jena*, et al, Carbon 97 (2016) 99-109

Polymer-drug conjugates that can actively target the tumor vasculature have emerged as an attractive technology for improving the therapeutic efficacy of cytotoxic drugs. We have recently provided, for the first time, in vivo evidence showing the significant advantage of the E-selectin-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin conjugate, P-(Esbp)-DOX, in inhibiting primary tumor growth and preventing the formation and development of cancer metastases. Here, we describe the design of a vascular endothelial growth factor receptor (VEGFR)-1-targeted HPMA copolymer-DOX conjugate (P-(F56)-DOX) that can actively and simultaneously target different cell types in the tumor microenvironment, such as endothelial cells (ECs), bone marrow-derived cells and many human cancer cells of diverse tumor origin. The VEGFR-1-targeted copolymer was tested for its binding, internalization and in vitro cytotoxicity in ECs (bEnd.3 and cEND cells) and cancer cells (B16-F10, 3LL and HT29). The in vivo anti-cancer activity of P-(F56)-DOX was then tested in two tumor-bearing mice (TBM) models (i.e., primary Lewis lung carcinoma (3LL) tumors and B16-F10 melanoma pulmonary metastases), relative to that of the E-selectin-targeted system (P-(Esbp)-DOX) that solely targets ECs. Our results indicate that the binding and internalization profiles of the VEGFR-1-targeted copolymer were superior towards ECs as compared to cancer cells and correlated well to the level of VEGFR-1 expression in cells. Accordingly, the VEGFR-1-targeted copolymer (P-(F56)-DOX) was more toxic towards bEnd.3 cells than to cancer cells, and exhibited significantly higher cytotoxicity than did the non-targeted control copolymer. P-(F56)-DOX inhibited 3LL tumor growth and significantly prolonged the survival of mice with B16-F10 pulmonary metastases. When compared to a system that actively targets only tumor vascular ECs, P-(F56)-DOX and P-(Esbp)-DOX exhibited comparable efficacy in slowing the growth of primary 3LL tumors and prolonging the survival of these mice. Still, P-(Esbp)-DOX had more pronounced anti-tumor activity in mice bearing B16-F10 lung metastases after a single intravenous injection, at an equivalent DOX dose. Overall, our results indicate that the VEGFR-1- and E-selectin-targeted drug delivery systems evaluated here show enhanced anti-cancer activity, and prolonged the survival of mice after a single intravenous injection. This is thus the first study comparing the anti-tumor activity of VEGFR-1- and E-selectin-targeted polymer drug conjugates in the same TBM models at an equivalent drug dose.

Development of targeted nanoparticle drug carriers often requires complex synthetic schemes involving both supramolecular self-assembly and chemical modification. These processes are generally difficult to predict, execute, and control. We describe herein a targeted drug delivery system that is accurately and quantitatively predicted to self-assemble into nanoparticles based on the molecular structures of precursor molecules, which are the drugs themselves. The drugs assemble with the aid of sulfated indocyanines into particles with ultrahigh drug loadings of up to 90%. We devised quantitative structure-nanoparticle assembly prediction (QSNAP) models to identify and validate electrotopological molecular descriptors as highly predictive indicators of nano-assembly and nanoparticle size. The resulting nanoparticles selectively targeted kinase inhibitors to caveolin-1-expressing human colon cancer and autochthonous liver cancer models to yield striking therapeutic effects while avoiding pERK inhibition in healthy skin. This finding enables the computational design of nanomedicines based on quantitative models for drug payload selection. Molecular simulations reveal the self-assembly of small molecules into nanoparticle drug carriers. Targeting of colon and liver cancer cells by the nanoparticles via kinase inhibitors is employed in anti-tumour therapy in vivo.

