Targeting Intracranial Tumours with a Combination of RNA and Chemotherapy.

Glioblastoma multiforme (GBM) is a fast-growing and aggressive brain tumour, which remains largely resistant to treatment; the prognosis for patients is poor, with a median survival time of about 12-18 months, post diagnosis. In an effort to bring more efficacious treatments to patients, we targeted the down regulation of ITCH, an E3 ligase that is overexpressed in a variety of cancers, and which inhibits P73, a tumour suppressor gene. 6-O-glycolchitosan (GC) was used to deliver siRNA ITCH (GC60-siRNA-ITCH) and gemcitabine via the nose to brain route in CD-1 nude mice which had previously been implanted intracranially with U87-MG-luc2 cells. Prior to this in vivo study, an in vitro study established the synergistic effect of siRNA-ITCH in combination with a chemotherapy drug-gemcitabine. A downregulation of ITCH, an upregulation of p73 and enhanced apoptosis were observed in vitro in U87-MG cells, using qPCR, Western blot analysis, confocal laser scanning microscopy, flow cytometry and cytotoxicity assays. When GC60-siRNA-ITCH was combined with gemcitabine, there was a resultant decrease in cell proliferation in vitro. In CD1 mice, the administration of siRNA-ITCH (7 doses of 0.081 mg/kg) alone did not significantly affect animal survival (increasing mean survival from 29 to 33 days when compared to untreated animals), whereas intranasal gemcitabine had a significant effect on survival (increasing survival from 29 to 45 days when compared to untreated animals, p < 0.01). The most significant effect was seen with combination therapy (GC60-siRNA-ITCH plus gemcitabine), where survival increased by 89%, increasing from 29 to 54 days (p < 0.01). Our data demonstrate that siRNA chemosensitises brain tumours to gemcitabine and that the nose-to-brain delivery route may be a viable route for the treatment of intracranial tumours.

Quaternary Ammonium Palmitoyl Glycol Chitosan (GCPQ) Loaded with Platinum-Based Anticancer Agents-A Novel Polymer Formulation for Anticancer Therapy.

Quaternary ammonium palmitoyl glycol chitosan (GCPQ) has already shown beneficial drug delivery properties and has been studied as a carrier for anticancer agents. Consequently, we synthesised cytotoxic platinum(IV) conjugates of cisplatin, carboplatin and oxaliplatin by coupling via amide bonds to five GCPQ polymers differing in their degree of palmitoylation and quaternisation. The conjugates were characterised by 1H and 195Pt NMR spectroscopy as well as inductively coupled plasma mass spectrometry (ICP-MS), the latter to determine the amount of platinum(IV) units per GCPQ polymer. Cytotoxicity was evaluated by the MTT assay in three human cancer cell lines (A549, non-small-cell lung carcinoma; CH1/PA-1, ovarian teratocarcinoma; SW480, colon adenocarcinoma). All conjugates displayed a high increase in their cytotoxic activity by factors of up to 286 times compared to their corresponding platinum(IV) complexes and mostly outperformed the respective platinum(II) counterparts by factors of up to 20 times, also taking into account the respective loading of platinum(IV) units per GCPQ polymer. Finally, a biodistribution experiment was performed with an oxaliplatin-based GCPQ conjugate in non-tumour-bearing BALB/c mice revealing an increased accumulation in lung tissue. These findings open promising opportunities for further tumouricidal activity studies especially focusing on lung tissue.

High-capacity glycol chitosan-based nanoemulsion for efficient delivery of disulfiram.

