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Immunotherapy is a valuable approach to cancer treatment as it is able to activate the immune system. However, the curative methods currently in clinical practice, including immune checkpoint inhibitors, present some limitations. Dendritic cell vaccination has been investigated as an immunotherapeutic strategy, and nanotechnology-based delivery systems have emerged as powerful tools for improving immunotherapy and vaccine development. A number of nanodelivery systems have therefore been proposed to promote cancer immunotherapy. This work aims to design a novel immunotherapy nanoplatform for the treatment of HER2 + breast cancer, and specially tailored chitosan-shelled nanobubbles (NBs) have been developed for the delivery of a DNA vaccine. The NBs have been functionalized with anti-CD1a antibodies to target dendritic cells (DCs). The NB formulations possess dimensions of approximately 300 nm and positive surface charge, and also show good physical stability up to 6 months under storage at 4 °C. In vitro characterization has confirmed that these NBs are capable of loading DNA with good encapsulation efficiency (82%). The antiCD1a-functionalized NBs are designed to target DCs, and demonstrated the ability to induce DC activation in both human and mouse cell models, and also elicited a specific immune response that was capable of slowing tumor growth in mice in vivo. These findings are the proof of concept that loading a tumor vaccine into DC-targeted chitosan nanobubbles may become an attractive nanotechnology approach for the future immunotherapeutic treatment of cancer. Graphical abstract

Development of resistance to chemo- and immunotherapies often occurs following treatment of melanoma brain metastasis (MBM). The brain microenvironment (BME), particularly astrocytes, cooperate toward MBM progression by upregulating secreted factors, among which we found that monocyte chemoattractant protein-1 (MCP-1) and its receptors, CCR2 and CCR4, were overexpressed in MBM compared with primary lesions. Among other sources of MCP-1 in the brain, we show that melanoma cells altered astrocyte secretome and evoked MCP-1 expression and secretion, which in turn induced CCR2 expression in melanoma cells, enhancing in vitro tumorigenic properties, such as proliferation, migration, and invasion of melanoma cells. In vivo pharmacological blockade of MCP-1 or molecular knockout of CCR2/CCR4 increased the infiltration of cytotoxic CD8+ T cells and attenuated the immunosuppressive phenotype of the BME as shown by decreased infiltration of Tregs and tumor-associated macrophages/microglia in several models of intracranially injected MBM. These in vivo strategies led to decreased MBM outgrowth and prolonged the overall survival of the mice. Our findings highlight the therapeutic potential of inhibiting interactions between BME and melanoma cells for the treatment of this disease.

Immunotherapy is a valuable approach for the treatment of cancer. Nanotechnology-based delivery systems emerged as a powerful tool for improving immunotherapeutics. Therefore, their association have been proposed to overcome some biopharmaceutical limitations of immunotherapy. This work aims at designing a novel immunotherapeutic nanoplatform for the treatment of HER2+ breast cancer. Here, purposely-tailored chitosan-shelled nanobubbles (NBs) were developed for the loading of DNA vaccine. The NBs were then functionalized with anti-CD1a antibody to target dendritic cells (DCs). The NB formulations showed sizes of about 300 nm and a good physical stability up to 6 months stored at 4 °C. The in vitro characterization confirmed that these NBs were able to load DNA with a good encapsulation efficiency (82%). The antiCD1a-functionalized NBs targeted to DCs demonstrated the capability to induce activation of DCs both in human and mouse models, and elicit a specific immune response able to delay tumor growth in vivo in mice. The results are the proof of concept that DC-targeted chitosan nanobubbles loaded with tumor vaccine may provide an attractive nanotechnology approach for the future immunotherapeutic treatment of cancer.

A low response rate, acquired resistance and severe side effects have limited the clinical outcomes of immune checkpoint therapy. Here, we show that combining cancer nanovaccines with an anti-PD-1 antibody (αPD-1) for immunosuppression blockade and an anti-OX40 antibody (αOX40) for effector T-cell stimulation, expansion and survival can potentiate the efficacy of melanoma therapy. Prophylactic and therapeutic combination regimens of dendritic cell-targeted mannosylated nanovaccines with αPD-1/αOX40 demonstrate a synergism that stimulates T-cell infiltration into tumours at early treatment stages. However, this treatment at the therapeutic regimen does not result in an enhanced inhibition of tumour growth compared to αPD-1/αOX40 alone and is accompanied by an increased infiltration of myeloid-derived suppressor cells in tumours. Combining the double therapy with ibrutinib, a myeloid-derived suppressor cell inhibitor, leads to a remarkable tumour remission and prolonged survival in melanoma-bearing mice. The synergy between the mannosylated nanovaccines, ibrutinib and αPD-1/αOX40 provides essential insights to devise alternative regimens to improve the efficacy of immune checkpoint modulators in solid tumours by regulating the endogenous immune response.

Summary: Melanoma is the deadliest skin cancer due to its high rate of metastasis, frequently to the brain. Brain metastases are incurable; therefore, understanding melanoma brain metastasis is of great clinical importance. We used a mouse model of spontaneous melanoma brain metastasis to study the interactions of melanomas with the brain microenvironment. We find that CXCL10 is upregulated in metastasis-associated astrocytes in mice and humans and is functionally important for the chemoattraction of melanoma cells. Moreover, CXCR3, the receptor for CXCL10, is upregulated in brain-tropic melanoma cells. Targeting melanoma expression of CXCR3 by nanoparticle-mediated siRNA delivery or by shRNA transduction inhibits melanoma cell migration and attenuates brain metastasis in vivo. These findings suggest that the instigation of pro-inflammatory signaling in astrocytes is hijacked by brain-metastasizing tumor cells to promote their metastatic capacity and that the CXCL10-CXCR3 axis may be a potential therapeutic target for the prevention of melanoma brain metastasis. : Melanoma brain metastases are incurable. Doron et al. find that astrocyte-secreted CXCL10 is functional in melanoma chemoattraction to the brain. CXCR3, the CXCL10 receptor, is upregulated in brain-seeking melanoma cells. Silencing CXCR3 expression attenuates brain metastasis, suggesting that the CXCL10-CXCR3 axis may be a therapeutic target for melanoma brain metastasis. Keywords: melanoma, brain metastasis, astrocytes, CXCL10, CXCR3, brain tropism

A novel unconventional supramolecular oligo-cationic structure (Agm6-M-PEG-OCH3) has been synthesized to yield high efficiency therapeutic oligonucleotide (ON) delivery. Agm6-M-PEG-OCH3 was obtaine...