Cell-penetrating peptides (CPPs) can be used for intracellular delivery of a broad variety of cargoes, including various nanoparticulate pharmaceutical carriers. However, the cationic nature of all CPP sequences, and thus lack of cell specificity, limits their in vivo use for drug delivery applications. Here, we have devised and tested a strategy for site-specific delivery of dyes and drugs into cancer cells by using polymers bearing a light activated caged CPP (cCPP). The positive charge of Lys residues on the minimum sequence of the CPP penetratin ((52)RRMKWKK(58)) was masked with photo-cleavable groups to minimize non-specific adsorption and cellular uptake. Once illuminated by UV light, these protecting groups were cleaved, the positively charged CPP regained its activity and facilitated rapid intracellular delivery of the polymer-dye or polymer-drug conjugates into cancer cells. We have found that a 10-min light illumination time was sufficient to enhance the penetration of the polymer-CPP conjugates bearing the proapoptotic peptide, (D)(KLAKLAK)(2), into 80% of the target cells, and to promote a 'switch' like cytotoxic activity resulting a shift from 100% to 10% in cell viability after 2 h. This report provides an example for tumor targeting by means of light activation of cell-penetrating peptides for intracellular drug delivery.

The treatment of cancer using pro-apoptotic peptides requires efficient intracellular delivery to trigger mitochondrial disruption. When the widely used non-toxic heptapeptide dimer D(KLAKLAK)2 is coupled with tumor targeting peptides that allow receptor-mediated internalization, it disrupts negatively charged membranes and induces mitochondrial-dependent apoptosis. Here, we describe the design, synthesis, and characterization of novel water-soluble HPMA copolymers bearing a multivalent display of NGR motifs that can target the pro-apoptotic drug D(KLAKLAK)2 to CD13-overexpressing cells. D(KLAKLAK)2 was attached to the polymeric backbone through an acid-sensitive hydrazone linker to facilitate intracellular drug release. The ability of an array of NGR motifs, with different structural conformations, to target several CD13 receptors and thus enhance the binding avidity and the pro-apoptotic activity of D(KLAKLAK)2 in human cell lines was tested. HPMA copolymer conjugates bearing the dimeric (GNGRG)2 and cyclic (CNGRC) NGR motifs exhibited preferential binding to CD13-overexpressing cells relative to the linear (GNGRG) targeting peptide. The binding of D(KLAKLAK)2 to the polymer facilitated rapid internalization and translocation into endosomal/lysosomal compartments. The polymer-bound D(KLAKLAK)2 markedly increased the cytotoxicity and the pro-apoptotic activity of the drug, relative to free D(KLAKLAK)2 and the targeted pro-apoptotic peptide (GNGRGG-D(KLAKLAK)2) in CD13(+) cells. This research is the first attempt to apply polymer-conjugates with NGR motifs as targeting ligand to deliver the pro-apoptotic D(KLAKLAK)2 sequence into CD13-overexpressing cells and thereby to promote the death of cancer cells. Copyright © 2010 John Wiley & Sons, Ltd.

Cell adhesion is a protein-mediated process intrinsic to most living organisms. Dysfunction in cell adhesion processes is implicated in various diseases, including thrombosis and metastatic cancers. Using an approach to resolve spectral features from cell membrane-associated photoluminescent single-walled carbon nanotubes, we found that nanotube optical bandgaps respond to the electrostatic potential of the cell surface, which corresponds to cell adhesion properties. We studied the carbon nanotube emission energy response to solution ionic potentials, which suggests sensitivity to local charge accumulation. We conclude that nanotubes respond to cell surface electrostatic potentials that are mediated by membrane proteins, which vary significantly across cell types. These findings portend the optical measurement of surface electrostatic potentials for biophysical measurements and biomedical applications.