Disulfiram (DS) is an anti-alcoholism drug capable of acting against important and hard-to-treat cancers. The drug's relative instability and variable absorption/distribution have led to its variable pharmacokinetics and suboptimal exposure. Hence, it was hypothesised that a nano-enabled form of DS might be able to overcome such limitations. Encapsulation of the labile DS was achieved with quaternary ammonium palmitoyl glycol chitosan (GCPQ) to form a high-capacity, soybean oil-based DS-GCPQ nanoemulsion. DS-GCPQ showed capability of oil-loading up to 50% v/v for a stable entrapment of high drug content. With increasing oil content (10 to 50% v/v), the mean particle size and polydispersity index were also increased (166 to 351 nm and 0.14 to 0.22, respectively) for a given amount of GCPQ. Formulations showed a highly positive particle surface charge (50.9 ± 1.3 mV), contributing to the colloidal stability of the individual particles. DS-GCPQ showed marked cytotoxicity against pancreatic cancer cell lines with enhanced activity in the presence of copper. An intravenous pharmacokinetic study of DS-GCPQ in vivo showed improved plasma drug stability with a DS half-life of 17 min. Prolonged survival was seen in tumour-bearing animals treated with DS-GCPQ supplemented with copper. In conclusion, DS-GCPQ nanoemulsion has the potential to be developed further for cancer therapeutic purposes.

Platinum(IV)-Loaded Degraded Glycol Chitosan as Efficient Platinum(IV) Drug Delivery Platform.

A new class of anticancer prodrugs was designed by combining the cytotoxicity of platinum(IV) complexes and the drug carrier properties of glycol chitosan polymers: Unsymmetrically carboxylated platinum(IV) analogues of cisplatin, carboplatin and oxaliplatin, namely (OC-6-44)-acetatodiammine(3-carboxypropanoato)dichloridoplatinum(IV), (OC-6-44)-acetaodiammine(3-carboxypropanoato)(cyclobutane-1,1-dicarboxylato)platinum(IV) and (OC-6-44)-acetato(3-carboxypropanoato)(1R,2R-cyclohexane-1,2-diamine)oxalatoplatinum(IV) were synthesised and conjugated via amide bonding to degraded glycol chitosan (dGC) polymers with different chain lengths (5, 10, 18 kDa). The 15 conjugates were investigated with 1H and 195Pt NMR spectroscopy, and average amounts of platinum(IV) units per dGC polymer molecule with ICP-MS, revealing a range of 1.3-22.8 platinum(IV) units per dGC molecule. Cytotoxicity was tested with MTT assays in the cancer cell lines A549, CH1/PA-1, SW480 (human) and 4T1 (murine). IC50 values in the low micromolar to nanomolar range were obtained, and higher antiproliferative activity (up to 72 times) was detected with dGC-platinum(IV) conjugates in comparison to platinum(IV) counterparts. The highest cytotoxicity (IC50 of 0.036 ± 0.005 µM) was determined in CH1/PA-1 ovarian teratocarcinoma cells with a cisplatin(IV)-dGC conjugate, which is hence 33 times more potent than the corresponding platinum(IV) complex and twice more potent than cisplatin. Biodistribution studies of an oxaliplatin(IV)-dGC conjugate in non-tumour-bearing Balb/C mice showed an increased accumulation in the lung compared to the unloaded oxaliplatin(IV) analogue, arguing for further activity studies.

Brain Gene Silencing with Cationic Amino-Capped Poly(ethylene glycol) Polyplexes.

Therapeutic gene silencing in the brain is usually achieved using highly invasive intracranial administration methods and/or comparatively toxic vectors. In this work, we use a relatively biocompatible vector: poly(ethylene glycol) star-shaped polymer capped with amine groups (4APPA) via the nose to brain route. 4APPA complexes anti- itchy E3 ubiquitin protein ligase (anti-ITCH) siRNA to form positively charged (zeta potential +15 ± 5 mV) 150 nm nanoparticles. The siRNA-4APPA polyplexes demonstrated low cellular toxicity (IC50 = 13.92 ± 6 mg mL-1) in the A431 cell line and were three orders of magnitude less toxic than Lipofectamine 2000 (IC50 = 0.033 ± 0.04 mg mL-1) in this cell line. Cell association and uptake of fluorescently labelled siRNA bound to siRNA-4APPA nanoparticles was demonstrated using fluorescent activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), respectively. Gene silencing of the ITCH gene was observed in vitro in the A431 cell line (65% down regulation when compared to the use of anti-ITCH siRNA alone). On intranasal dosing with fluorescently labelled siRNA-4APPA polyplexes, fluorescence was seen in the cells of the olfactory bulb, cerebral cortex and mid-brain regions. Finally, down regulation of ITCH was seen in the brain cells (54 ± 13% ITCH remaining compared to untreated controls) in a healthy rat model, following intranasal dosing of siRNA-4APPA nanoparticles (0.15 mg kg-1 siRNA twice daily for 3 days). Gene silencing in the brain may be achieved by intranasal administration of siRNA- poly(ethylene glycol) based polyplexes.