Cyclodextrin-based nanosponges have been found to be promising drug delivery systems. This paper investigates an application that still needs to be studied in depth, that is, the oral delivery of peptides and proteins, choosing insulin as a case study. The nanosponge was synthesized by crosslinking β-cyclodextrins with pyromellitic dianhydride, adopting a top-down approach for its subsequent formulation. A physicochemical characterization, in-vitro and in-vivo tests were carried out on the formulation developed. It was nanometric (around 250 nm) with high negative zeta potential, mucoadhesion and swelling properties, good loading capability (about 14%) and encapsulation efficiency (above 90%). The in-vitro release of insulin was negligible at a gastric pH (below 2%) while sustained at an intestinal pH, thus showing a pH-sensitive behaviour of the nanosponge. The Caco-2 cell permeability assay proved that the intestinal permeation of insulin was enhanced when loaded inside the nanosponge. The in-vivo studies confirmed the presence of insulin in rat plasma and a marked hypoglycemic effect in diabetic mice after duodenal and oral administrations, respectively. These preliminary results are encouraging with a view to continuing to study this β-cyclodextrin nanosponge technology for the oral administration of insulin and extending this approach to other proteins of pharmaceutical interest.

Targeted therapies against cancer can relieve symptoms and induce remission, however, they often present limited duration of disease control, cause side effects and often induce acquired resistance. Therefore, there is a great motivation to develop a unique delivery system, targeted to the tumor, in which we can combine several active entities, increase the therapeutic index by reducing systemic exposure, and enhance their synergistic activity. To meet these goals, we chose the biocompatible and biodegradable poly(α,L-glutamic acid) (PGA) as a nanocarrier that facilitates extravasation-dependent tumor targeting delivery. The RAS/RAF/MEK/ERK pathway when aberrantly activated in melanoma, can lead to uncontrolled cell proliferation, induced invasion, and reduced apoptosis. Here, we selected two drugs targeting this pathway; a MEK1/2 inhibitor (selumetinib; SLM) and a modified BRAF inhibitor (modified dabrafenib; mDBF), that exhibited synergism in vitro. We synthesized and characterized our nanomedicine of PGA conjugated to SLM and mDBF (PGA-SLM-mDBF). PGA-SLM-mDBF inhibited the proliferation of melanoma cells and decreased their migratory and sprouting abilities without inducing a hemolytic effect. Moreover, the polymer-2-drugs conjugate exhibited superior anti-tumor activity in comparison with the two separate polymer-drug conjugates in vitro and with free drugs in a mouse model of primary melanoma and prolonged survival at a lower dose.

The majority of theranostic prodrugs reported so far relay information through a fluorogenic response generated upon release of the active chemotherapeutic agent. A chemiluminescence detection mode offers significant advantages over fluorescence, mainly due to the superior signal-to-noise ratio of chemiluminescence. Here we report the design and synthesis of the first theranostic prodrug monitored by a chemiluminescence diagnostic mode. As a representative model, we prepared a prodrug from the chemotherapeutic monomethyl auristatin E, which was modified for activation by β-galactosidase. The activation of the prodrug in the presence of β-galactosidase is accompanied by emission of a green photon. Light emission intensities, which increase with increasing concentration of the prodrug, were linearly correlated with a decrease in the viability of a human cell line that stably expresses β-galactosidase. We obtained sharp intravital chemiluminescent images of endogenous enzymatic activity in β-galactosidase-overexpressing tumor-bearing mice. The exceptional sensitivity achieved with the chemiluminescence diagnostic mode should allow the exploitation of theranostic prodrugs for personalized cancer treatment.

Nanotechnology-based strategies can dramatically impact the treatment, prevention and diagnosis of a wide range of diseases. Despite the unprecedented success achieved with the use of nanomaterials to address unmet biomedical needs and their particular suitability for the effective application of a personalized medicine, the clinical translation of those nanoparticulate systems has still been impaired by the limited understanding on their interaction with complex biological systems. As a result, unexpected effects due to unpredicted interactions at biomaterial and biological interfaces have been underlying the biosafety concerns raised by the use of nanomaterials. This review explores the current knowledge on how nanoparticle (NP) physicochemical and surface properties determine their interactions with innate immune cells, with particular attention on the activation of pattern-recognition receptors and inflammasome. A critical perspective will additionally address the impact of biological systems on the effect of NP on immune cell activity at the molecular level. We will discuss how the understanding of the NP-innate immune cell interactions can significantly add into the clinical translation by guiding the design of nanomedicines with particular effect on targeted cells, thus improving their clinical efficacy while minimizing undesired but predictable toxicological effects.

Polymer-directed enzyme prodrug therapy (PDEPT) and polymer enzyme liposome therapy (PELT) are two-step therapies developed to provide anticancer drugs site-selective intratumoral accumulation and release. Nanomedicines, such as polymer-drug conjugates and liposomal drugs, accumulate in the tumor site due to extravasation-dependent mechanism (enhanced permeability and retention - EPR - effect), and further need to cross the cellular membrane and release their payload in the intracellular compartment. The subsequent administration of a polymer-enzyme conjugate able to accumulate in the tumor tissue and to trigger the extracellular release of the active drug showed promising preclinical results. The development of polymer-enzyme, polymer-drug conjugates and liposomal drugs had undergone a vast advancement over the past decades. Several examples of enzyme mimics for in vivo therapy can be found in the literature. Moreover, polymer therapeutics often present an enzyme-sensitive mechanism of drug release. These nanomedicines can thus be optimal substrates for PDEPT and this review aims to provide new insights and stimuli toward the future perspectives of this promising combination.