Dendritic cells (DCs) are a family of specialized antigen presenting cells (APCs) that detect antigens and initiate a wide spectrum of immune responses against them. These characteristics make them promising candidates for immunotherapy manipulations. In this study we designed and synthesized DC-targeted block copolymers composed of linear polyethylenimine (PEI) conjugated to hydrophilic polyethylene glycol (PEG) installed with a DC-targeting peptide (DC3, primary sequence FYPSYHSTPQRP). Two different conjugation procedures (basic and modified) were employed to synthesize the DC3-PEG-b-PEI and the control SCRM-PEG-b-PEI (with a scrambled DC3 peptide sequence) by one-pot synthesis, in two steps. In the first, basic conjugation procedure, PEG with N-hydroxysuccinimide (NHS) ester and maleimide (MAL) groups (NHS-PEG-MAL, 3.5 kDa) was first coupled to linear PEI (25 kDa) via the NHS group to yield the intermediate MAL-PEG-b-PEI, that was then conjugated to N-terminus-cysteine harboring peptides DC3 or SCRM via the MAL double bond to yield the final DC3-PEG-b-PEI or SCRM-PEG-b-PEI copolymers, respectively. In the second, modified conjugation procedure, Fmoc-cysteine harboring DC3 or SCRM peptides were first conjugated to NHS-PEG-MAL via the MAL group followed by coupling to linear PEI via the NHS functional group. Fmoc cleavage yielded the same final product as in the basic procedure. The modified conjugation procedure was capable of yielding block copolymers richer with peptides, as determined by (1)H NMR analysis. Self-assembly of DC3-PEG-b-PEI copolymers and DNA molecules yielded nanosized polyion complexes (polyplexes), with lower surface charge and limited cytotoxicity when compared to the PEI building block. Significant transfection efficiency of the DC-targeted polyplexes by murine dendritic DC2.4 cells was observed only in DC3-PEG-b-PEI/DNA polyplexes synthesized by the modified conjugation procedure. These polyplexes, with higher peptide-load, showed greater transfection capability in DC2.4 cells relative to the control nontargeted SCRM-PEG-b-PEI/DNA polyplexes, but not in endothelial cells. The transfection efficiency was comparable to or higher than that of the PEI/DNA positive control. The results indicate that PEGylated-PEI polyplexes show significant transfection efficiency into DCs when decorated with DC3 peptide, and are attractive candidates for immunotherapy via DCs.

Purpose: PI3K-pathway activation is the second most common genetic abnormality occurring in head and neck squamous cell carcinoma (HNSCC). Mutation or amplification of PIK3CA, the gene coding for the p110α subunit of PI3K, occur in 34%-56% of HNSCC and there is evidence that targeting PI3Kα in these tumors can be radiosensitizing. Small molecule kinase inhibitors of PI3Kα are showing promising activity. However, the use of this molecule is limited by serious side effects such as hyperglycemia, diarrhea and rush. In this study we aimed to apply novel nanotechnology for targeted drug delivery via the cell adhesion molecule P-selectin, which was previously shown to be overexpressed in tumor microvasculature. Furthermore, P-selectin is up regulated following radiation therapy (RT), which could enhance drug delivery using targeted nanoparticles. Experimental design: We explored the efficacy and specificity of targeted delivery of BYL719, a potent alpha-specific PI3K inhibitor, using nanoparticles that selectively target P-selectin present in the tumor microvasculature in PIK3CA-mutated HNSCC. We compared the antitumor effects of nanoparticle delivery versus standard oral gavage with free drug in PIK3CA mutated HNSCC cell line- and patient-derived xenografts (PDXs). In addition, we compared blood glucose and insulin levels between standard BYL719 administration and nanoparticle drug delivery at different time points after treatment. Furthermore, we sought to evaluate the radiosensitizing properties of BYL719 when combined with fractionated RT of 5X4Gy. Results: P-selectin targeted delivery of 50 mg/kg/week BYL719 resulted in a significant tumor growth delay, which was comparable to the standard drug administration of 350 mg/kg/week (50 mg/kg/daily). While blood glucose and insulin levels were spiking after standard oral gavage of BYL719, these parameters were virtually unchanged upon nanoparticle administration of the drug. The radiosensitizing abilities of low-dose nanoparticle-linked BYL719 were comparable to the drug concentrations used in standard daily BYL719 administration. When given concomitantly with fractionated RT, both methods showed robust enhancement of radiotherapy response in all the models tested resulting in durable control of tumor growth. Conclusions: P-selectin targeted delivery of a PI3Kα inhibitor resulted in inhibition of the PI3K/Akt/mTOR pathway without affecting glucose or insulin levels. The magnitude of pathway inhibition was sufficient to radiosensitize several HNSCC animal models. This novel targeting strategy could be translated to the clinic to treat patients with PIK3CA activated and radioresistant HNSCC tumors sparing most of the systemic adverse effects of PI3K inhibition. Citation Format: Aviram Mizrachi, Yosi Shamay, Janki Shah, Samuel I. Brook, Joanne Soong, Vinagolu K. Rajasekhar, John L. Humm, Simon N. Powell, José Baselga, Daniel A. Heller, Adriana Haimovitz-Friedman, Maurizio Scaltriti. Tumor-specific PI3K inhibition via nanoparticle targeted delivery in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4122. doi:10.1158/1538-7445.AM2017-4122