Ultrasmall-in-Nano: Why Size Matters.

Gold nanoparticles (AuNPs) are continuing to gain popularity in the field of nanotechnology. New methods are continuously being developed to tune the particles' physicochemical properties, resulting in control over their biological fate and applicability to in vivo diagnostics and therapy. This review focuses on the effects of varying particle size on optical properties, opsonization, cellular internalization, renal clearance, biodistribution, tumor accumulation, and toxicity. We review the common methods of synthesizing ultrasmall AuNPs, as well as the emerging constructs termed ultrasmall-in-nano-an approach which promises to provide the desirable properties from both ends of the AuNP size range. We review the various applications and outcomes of ultrasmall-in-nano constructs in vitro and in vivo.

Gene Targeting to the Cerebral Cortex Following Intranasal Administration of Polyplexes.

Gene delivery to the cerebral cortex is challenging due to the blood brain barrier and the labile and macromolecular nature of DNA. Here we report gene delivery to the cortex using a glycol chitosan-DNA polyplex (GCP). In vitro, GCPs carrying a reporter plasmid DNA showed approximately 60% of the transfection efficiency shown by Lipofectamine lipoplexes (LX) in the U87 glioma cell line. Aiming to maximise penetration through the brain extracellular space, GCPs were coated with hyaluronidase (HYD) to form hyaluronidase-coated polyplexes (GCPH). The GCPH formulation retained approximately 50% of the in vitro hyaluronic acid (HA) digestion potential but lost its transfection potential in two-dimensional U87 cell lines. However, intranasally administered GCPH (0.067 mg kg-1 DNA) showed high levels of gene expression (IVIS imaging of protein expression) in the brain regions. In a separate experiment, involving GCP, LX and naked DNA, the intranasal administration of the GCP formulation (0.2 mg kg-1 DNA) resulted in protein expression predominantly in the cerebral cortex, while a similar dose of intranasal naked DNA led to protein expression in the cerebellum. Intranasal LX formulations did not show any evidence of protein expression. GCPs may provide a means to target protein expression to the cerebral cortex via the intranasal route.

Tissue-Engineering the Fibrous Pancreatic Tumour Stroma Capsule in 3D Tumouroids to Demonstrate Paclitaxel Response.

Pancreatic cancer is a unique cancer in that up to 90% of its tumour mass is composed of a hypovascular and fibrotic stroma. This makes it extremely difficult for chemotherapies to be delivered into the core of the cancer mass. We tissue-engineered a biomimetic 3D pancreatic cancer ("tumouroid") model comprised of a central artificial cancer mass (ACM), containing MIA Paca-2 cells, surrounded by a fibrotic stromal compartment. This stromal compartment had a higher concentration of collagen type I, fibronectin, laminin, and hyaluronic acid (HA) than the ACM. The incorporation of HA was validated with alcian blue staining. Response to paclitaxel was determined in 2D MIA Paca-2 cell cultures, the ACMs alone, and in simple and complex tumouroids, in order to demonstrate drug sensitivity within pancreatic tumouroids of increasing complexity. The results showed that MIA Paca-2 cells grew into the complex stroma and invaded as cell clusters with a maximum distance of 363.7 µm by day 21. In terms of drug response, the IC50 for paclitaxel for MIA Paca-2 cells increased from 0.819 nM in 2D to 3.02 nM in ACMs and to 5.87 nM and 3.803 nM in simple and complex tumouroids respectively, indicating that drug penetration may be significantly reduced in the latter. The results demonstrate the need for biomimetic models during initial drug testing and evaluation.