Novel bioconjugates (Agm6-M-PEG-FA) for active oligonucleotide (ON) delivery have been developed by conjugating a cationic oligo-guanidyl star-like shaped “head” (Agm6-M) to a polymeric “tail” (PEG) terminating with folic acid (FA) as targeting agent or methoxy group (Agm6-M-PEG-FA and Agm6-M-PEG-OCH3, respectively). Gel electrophoresis showed that the bioconjugates completely associated with ONs at 3 nitrogen/phosphate (N/P) ratio. Studies performed with folate receptor (FR)-overexpressing HeLa cells, showed that optimal cell up-take was obtained with the 75:25 w/w Agm6-M-PEG-OCH3:Agm6-M-PEG-FA mixture. Dynamic light scattering and transmission electron microscopy showed that the polyplexes had size

Colorectal cancer (CRC) is among the five most commonly diagnosed cancers worldwide, constituting 6% of all cancers and the third leading cause of cancer death. CRC is the third and second most frequent cancer in men and women worldwide, accounting for 14% and 13% of all cancer incidence rates, respectively. CRC incidence is decreasing in older populations, but it has been significantly rising worldwide in adolescents and adults younger than 50 years old. Significant advances in the screening methods and surgical procedures have been underlying the reduction of the CRC incidence rate in older populations. However, there is an urgent demand for the development of alternative effective therapeutic options to overcome advanced metastatic CRC, while preventing disease recurrence. This review addresses the immune and CRC biology, summarizing the recent advances on the immune and/or therapeutic regimens currently in clinical use. We will focus on the emerging role of nanotechnology in the development of combinational therapies targeting and thereby regulating the function of the major players in CRC progression and immune evasion.

Chemiluminescence is being considered an effective imaging modality as it offers low background and high sensitivity. Recent discovery by our group has led to development of new phenoxy-dioxetane chemiluminescence luminophores, which are highly bright under physiological conditions. However, the current scope of probes based on these luminophores is limited, as they can only be turned on by phenol protecting group removal. Here we present a new chemiluminescence resonance energy transfer (CRET) system, Glow-CRET, in which light emission is triggered by proteolytic cleavage of a peptide substrate that links a dioxetane luminophore and a quencher. In order to compose such system, a new phenoxy-dioxetane luminophore, 7-HC-CL, was developed. This luminophore exhibits intense and persistent glow chemiluminescence; it undergoes very slow chemiexcitation, and it has the highest chemiluminescence quantum yield ever reported under physiological conditions. Based on 7-HC-CL, a Glow-CRET probe for matrix metalloproteinases, MMP-CL, was synthesized. Incubation of MMP-CL with its cognate protease resulted in 160-fold increase in chemiluminescence signal. MMP-CL was also able to detect matrix metalloproteinase activity in cancer cells with significantly higher signal-to-background ratio than an analogous fluorescence resonance energy transfer (FRET)-based probe. This work is expected to open new horizons in chemiluminescence imaging, as it enables to use the dioxetanes in ways that had not been possible. We anticipate that 7-HC-CL and future derivatives will be utilized not only for the construction of further Glow-CRET probes, but also for other applications, such as chemiluminescence tagging of proteins.

Glioblastoma Multiforme (GBM) is one of the most aggressive forms of all cancers. The median survival with current standard-of-care radiation and chemotherapy is about 14months. GBM is difficult to treat due to heterogeneity in cancer cell population. MicroRNA-based drugs have rapidly become a vast and burgeoning field due to the ability of a microRNA (miRNA) to target many genes involved in key cellular pathways. However, in vivo delivery of miRNA remains a crucial challenge for its therapeutic success. To bypass this shortcoming, we designed polymeric nanogels (NGs), which are based on a polyglycerol-scaffold, as a new strategy of miRNA delivery for GBM therapy. We focused on miR-34a, which is known for its key role in important oncogenic pathways and its tumor suppression ability in GBM and other cancers. We evaluated the capability of six NG derivatives to complex with miR-34a, neutralize its negative charge and deliver active miRNA to the cell cytoplasm. Human U-87 MG GBM cells treated with our NG-miR-34a nano-polyplexes showed remarkable downregulation of miR-34a target genes, which play key roles in the regulation of apoptosis and cell cycle arrest, and induce inhibition of cells proliferation and migration. Administration of NG-miR-34a nano-polyplexes to human U-87 MG GBM-bearing SCID mice significantly inhibited tumor growth as opposed to treatment with NG-negative control miR polyplex or saline. The comparison between different polyplexes highlighted the key features for the rational design of polymeric delivery systems for oligonucleotides. Taken together, we expect that this new therapeutic approach will pave the way for safe and efficient therapies for GBM.

We have developed a new difluoroalkyl ketal sulfinate salt reagent suitable for direct derivatization of heteroarene C-H bonds. The reagent is capable of introducing a ketone functional group on heteroarene bioactive compounds via a one-pot reaction. Remarkably, in three examples the ketone analog and its parent drug had almost identical cytotoxicity. In a representative example, the ketone analog was bioconjugated with a delivery vehicle via an acid-labile semicarbazone linkage and with a photolabile protecting group to produce the corresponding prodrug. Controlled release of the drug-ketone analog was demonstrated in vitro for both systems. This study provides a general approach to obtain taggable ketone analogs directly from bioactive heteroarene compounds with limited options for conjugation. We anticipate that this sodium ketal-sulfinate reagent will be useful for derivatization of other heteroarene-based drugs to obtain ketone-taggable analogs with retained efficacy.