There is increasing interest in the use of nanoparticle imaging probes for cancer diagnosis. However, various biological barriers limit the efficient delivery of nanoparticles to tumors following parenteral administration. We have investigated the applicability of a water-soluble polymeric imaging probe for improving the detection of gastrointestinal (GI) tumors after intra-luminal (colonic) administration. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing either fluorescein-isothiocyanate (FITC) or near-infrared fluorescence (NIRF) dye (IR-783) were conjugated with EPPT1 peptide, derived from the CDR3 Vh region of a monoclonal antibody (ASM2) raised against human epithelial cancer cells, for targeting under-glycosylated mucin-1 (uMUC-1) expressed in neoplastic tissues. The targeted FITC-labeled copolymer, P-(EPPT1)-FITC, was investigated for its ability to bind human CRC cells and tissue specimens in vitro. The uMUC-1-targeted NIRF-labeled copolymer, P-(EPPT1)-IR783, was assessed for its ability to detect colonic lesions in vivo. P-(EPPT1)-FITC demonstrated superior binding to colorectal cancer (CRC) cells that over-express the uMUC-1 antigen and exhibited selectivity towards human CRC tissue specimens, as compared to adjacent normal tissues from the same patient. When applied intra-colonically, P-(EPPT1)-IR783 significantly accumulated in cancerous tissue, relative to the adjacent normal mucosa of HT29 and LS174T tumor-bearing mice, and demonstrated higher signal intensities in colonic tumors, as compared to the non-targeted P-(GG-OH)-IR783 probe (i.e., without EPPT1). We found that P-(GG-OH)-IR783 can also accumulate specifically at tumor sites. The cancer-specific uptake and retention of P-(GG-OH)-IR783 was not mediated by organic anion transporting peptides (OATPs). Our findings indicate that the polymer-bound NIRF probe can successfully detect solid tumors in the GI tract following intra-colonic administration, and could be used in conjunction with colonoscopic procedures to improve the sensitivity of colonoscopies for polyp detection.

Cell penetrating peptides (CPPs) can mediate effective delivery of their associated drugs and drug carriers intracellularly, however their lack of cell specificity remains a major obstacle for their clinical development. We aimed at improving the cell specificity and therapeutic efficacy of HPMA copolymer-octaarginine (R8) conjugate (P-R8) in cells at the tumor micro-environment.To avoid premature cell-penetration, the positively charged R8 moieties were masked via electrostatic complexation with various polyanionic molecules (heparin sulfate, hyaluronic acid, fucoidan and poly-glutamic acid). We followed the kinetics of the FITC-labeled P-R8 penetration into endothelial and cancer cells over-time after its complexation in vitro and further tested whether the in situ addition of a stronger polycation can trigger the release of P-R8 from the complexes to resume cell penetration activity. A murine model of B16-F10 lung metastasis was then used as an in vivo model for assessing the therapeutic efficacy of the P-R8, loaded with doxorubicin (P-R8-DOX), after its complexation with PGA.The intracellular penetration of P-R8-FITC was reversibly inhibited by forming electrostatic interactions with counter polyanions, and can be restored either gradually over time by dissociation from the polyanions, or promptly following the addition of protamine sulfate. Mice injected with B16-F10 cells and treated with P-R8-DOX/PGA complexes, exhibited a significant prolonged survival times when compared with DOX-treated mice or relative to mice treated with either P-R8-DOX or P-DOX alone.The gradual release of P-R8 from P-R8-DOX/PGA may improve the therapeutic efficacy of water-soluble based nanomedicines for the treatment of solid lung tumors.

Synthetic oligopeptides have emerged as a promising class of targeting ligands, providing a variety of choices for the construction of conjugates for desired ligand functionality. To explore the potential of short peptides as ligands for targeted delivery of macromolecular therapeutics for colorectal cancer (CRC), fluorescently labelled HPMA copolymers--bearing either G3-C12 or GE11 for targeting galectin-3 and epidermal growth factor receptor (EGFR), respectively--were synthesised and the mechanisms of their internalisation and subcellular fate in CRC cells were studied. The targetability of the G3-C12 bearing copolymers towards galectin-3 was further compared to that of galactose-containing copolymers. The resulting G3-C12-bearing conjugate actively and selectively targets CRC tumour cells over-expressing galectin-3 and exhibits superior targetability to galectin-3 when compared to the galactose-bearing copolymer. GE11 copolymer conjugate binds specifically and efficiently to EGFR over-expressing cells, thus mediating internalisation to a significantly higher extent relative the copolymer conjugated to a scrambled sequence peptide. We further incorporated doxorubicin (DOX) into GE11 bearing copolymer via an acid-labile hydrazone bond. The GE11-DOX copolymer conjugate demonstrated higher cytotoxicity toward EGFR over-expressing cells relative to the control non-targeted DOX conjugate. Altogether, our results show a proof of principle for the selective delivery of DOX to the target CRC cells.