Down-regulation of GP130 signaling sensitizes bladder cancer to cisplatin by impairing Ku70 DNA repair signaling and promoting apoptosis.

Chemoresistance is one of the barriers for the development of bladder cancer treatments. Previously, we showed that glycoprotein-130 (GP130) is overexpressed in chemoresistant bladder cancer cells and that knocking down GP130 expression reduced cell viability. In our current work, we showed that down-regulation of GP130 sensitized bladder cancer cells to cisplatin-based chemotherapy by activating DNA repair signaling. We performed immunohistochemistry and demonstrated a positive correlation between the levels of Ku70, an initiator of canonical non-homologous end joining repair (c-NHEJ) and suppressor of apoptosis, and GP130 in human bladder cancer specimens. GP130 knockdown by SC144, a small molecule inhibitor, in combination with cisplatin, increased the number of DNA lesions, specifically DNA double-stranded breaks, with a subsequent increase in apoptosis and reduced cell viability. Furthermore, GP130 inhibition attenuated Ku70 expression in bladder and breast cancer cells as well as in transformed kidney cells. In addition, we fabricated a novel polymer-lipid hybrid delivery system to facilitate GP130 siRNA delivery that had a similar efficiency when compared with Lipofectamine, but induced less toxicity.

Hyaluronidase Coated Molecular Envelope Technology Nanoparticles Enhance Drug Absorption via the Subcutaneous Route.

Parenteral chemotherapy is usually administered intravenously, although patient preference and health economics suggest the subcutaneous (sc) route could be an attractive alternative. However, due to the low aqueous solubility of hydrophobic drugs and injection volume limitations, the total amount of drug that can be administered in a single sc injection is frequently insufficient. We have developed hyaluronidase coated nanoparticles (NPs) that efficiently encapsulate such drugs, thus addressing both issues and allowing sufficient amounts of hydrophobic drug to be administered and absorbed effectively. CUDC-101, a poorly water-soluble multitargeted anticancer drug that simultaneously inhibits the receptor tyrosine kinases (RTKs) EGFR and HER2, as well as histone deacetylase (HDAC), was encapsulated in polymeric Molecular Envelope Technology (MET) NPs. The role of polymer chemistry, formulation parameters, and coating with hyaluronidase (HYD) on MET-CUDC-101 NP formulations was examined and optimized to yield high drug loading and colloidal stability, and, after freeze-drying, stable storage at room temperature for up to 90 days. The pharmacokinetic studies in healthy rats showed that plasma AUC0-24h after sc administration correlates tightly with formulation physical chemistry, specifically in vitro colloidal stability. Compared to uncoated NPs, the HYD-coating doubled the drug plasma exposure. In a murine A431 xenograft model, the coated HYD-MET-CUDC-101 NPs at a dose equivalent to 90 mg kg-1 CUDC-101 increased the survival time from 15 days (control animals treated with hyaluronidase alone) to 43 days. Polymer MET nanoparticles coated with hyaluronidase enabled the subcutaneous delivery of a hydrophobic drug with favorable therapeutic outcomes.

Clustering superparamagnetic iron oxide nanoparticles produces organ-targeted high-contrast magnetic resonance images.

Aim: Superparamagnetic iron oxide nanoparticles (SPIONs) have been used as magnetic resonance imaging (MRI) contrast agents; however, a number of T2-weighted imaging SPIONs have been withdrawn due to their poor clinical contrast performance. Our aim was to significantly improve SPION T2-weighted MRI contrast by clustering SPIONs within novel chitosan amphiphiles. Methods: Clustering SPIONs was achieved by encapsulation of hydrophobic-coated SPIONs with an amphiphilic chitosan polymer (GCPQ). Results: Clustering increases the spin-spin (r2) to spin-lattice (r1) relaxation ratio (r2/r1) from 3.0 to 79.1, resulting in superior contrast. Intravenously administered clustered SPIONs accumulated only in the liver and spleen; with the reduction in T2 relaxation confined, in the liver, to the extravascular space, giving clear MRI images of the liver vasculature.