Chemiluminescence offers advantages over fluorescence for bioimaging, since an external light source is unnecessary with chemiluminescent agents., Chemiluminescence offers advantages over fluorescence for bioimaging, since an external light source is unnecessary with chemiluminescent agents. This report demonstrates the first encapsulation of chemiluminescence phenoxy-adamantyl-1,2-dioxetane probes with trimethyl β-cyclodextrin. Clear proof for the formation of a 1 : 1 host–guest complex between the adamantyl-1,2-dioxetane probe and trimethyl β-cyclodextrin was provided by mass spectroscopy and NMR experiments. The calculated association constant of this host–guest system, 253 M–1, indicates the formation of a stable inclusion complex. The inclusion complex significantly amplified the light emission intensity relative to the noncomplexed probe under physiological conditions. Complexation of adamantyl-dioxetane with fluorogenic dye-tethered cyclodextrin resulted in light emission through energy transfer to a wavelength that corresponds to the fluorescent emission of the conjugated dye. Remarkably, the light emission intensity of this inclusion complex was approximately 1500-fold higher than that of the non-complexed adamantyl-dioxetane guest. We present the first demonstration of microscopic cell images obtained using a chemiluminescence supramolecular dioxetane probe and demonstrate the utility of these supramolecular complexes by imaging of enzymatic activity and bio-analytes in vitro and in vivo. We anticipate that the described chemiluminescence supramolecular dioxetane probes will find use in various biological applications.

RNA interference (RNAi) can contribute immensely to the area of personalized medicine by its ability to target any gene of interest. Nevertheless, its clinical use is limited by lack of efficient delivery systems. Polymer therapeutics can address many of the challenges encountered by the systemic delivery of RNAi, but suffer from inherent drawbacks such as polydispersity and batch to batch heterogeneity. These characteristics may have far-reaching consequences when dealing with therapeutic applications, as both the activity and the toxicity may be dependent on the length of the polymer chain. To investigate the consequences of polymers’ heterogeneity, we have synthesized two batches of aminated poly(α)glutamate polymers (PGAamine), differing in their degree of polymerization, but not in the monomer units or their conjugation. Isothermal titration calorimetry study was conducted to define the binding affinity of these polymers with siRNA. Molecular dynamics simulation revealed that Short PGAamine:siRNA polyplexes exposed a higher amount of amine moieties to the surroundings compared to Long PGAamine. This resulted in a higher zeta potential, leading to faster degradation and diminished gene silencing. Altogether, our study highlights the importance of an adequate physico-chemical characterization to elucidate the structure–function-activity relationship, for further development of tailor-designed RNAi delivery vehicles.

The modulation of immune checkpoint receptors has been one of the most successful, exciting, and explored approaches for cancer immunotherapy. Currently, several immune checkpoint modulators, mainly monoclonal antibodies, are showing remarkable results. However, the failure to show a response in most patients and the induction of severe immune-related adverse effects are the major drawbacks. Novel approaches concerning the development of immune modulatory small molecules have emerged as an alternative. Nevertheless, the lack of structural information about immune checkpoint receptors has hindered the rational design of those small-molecule modulators by preventing the use of methodologies such as computer-aided drug design. Herein, we provide an overview and critical analysis of the structural and dynamic details of immune checkpoint receptors (cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and glucocorticoid-induced TNFR-related protein (GITR)) and their interaction with known modulators. This knowledge is essential to advance the understanding of their binding mode and guide the design of novel effective targeted anticancer medicines.

RNAi therapeutics carried a great promise to the area of personalized medicine: the ability to target "undruggable" oncogenic pathways. Nevertheless, their efficient tumor targeting via systemic administration had not been resolved yet. Amphiphilic alkylated poly(α)glutamate amine (APA) can serve as a cationic carrier to the negatively-charged oligonucleotides. APA polymers complexed with siRNA to form round-shaped, homogenous and reproducible nano-sized polyplexes bearing ~50 nm size and slightly negative charge. In addition, APA:siRNA polyplexes were shown to be potent gene regulators in vitro. In light of these preferred physico-chemical characteristics, their performance as systemically-administered siRNA nanocarriers was investigated. Intravenously-injected APA:siRNA polyplexes accumulated selectively in tumors and did not accumulate in the lungs, heart, liver or spleen. Nevertheless, the polyplexes failed to induce specific mRNA degradation, hence neither reduction in tumor volume nor prolonged mice survival was seen.

Complete tumor removal during surgery has a great impact on patient survival. To that end, the surgeon should detect the tumor, remove it and validate that there are no residual cancer cells left behind. Residual cells at the incision margin of the tissue removed during surgery are associated with tumor recurrence and poor prognosis for the patient. In order to remove the tumor tissue completely with minimal collateral damage to healthy tissue, there is a need for diagnostic tools that will differentiate between the tumor and its normal surroundings. Methods: We designed, synthesized and characterized three novel polymeric Turn-ON probes that will be activated at the tumor site by cysteine cathepsins that are highly expressed in multiple tumor types. Utilizing orthotopic breast cancer and melanoma models, which spontaneously metastasize to the brain, we studied the kinetics of our polymeric Turn-ON nano-probes. Results: To date, numerous low molecular weight cathepsin-sensitive substrates have been reported, however, most of them suffer from rapid clearance and reduced signal shortly after administration. Here, we show an improved tumor-to-background ratio upon activation of our Turn-ON probes by cathepsins. The signal obtained from the tumor was stable and delineated the tumor boundaries during the whole surgical procedure, enabling accurate resection. Conclusions: Our findings show that the control groups of tumor-bearing mice, which underwent either standard surgery under white light only or under the fluorescence guidance of the commercially-available imaging agents ProSense® 680 or 5-aminolevulinic acid (5-ALA), survived for less time and suffered from tumor recurrence earlier than the group that underwent image-guided surgery (IGS) using our Turn-ON probes. Our "smart" polymeric probes can potentially assist surgeons' decision in real-time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome.