To evaluate the effect of the size of low molecular weight hyaluronan (LMW-HA) oligomers on the targeting ability of the HA-containing copolymers to the CD44-overexpressing cells for delivering Paclitaxel (PTX) to ovarian cancer.LMW-HA oligosaccharides of 4, 6, 8, 10, 12 and 14 sugar residues were attained by digestion of HMW-HA using hyaluronate lyase at different incubation times and then attached to FITC-labeled HPMA copolymer precursor. The binding and uptake of the HA-modified HPMA-copolymer into CD44-expressing cells was studied by flow cytometry and confocal microscopy. PTX was further attached to HPMA-copolymer precursor bearing HA oligosaccharide at the size of 34 monosaccharides, through an acid-sensitive hydrazone linker. The cytotoxicity of the polymer was tested using cell viability assay.Polymer conjugates bearing HA oligomers at the size of 10 oligosaccharides and above (HA(10-14)) bind actively and profoundly to CD44-overexpressing ovarian cancer cells (SK-OV-3) and internalize to the greatest extent relative to HA-polymer conjugates of 8 oligomers and below (HA(4-8)). The HA-modified HPMA-copolymer PTX conjugate (P-(HA)(34)-PTX) exhibited 50-times higher cytotoxicity towards CD44-overexpressing cells relative to the control, non-targeted, HPMA-copolymer PTX conjugate (P-PTX).P-(HA)(34)-PTX was significantly more toxic than the non-targeted P-PTX in cells expressing high levels of CD44.

The site-specific expression of molecular markers on endothelial cells of blood vessels during inflammatory response and angiogenesis provides an opportunity to target drugs and imaging molecules to the vascular endothelium of diseased tissues. This paper describes an innovative strategy for selective delivery of polymer conjugates to E- and P-selectin expressing cells using a series of quinic acid (Qa) based non-carbohydrate analogues of the natural ligand sialyl Lewis(x) (sLe(x)) as targeting moieties. We demonstrate that such analogues antagonize the adhesion of sLe(x) expressing HL-60 cells to both E- and P-selectin. Significantly, the apparent avidity of polymer conjugates carrying multiple Qa copies has increased by 3 orders of magnitude relative to their monomeric forms. Furthermore, we found that the major mechanism of copolymer entry and delivery into E-selectin expressing cells is endocytosis. These selectin-targetable copolymers provide the foundation to support controlled delivery of anticancer drugs and imaging agents to tumor vasculature for therapeutic and diagnostic applications.

The hypothesis that E-selectin on activated endothelial cells could be exploited to selectively target drug delivery systems to tumor vasculature was investigated. HPMA copolymer-doxorubicin (DOX) conjugates displaying the high affinity E-selectin binding peptide (Esbp, primary sequence DITWDQLWDLMK) as targeting ligand were synthesized and tested for their cytotoxicity and intracellular fate in human immortalized vascular endothelial cells (IVECs). The targeted copolymers displaying multiple copies of Esbp are bound to surface-associated E-selectin with affinity at the low nano-molar range, three orders of magnitude stronger than the free Esbp. In addition, the binding affinity of the HPMA-Esbp copolymers to E-selectin expressing IVECs was found to be 10-fold superior relative to non-targeted copolymers. Once bound, E-selectin facilitated rapid internalization and lysosomal trafficking of the copolymers. This lysosomotropism of HPMA-Esbp-bound DOX copolymers was then correlated with a 150-fold higher cytotoxicity relative to non-targeted HPMA-DOX conjugates. These findings strongly support the emerging role of E-selectin as a viable target for controlled drug delivery in cancer therapy.