Nose-to-Brain Delivery.

The global prevalence of neurologic disorders is rising, and yet we are still unable to deliver most drug molecules, in therapeutic quantities, to the brain. The blood brain barrier consists of a tight layer of endothelial cells surrounded by astrocyte foot processes, and these anatomic features constitute a significant barrier to drug transport from the blood to the brain. One way to bypass the blood brain barrier and thus treat diseases of the brain is to use the nasal route of administration and deposit drugs at the olfactory region of the nares, from where they travel to the brain via mechanisms that are still not clearly understood, with travel across nerve fibers and travel via a perivascular pathway both being hypothesized. The nose-to-brain route has been demonstrated repeatedly in preclinical models, with both solution and particulate formulations. The nose-to-brain route has also been demonstrated in human studies with solution and particle formulations. The entry of device manufacturers into the arena will enable the benefits of this delivery route to become translated into approved products. The key factors that determine the efficacy of delivery via this route include the following: delivery to the olfactory area of the nares as opposed to the respiratory region, a longer retention time at the nasal mucosal surface, penetration enhancement of the active through the nasal epithelia, and a reduction in drug metabolism in the nasal cavity. Indications where nose-to-brain products are likely to emerge first include the following: neurodegeneration, post-traumatic stress disorder, pain, and glioblastoma.

Nanomedicines for the Delivery of Biologics.

A special symposium of the Academy of Pharmaceutical Sciences Nanomedicines Focus Group reviewed the current status of the use of nanomedicines for the delivery of biologics drugs. This meeting was particularly timely with the recent approval of the first siRNA-containing product Onpattro™ (patisiran), which is formulated as a lipid nanoparticle for intravenous infusion, and the increasing interest in the use of nanomedicines for the oral delivery of biologics. The challenges in delivering such molecules were discussed with specific emphasis on the delivery both across and into cells. The latest developments in Molecular Envelope Technology® (Nanomerics Ltd, London, UK), liposomal drug delivery (both from an academic and industrial perspective), opportunities offered by the endocytic pathway, delivery using genetically engineered viral vectors (PsiOxus Technologies Ltd, Abingdon, UK), Transint™ technology (Applied Molecular Transport Inc., South San Francisco, CA, USA), which has the potential to deliver a wide range of macromolecules, and AstraZeneca's initiatives in mRNA delivery were covered with a focus on their uses in difficult to treat diseases, including cancers. Preclinical data were presented for each of the technologies and where sufficiently advanced, plans for clinical studies as well as early clinical data. The meeting covered the work in progress in this exciting area and highlighted some key technologies to look out for in the future.

T-shaped Peptide Amphiphiles Self Assemble into Nanofiber Networks.

BACKGROUND: Conventional nanofiber forming peptide amphiphiles comprise a beta sheet forming, short peptide sequence with an alkyl chain attached at one terminus. We report the selfassembly of a peptide amphiphile possessing a mid-chain located alkyl substituent (a T-shaped peptide amphiphile) into nanofiber networks. METHOD: Peptide synthesis was carried out using standard 9-fluorenylmethoxycarbonyl solid phase peptide synthesis protocols, followed by covalent attachment of the alkyl chains to yield target peptide amphiphiles. Self-assembly was then studied using electron microscopy and coarse-grained molecular dynamics simulations. RESULTS: T-shaped peptide amphiphiles self-assembled into nanofibers just like linear peptide amphiphiles, but then unlike linear peptide amphiphiles, T-shaped peptide amphiphiles formed inter-fiber associations and ultimately nanofiber networks. CONCLUSION: Changing the position of the alkyl chain in a peptide amphiphile from the terminal end of the peptide to the middle part of the peptide, to form a T-shaped peptide amphiphile, does not disrupt the molecular interactions required for the self-assembly of the peptide amphiphiles into nanofibers.