RNA interference is one of the most promising fields in modern medicine to treat several diseases, ranging from cancer to cardiac diseases, passing through viral infections and metabolic pathologies. Since the discovery of the potential therapeutic properties of non-self oligonucleotides, it was clear that it is important to develop delivery systems that are able to increase plasma stability and bestow membrane-crossing abilities to the oligonucleotides in order to reach their cytoplasmic targets. Polymer therapeutics, among other systems, are widely investigated as delivery systems for therapeutic agents, such as oligonucleotides. Physico-chemical characterization of the supramolecular polyplexes obtained upon charge interaction or covalent conjugation between the polymeric carrier and the oligonucleotides is critical. Appropriate characterization is fundamental in order to predict and understand the in vivo silencing efficacy and to avoid undesired side effects and toxicity profile. Shedding light on the physico-chemical and in vitro requirements of a polyplex leads to an efficient in vivo delivery system for RNAi therapeutics. In this review, we will present the most common techniques for characterization of obtained polymer/oligonucleotide polyplexes and an up-to-date state of the art in vivo preclinical and clinical studies. This is the first review to deal with the difficulties in appropriate characterization of small interfering RNA (siRNA) or microRNA (miRNA) polyplexes and conjugates which limit the clinical translation of this promising technology.

Glioblastoma is a highly aggressive brain tumor. Current standard-of-care results in a marginal therapeutic outcome, partly due to acquirement of resistance and insufficient blood-brain barrier (BBB) penetration of chemotherapeutics. To circumvent these limitations, we conjugated the chemotherapy paclitaxel (PTX) to a dendritic polyglycerol sulfate (dPGS) nanocarrier. dPGS is able to cross the BBB, bind to P/L-selectins and accumulate selectively in intracranial tumors. We show that dPGS has dual targeting properties, as we found that P-selectin is not only expressed on tumor endothelium but also on glioblastoma cells. We delivered dPGS-PTX in combination with a peptidomimetic of the anti-angiogenic protein thrombospondin-1 (TSP-1 PM). This combination resulted in a remarkable synergistic anticancer effect on intracranial human and murine glioblastoma via induction of Fas and Fas-L, with no side effects compared to free PTX or temozolomide. This study shows that our unique therapeutic approach offers a viable alternative for the treatment of glioblastoma.

‘Polymer Enzyme Liposome Therapy’ (PELT) is a two-step anticancer approach in which a liposomal drug and polymer-phospholipase conjugate are administered sequentially to target the tumour interstitium by the enhanced permeability and retention effect, and trigger rapid, local, drug release. To date, however, the concept has only been described theoretically. We synthesised two polymer conjugates of phospholipase C (PLC) and A2 (PLA2) and evaluated their ability to trigger anthracycline release from the clinically used liposomes, Caelyx® and DaunoXome®. N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer–PLC and a dextrin-PLA2 were synthesised and their enzymatic activity characterised. Doxorubicin release from polyethyleneglycol-coated (PEGylated) Caelyx® was relatively slow (

The ability to specifically silence genes using RNA interference (RNAi) has wide therapeutic applications for the treatment of disease. Numerous studies have demonstrated global gene and protein signatures distinguishing malignant and nonmalignant tissues. This worldwide pursuit of optimal cancer targets has so far provided a wide list of potential targets for each cancer type and for each patient, for which RNAi-based therapies can be applied. Nevertheless, due to poor stability of RNAi molecules in physiological conditions and their inability to cross cellular membranes, the delivery of siRNA and microRNA (miRNA) in vivo holds a great challenge and remains a crucial issue for their therapeutic success. Supramolecular carriers are often used in order to improve the physicochemical and biopharmaceutical properties of RNAi. Nano-sized delivery systems enable the accumulation of drugs and oligonucleotides (ONTs) in angiogenesis-dependent areas due to the enhanced permeability and retention (EPR) effect, and are able to cross cellular membranes and release the siRNA/miRNA only inside the target cell. In addition, a targeting moiety can increase the selectivity and specific uptake in the target tissue. Several vehicles (dendrimers, nanoparticles, liposomes, polyplex, lipoplex, polymeric nanoconjugates) are being developed for siRNA/miRNA delivery. These vehicles provide an important tool for exploiting the full potential of ONTs as therapeutic agents. In this review we will focus on the polymer-based approaches to deliver siRNA to cancer in vivo.

MicroRNAs (miRNAs) are small noncoding RNAs found to govern nearly every biological process. They frequently acquire a gain or a loss of function in cancer, hence playing a causative role in the development and progression of cancer. There are major obstacles on the way for the successful delivery of miRNA, which include low cellular uptake of the RNA and endosomal escape, immunogenicity, degradation in the bloodstream, and rapid renal clearance. The delivered miRNA needs to be successfully routed to the target organ, enter the cell and reach its intracellular target in an active form. Consequently, in order to exploit the promise of RNA interference, there is an urgent need for efficient methods to deliver miRNAs. These can be divided into three main categories: complexation, encapsulation, and conjugation. In this review, we will discuss the special considerations for miRNA delivery for cancer therapy, focusing on nonviral delivery systems: lipid, polymeric, and inorganic nanocarriers.

It has been two decades since cationic polymers were introduced to the world of oligonucleotides delivery. However, the optimal physicochemical properties to make them a successful delivery vehicle are yet unknown. An ideal system became particularly interesting and necessary with the introduction of RNA interference as a promising therapeutic approach. Such nanocarrier should overcome challenges such as low plasma stability, poor cellular internalization and endosomal escape to induce gene silencing. To that end, we synthesized a library of biodegradable aminated poly(α)glutamate varied by amine moieties. In an attempt to elucidate the structure-function relationship, our polyplexes were physicochemically characterized and their silencing activity and cytotoxicity were evaluated. We found several structures that demonstrated improved cellular internalization. These candidates silenced gene expression to less than 50% of their initial levels, while being safe to cells and mice. Based on our research, an improved and promising tailor-designed siRNA delivery platform can be developed.

Small interfering RNA (siRNA) can silence the expression of a targeted gene in a process known as RNA interference (RNAi). As a consequence, RNAi has immense potential as a novel therapeutic approach in cancer targeted therapy. However, successful application of siRNA for therapeutic purposes is challenging due to its rapid renal clearance, degradation by RNases in the bloodstream, poor cellular penetration, immunogenicity and aggregation in the blood. In addition, the few oligonucleotide-based nanomedicines that reached clinical trials either go to the liver following systemic administration or are applied topically. Treatment of solid tumors requires selective distribution of siRNA to the target tissue, hence there is an unmet medical need for an efficacious and safe nano-sized delivery system for their clinical use. To overcome these hurdles, we have designed, synthesized and physico-chemically characterized a novel nanocarrier based on aminated poly(α)glutamate (PGAamine). This cathepsin B-biodegradable polymer interacts electrostatically with the siRNA to form a nano-sized polyplex stable in plasma. Treatment with PGAamine-Rac1 siRNA polyplex (siRac1-polyplex) caused specific gene silencing by 80% in HeLa and SKOV-3 human ovarian adenocarcinoma cells as opposed to PGAamine-control non-targeting siRNA polyplex (siCtrl-polyplex) leading to inhibition of cell migration and wound healing abilities. A stepwise dose escalation was performed in order to determine the in vivo maximum tolerated dose (MTD). This was followed by intraperitoneal administration of siRac1-polyplex to mCherry-labeled ovarian adenocarcinoma-bearing mice leading to preferred tumor accumulation of siRac1 (8-fold) which resulted in 38% Rac1 knockdown. Furthermore, the polyplex was administered intravenously to lung carcinoma-bearing mice in which it caused 33% Rac1 knockdown. These promising results led to efficacy studies administering systemic treatment with an anticancer siRNA, siPlk1-polyplex, which inhibited tumor growth by 73% and 87% compared with siCtrl-polyplex or saline-treated mice, respectively, leading to prolonged overall survival. These findings represent the first time that a polyaminated poly(α)glutamate polymer is used for an efficacious and safe tumor delivery of RNAi following systemic administration.

In recent years, siRNA technology has emerged as a promising strategy for gene silencing in cancer therapy. We have designed novel CD44-targeted polyion complexes (PICs) composed of poly(ethylene glycol)-block-polyethylenimine (PEG-b-PEI) and laminin-derived peptides (mA5G27D or mA5G27F) for in vivo siRNA delivery and gene silencing in tumors. The full-length A5G27 peptide (RLVSYNGIIFFLK), from which mA5G27D and mA5G27F are derived, binds to CD44v3 and CD44v6 and inhibits tumor cell migration, invasion, and angiogenesis. Thus, when attached to the surface of PICs, A5G27-based peptides can serve both as targeting ligands to navigate siRNA molecules directly to CD44-overexpressing tumors, and as anti-migratory agents to inhibit tumor progression. The mA5G27D- or mA5G27F-harboring PEG-b-PEI copolymers strongly condensed siRNA molecules into nanosized PICs presenting positive surface charges, low in vitro cytotoxicity, and high serum stability. mA5G27D- or mA5G27F-bearing PICs demonstrated high efficacy and selectivity in delivering siRAC1 into CD44-overexpressing cells, thereby silencing RAC1 mRNA and protein levels in such cells. These PICs presented substantial anti-migratory features in vitro and accumulated significantly in SK-OV-3 tumor-bearing mice, following 3 sequential intraperitoneal (i.p.) injections. Treatment of mice with 8 or 9 sequential parenteral (intravenous, (i.v.) or i.p.) injections of mA5G27F-PEG-b-PEI/siRNA efficiently inhibited tumor growth in two different CD44-overexpressing tumor mouse models (A549 and SK-OV-3), regardless of the type of siRNA (siPLK1 or siLUC) used. The results thus reveal the potential utility of this system for targeted delivery of siRNA molecules into solid tumors to prolong the survival time of mice, while at the same time reducing potential toxicity.

Introduction The brain microenvironment consists of various cell types such as astrocytes, microglia, neurons and brain endothelial cells. It sustains the normal brain functions and the structures of the cerebral capillaries that form the blood brain barrier (BBB). During tumour progression, malignant cells release vascular endothelial growth factor and other cytokines that increase the permeability of blood vessels allowing them to intravasate into the circulation. In addition, the cells can take advantage of the patho-physiology of the tumour angiogenenic leaky blood vessels at distant organs and extravasate from them at inflamed sites. Such activated site can be the brain parenchyma, which may become a pro-inflammatory metastatic niche. Although little is known about the precise mechanisms and the factors that contribute to the brain metastatic process, there is increasing evidence that astrocytes are involved by mediating a neuroinflammation. They respond by releasing pro-inflammatory cytokines that compromise the integrity of the BBB, increasing the permeability of the capillary endothelium and preparing a metastatic niche for melanoma colonisation. Material and methods We established three spontaneous melanoma brain metastasis mouse models that present a pro-inflammatory microenvironment and a pro-metastatic niche preceding the colonisation of melanoma to the brain. siRNA targeting specific chemokines, neutralizating antibody, or a CRISPR/Cas9 system were used to downregulate the expression and secretion of chemokines for further in vitro studies. Results and discussions We identified MCP-1, a pro-inflammatory chemokine, implicated in the paracrine interaction between melanoma cells and the brain. The positive staining for GFAP and MCP-1 in the tumour area suggests a cooperation between melanoma and the tumour microenvironment, in which MCP-1 plays a pro-tumorigenic role during cell migration. Astrocytes-secreted MCP-1 expression levels increase when co-cultured with melanoma cells or their conditioned media. Downregulation of MCP-1 leads to decreased migration of melanoma cells in vitro . Moreover, neutralising antibody-targeting MCP-1 reduces melanoma invasion in matrigel towards sprouting aortas. Conclusion Activated astrocytes promote the migration of circulating melanoma cells to the brain by increased secretion of pro-inflammatory cytokines, such as MCP-1. Consequently, investigating the changes in melanoma and astrocytes gene-expression could be the key for interfering and preventing melanoma colonisation to the brain.