Limiting the level of tertiary amines on polyamines leads to biocompatible nucleic acid vectors.

We have designed an efficient, synthetic nucleic acid vector, which is relatively non-toxic. [N-(2-ethylamino)-6-O-glycolchitosan - EAGC] polymers were 10-50 fold less toxic than Lipofectamine 2000, able to complex DNA, mRNA and siRNA into positively charged (zeta potential=+40 - 50mV), 50-450nm nanoparticles. The level of tertiary amine N-2-ethylamino substitution (DStert) was inversely proportional to the IC50 of the EAGC polymers in the A431 cell line: IC50=6.18DStert-0.9, r2=0.9991. EAGC polyplexes were stable against a heparin challenge, able to protect the nucleic acids from nuclease degradation and achieve levels of transfection comparable to Lipofectamine 2000 formulations. The relative biocompatibility of the vector allowed 10 fold higher doses of DNA (1µg compared to 0.1µg per well with Lipofectamine 2000) and siRNA (10.7µg per well vs 1.3µg with Lipofectamine 2000) to be applied to cells, when compared to Lipofectamine 2000. Finally intranasal application of EAGC - siRNA complexes resulted in siRNA transfer to the neurons of the olfactory bulb.

Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels - A Strategy for Brain Cancer Treatments.

PURPOSE: The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression. METHODS: Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg(-1)) or ethanolic lomustine (6.5 mg kg(-1)) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg(-1)) or ethanolic lomustine (daily 1.2 mg kg(-1) - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated. RESULTS: The MET formulation resulted in modest brain targeting (brain/ bone AUC0-4h ratios for MET and ethanolic lomustine = 0.90 and 0.53 respectively and brain/ liver AUC0-4h ratios for MET and ethanolic lomustine = 0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes. CONCLUSIONS: Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity.

Strategies to deliver peptide drugs to the brain.

Neurological diseases such as neurodegeneration, pain, psychiatric disorders, stroke, and brain cancers would greatly benefit from the use of highly potent and specific peptide pharmaceuticals. Peptides are especially desirable because of their low inherent toxicity. The presence of the blood brain barrier (BBB), their short duration of action, and their need for parenteral administration limits their clinical use. However, over the past decade there have been significant advances in delivering peptides to the central nervous system. Angiopep peptides developed by Angiochem (Montreal, Canada), transferrin antibodies developed by ArmaGen (Santa Monica, USA), and cell penetrating peptides have all shown promise in delivering therapeutic peptides across the BBB after intravenous administration. Noninvasive methods of delivering peptides to the brain include the use of chitosan amphiphile nanoparticles for oral delivery and nose to brain strategies. The uptake of the chitosan amphiphile nanoparticles by the gastrointestinal epithelium is important for oral peptide delivery. Finally protecting peptides from plasma degradation is integral to the success of most of these peptide delivery strategies.

Nanofiber-based delivery of therapeutic peptides to the brain.

The delivery of therapeutic peptides and proteins to the central nervous system is the biggest challenge when developing effective neuropharmaceuticals. The central issue is that the blood-brain barrier is impermeable to most molecules. Here we demonstrate the concept of employing an amphiphilic derivative of a peptide to deliver the peptide into the brain. The key to success is that the amphiphilic peptide should by design self-assemble into nanofibers wherein the active peptide epitope is tightly wrapped around the nanofiber core. The nanofiber form appears to protect the amphiphilic peptide from degradation while in the plasma, and the amphiphilic nature of the peptide promotes its transport across the blood-brain barrier. Therapeutic brain levels of the amphiphilic peptide are achieved with this strategy, compared with the absence of detectable peptide in the brain and the consequent lack of a therapeutic response when the underivatized peptide is administered.