Ligand-receptor mediated targeting may affect differently the performance of supramolecular drug carriers depending on the nature of the nanocarrier. In this study, we compare the selectivity, safety and activity of doxorubicin (Dox) entrapped in liposomes versus Dox conjugated to polymeric nanocarriers in the presence or absence of a folic acid (FA)-targeting ligand to cancer cells that overexpress the folate receptor (FR). Two pullulan (Pull)-based conjugates of Dox were synthesized, (FA-PEG)-Pull-(Cyst-Dox) and (NH2-PEG)-Pull-(Cyst-Dox). The other delivery systems are Dox loaded PEGylated liposomes (PLD, Doxil®) and the FR-targeted version (PLD-FA) obtained by ligand post-insertion into the commercial formulation. Both receptor-targeted drug delivery systems (DDS) were shown to interact in vitro specifically with cells via the folate ligand. Treatment of FR-overexpressing human cervical carcinoma KB tumor-bearing mice with three-weekly injections resulted in slightly enhanced anticancer activity of PLD-FA compared to PLD and no activity for both pullulan-based conjugates. When the DDS were administered intravenously every other day, the folated-Pull conjugate and the non-folated-Pull conjugate displayed similar and low antitumor activity as free Dox. At this dosing regimen, the liposome-based formulations displayed enhanced antitumor activity with an advantage to the non-folated liposome. However, both liposomal formulations suffered from toxicity that was reversible following treatment discontinuation. Using a daily dosing schedule, with higher cumulative dose, the folated-Pull conjugate strongly inhibited tumor growth while free Dox was toxic at this regimen. For polymeric constructs, increasing dose intensity and cumulative dose strongly affects the therapeutic index and reveals a major therapeutic advantage for the FR-targeted formulation. All DDS were able to abrogate doxorubicin-induced cardiotoxicity. This study constitutes the first side-by-side comparison of two receptor-targeted ligand-bearing systems, polymer therapeutics versus nanoparticulate systems, evaluated in the same mouse tumor model at several dosing regimens.

A novel polysaccharide bioconjugate was designed to selectively target breast cancer bone metastases using a bisphosphonate moiety (alendronate, ALN). Paclitaxel (PTX) was first covalently conjugated to pullulan (Pull) through a Cathepsin K-sensitive tetrapeptide spacer followed by a self-immolative aminobenzyl alcohol spacer to obtain Pull-(GGPNle-φ-PTX). ALN was then conjugated to the polymeric backbone of Pull-(GGPNle-φ-PTX) via a PEG spacer. The final bioconjugate Pull-(GGPNle-φ-PTX)-(PEG-ALN) was found to assemble into colloidal spherical structures, which were physically and chemically stable under physiological conditions. In vitro studies showed that Pull-(GGPNle-φ-PTX)-(PEG-ALN) had strong affinity for hydroxyapatite, which simulates the bone tissue. Paclitaxel was rapidly released from the bioconjugate by Cathepsin K cleavage under pathological conditions. All studies performed using human MDA-MB-231-BM (bone metastases-originated clone), murine 4T1 breast cancer cells, murine K7M2, and human SAOS-2 osteosarcoma cells showed that the bioconjugate exerted an enhanced antiproliferative activity compared to the conjugate without the ALN. Furthermore, the nanoconjugate inhibited the migration of cancer cells and further displayed potent anti-angiogenic activity. In conclusion, the results showed that this conjugate has an excellent potential for selective treatment of bone neoplasms such as breast cancer bone metastases and osteosarcoma.

The standard of care for cancer patients comprises more than one therapeutic agent. Treatment is complex since several drugs, administered by different routes, need to be coordinated, taking into consideration their side effects and mechanisms of resistance. Drug delivery systems (DDS), such as polymers and liposomes, are designed to improve the pharmacokinetics and efficacy of bioactive agents (drugs, proteins or oligonucleotides), while reducing systemic toxicity. Using DDS for co-delivery of several agents holds great potential since it targets simultaneously synergistic therapeutic agents increasing their selective accumulation at the tumor site and enhancing their activity allowing administration of lower doses of each agent, thus reducing their side effects. Taken together, implementation of smart DDS will hopefully result in increased patient's compliance and better outcome. This review will focus on the latest developments of combination therapy for cancer using DDS.

The ability to detect pathways that malignant tissue depends on, combined with the specific gene-knockdown ability of siRNA/miRNA, may revolutionize cancer treatment. The capability of providing personalized care to cancer patients allows therapy to be specifically tailored for each case. Several studies of malignant and nonmalignant tissues have been performed in the context of global protein interaction networks in order to find the optimal targets, providing a list of potential gene and protein targets for each cancer type and for each patient. Nevertheless, due to poor stability of RNAi molecules in physiological conditions and its inability to cross cellular membranes, the in vivo delivery of siRNA and miRNA holds a great challenge and remains a crucial issue for its therapeutic success. Supramolecular carriers are often used in order to improve the physicochemical and biopharmaceutical properties of RNAi. Nanoscale drug delivery systems will enable the accumulation of the drugs in the tumors due to the enhanced permeability and retention (EPR) effect, and release the siRNA/miRNA only inside the target cell. In addition, a targeting moiety can increase the selectivity and specific uptake in the target tissue. Several vehicles (dendrimers, nanoparticles, polyplex, lipoplex, polymeric nanoconjugates) are being developed for siRNA/miRNA delivery. These vehicles provide an important tool for exploiting the full potential of nucleic acids as therapeutic agents. In this chapter, we will review the different approaches to deliver oligonucleotides in vivo.

Polymer conjugation is an efficient approach to improve the delivery of drugs and biological agents, both by protecting the body from the drug (by improving biodistribution and reducing toxicity) and by protecting the drug from the body (by preventing degradation and enhancing cellular uptake). This review discusses the journey that polymer therapeutics make through the body, following the ADME (absorption, distribution, metabolism, excretion) concept. The biological factors and delivery system parameters that influence each stage of the process will be described, with examples illustrating the different solutions to the challenges of drug delivery systems in vivo.

Two new anticancer polymer therapeutics were designed for tumour cell targeting. The bioconjugates were synthesised by pullulan derivatisation with either doxorubicin or doxorubicin and folic acid. Pullulan was activated by periodate oxidation and functionalised by reductive conjugation with cysteamine and 1.9 kDa PEG(NH(2))(2). The cysteamine thiol groups were conjugated to doxorubicin through a pH-sensitive hydrazone spacer while the pending PEG-NH(2) functions of one derivatised pullulan batch were conjugated to folic acid to obtain one of the two polymer therapeutics. The reaction intermediates and the final products were characterised by mass spectrometry, UV-vis analysis and reverse phase and gel permeation chromatography. The folic acid-free derivative [(NH(2) PEG)-Pull-(Cyst-Dox)] contained 6.3% (w/w) doxorubicin while the folic acid-doxorubicin-coupled derivative [(FA-PEG)-Pull-(Cyst-Dox)] contained 6% (w/w) doxorubicin and 4.3% (w/w) folic acid. Photon correlation spectroscopy showed that (NH(2) PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) assembled into particles of about 150 and 100 nm diameter, respectively. The two bioconjugates displayed similar drug release profiles either at pH 7.4 buffer or in plasma, where less than 20% of doxorubicin was released within three days. At pH 5.5, both conjugates underwent complete drug release in about 40 h. In vitro studies carried out with KB tumour cells over-expressing folic acid receptor showed that both free doxorubicin and (FA-PEG)-Pull-(Cyst-Dox) were rapidly taken up by the cells, while the internalisation of the non-folated derivative was significantly slower. Cell viability studies did not show relevant difference between the two bioconjugates. After 72 h of incubation with folic acid receptor non-expressing MCF7 cells, the IC(50) values of doxorubicin, (NH(2)PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) were 0.3 μM, 1.2 μM and 3.1 μM, respectively. After incubation with KB cells over-expressing folic acid receptor, the IC(50) values were 0.4 μM, 1.8 μM and 1.1 μM, respectively. Pharmacokinetic studies showed that 4 h after intravenous administration of the conjugates to Balb/c mice about 40% of the administered drug equivalent dose was present in the bloodstream while in the case of unconjugated doxorubicin, 80% of the drug was cleared within 30 min. These findings suggest that the novel doxorubicin-pullulan bioconjugates possess suitable properties for passive tumour targeting. On the other hand, folic acid conjugation has been found to have limited effect on selective cell up-take.

Supramolecular conjugation techniques have been developed to produce novel nanosized systems by assembling materials with diverse physicochemical and biological features. These techniques have been adapted to obtain innovative bioconjugates to deliver drugs with poor biopharmaceutical properties and nano-devices with potential “theranostic” activity. Supramolecular drug delivery systems include polymer therapeutics such as drug–polymer bioconjugates, and colloidal carriers such as micelles, liposomes, polyplexes, and organic and inorganic nanoparticles. By virtue of their wide array of chemical composition and properties, polymers represent key elements for the construction of novel supramoelcular formulations. Polymer bioconjugation is a fledged technique for fabrication of protein–polymer conjugates. PEGylation, in particular, produces derivatives with enhanced pharmacokinetic, immunological, and stability properties as compared to the parent protein. Over the years, new methods have been set up to obtain site-directed polymer conjugation. In this review we report few grafting to and growing from PEGylation examples for the preparation of therapeutically effective protein bioconjugates. Supramolecular formulations with unique properties can be also obtained by assembling functional polymers, targeting agents, physicochemical modifiers, and biomodulators. These systems may be designed for disease tissue disposition and cell recognition/penetration. Cyclodextrins, for example, have been functionalized with polyethylene glycol and folic acid to produce tumor-targeted drug carriers. Interesting results have been obtained with this novel class of drug delivery systems. In addition, responsive polymers have been conjugated to gold nanoparticles to endow a new colloidal platform with triggerable cell disposition properties, which can be exploited either in biomedicine or diagnosis.

A new end-tailored monomethoxypoly(ethylene glycol) (PEG) for site-directed protein conjugation was synthesized according to a three-step procedure: (1) linear 20 kDa PEG-NH(2) was conjugated to 12-(Boc-amino)dodecanoic acid; (2) PEG-NHCO(CH(2))(11)-Boc was deprotected by TFA treatment; (3) PEG-NHCO(CH(2))(11)-NH(2) was conjugated to 6-maleimidohexanoic acid to yield PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal (PEG-C(18)-Mal). The chemical intermediates as well as the final product were purified by solvent precipitation/extraction and characterized by (1)H NMR spectroscopy and colorimetric analysis. The synthesis procedure yielded over 90% activated product [PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal/PEG-NH(2) molar ratio, %]. Both PEG-C(18)-Mal and the commercial maleimido activated 20 kDa linear PEG (PEG-Mal) were used for conjugation to (17)Cys of recombinant human granulocyte colony stimulating factor (rh-G-CSF). Under denaturing conditions, at pH 7.0, both activated PEGs yielded over 90% protein conjugation. Under native conditions, about 55% and 7% PEGylated protein were obtained with PEG-C(18)-Mal and PEG-Mal, respectively. Circular dichroism analysis showed that the PEGylation does not induce detectable alteration of the protein secondary structure. On the other hand, the PEGylation conditions were found to affect significantly the protein stability. The derivatives obtained either with the two polymers by unfolding/refolding process or with PEG-Mal under native conditions displayed rapid aggregation with half-life ranging from 30 to 90 min. The derivative obtained with PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal in the absence of guanidinium chloride displayed remarkably higher stability with aggregation half-life of about 60 h.